CELLS, COMPOSITIONS AND METHODS

The present invention relates to induced T-to-Natural-Killer cells [herein “ITNK” cells], methods for their production and use of such cells, as well as methods for producing T cells.

Natural killer (NK) cells are a type of cytotoxic lymphocyte that constitute a major component of the innate immune system. NK cells play a major role in the rejection of tumors and cells infected by viruses and microbes. NK-cells are large granular lymphocytes (LGL) and constitute cells differentiated from stem cells or multipotent progenitors. The molecular mechanisms controlling the development of different cell types from stem cells is not fully understood.

STATEMENTS OF INVENTION

The invention provides a method of producing induced T-to-Natural-Killer [ITNK] cells from T cells and/or pro-T cells, the method comprising modulating the activity and/or effect of the Bcl11b gene and/or Bcl11b protein present in a T cell or pro-T cell, thereby converting said T cell and/or pro-T cell to an ITNK cell.

The invention provides a method of producing target T cells and/or target pro-T cells, the method comprising modulating the activity and/or effect of at least one Bcl11b gene and/or protein product present in a T cell and/or pro-T cell, and converting said T cell and/or pro-T cell to said target T cells and/or target pro-T cells.

The invention provides an ITNK cell obtainable, or obtained, from a T cell or pro-T cell. Suitably the T cell or pro-T cell includes a Bcl11b gene and/or gene product the activity and/or effect of which has been modulated so that the T cell or pro-T cell is capable of conversion to a ITNK cell.

The invention also relates to mature activated T cells in which Bcl11b expression is downregulated or absent (hereafter referred to as TBcl11b-cells), for use in medicine, such as prophylaxis or treatment of disease. The invention also relates to isolated or purified mature activated T cells in which Bcl11b expression is downregulated or absent.

The invention provides a target T cell or target pro-T cell obtainable, or obtained, from a T cell or pro-T cell respectively. Suitably the target cell comprises at least one Bcl11b gene and/or gene product the activity and/or effect of which has been modulated when compared to the wild type cell, so that the target T cell or target pro-T cell is capable of conversion to an ITNK cell. Wild type cells in the context of this disclosure does not refer to cancerous or transformed cells.

The invention provides a pharmaceutical composition comprising ITNK cells, or target T cells, or target pro-T cells together with a pharmaceutically acceptable excipient.

The invention provides ITNK cells or target T cells or target pro-T cells for use in therapy.

The invention provides a method of treating a human or non-human mammal subject suffering from, or susceptible to disease such as cancer or viral infection, the method comprising administering to the subject a therapeutically effective amount of ITNK cells or target T cells/pro-T cells, preferably ITNK cells or target T cells/pro-T cells which are derived from T cells or pro-T cells that have been obtained from that subject.

The invention provides a method of treating a human or non-human mammal subject suffering from, or susceptible to disease such as cancer or viral infection, the method comprising administering to the subject a therapeutically effective amount of a compound which modulates or inhibits the expression, activity and/or effect of Bcl11b gene or protein in T cells or pro-T cells and leads to the conversion of these T cells or pro-T cells to ITNK cells.

The invention provides an assay for identifying a target with which the Bcl11b gene product and/or protein product interacts or has an effect thereon, which assay comprises modulating the activity of a Bcl11b gene and/or gene product and monitoring the interaction or effect on a potential downstream target. Optionally a downstream target thus identified is modified to cause or assist in ITNK cell production.

The invention also relates to upstream modulators of Bcl11b activity, suitably those capable of causing or assisting in the conversion of T cells or pro-T cells to ITNK cells or target T cells/pro T cells. The invention also relates to methods for identification of upstream modulators of Bcl11b comprising identification of compounds that are able affect Bcl11b gene or protein expression or activity or effect, suitably as assessed by an effect of the upstream modulator on ITNK formation as disclosed herein.

In one aspect the invention relates to the use of factors which regulate the Bcl11b gene or protein expression or activity, or which are functionally downstream of the Bcl11b gene or protein, or which are functionally upstream of the Bcl11b gene, to effect the conversion of T cells to ITNK cells, and to the use of modulators of these factors to effect the conversion of T cells to ITNK cells.

The invention provides an assay for identification of a compound which assists in the reprogramming of T cells or pro-T cells to ITNK cells, the method comprising contacting T cells or pro-T cells with a test compound and monitoring or selecting for the conversion of T cells to ITNK cells or target T/pro T cells.

The invention provides an assay for identification of a mutation which results in or contributes to the reprogramming of T cells or pro-T cells to ITNK cells, the method comprising mutagenesis of T cells or pro-T cells and monitoring or selecting for the conversion of T cells to ITNK cells, followed by identification of the location of the mutation.

The invention provides an assay for identification of a compound which assists in the reprogramming of T cells to ITNK cells, the method comprising screening for compounds that bind to the Bcl11b DNA or RNA or the Bcl11b protein, and assessing whether said compounds are able to promote the conversion of T cells to ITNK cells.

The invention further provides use of compounds so discovered in the conversion of T cells or pro-T cells to ITNK cells.

The invention further provides a non-human animal carrying ITNK cells, and/or target T cells or target pro-T cells.

FIGURES AND TABLES

FIG. 1. Bcl11b is essential for T cell development and for maintaining T cell identity.

(A) Flow cytometry profiles of cultured DN1 and DN2 thymocytes (+OHT) in the absence of IL-2.

(B) Flow cytometry profiles of cultured flox/flox DN3 thymocytes (±OHT) supplemented with IL-2.

(C) Killing of OP9-DLI stromal cells by OHT-treated flox/flox DN3 thymocytes.

(D) DNA from purified NKp46⁺ cells was prepared and subjected to PCR to detect DJ (top) and VDJ (bottom) recombination at the TCRβlocus.

(E-G) Microarray analysis of gene expression in NKp46⁺CD3⁺ ITNK cells from DN3 thymocytes.

(E) Two-way hierarchical cluster map of the array data.

(F) and (G) qRT-PCR validation of gene expression of selected genes among ITNKs, LAKs and DN3 cells.

FIG. 2. Efficient reprogramming of T cells to ITNKs.

(A) Representative flow cytometry profiles of ITNKs reprogrammed from single flox/flox DN3 cells.

(B) PCR genotyping of Bcl11b deletion in two representative T cell (T1, T2) and ITNK (I1, I2) wells.

(C) DJ recombination at the TCRβ locus of five ITNK wells (I1-I5) showing unique DJ recombination.

(D) Giemsa stain of parental DN3 thymocytes (T) and ITNK cells.

(E) and (e) Transmission electron micrographs of an ITNK cell.

(F) Cytotoxicity of ITNKs (labeled as “+OHT”) and LAKs measured in standard ⁵¹Cr release assays with B16F10, RMA and RMA-S tumor cell targets at the indicated effector-to-target (E:T) ratios. −OHT: flox/flox T cells.

FIG. 3. ITNKs reprogrammed in vivo were potent tumour cell killers.

(A) Flow cytometric analysis of thymocytes and splenocytes from OHT treated flox/flox and flox/+ mice.

(B) Analysis of ITNKs from thymic γδ T cells in OHT treated flox/flox mice.

(C) ITNKs production in Rag2^−/−Il2rg^−/− recipients injected with flox/flox DP thymocytes.

(D) Ex vivo expansion of ITNKs in IL-2 from splenocytes of the recipient mice.

(d) Ex vivo expansion of in vivo reprogrammed iTNK cells starting from splenotypes of four Rag2^−/−Il2γc^−/− recipient mice.

(E) The ex vivo-expanded ITNKs (labeled as “+OHT”) were used in ⁵¹Cr release killing assays with B16F10, RMA and RMA-S tumor cell targets at the indicated effector-to-target (E:T) ratios. −OHT: flox/flox T cells.

(F) ITNKs prevented tumour metastasis. Rag2^−/−Il2rg^−/− recipients transplanted with treated (+OHT) or untreated (−OHT) flox/flox DP thymocytes or PBS and subsequently injected intravenously with 50,000 B16F10 melanoma cells.

(G) In vivo iTNKs effectively eliminated B16F10 melanoma cells in mice.

FIG. 4. Bcl11b is a direct downstream target gene of Notch signaling.

(A). Bcl11b protein in T cells following OHT treatment detected by Western blot.

(B) Schematic of the Bcl11b locus showing putative CSL binding sites (BS) and that of an irrelevant control binding site (CTL).

(C) Genomic DNA was prepared from immunoprecipitation of thymocytes, using CSL or control IgG antibodies, and was amplified using primers flanking the putative CSL or the control binding sites at the Bcl11b locus.

FIG. 5. Generation of the Bcl11b-tdTomato reporter mouse.

(A) The tdTomato cassette was targeted to the 3′ UTR of the Bcl11b locus.

(B) Insertion of the tdTomato cassette at the Bcl11b 3′ UTR did not affect T cell development.

FIG. 6. Detection of Bcl11b expression in hematopoietic lineages using the Bcl11b-tdTomato reporter mice.

(A) CD4 CD8 double negative (DN; DN1-DN4) thymocyte subsets.

(B) Double positive (DP) thymocytes (CD4⁺CD8⁺), splenic CD4⁺ and CD8⁺ T cells, thymic γδ T cells, and splenic NKT cells (CD3⁺CD1d⁺).

(C) Bone marrow B cells (CD19⁺B220⁺) and myeloid cells (CD11b⁺Gr-1⁺).

(D) Splenic (CD3), and thymic (CD3CD4CD8) NK cells.

(E) qRT-PCR of Bcl11b expression in sorted splenic naïve (CD44⁻CD62L⁺) and activated (CD44⁺CD62L⁻) T cells population.

(F) Quantification of Bcl11b expression in naïve and activated T cells in the Bcl11b^td/+ mice.

FIG. 7. Strategies for identification of cell populations for flow sorting and analysis.

(A) Identification of double negative (DN) thymocyte (DN1-DN4) populations defined by Lin and expression of CD25 and CD44.

(B) Identification of γδ T cells.

(C) Identification of NKT cells in the spleen by first gating (or, prior to FACS sorting, magnetically depleting) out B cells.

(D) Identification of NK precursors and NK cell subsets cells.

(E) Thymic NK cells were defined as NK1.1⁺CD127⁺ thymocytes.

(F) Identification of naïve (CD44⁻CD62L⁺) and activated (CD44⁺CD62L⁻) T cells.

FIG. 8. In vitro analysis of Bcl11b-deficient T cells.

(A) Schematic diagram of the Bcl11b conditional knockout allele. (B) Experimental design for the analysis of Bcl11b-deficient DN thymocytes. (C) NKp46⁺CD3 cells from DN1 and DN20HT-treated flox/flox thymocytes did not express TCRβ.

(D) Homozygous Bcl11b deletion in ITNK (NKp46⁺CD3) but not in T (NKp46⁻CD3⁺) cell populations from DN1 and DN2 cultures.

(E) No NKp46⁺ cells but T cells were obtained from untreated flox/flox thymocytes.

(F) NKp46⁺TCRβ⁻ cells from OHT-treated DN1 and DN2 flox/flox thymocytes in the absence of IL-2 or IL-15 cultured on OP9 stromal cells.

(G) NKp46⁺TCRβ⁻ cells were detected in OHT-treated DN3 flox/flox, but not flox/+, thymocytes in T cell media.

(H) Reprogramming of Bcl11b-deficient DN3 thymocytes to NKp46⁺ cells in myeloid cell culture condition.

(I) Reprogramming of Bcl11b-deficient DN3 thymocytes to NKp46⁺CD19⁻ cells in B cell culture condition.

(J) Venn diagram comparison of the upregulated (>2-fold) genes between LAK vs DN3 (green) and ITNK vs DN3 (purple).

(K) ITNKs from DP flox/flox thymocytes treated with OHT and cultured on OP9-DL1 in the presence of IL-2.

(L) ITNKs from splenic flox/flox CD8⁺ T cells treated with OHT cultured on OP9-DL1 in the presence of IL-2.

FIG. 9. Characterization of in vitro reprogrammed ITNK phenotype.

(A) and (a) Experimental design for reprogramming of single DN3 thymocytes to ITNK.

(B-C) Expression of intracellular and NK cell surface markers by the reprogrammed ITNK from DN3 thymocytes in vitro.

(D) Expression of NK cell markers by ITNKs reprogrammed from Bcl11b-deficient DP thymocytes in vitro.

(E) ITNKs did not express CD127 and thus were not thymic NK cells.

(F) Analysis of CD27 and CD11b in bulk-cultured ITNKs reprogrammed from DN3 thymocytes.

FIG. 10. Analysis of in vivo reprogrammed ITNK cells in the flox/flox mouse.

(A) Experimental design for the analysis of in vivo reprogrammed ITNK cells.

(B) PCR of Bcl11b deletion in ITNK (NKp46⁺CD3⁺ and NKp46⁺CD3⁻) cell populations in flox/flox mice.

(C) Flow cytometric analysis of CD4 and CD8 expression in NKp46⁺ ITNKs.

(D) Flow cytometric analysis of cells following ex vivo expansion of whole thymocytes or splenocytes from OHT treated mice.

(E) Flow cytometric analysis of CD1d-restriced NKT cells in thymus and spleen.

(F) Analysis of CD1d-restricted cells in the ex vivo-expanded ITNK culture.

(G) qRT-PCR analysis of several key T or NK cell-associated genes in CD8⁺ T cells, CD8⁺ ITNKs and LAKs.

(H) Splenocytes from flox/flox or flox/+ mice treated with Tamoxifen were stained with NKp46, NK1.1, CD8 and CD3 to confirm expression of CD3 on ITNKs.

FIG. 11. In vivo reprogrammed ITNKs from DP thymocytes prevented tumour metastasis.

(A) Experimental design for the analysis of in vivo reprogramming of DP thymocytes to ITNKs.

(B) Most ITNKs in the spleen were CD8⁺.

(C) ITNKs had complete Bcl11b deletion whereas donor derived NKp46 cells still retained at least one copy of the foxed allele. (D) ITNKs were also found in bone marrow and peripheral blood.

(E) Expression of additional NK cell surface markers on the in vivo reprogrammed ITNKs.

(F) ITNKs prevented tumour metastasis. Rag2^−/−Il2rg^−/− recipients were transplanted with treated (+OHT) or untreated (−OHT) flox/flox DP thymocytes or PBSand subsequently injected intravenously with 5×10⁴B16F10 melanoma cells.

(G) Plot shows inverse correlation between the percentage of ITNK cells (squares) obtained from recipient mice following in vivo reprogramming and tumor challenge and the number of lung colonies (circles) observed.

FIG. 12. A working model showing that Bcl11b acts downstream of Notch signaling and promotes T cell development and maintains T cell identity.

FIG. 13.

- a. Expression of Bcl11b in thymocytes from Bcl11b-lacZ knock-in mice using the fluorescent substrate FDG.
- b. Detection of Bcl11b expression in the five DN1 subpopulations.
- c. Top, acute loss of Bcl11b caused DN1 thymocytes to express NK-specific genes. Bottom, deleting Bcl11b in DN2 thymocytes gave rise to the same phenotype of converting to NK-like cells.

FIG. 14.

- a. Left panel: different double negative (DN) thymocyte populations; Right panel: five subpopulations of DN1 thymocytes.
- b. Flow chart of analyzing Bcl11b-deficient DN1 thymocytes.

FIG. 15.

- a. Double positive (DP) thymocytes expressed NKp46 after Bcl11b deletion.
- b. Purified CD8 single positive cells (−OHT) proliferated on OP9-DL1 stromal cells. They did not express NKp46. Once Bcl11b was deleted, 38% of the cells now expressed NKp46 which killed the stromal cells.
- c. Purified CD4 single positive cells (−OHT) growing in T cell media (left). Bcl11b deletion (+OHT) caused these CD4 T cells to express NKp46 and NKG2D.
  
  Table 1. Comparison of gene expression profiles of ITNK, DN3 and LAK cells in microarray analysis.
  
  Table 2. Comparison of cell surface receptor repertoire of LAK and ITNKs.
  
  Table 3. Changes of gene expression profiles in thymocytes at 24 hours and 48 hours after deletion of Bcl11b in microarray analysis.
  
  Table 4. PCR primers

GENERAL DESCRIPTION

T cells develop from early T cell progenitors which have NK and myeloid potential through a series of steps, known as DN1 (double negative stage 1), DN2, DN3 and DN4, DP (double positive), and then into single positive (SP) mature CD4 or CD8 positive T cells. There are many different types of T cells including helper, cytotoxic and regulatory T cells.

Activation of T cells is brought about by interaction with appropriate antigen MHC complex. For example, helper T cells become activated when they are presented with peptide antigens by MHC class II molecules that are expressed on the surface of Antigen Presenting Cells (APCs). The process of activation of T cells is known to the skilled person.

In the present invention we show that modulation of the Bcl11b gene/gene pathway allows T cells and pro-T cells to be reprogrammed into induced T-to-Natural-Killer (ITNK) cells. Data is presented for DN, DP and SP T cells. In addition, we show that such ITNK cells are effective in the amelioration of disease in an in vivo model and do not shown any adverse effects on the animal model. The Bcl11b protein in mice and humans is highly conserved, also, T cell development in both humans and mice is very similar. This information indicates that findings in mice may be extrapolated to the treatment or prevention of human diseases.

Reference to Bcl11b herein includes any Bcl11b homologues that may be identified in other species, suitably homologues that when deleted in whole or in part can result in the generation of ITNK cells in that species.

The invention provides a method of producing induced T-to-Natural-Killer [ITNK] cells from T cells and/or pro-T cells, the method comprising modulating the activity and/or effect of at least one Bcl11b gene and/or gene product present in a T cell or pro-T cell, thereby converting said T cell and/or pro-T cell to an ITNK cell.

Reference to T cells includes, for example, DN, DP or SP T cells such as DN1, DN2, DN3, DN4, DP thymocytes, CD4 or CD8 single positive mature T cells or γδ-T cells. Reference to pro-T cells includes common lymphoid precursor cells, stem cells and other non-T hematopoietic cells or non-hematopoietic cells which can be converted to T cells

Target T cells or target proT cells are cells which have the potential to convert into ITNK cells as a result of the modulation of the activity and/or effect of at least one Bcl11b gene and/or gene product in the T cell or pro T cell, but which have not yet converted to give the ITNK like phenotype.

Modulation of the activity or the effect of the Bcl11b gene or protein is suitably achieved by inhibiting the activity or effect of Bcl11b, either directly or indirectly.

Suitably the inhibition comprises deletion of at least part of said Bcl11b gene, suitably at least a single exon of the Bcl11b gene, suitably at least exon 4 of the Bcl11b gene. In one aspect all of the gene is deleted. Suitably, inhibition of the activity or effect of Bcl11b may be achieved by disrupting the function of Bcl11b through insertion a genetic cassette to the Bcl11b locus. Suitably, inhibition of the activity or effect of Bcl11b may be achieved by modulating epigenetic changes at the Bcl11b locus or those gene loci that regulate Bcl11b or are regulated by Bcl11b. Suitably, inhibition of the activity or effect of Bcl11b may be achieved by using antibodies (conventional or peptide Abs) to neutralize gene products of Bcl11b or its upstream or down-stream genes.

In another aspect the invention relates to genomes comprising a Bcl11b conditional knockout (cko) allele, preferably T cells or pro T cells having such a conditional mutation. The generation of conditional alleles allows the growth of cells under conditions in which Bcl11b is expressed, followed by growth under different conditions that cause the Bcl11b gene to be deleted and the ITNK phenotype to be expressed. Thus the invention also relates to a process for the induction of ITNK cells comprising activation of a conditional mutation, suitable to modulation of the activity and/or effect of at least one Bcl11b gene and/or gene product in the T cell or pro T cell.

In one aspect the modulation is directly at the level of Bcl11b gene expression, where the expression of Bcl11b is preferably inhibited to stimulate ITNK cell production. In one aspect the sequences of the Bcl11b gene, or control sequences such as promoter or enhancer regions, may be mutated, such that transcription or translation are adversely affected.

In one aspect control of the expression of Bcl11b is achieved by control of mRNA expression or protein translation. In one aspect the expression of Bcl11b is modulated by antisense RNA or the use of small interfering RNA (sRNA) or miRNA.

In one aspect modulation of Bcl11b is at the protein level. The activity of the Bcl11b protein may be modulated, preferably inhibited, by Bcl11b binding proteins, for example.

In one aspect modulating or inhibiting of the activity and/or effect of said Bcl11b gene or protein produces a downstream modulation in a biological pathway (s) in which said Bcl11b protein is involved. In one aspect said downstream modulation regulates the presence and/or activity and/or effect of a downstream target in said biological pathway. Assessment of downstream elements regulated by Bcl11b allows alternative targets to be identified which may control ITNK production from T cells and pro-T cells. The present invention also relates to identification of downstream targets—see below.

The invention provides an ITNK cell obtainable, or obtained, from a T cell or pro-T cell, including from stem cells or progenitors, wherein the T cell or pro-T cell includes a Bcl11b gene and/or gene product the activity and/or effect of which has been modulated so that the T cell or pro-T cell is capable of conversion to a ITNK cell.

The invention also provides a target T cell or target pro-T cell including at least one Bcl11b gene and/or gene product the activity and/or effect of which has been modulated when compared to the wild type cell, so that the T cell or pro-T cell is capable of conversion to an ITNK cell. The target T cell or target pro-T cell may be an ES cell, or adult stem cell, or induced pluripotent stem cell (IPS cell).

In one aspect of the invention the ITNK cells or target T/pro T cells are obtained from T cells or pro-T cells in which all or part of the Bcl11B gene has been deleted. In one aspect there is a deletion in both alleles of the Bcl11b gene, or part thereof.

The invention also relates to a mammalian genome from which all or part of the Bcl11b gene has been deleted.

The invention also relates to mature activated T cells in which Bcl11b expression is downregulated or absent (also referred to as TBcl11b-cells). Mature T cells in this context refer to normal mature T cells and not to cancerous or transformed T cells. As shown in the example section below, it has been observed by the present inventors that at a single cell level about 10-20% of activated splenic T cells have very low level of Bcl11b expression (also FIG. 6 (F)). Hence, use of these cells in medicine, particularly in the treatment of cancers and viral infections forms an aspect of this invention.

The invention also relates to cells, such as T cells and pro T cells and stem cells and animals such as non-human animals, such as a mouse, the genome of which comprises a Bcl11b conditional knockout (cko) allele.

In one aspect all or part of Bcl11b gene is floxed or otherwise associated with recombinase target sequences, to allow the Bcl11b gene or part thereof to be deleted. In one aspect the cell comprising the floxed gene expresses Tamoxifen (OHT)-inducible Cre recombinase. Expression of the Cre recombinase by OHT induction suitably causes all or part of Bcl11b to be deleted.

The invention also relates to a cell or non-human mammal in which the Bcl11b gene or protein activity has been modulated, other than by deletion, to produce an ITNK cell or target ITNK cell.

ITNK cells suitably are obtained or obtainable from another cell type (such as T cells or pro-T cells, suitably DN1, DN2, DN3, DN4, DP thymocytes, CD4 or CD8 single positive mature T cells, common lymphoid precursor cells or stem cells) and suitably exhibit one or more or all of the following properties:

(a) a morphology comparable to natural killer cells, in comparison to T cells, for example as shown in FIG. 2D, FIG. 2E and FIG. 2e.

As shown below, reprogrammed thymocytes not only expressed NK cell surface receptors but morphologically do not look like T cells, rather, they were much similar to regular NK cells which are large size, large cytoplasm, have granules and high protein synthesis activity in the abundant endoplasmic reticulum (ER) (FIGS. 2D, 2E and 2e).

(b) TCR 6 specific genomic DNA re-arrangement, for example as shown in FIG. 2C;

As shown below, certain ITNK cells have a rearranged TCR 6 locus, indicative of their origin as T cells.

(c) a gene expression profile more similar to that of NK cells, such as LAK cells, than the parental cells from which they were developed, for example as shown in FIGS. 1E and 1G. Genes that showed an expression difference between the parental DN3 thymocytes and their Bcl11b-deficient derivatives are listed in Table 1. When considering this table of genes, ITNK cells suitably have at least 50%, suitably at least 60%, suitably at least 70% of genes differentially expressed (2 fold difference or more) in the same direction (increase or decrease) as LAK cells.

(d) cellular expression of one or more NK specific genes not found, or not expressed at high levels on non-effector or naïve T cells such as:

ZFP105, IL2Rβ, Id2, JAK1, NKG2D, NKG2A/C/E, B220, Rog (Zbtb32), Tnfrsf9, Cdkn1c, Trail, Perforin, Interferon-γ, NK1.1, NKp46, E4 bp4, NKG7, KLRD1, LTA, PLCG2, Ly49C/I and Ly49G2

(e) decreased or no expression of one or more T lineage genes, in comparison to the parent cells from which the ITNK cell was derived, such as decreased or no expression of Notch1, Est1, Hes1, Gata3, Deltaxi, TCRβ, CD3, Tcf1, IL7Ra, T-bet, CD8. In one aspect, ITNK cells are derived from CD8+ cells and do not express IL7R and/or T-bet and express low levels of CD8a.

(f) cell killing ability, for example the ability to prevent or ameliorate tumour formation or growth, the ability to kill stromal cells, tumour cells, or infected cells, suitably in comparison to the precursor cell used (parent T cells or proT cells). Cell killing may be assessed in vitro or in vivo by methods described in the Examples section herein. Additionally, the ITNKs can recognize MHC—I molecules. Moreover, the ITNK cells produced in vivo are not MHC—I restricted and are capable of killing MHC—I positive or negative cells. The ITNK cells whether produced in vitro or in vivo kill MHC—I low or negative cells.

(g) a mutation in the Bcl11b gene, or control sequences, affecting transcription, or translation or protein sequence, or otherwise affecting Bcl11b activity or effect, suitably promoting ITNK production.

Suitably the cells are capable of killing OP9-DL1 stromal cells, suitably within 2-20 days, such as 5-15 days such as 10 days after treatment to initiate the conversion from T cells or pro-T cells to ITNK cells, such as by OHT treatment. Suitably ITNKs retain a killing ability even when cultured in vitro for one month.

For the avoidance of doubt, ITNK cells produced by modulating Bcl11b activity and/or effect in a T cell and/or pro-T cell, remain ITNK cells according to the invention, if they retain cell killing ability even if Bcl11b returns to normal levels in such cells subsequently.

Suitably, ITNK cells of the invention exhibit the properties in (a), (c), (d), (e) and (f) above. Suitably, ITNK cells of the invention exhibit the properties in (a) or (c) or (d) or (e) and (f) above. ITNK cells may also possess one, or more, or all, of the following properties.

Suitably the proliferation and/or differentiation of the ITNK cells is promoted by a Supplement of IL-2 or IL-15 in the culture media.

Suitably ITNKs are able to grow out from T cell cultures within 2-20 days, such as 5-15 days, such as 10 days after Bcl11b is deleted or otherwise affected, or the Bcl11b pathway modulated suitably as assessed by the abundance of NKp46⁺ cells (FIG. 8K, 8L, 15a and 15b).

Suitably T cell/pro T cell to ITNK cell conversion from T cells/pro-T cells is greater than 50% efficient, such as greater than 60%, greater than 70%, greater than 80%, greater than 90%, greater than 95% efficient, suitably 100% efficient, by which it is meant that more than e.g. 50% of all cells in which the Bcl11b gene has been deleted, or in which the Bcl11b pathway has been otherwise modulated, go on to produce ITNK cells.

Suitably ITNK cells produced in vivo are detectable in the recipient host, such as a recipient mouse, for at least 1 month, preferably 2 months, preferably 3 months. Suitably recipient animals do not show any noticeable abnormality, indicating that the ITNK cells do not attack normal host cells in the recipient mice.

Suitably ITNK cells according to the invention possess functions of NK cells relating to regulation of the immune response, such as cytokine release.

Suitably ITNKs are able to continue proliferating for at least 3 weeks in cell culture.

In one aspect ITNK cells do not express NKp46.

Suitably ITNK cells or T cells can be independent of Notch signalling.

In one aspect the ITNK cells are not completely identical to NK cells. In one aspect ITNK cells do not express Ly49D. In one aspect ITNK cells do not express one or more T cell surface markers such as CD8, CD3e, and βTCR.

In another aspect ITNK cells express at least 20% of NK cell specific markers listed in table 2 as specific to LAK, preferably 40%, 60% or 80% of these known NK cell markers.

In one aspect, the ITNK cells produced in vivo are not MHC—I restricted and are capable of killing MHC—I positive or negative cells. The ITNK cells whether produced in vitro or in vivo kill MHC—I low or negative cells. This is explained in further detail in the example section below and shown in FIG. 3E where it is shown that unlike LAK, the in vivo produced ITNK cells killed RMA cells with almost the same efficiency as killing RMA-S. Such in vivo produced ITNKs have the advantage that their use has no risk of autoimmune diseases.

In one aspect the ITNK cells have at least 2, 3, 4 or more of the properties listed above, and preferably all such properties.

In one aspect ITNK cells demonstrate a rearranged TCR β locus, do not express all of the genes listed in the table 2 as specific to LAK, and exhibit cell killing as described herein.

In one aspect the invention provides an ITNK cell obtainable or obtained by the present invention having by a cell killing ability as assessed by methods such as those of examples 1.1.9 and 1.1.11 herein, but which do not express Ly49D.

In one aspect the NK cells comprise a suicide gene or other mechanism to allow ITNK cells to be eliminated. By way of example the genome of the ITNK cell, or T cell or pro-T cell may be engineered to contain a negative selection cassette.

The invention provides a pharmaceutical composition comprising ITNK cells together with a pharmaceutically acceptable excipient. Suitable excipients are well known in the art and include pharmaceutically acceptable buffers, preservatives, diluents and carriers and the like.

Also provided are mixtures of the ITNK cells of the invention with therapeutic agents such as anti-cancer agents or anti-infective agents e.g antiviral agents. The ITNK cells may be used in a combined preparation for simultaneous, separate or sequential use in disease therapy such as anticancer or antiviral therapy, although the use of ITNKs is not limited to cancer and antiviral therapy, and ITNKs might be useful for eliminating many types of abnormal cells. For example, ITNKs may also be used for treatment or prophylaxis of bacterial, yeast and parasite infections.

Suitable anticancer agents include alkylating agents, antimetabolites, anthracyclines, plant alkaloids, topoisomerase inhibitors, and other drugs affect cell division or DNA synthesis and function in some way. Other drugs include targeted therapies such as monoclonal antibodies and tyrosine kinase inhibitors and nanoparticles. Furthermore, also suitable are drugs that modulate tumor cell behaviour without directly attacking those cells, such as hormone treatments, known as an adjuvant therapy. As an alternative, agents for immunotherapy may also be included, such as use of interferons and other cytokines to induce an immune, and vaccines to generate specific immune responses.

Suitable anti-infectives include drugs that act to block viral entry into cells, drugs that prevent virus replication, such as reverse transcriptase inhibitors, integrase inhibitors, Protease inhibitors, and drugs that prevent virus release into the body.

Delivery of cells and compositions of the invention may be by any suitable route of administration including enteral or parenteral, such as by injection or infusion, for example in a once a day, once a week, once a month, or other suitable schedule. Multiple or single rounds of treatment may be employed.

The invention relates to a method for the preparation of a medicament for a human or non-human mammal comprising taking a sample of T cells, and converting the T cells to ITNK cells as described herein, optionally then using said cells in a medicament for treatment. Optionally the method comprises dilution or otherwise selection of a single T cell, and optionally manipulation of the T cell genome prior to use as a medicament.

The invention provides ITNK cells and target T/pro-T cells for use in medicine, and use of ITNK cells and target T/pro-T cells in the preparation of a medicament for the treatment or prophylaxis of disease, such as cancer or viral infection. ITNKs may also be used for treatment or prophylaxis of bacterial, yeast and parasite infections.

The invention also provides mature activated T cells in which Bcl11b expression is down-regulated or absent (also referred to as TBcl11b-cells) for use in medicine, and use of such cells in the preparation of a medicament for the treatment or prophylaxis of disease, such as cancer or viral infection.

NK cells play a major role in the rejection of tumors and cells infected by viruses and the ITNK cells of the present invention demonstrate anti cancer properties in vitro and in vivo. In one aspect ITNK cells produced from T cells or pro T cells are used to treat diseases such as cancer and infectious diseases such as viral infections.

The ability to convert T cells or pro-T cells into ITNK cells and use of TBcl11b-cells allows therapies to be developed using a patient's own cells, which can be used in the same patient without rejection.

The invention thus relates to use of a therapeutically effective amount of ITNK cells derived from the T cells or pro-T cells of a patient in the treatment or prevention of infection or disease in that individual. In a further aspect the cells may be used in another individual.

The invention provides a method of treating a patient, the method comprising administering to said patient a therapeutically effective amount of ITNK cells or TBcl11b-cells preferably wherein the ITNK cells are derived from T cells or pro-T cells that have been obtained from the patient.

Target T cells or pro-T cells may also be employed as above, in place of ITNK cells.

In one aspect, T cells/pro-T cells or target T cells or target pro-T cells of the invention do not refer to cancerous or transformed T cells.

In one aspect the ITNK cells according to the invention are obtained by modulating Bcl11b activity and/or effect in transformed or cancerous T cells, such as T cells from lymphoma patients, which may have different levels of Bcl11b as compared to wild type cells. In this aspect, the transformed or cancerous T cells are the T cells/pro-T cells or target T cells or target pro-T cells capable of conversion to ITNK cells.

In one aspect ITNK cells do not show any adverse effects on the patient.

In one aspect, the invention provides a method of isolating naturally occurring mature activated T cells in which Bcl11b expression is downregulated or absent (TBcl11b-cells) from a patient, expanding the cells in vitro and administering to the patient a therapeutically effective amount of the TBcl11b-cells for treatment of conditions such as cancer and viral infections.

In one aspect, the invention provides a method of isolating T cells/pro-T cells from a patient (human or non-human); modulating the activity and/or effect of the Bcl11b gene and/or gene product so that the T cell or pro-T cell is capable of conversion to ITNK cells; administering to the patient a therapeutically effective amount of ITNK cells or target T cells or target pro T cells for treatment of conditions such as cancer and viral infections.

In one aspect the ITNK cells are derived from a single T cell which is converted into ITNK cells using the methods described herein. This process suitably allows for a T cell specific for an antigen of interest, such as a disease specific antigen, such as a viral or microbial antigen or such as a tumour-specific antigen, to be converted into an NK-like cells.

From a single T cell up to 0.5 million ITNKs can be obtained. This is a much higher number as compared to human NK cells where approximately 1600 cells can be produced by proliferation of a single NK cell.

The invention relates to modulation of Bcl11b directly, and also use of components of the Bcl11b pathway and modulators thereof in the production of ITNK cells.

An appreciation that T cells and pro-T cells can be converted to ITNK cells allows this conversion to be used as an assay for compounds that might be used to control the conversion process. Thus the invention relates to an assay for identification of a compound which assists in the reprogramming of T cells to ITNK cells, the method comprising contacting T cells or pro-T cells with a test compound and then monitoring or selecting for the conversion of T cells to ITNK cells. Such compounds could include small chemical molecules, proteins (including but not limited to growth factors, cytokines, antibodies) or nucleic acid based therapies, and libraries of any of these compounds. The invention also relates to use of compounds so identified in the conversion of T cells or pro-T cells to ITNK cells and additionally to those compounds per se.

In addition the invention relates to an assay for identification of a genetic mutation which controls the reprogramming of T cells to ITNK cells, the method comprising random or targeted mutation of T cells or pro-T cells and screening for ITNK cells or selection of ITNK cells under conditions where T cells or pro-T cells are not viable.

An appreciation that Bcl11b plays a role in the conversion of T cells and proT cells to ITNK cells allows the Bcl11b gene and protein to be used directly as probes to identify other components in the Bcl11b signaling pathway, which may then be tested for an effect on conversion of T cells to ITNK cells. Thus the invention relates to an assay for identification of a compound which assists in the reprogramming of T cells to ITNK cells, the method comprising screening for compounds that bind to the Bcl11b gene or the Bcl11B protein, and further optionally assessing whether said compounds are able to promote the conversion of T cells to ITNK cells. The invention further relates to use of compounds so identified in the conversion of T cells or pro-T cells to ITNK cells and those compounds per se.

In a yet further aspect the invention relates to the use of factors which regulate the Bcl11b gene or protein expression or activity, or which are functionally downstream of the Bcl11b gene or protein, or which are functionally upstream of the Bcl11b gene, to effect the conversion of T cells to ITNK cells, and to the use of modulators of these factors to effect the conversion of T cells to ITNK cells. Suitably, the modulators are antibodies targeting Bcl11b or factors which regulate the Bcl11b gene or protein expression or activity or downstream gene products or upstream gene products. Suitably, the modulators are administered to human or non-human diseased subjects.

For example, Notch is upstream of Bcl11b. In one aspect modulators of Notch signalling are used to effect a conversion of T cells and proT cells to ITNK cells.

CSI acts upstream of Bcl11b. In one aspect modulators of CSL are used to effect a conversion of T cells and proT cells to ITNK cells.

In another aspect the invention relates to an assay for identifying a downstream target for Bcl11b, the assay comprising monitoring the effect of modulating the Bcl11b gene and/or protein product on a putative downstream target. Such an assay may further comprise monitoring conversion of T cells or pro-T cells to ITNK cells when the downstream target per se has been modified. Such an assay may further comprise identifying a modulator which either interacts with said downstream target so as to modulate the activity and/or effect thereof, to result in the conversion of a T cell or pro-T cell to one or more ITNK cells.

The invention further provides for a non-human animal carrying ITNK cells, and/or target T cells or target pro-T cells.

In one aspect ITNK are independent of Notch signalling.

In a further aspect the invention relates to a method of stimulating T cell production, the method comprising modulating the activity and/or effect of at least one Bcl11b gene and/or protein present in a pro-T cell, such as a human or embryonic stem cell, or IPS cell. Suitably the method comprises stimulating the Bcl11b expression or activity.

An understanding of the importance of Bcl11b in the T cell maturation pathway suggests that manipulation of the Bcl11b gene or protein expression or activity can help to stimulate T cell production. The present invention thus relates to use of activators of the Bcl11b pathway, either upstream or downstream, in the stimulation of T cells production, either in vivo or in vitro, and use of T cells so produced in medicine.

EXAMPLES

T cells develop in the thymus and are critical for adaptive immunity. Natural killer (NK) lymphocytes constitute an essential component of the innate immune system in tumor surveillance and defense against microbes and viruses.

General Introduction to T and NK cell development

T cell development involves progenitor homing, lineage specification and commitment, and requires a complex interplay among key transcription factors (1, 2). The earliest populations of thymocytes, which lack T cell receptor (TCR) co-receptors CD4 and CD8 (double negative or DN cells) (28), can be further subdivided by cell surface markers as DN1-4 (29). The DN1 (CD44⁺CD25⁻) thymocyte population contains multipotent progenitors (30, 31) whereas DN2 thymocytes (CD44⁺CD25⁺) have NK and myeloid potential (30, 31). These non-T cell developmental potentials are lost in the DN3 (CD44CD25⁺) thymocytes. DN4 thymocytes (CD44⁻CD25⁻) have undergone have undergone β-selection after successful Tcrβ gene rearrangement (32) and already initiated the process of differentiating to the CD4⁺CD8⁺ double positive (DP) stage (33, 34).

In the periphery, the cytokine IL-7 and the constant interaction of T cells with self peptide-MHC play a critical role in T cell maintenance (3). RT-PCR analysis indicates that many genes important for T cell commitment start to increase their expression in the transition from DN1 to DN2, with Bcl11b being the most upregulated transcription factor (4). In bony fish, Bcl11b is shown to be required for T cell precursor homing to the thymus (5). In the mouse, Bcl11b has critical roles in fetal thymocyte development and survival, and in positive selection and survival of double-positive thymocytes (6, 7).

NK cell committed precursors (CD122⁺) differentiate from multipotent haematopoietic progenitors primarily in the bone marrow but differentiation can also occur in the thymus and secondary lymphoid tissues (35). These precursors give rise to NKp46⁺ immature NK cells, which subsequently express additional receptors as they differentiate, including MHC receptors, NKG2A/C/E and Ly49s (36, 12). Besides their participation in innate immune responses, NK cells have recently been shown to possess some adaptive immune features (37).

Although NK developmental pathways are not entirely clear, two subsets of NK cells, bone marrow-derived (CD127⁻) and thymic (CD127⁺) NK cells have been identified in the mouse that differ in development sites and origins (Huntington et al., 2007). Previous studies have identified molecules important for NK cell development and homeostasis. For example, Id2, which antagonizes the bHLH E proteins E2A and HEB, is essential for the NK lineage since the Id2-knockout mice lack NK cells (Ikawa et al., 2001; Yokota et al., 1999). Conversely, forced expression of Id2 or Id3 is able to re-direct pro-T cells to NK cell differentiation (Blom et al., 1999; Fujimoto et al., 2007). A recent study also identifies Zfp105 as a NK specific transcription factor since overexpressing it promotes differentiation from hematopoietic stem cells to the NK lineage (Chambers et al., 2007).

Several genes or pathways important for T cell development genes also have functions for NK cells. For example, Gata3 and T-bet plays important roles in NK development, maturation and homeostasis (Samson et al., 2003; Vosshenrich et al., 2006) (Townsend et al., 2004). Notch triggers initiation of T cell program, and is required to sustain or protect the cells throughout the pro-T cell stages (Maillard et al., 2005; Radtke et al., 1999; Rothenberg, 2007). Loss of Notch signalling in DN1 thymocytes convert them into dendritic cells (Feyerabend et al., 2009). Deleting of Notch in the thymus leads to accumulation of B cells in the thymus possibly by a cell-extrinsic pathway (Feyerabend et al., 2009; Radtke et al., 1999).

In contrast to its role in T cells, Notch generally suppresses NK potential in DN1 and DN2 pro-T cells until the cells progress to the committed DN3 stage (Carotta et al., 2006; De Smedt et al., 2005; Garcia-Peydro et al., 2006; Rolink et al., 2006; Schmitt et al., 2004; Taghon et al., 2007; van den Brandt et al., 2004). Nevertheless, it is proposed that transient Notch signaling is required for NK differentiation from early progenitors or stem cells (Benne et al., 2009; Haraguchi et al., 2009; Rolink et al., 2006). This may reflect the role of Notch in promoting T/NK bipotent progenitors (DeHart et al., 2005).

Bcl11b is a C₂H₂zinc finger transcription repressor (Avram et al., 2000; Cismasiu et al., 2005). Germline mutation of Bcl11b in the mouse causes thymocyte developmental block at the DN3 stage secondary to apoptosis induced by defective β-selection in thymocytes (Wakabayashi et al., 2003). Bcl11b is recently shown to be required for positive selection and survival of double-positive thymocytes (Albu et al., 2007). However, suppression of Bcl11b expression by RNA interference selectively induces apoptosis in transformed T cells but does not appear to affect normal mature T cells (Grabarczyk et al., 2007).

Here we show that the transcription factor Bcl11b was expressed in all T cell compartments, and was indispensable for T lineage development. When Bcl11b was deleted, T cells from all developmental stages acquired NK cell properties and concomitantly lost or decreased T cell-associated gene expression. These Induced T-to-Natural-Killer (ITNK) cells, which were morphologically and genetically similar to conventional NK cells, killed tumor cells in vitro and effectively prevented tumor metastasis in vivo. Therefore ITNKs may represent a new cell source for cell-based therapies.

Bcl11b is Expressed and Required in the Early T Cell Progenitors

Microarray studies indicate that expression of many genes important in T cell commitment, including Bcl11b, starts to increase in DN2 thymocytes. Among transcription factors, Bcl11b is the most drastically upregulated in the transition from DN1 to DN2 (Rothenberg, 2007). To determine Bcl11b expression in early T cells at the single cell level, we produced a lacZ knock-in allele of Bcl11b where a SA-lacZ cassette is inserted into the intron 3 to trace its expression (Song-Choon Lee, et al, unpublished). Therefore, Bcl11b expression can be traced indirectly by using Fluorescein di-3-D-galactopyranoside (FDG), a fluorescent substrate of 3-galactosidase, in flow cytometry. In hematopoietic lineages, expression of Bcl11b was only detectable in T cells (data not shown). In the thymus, almost all DN2-DN4 thymocytes expressed Bcl11b (FIG. 13a and FIG. 14a). In contrast, only about 80% of DN1 thymocytes expressed Bcl11b. Further analysis using a CD117 antibody identified that 60% of DN1a and DN1b thymocytes, which are thought to be the earliest T cell progenitors (Porritt et al., 2004), already expressed Bcl11b (FIGS. 13B and 14a), suggesting a possible role of Bcl11b at the earliest T lineage specification steps.

To determine Bcl11b expression in T cells at the single cell level, we produced and analyzed a Bcl11b tdTomato knock-in mouse (FIG. 5A-B). In hematopoietic lineages, Bcl11b was not expressed in B or myeloid cells whereas almost all DN2-DN4 and DP thymocytes, CD4⁺ and CD8⁺ T cells, γδ-T cells and Natural Killer T cells (NKT) expressed Bcl11b (FIG. 6, A-C and 7, A-C). In DN1 thymocytes, very little to no expression of Bcl11b was detected in CD117⁺⁺ cells (known as Early T-cell-lineage Progenitors (2)) (FIGS. 6A and 7A). During NK development, transient, low Bcl11b expression was observed in immature NK cells but not in NK precusors (NKP) or mature NK cells (FIGS. 6D and 7D). In contrast, the majority of thymic NK cells, identified by CD127 (8), expressed Bcl11b (FIGS. 6D and 7E). Moreover, in both CD4⁺ and CD8⁺ splenic T cells, Bcl11b transcript was reduced roughly two-fold in activated T cells (CD44⁺CD62⁻L) compared to naïve (CD44⁻CD62⁺L) cells in quantitative real time-polymerase chain reaction (qRT-PCR) analysis (FIGS. 6E and 7F) and exhibited a bimodal pattern of expression (FIG. 6F).

Bcl11b Deletion Caused Loss of T Cell Identity and Acquisition of Nk-Specific Properties in T cells

The above expression and function data have demonstrated that Bcl11b is expressed in T cell precursors and required for differentiation to T cell lineage. Germline deletion of Bcl11b caused apoptosis in DN3 thymocytes in the fetal thymus but did not obviously affect DN1/2 cells (Wakabayashi et al., 2003). To further determine Bcl11b functions in T cells, we generated the conditional knockout mice (Bcl11b^flox/flox) where exon 4 was floxed (FIG. 8A), which were crossed to the Rosa26Cre-ERT2 mice (9). All the thymocytes from CreERT2; Bcl11b^flox/floxmice express Tamoxifen (OHT)-inducible Cre recombinase. Consequently, in CreERT2; Bcl11b^flox/floxmice (PLBD line. Referred to as flox/flox in the manuscript), Bcl11b could be deleted by treating cultured cells or mice with Tamoxifen (OHT). From OHT-treated whole thymocytes from these and the control (CreERT2; Bcl11b^flox/+, referred to flox/+) mice, we sorted and subsequently cultured DN1 and DN2 cells in T cell media (Flt3 ligand and 11-7) for 2 weeks (FIG. 14b) on OP9-DL1 stromal cells (FIG. 8B) (10), which support T cell development but suppress NK cell development from the progenitors (11). OP9-DL1 stromal cells express Delta-Like-1 Notch ligand and support robust T cell development (Schmitt and Zuniga-Pflucker, 2002) while normally suppressing NK cell development (Rolink et al., 2006; van den Brandt et al., 2004). All stromal cells were killed in the OHT-treated flox/flox DN1 thymocyte culture.

Flow cytometry showed that 18% of the cultured thymocytes now expressed the NK cell marker NK1.1 (DN1 in FIG. 13c). 24% of cells in this culture expressed NKp46, which is primarily expressed on NK cells (FIG. 1A) (12). These NKp46⁺ cells did not express T cell genes CD3 or TCRβ (FIG. 8C), and had lost both alleles of the Bcl11b exon 4 (FIG. 8D), indicating that they did not acquire or had lost T cell features despite being co-cultured with OP9-DL1 stromal cells for 14 days. PCR genotyping of these NK1.1⁺CD3⁻ and NKp46⁺CD3⁻ cells showed that they had deleted both alleles of the Bcl11b exon 4 while those NKp46⁻CD3⁺ cells from the same OHT treated culture were found to still retain at least one copy of the Bcl11b cko allele. On the other hand, the control OHT-treated flox/+ and untreated flox/flox DN1 cells proliferated rapidly, and many (36%) acquired CD3 expression but not NK1.1⁺ or NKp46⁺ (FIG. 1A and FIG. 8E) consistent with Notch signalling suppressing NK development and excluding the possibility that the NKp46⁺ cells in OHT treated DN1 cell culture were derived from NK precursor contamination (FIG. 13c). These data thus demonstrated that Bcl11b deficiency caused production of the NKp46⁺ cells from DN1 thymocytes and that Bcl11b was required in early T cell development.

T cell lineage commitment is thought to occur in DN2 cells with increased expression of T cell specification genes such as Gata3, Tcf1 and Bcl11b (Ciofani and Zuniga-Pflucker, 2007; Rothenberg, 2007). Nevertheless, recent data suggest that even DN2 thymocytes still retain differentiation potentials of myeloid and NK lineages (Bell and Bhandoola, 2008). We next investigated Bcl11b function during T cell lineage commitment by deleting Bcl11b in purified DN2 thymocytes. Wild type DN2 thymocytes (−OHT) proliferated extensively on OP9-DL1 cells and gave rise to CD3⁺ cells but no NK cells (−OHT DN2 in FIG. 13c). Similar to cultured DN1 thymocytes, OHT-treated flox/flox DN2 thymocytes also produced NKp46⁺CD3⁻ cells which killed the stromal cells, whereas control DN2 thymocytes did not (FIG. 1A and FIG. 8E). Similar to that in DN1 thymocyte culture, NK1.1⁺CD3⁻ and NKp46⁺CD3⁻ cells also grew out from Bcl11b-deficient DN2 thymocytes culture on OP9-DL1 stromal cells (+OHT DN2 in FIG. 13c), demonstrating rapid loss of T cell differentiation potential upon Bcl11b loss in the DN2 thymocytes.

Growth of NK-like cells from Bcl11b-deficient DN1 or DN2 thymocytes appeared to be Notch signaling independent since NKp46⁺ cells were readily produced from DN1 or DN2 thymocytes cultured on OP9 stromal cells without IL-2 (FIG. 8F). Hence, Bcl11b has an essential function in the initial specification of the T cell lineage.

We subsequently deleted Bcl11b in DN3 thymocytes. Again, stromal cell-killing NKp46⁺CD3⁻ cells appeared (FIG. 1B-C; FIG. 8G). We purified DN3 thymoytes from OHT treated whole thymocytes from CreERT2; Bcl11b^flox/floxand cultured them on OP9-DL1 stromal cells. Within 14 days of culturing, most of the cells became NKp46⁺CD3⁻ and were able to kill stromal cells. Supplement of IL-2 or IL-15 in the culture media greatly promoted proliferation and/or differentiation of these cells. Consequently most cells in the culture were NKp46′ and they started to kill stromal cells within 10 days after OHT treatment (FIGS. 1B and 1C). NK progenitors normally do not differentiate on OP9-DL1 stromal cells. (FIG. 1D).

The reprogramming also worked in myeloid or B cell culture media (FIG. 8H-I), demonstrating that reprogramming to NKp46⁺ cells was intrinsic to the Bcl11b-deficient thymocytes. To further confirm that the NKp46⁺CD3⁻ cells came from T cells, we purified them and examined their TCRβ locus for DNA rearrangements. These NKp46⁺CD3⁻ cells retained TCR β V(D)J recombination even though they no longer expressed Tcr β on the cell surface, thus genetically confirming the T cell origin of these NKp46⁺CD3⁻ cells (FIG. 1D). We thus named these killer cells that were reprogrammed from T cells as Induced T-to-Natural-Killer or ITNK cells.

We next compared using microarray analysis the expression profiles of DN3 thymocytes, normal splenic NK cells that were expanded in vitro after enrichment (lymphokine-activated killer, or LAK cells, composed of >90% NK cells), and ITNKs reprogrammed from DN3 cells (FIG. 1E). Consistent with the killing ability of ITNK cells, their expression profile was much more similar to that of LAK cells than to their parental DN3 thymocytes. Genes that showed expression difference between the parental DN3 thymocytes and their Bcl11b-deficient derivatives were listed in Table 2. qRT-PCR analysis was subsequently performed to confirm the array results (FIG. 13F). qRT-PCR validation showed that expression of many T lineage genes, such as Notch1, Est1, Hest Gata3, Dtx1 and Tcf1 was decreased, whereas expression of genes usually associated with NK cells such as Id2 (13), IL2rβ (CD122), Zfp105 (14) and E4 bp4 (15) was upregulated (FIG. 1F and table 1). Zbtb32 (Rog, Repressor of GATA), which is not normally expressed in DN3 cells, but plays important roles in regulating T cell activation and suppresses Gata3 activity (16), was highly expressed in ITNKs. Expression of Cdkn1c (p57KIP2), a putative direct downstream target gene of Bcl11b (17), was also drastically increased in ITNKs (FIGS. 1F and 1G). Indeed, p57KIP2 expression was not barely detectable in DN3 cells but drastically increased in DN3 derived iTNKs (FIGS. 1F and 1G). Further analysis from the array data identified 504 genes that were expressed at least two folds higher in LAKs vs DN3 thymocytes, and 366 genes in DN3 thymocyte-derived NKp46⁺CD3⁻ cells vs their parental DN3 thymocytes (Table 2). 70% of these 366 genes in iTNKs were found overexpressed in LAKs (FIG. 8J). These results thus collectively demonstrated that Bcl11b was essential for maintaining the T cell expression profile and for suppressing NK cell gene expression.

We next investigated whether Bcl11b was required for T cell identity maintenance in all T cells by subjecting purified double positive (DP) thymocytes, CD4 or CD8 single positive mature T cells, to OHT treatment. These cells were then cultured on OP9-DL1 stromal cells. Similar to cultured Bcl11b-deficient DN3 thymocytes, iTNKs grew out from all T cell cultures within 10 days after Bcl11b was deleted, as demonstrated by many NKp46⁺ cells (FIG. 15a, 15b, 15c). Interestingly, these iTNKs that were derived from Tcrβ-expressing T cells, still retained Tcrδ on the cell surface. In contrast to iTNKs from CD8⁺ T cells that still expressed CD8, the CD4⁺ single-positive T cell-derived iTNKs did not express CD4 anymore (FIG. 15c).

ITNKs could also be produced from mature T cells. We OHT-treated sorted double positive (DP) thymocytes, CD4⁺ and CD8⁺ T cells, and γδ-T cells from flox/flox mice. Many ITNKs (NKp46⁺) were found growing in DP thymocytes and CD8⁺ T cell cultures (FIG. 8K-L), which effectively killed stromal cells. These ITNKs, in contrast to those reprogrammed from DN1-3 thymocytes, retained TCRβ on the cell surface. We were unable to obtain consistent production of NKp46⁺ cells from splenic or thymic CD4⁺ T cells, or from γδ T cells, because these cells appeared prone to cell death in vitro once Bcl11b was deleted.

Once Bcl11b Deleted, all DN3 Thymocytes Lost T Cell Identity and Became ITNK

To estimate the reprogramming (T to NK conversion upon Bcl11b deletion) efficiency, we sorted single DN3 thymocytes from OHT-treated flox/flox thymocytes into individual wells of 96-well plates pre-seeded with OP9-DL1 stromal cells in T cell media (FIG. 9A). Out of the 79 wells that had cells growing, 36 wells had many fast-proliferating T cells which expressed T cell surface markers including CD3 and Tcrβ (FIG. 2A). PCR genotyping confirmed that cells in these wells did not have complete Bcl11b deletion—but deleted only one fox Bcl11b allele (FIG. 2B, lanes T1 and T2). These cells (flox/−) nevertheless served as excellent controls for Cre toxicity because they had activated Cre recombinase. In the other 43 wells, thymocytes were reprogrammed to NKp46⁺ stromal cell-killing ITNKs (FIG. 2A). In these 43 wells, cells grew relatively slow but killed stromal cells. Still, from one DN3 thymocyte, up to 0.5 million of stromal-killing cells were readily obtained 14 days post OHT treatment. Flow cytometry analysis showed that almost all the cells in these wells expressed NK-specific markers NKp46 and thus were ITNKs (FIG. 2A). IL-2 was clearly able to greatly promote proliferation of ITNKs because from one DN3 thymocyte, up to 0.5 million ITNKs were obtained with IL-2, but only about 50,000 cells without IL-2. All ITNK cells had lost both Bcl11b alleles (FIG. 2B, lanes 11 and 12), and ITNKs of individual wells possessed unique rearranged TCRβ loci thus confirming their independent origins (FIG. 2C). Therefore, once Bcl11b was deleted, the reprogramming efficiency of DN3 thymocytes to ITNKs could reach 100%. ITNKs from DN3 thymocytes not only expressed NK cell surface receptors and possessed similar cytotoxic functions, but were morphologically similar to LAK cells which are larger than T cells, have granules and high protein synthesis activity with abundant endoplasmic reticulum (FIG. 2, D-E).

ITNKs were larger than thymocytes and had granules and showed evidence of high protein synthesis activity with abundant endoplasmic reticulum (FIG. 2, D-E). Besides NK1.1 and NKp46, ITNKs expressed NKG2A/C/E, TRAIL, perforin and interferon-γ, but not some other key NK cell function genes, such as members of the Ly49 family or FasL (CD178) (FIG. 9B-C). Similar observations were made with in vitro reprogrammed ITNK cells from DP thymocytes (table 2 and FIG. 9D). ITNKs were unlikely to be related to thymic NK cells since they did not express CD127 (FIG. 9E). Moreover, unlike conventional mature NK cells, most ITNKs did not express CD11b, rather, they expressed CD27, and retained killing ability even after being cultured in vitro for one month (FIG. 9F). The iTNKs from in vitro cultured Bcl11b deficient DN3 thymocytes killed OP9-DL1 stromal cells after overnight co-culture. In fact, iTNKs retained the killing ability even cultured in vitro for at least a month. Transferring of supernatant of the iTNK cells culture to fresh stromal cells did not kill these cells, therefore cytokines secreted by iTNK cells were not sufficient, and cell-cell contact was required, for efficient killing.

We next measured the killing ability of the DN3-reprogrammed ITNKs by performing standard ⁵¹Cr-release assays with three NK-sensitive cell lines: B16F10 melanoma (MHC—I low or negative) (18), RMA lymphoma, which express MHC class I molecules, and RMA-S lymphoma (TAP-1-deficient variant), which have reduced MHC class I presentation (19, 20). LAK cells generally only killed MHC-class I negative cells (FIG. 2F). Similar to LAKs, ITNKs also selectively killed MHC—I negative B16F10 and RMA-S cells, but did not kill MHC—I positive RMA lymphoma cells (FIG. 2F). Compared to regular LAKs, iTNKs appeared to have relatively lower killing potency. This is consistent with a lack of the full NK cell surface repertoire in the in vitro derived ITNKs (Table 2). We speculated that an in vivo microenvironment might be required for fully converting Bcl11b deficient T cells to more potent tumour cell killers.

In Vivo Reprogrammed NK Cells are More Potent Tumour Cell Killers

To exclude the possibility that ITNKs were in vitro artifacts, we deleted Bcl11b in vivo (FIG. 10A). Two to three weeks after OHT treatment, ITNKs were detected in both the spleen (NKp46⁺CD3⁺) and the thymus (NKp46⁺) from flox/flox mice but not the fox/+ controls (FIG. 3A). Bcl11b was found deleted in these in vivo reprogrammed ITNKs (FIG. 10B). Importantly, both CD4⁺ and CD8⁺ ITNKs (NKp46⁺) were found (FIG. 10C). Some wild type γδ-T cells expressed NKp46, however, Bcl11b deletion caused a 3-fold increase in the NKp46⁺γδ-T cells (FIG. 3B), which suggested that all T cell populations have reprogramming potential. The in vivo reprogrammed ITNKs could readily be expanded in NK culture conditions (FIG. 10D), but they were not NKT cells (FIG. 10E-F). Besides expressing NK cell-associated genes, the in vivo reprogrammed ITNKs also lost or decreased some key T cell genes such as Il7ra, Tbx21 (T-bet), Cd8 (FIG. 10G). Consequently, TCR signaling in ITNKs appeared to be compromised (FIG. 10H).

The in vivo analysis of ITNKs in flox/flox mice was complicated by the presence of many host T cells and NK cells (FIG. 3A). To address this problem, and also to investigate whether in vivo reprogramming upon Bcl11b loss is cell autonomous, we transplanted 2-4 million OHT-treated DP thymocytes from flox/flox mice (CD45.2⁺) into Rag2^−/−Il2rg^−/− mice (CD45.1⁺) that lack B, T and NK cells (FIG. 11A) (21). We chose DP thymocytes because they usually account for more than 75% of total thymocytes and could be efficiently reprogrammed in vitro to ITNKs (FIG. 8K). Two weeks after transplantation, around 5% of splenocytes were found to be from the donor cells (CD45.2⁺) (FIG. 3C), and approximately 47% of them expressed NKp46 and thus were ITNKs. ITNKs lost both copies of Bcl11b and the majority of them expressed CD8 (FIG. 11B-C). The other 53% cells (NKp46⁻) were T cells and still retained the Bcl11b floxed allele (FIG. 11C). The ITNKs usually accounted for 2-3% of total splenocytes. Interestingly, the majority of the splenic NKp46⁺ ITNKs expressed CD8 (FIG. 11B). Significant amount of NKp46⁺ ITNKs were also present in the bone marrow and peripheral blood (FIG. 11D). We estimated there were about 200,000 iTNK cells in the spleen alone. Nevertheless, this low ITNK number was unexpected because 2-4 millions of DP thymocytes were initially transplanted and because the T to ITNK conversion in vitro was 100%. It is possible that most of the Bcl11b-deficient DP thymocytes died either before or immediately following the conversion due to the difference between the in vivo microenvironment and in vitro culture condition, for example, the relative low levels of cytokines in the mice. No NKp46⁺ cells were found in control mice transplanted with untreated DP thymocytes (FIG. 3C). ITNK cells were maintained in the recipients for at least 3 months without change in cell number, perhaps representing a dynamic balance in their numbers. Importantly the recipient mice did not show any noticeable abnormality, indicating that ITNK cells did not indiscriminately kill normal cells nor were malignantly transformed.

The in vivo iTNKs were further phenotyped by flow cytometry. Compared to those reprogrammed in vitro, the in vivo reprogrammed ITNKs appeared to express more NK surface receptors such as NKG2A/C/E and most receptors of the Ly49 family including Ly49C/I and Ly49G2 (FIG. 11E) (table 2), and could be extensively expanded ex vivo with IL-2 or IL-15 for at least 3 weeks while still retaining their killing ability (FIG. 3D). NK surface receptors such as Ly49 family genes including Ly49C/I, Ly49G2 were absent in the in vitro derived iTNK cells. Importantly, these iTNK cells were not NKT cells because CD1d-restricted NKT cells do not express NKp46 (Walzer et al., 2007), and the iTNKs examined in this study did not express V β 2TCR which is present in many NKT cells and recognizes non-polymorphic CD1d molecule (data not shown) (Bendelac et al., 2007).

Regular NK cells become LAKs in culture with cytokines and can be expanded for up to 7 days. After that, LAKs gradually lose proliferation and killing ability. To test the proliferation capacity of the in vivo iTNK, we cultured 2 millions splenocytes (containing approximately 50,000 iTNKs) from recipient mice in LAK condition. Most cells died in the first 3 days (FIG. 3d). However, within 7 days of culturing, we obtained about 2 millions NKp46⁺Tcrβ⁺ ITNKs which accounted for 80-90% of the cell population and were able to continue proliferating for at least 3 weeks (FIG. 3d).

To assess functions of the in vivo iTNK cells, we used the ex vivo expanded iTNKs from the recipient mice to investigate their tumour-cell killing ability. Consistent with their expressing more killer effectors and receptors, the in vivo iTNK cells were much more potent in killing tumour cells than the regular LAKs, even after extensive ex vivo expansion These cells exhibited elevated cytotoxic potential and were also generally more potent than both in vitro ITNKs and LAKs against each of the target cells (FIG. 3E, and FIG. 2F). Unexpectedly, these in vivo iTNK were potent killers for all three tumour cell lines tested, regardless of their MHC—I expression status. They killed RMA cells with almost the same efficiency as killing RMA-S cells (FIG. 3E), despite expression of some inhibitory Ly49 receptors which recognize MHC—I. Transplantable murine melanoma B16 cell lines are well-established models for studying experimental cancer therapies and NK cell tumour surveillance function (22). Injection of B16 cells into Rag2^−/−Il2rg^−/− mice leads to rapid formation of metastatic foci in the lungs (23). To investigate the tumour-killing ability of the ITNK cells in vivo, we injected two million OHT-treated or -untreated DP thymocytes from flox/flox mice into Rag2^−/−Il2rg^−/− recipients to allow reprogramming of thymocytes to ITNKs in vivo (FIG. 11F). Two weeks later, each recipient was injected with 50,000 B16F10 melanoma cells. Four weeks after the initial thymocyte transplantation, recipients were sacrificed and analyzed. Mice injected with PBS or with untreated DP cells had about 200 metastatic foci in the lungs. In contrast, mice injected with OHT-treated DP thymocytes had approximately 20 tumour colonies in the lung (FIG. 3F and FIG. 11G). Therefore ITNKs were potent killers of tumour cells in vivo and prevented cancer progression.

Bcl11b Regulated by Notch Signalling in T Cells

Western blot indicated that in the thymocytes from CreERT2; Bcl11b^flox/flox, the Bcl11b protein levels decreased drastically 24 hours after OHT treatment. And 48 hours later, Bcl11b protein was undetectable. Hence, deletion of BcIllbled to rapid disappearance of Bcl11b protein (FIG. 4A). To probe gene expression changes immediately following Bcl11b deletion in T cells, we performed expression array analysis 24 and 48 hours following OHT treatment. Microarray analysis showed that in OHT-treated flox/flox thymocytes, expression of T cell genes such as TCRβ and CD3 was already down-regulated within 24 hours (table 3). In another 24 hours, many genes associated with NK cells were expressed (table 3). Table 3 lists genes that Bcl11b loss significantly affected their expression (2 folds). Expression of several genes that are important for NK cell functions, such as NKG7, KLRD1 (CD94), PLCG and IFNG, were already increased 48 hours after OHT treatment.

Bcl11b is proposed to be regulated by Notch signaling in T cell development (24). Recent genome-wide ChIP-seq in Drosophila has indeed identified CG6530, the Drosophila orthologue of Bc111 gene, is a direct downstream target gene of Notch signalling (Krejci et al., 2009). Notch signalling normally plays an inhibitory role in NK lineage differentiation and no NK cells would grow out from bone marrow or thymocytes cultured on OP9-DL1 stromal cells. Consistent with the idea that Bcl11b acts downstream of Notch signalling in T cells, once Bcl11b was deleted, iTNK production from T cells was independent of Notch signalling because T to NK conversion occurred using either OP9 or OP9-DL1 stromal cells (data not shown).

To confirm that Bcl11b is directly regulated by Notch signalling in mouse T cells at the molecular level, we first searched within the Bcl11b gene locus for putative CSL-binding sites (CGTGGGAA) (26) at the Bcl11b locus, which were conserved between mouse and human Bcl11b genes (FIG. 4B) (table 4). Several CSL sites were identified but we focused our attention on the ones that were conserved between mouse and human Bcl11b genes. Chromatin immunoprecipitation (ChIP) assay was subsequently performed using a CSL polyclonal antibody pulled down genomic DNA fragments from T cells. Three genomic regions were greatly enriched in the T cell samples using the CSL antibody compared to the control (FIG. 4C). Primers flanking the putative CSL binding regions were designed to amplify the ChIP pull-down genomic DNA (FIG. 4B). Regions 3, 4, 7 were greatly enriched in the T cell samples using the CSL antibody compared to using the antibody control (FIG. 4C and Table 4). Region 3 is about 1.8 kb from start of the transcription. Region 4 was located 5.4 kb downstream of exon 1; and region 7 was at about 600 bp downstream of exon 2 The ChIP result thus confirmed that the canonical Notch signaling directly regulated Bcl11b in T cells (FIG. 12).

However, it is reported that deleting CSL (RBPJk) using either CD4-Cre or Lck-Cre did not cause total T cell loss or lead to production of ITNKs (38). This discrepancy likely reflects that we acutely deleted Bcl11b in T cells for immediate functional consequences whereas if CD4-Cre is used, the deletion can occur in progenitors. Consequently, in CD4-Cre mice, the cells having defects are those from mutant progenitors and have developed mechanisms to compensate for the loss of Bcl11b. We propose that Bcl11b is a downstream target gene of Notch signalling, and that Bcl11b, together with other Notch downstream transcription factors Gata3 and Tcf1, play pivotal roles in specification, commitment and maintenance of the T cell lineage.

We show that Bcl11b was essential for T cell development and maintenance of T cell identity. Unlike loss of Pax5 in B cells (39), however, deletion of Bcl11b did not appear to have detectable de-differentiation steps because both lymphocytes and mature T cells were readily reprogrammed to ITNKs, and ITNKs from DP thymocytes and mature T cells still retained expression of TCRβ, CD4 or CD8. This “transdifferentiation” might reflect the fact that T and NK lineages were diverted late in hematopoiesis and thus loss of one transcription factor, Bcl11b, was sufficient to cause lineage switch with 100% efficiency.

Because ITNKs reprogrammed from mature T cells retain TCRβ expression, it is possible that Bcl11b mainly functions as a suppressor of NK lineage rather than promoting and maintaining the T cell linage. Our data however do not support this possibility: ITNKs are different from NK cells, even those reprogrammed from DN1-DN2 thymocytes; Bcl11b is expressed at certain stages of NK development; Although ITNKs from mature T cells retain more T cell properties, they are still vastly different from either T cells or NK cells, and have no or diminished expression of IL7Ra, CD4, CD8, CD3 and T-bet (FIG. 10G); Microarray data show that in OHT-treated thymocytes (Bcl11b deletion), in the first 24 hours, down-regulation of T cell-associated genes account for almost all the gene expression changes. NK-associated genes expression follows down-regulation of T cell genes and starts after 48 hours following Bcl11b deletion. Master regulators that promote a cell lineage and that are required to maintain lineage identity have been identified for several cell lineages. For example, ectopically expressing Cebpa in pro-B and pro-T cells transforms them into macrophages at a frequency of around 60% (Laiosa et al., 2006; Xie et al., 2004). 25-50% of fibroblast cells expressing MyoD convert to myogenic colonies (Davis et al., 1987). Recently, it is shown that pancreatic acinar cells expressing three TFs, Pdx1, Ngn3 and Mafa is able to convert them into insulin-expressing β cells in vivo at an estimated frequency of 20%. Additionally, loss of Pax5 in B cells enables de-differentiation of B cells to become multi-potent progenitors (Mikkola et al., 2002). Similar to Pax5 in B cells, we show here that Bcl11b is essential for T cell development and currently the only known transcription factor for T cell identity maintenance. However, unlike de-differentiation in B cell upon loss of Pax5 (Cobaleda et al., 2007), deletion of Bcl11b in T cells does not appear to have obvious or prolonged de-differentiation steps because both pro-T and mature T cells readily convert to ITNKs. Moreover, ITNKs from DP thymocytes and mature T cells still retained Tcrβ expression. This may reflect the fact that T and NK lineages are diverted late during T cell development in the thymus and thus loss of one transcription factor, Bcl11b, is sufficient to convert T cells into iTNK cells with 100% efficiency. Our study therefore adds Bcl11b to the collection of transcription factors that play pivotal roles in hematopoietic lineage specification, commitment and maintenance.

NK cell-based therapies hold promise in cancer treatment. We are now able to reprogramme T cells to ITNKs, which can be extensively expanded but are not malignantly transformed. Rather, they effectively killed tumour cells in vitro and eliminated metastatic cells in mice but did not appear to attack normal cells. Therefore, ITNK cells may serve as a new cell source for cancer immunotherapy and other cell-based therapies.

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FIGURE LEGENDS

FIG. 1. Bcl11b is essential for T cell development and for maintaining T cell identity. Thymocytes from flox/flox or flox/+ control mice were treated, or not, with OHT then sorted into DN1 or DN2 subsets, and cultured on OP9-DL1 stromal cells. (A) Flow cytometry profiles of cultured DN1 and DN2 thymocytes (+OHT) in the absence of IL-2. Numbers refer to percentage of cells in the gate. Data are representative of three experiments. (B) Flow cytometry profiles of cultured flox/flox DN3 thymocytes (±OHT) supplemented with IL-2. Data are representative of three experiments. Bcl11b-deficient DN3 thymocytes lost T cell identity and converted to NKp46 expressing cells. −OHT: non-treated cells; +OHT: treated cells. (C) Killing of OP9-DLI stromal cells by OHT-treated flox/flox DN3 thymocytes. Scale bar: 40 μm. The NKp46⁺ cells from Bcl11b deficient DN3 thymocytes (+OHT) killed OP9-DL1 stromal cells effectively. (D) DNA from purified NKp46⁺ cells was prepared and subjected to PCR to detect DJ (top) and VDJ (bottom) recombination at the TCRβ locus. T: T cells growing from untreated DN3 thymocytes; N1 and N2: sorted NKp46⁺ cells growing from OHT-treated flox/flox DN3 thymocytes; Thy: wild type whole thymocytes; B: B cells; GL: germline band; H₂O: no DNA template in PCR. Numbers indicate DJ recombination products. The NKp46⁺ cells from Bcl11b deficient DN3 thymocytes still retained V(D)J recombination at the Tcrβ locus even though they did not express Tcrβ. (E-G) Microarray analysis of gene expression in NKp46⁺CD3⁺ ITNK cells from DN3 thymocytes (I1-I4), IL-2-expanded NK cells (LAK; L1-L4) and sorted DN3 flox/flox thymocytes (DN3; D1-D4) were subjected to expression. (E) Two-way hierarchical cluster map of the array data. Column numbers (I1-I4 for instance) refer to 4 independent RNA samples for each cell type and rows represent individual transcripts. Scale indicates the log2 value of normalized signal level. Comparison of expression profiles of parental DN3 thymocytes, iTNK cells derived from DN3 thymocytes and regular NK cells (LAKs). RNA samples were made from 4 mice for each cell type. (F) qRT-PCR validation of gene expression of selected genes among ITNKs, LAKs and DN3 cells. Bars are mean+SD of 3 samples. In each histogram in FIG. 1 (F), the first bar represents DN3 cells, the second bar represents ITNKs and the third bar represents LAKs. (G) qRT-PCR validation of gene expression difference among DN3, iTNK and LAK cells. Expression of T cell specific genes was generally decreased, and expression of NK-specific genes was greatly increased in the NK-like cells. Zbtb23 (Rog) and Cdkn1c (p57Kip) were not normally expressed in DN3 thymocytes. In each histogram in FIG. 1 (G), the first bar represents LAK cells, the second bar represents ITNKs and the third bar represents DN3 cells.

FIG. 2. Efficient reprogramming of T cells to ITNKs. (A) Representative flow cytometry profiles of ITNKs reprogrammed from single flox/flox DN3 cells. Numbers refer to percentage in total cells. T: T cells that did not have complete Bcl11b deletion. Data are representative of three experiments. NKp46⁺ iTNKs derived from single Bcl11b-deficient DN3 thymocytes in individual wells (96-well plate) co-cultured with OP9-DL1 stromal cells. T: cells that expressed T cell genes and Bcl11b was not completely deleted; iTNK: cells that had deleted both copies of Bcl11b and expressed NKp46.

(B) PCR genotyping of Bcl11b deletion in two representative T cell (T1, T2) and ITNK (I1, I2) wells. flox: floxed allele; del; deletion allele. −OHT: no OHT treatment; H₂O: no template control. PCR-genotyping indicated that cells in some wells did not have complete Cre-loxP recombination (T1 and T2). These cells had one deletion allele and one cko allele at the Bcl11b locus. On the other hand, all the NKp46⁺ cells had Bcl11b completely deleted (11 and 12). No deletion was detected in cells without OHT treatment (−OHT).

(C) DJ recombination at the TCRβ locus of five ITNK wells (I1-I5) showing unique DJ recombination. L: DNA ladder; Thy: wild type thymocytes. (D) Giemsa stain of parental DN3 thymocytes (T) and ITNK cells. Scale bar: 20 μm. (E) Transmission electron micrograph of an ITNK cell. 1: Nucleus; 2. Golgi body; 3. Granule; 4. ER. Scale bar: 2 μm. (e) Electron Transmission Microscopy image of ITNK cells shows prominent Golgi and ERs, and granules. Arrows: 1=nucleus; 2=ER; 3=granule; 4=golgi. (F) Cytotoxicity of ITNKs (labeled as “+OHT”) and LAKs measured in standard ⁵¹Cr release assays with B16F10, RMA and RMA-S tumor cell targets at the indicated effector-to-target (E:T) ratios. −OHT: flox/flox T cells. Data are mean of triplicate wells. In vitro derived ITNK cells from DN3 thymocytes killed tumour cells effectively. Both LAK and ITNK cells killed MHC—I negative B16F10 melanoma and RMA-S lymphoma cells.

FIG. 3. ITNKs reprogrammed in vivo were potent tumour cell killers. (A) Flow cytometric analysis of thymocytes and splenocytes from OHT treated flox/flox and flox/+ mice. Numbers refer to percentage in lymphocyte gate. Data are representative of four mice. (B) Analysis of ITNKs from thymic γδ cells in OHT treated flox/flox mice. Data are representative of two mice. (C) ITNKs production in Rag2^−/−Il2rg^−/− recipients injected with flox/flox DP thymocytes. Two weeks after injection, donor (CD45.2⁺) and host (CD45.1⁺) splenocytes were analyzed. Numbers refer to percentage of lymphocyte gate. Plots are representative of 15 mice from three independent experiments. Donor cells were identified by CD45.2 staining. About 5% of splenocytes were donor derived and roughly half of these donor-derived cells were NKp46⁺ iTNKs. (D) Ex vivo expansion of ITNKs in IL-2 from splenocytes of the recipient mice. Viable cells were counted and analyzed (bottom panel) at the indicated time points. Numbers refer to percentages. Most cells in the culture were ITNKs because they expressed NKp46, TCRβ, NK1.1 and NKG2D. Bars are mean±SD of 4 samples. Data are representative of three experiments. (d) Ex vivo expansion of in vivo reprogrammed iTNK cells starting from splenotypes of four Rag2^−/−Il2γc^−/− recipient mice. These cells were able to proliferate extensively in the culture for up to 3-4 weeks. Bottom panel: iTNK cells (NK1.1⁺ and/or NKp46⁺) accounted for the majority of the cells in the culture after one week culturing. (E) The ex vivo-expanded ITNKs (labeled as “+OHT”) were used in ⁵¹Cr release killing assays with B16F10, RMA and RMA-S tumor cell targets at the indicated effector-to-target (E:T) ratios. −OHT: flox/flox T cells. Data are mean of triplicate wells. Results are representative of three experiments. Ex vivo expanded iTNKs were more potent killers for tumour cells than LAKs. iTNKs effectively killed tumour cells of either MHC—I positive or negative.

(F) ITNKs prevented tumour metastasis. Rag2^−/−Il2rg^−/− recipients first transplanted with treated (+OHT) or untreated (−OHT) flox/flox DP thymocytes or PBS. Recipients were subsequently injected intravenously with 50,000 B16F10 melanoma cells. Lung tumour colonies were enumerated two weeks after tumour challenge. Data are from individual mice and bar represents the mean. (G) In vivo iTNKs effectively eliminated B16F10 melanoma cells in mice. Many metastatic colonies were visible in the lung of the control mice that were injected with either PBS (no cells) or untreated DP thymocytes (−OHT). Very few metastatic colonies existed if OHT-treated DP thymocytes were injected and hence iTNK were produced (+OHT).

FIG. 4. Bcl11b is a direct downstream target gene of Notch signaling. (A). Bcl11b protein in T cells following OHT treatment detected by Western blot. (B) Schematic of the Bcl11b locus showing putative CSL binding sites (BS) and that of an irrelevant control binding site (CTL). (C) Genomic DNA was prepared from immunoprecipitation of thymocytes, using CSL or control IgG antibodies, and was amplified using primers flanking the putative CSL or the control binding sites at the Bcl11b locus. Three Bcl11b binding regions: Region 1, about 1.8 kb from start of the transcription; Region 2, 5.4 kb downstream of exon 1; region 3, about 600 bp downstream of exon 2. CSL: CSL antibody; IgG: control IgG. Fold-enrichment was calculated relative to the IgG control (set to 1). Bars are mean±SD of triplicate. In the histogram in FIG. 4 (c), the first bar represents CSL and the second bar represents IgG.

FIG. 5. Generation of the Bcl11b-tdTomato reporter mouse. (A) The tdTomato cassette was targeted to the 3′ UTR of the Bcl11b locus. (B) Insertion of the tdTomato cassette at the Bcl11b 3′ UTR did not affect T cell development. Numbers refer to percentage of lymphocytes gate. Data are representative of three mice.

FIG. 6. Detection of Bcl11b expression in hematopoietic lineages using the Bcl11b-tdTomato reporter mice. Leukocytes from the thymus, spleen and bone marrow of Bcl11b^fd/+ mice were labeled with antibodies for flow cytometric analysis. Bcl11b-expressing cells had red fluorescence. Solid line refers to Bcl11b^fd/+ mice and dashed line refers to wild type mouse. (A) CD4⁻CD8⁻ double negative (DN; DN1-DN4) thymocyte subsets. DN1: CD44⁺CD25⁻; DN2: CD44⁺CD25⁺; DN3: CD44⁻CD25⁺; DN4: CD44⁻CD25⁻. (B) Double positive (DP) thymocytes (CD4⁺CD8⁺), splenic CD4⁺ and CD8⁺ T cells, thymic γδ T cells, and splenic NKT cells (CD3⁺CD1d⁺). (C) Bone marrow B cells (CD19⁺B220⁺) and myeloid cells (CD11b⁺Gr-1⁺). (D) Splenic (CD3⁻), and thymic (CD3⁻CD4⁻CD8⁻) NK cells. NKP: NK cell precursor; Immature: NK1.1⁺CD27⁺CD11b⁻ and NK1.1⁺CD27⁺CD11b⁺. (E) qRT-PCR of Bcl11b expression in sorted splenic naïve (CD44CD62⁻L⁺) and activated (CD44⁺CD62L⁻) T cells population. Bcl11b expression was calculated relative to that in CD8⁺CD44⁺CD62L⁻ (set to 1). Bars are mean±SEM of 3 samples. (F) Quantification of Bcl11b expression in naïve and activated T cells in the Bcl11b^fd/+ mice. Percentages refer to the indicated T cell subsets in Bcl11b^fd/+ mice. All FACS data in this figure are representative of three experiments.

FIG. 7. Strategies for identification of cell populations for flow sorting and analysis. (A) Identification of double negative (DN) thymocyte (DN1-DN4) populations defined by Lin⁻ and expression of CD25 and CD44. DN1 subpopulations were defined by expression of CD117 (c-Kit). Numbers refer to percentages. (B) Identification of γδ T cells. (C) Identification of NKT cells in the spleen by first gating (or, prior to FACS sorting, magnetically depleting) out B cells. INKTs were CD3⁺ and stained positively by CD1d dimer. (D) Identification of NK precursors (CD3⁻CD122⁺NK1.1⁻) and NK cell subsets (NK1.1⁺CD27⁺CD11 b⁻, NK1.1⁺CD27⁺CD11b⁺, NK1.1⁺CD27⁻CD11b⁺) cells. (E) Thymic NK cells were defined as NK1.1⁺CD127⁺ thymocytes. (F) Identification of naïve (CD44⁻CD62L⁺) and activated (CD44⁺CD62L⁻) T cells.

FIG. 8. In vitro analysis of Bcl11b-deficient T cells. (A) Schematic diagram of the Bcl11b conditional knockout allele. Bcl11b exon 4 was flanked by loxP sites. Indicated DNA fragments were detected by the 5′ probe in Southern blot analysis of targeted ES cells. Southern blot analysis of the targeted ES cell clones using a 5′ probe which detected a 27 kb wild type BamHI band. The same probe hybridized to a 12.6 kb fragment in the conditional knockout clones (cko/+) and a 17.5 kb fragment in clones that did not have the 5′ IoxP site (+/−). (B) Experimental design for the analysis of Bcl11b-deficient DN thymocytes. Whole thymocytes from CreERT2; Bcl11b^flox/flox(fox/fox) or CreERT2; Bcl11b^flox/+ (flox/+) mice were treated with OHT (+OHT) or left untreated (−OHT) for 48 hr then sorted into the indicated subset and cultured on OP9-DL1 stromal cells for 2 weeks. (C) NKp46⁺CD3⁻ cells from DN1 and DN20HT-treated flox/flox thymocytes did not express TCRβ. Numbers refer to percentage of cells. Data are representative of two experiments. (D) Homozygous Bcl11b deletion in ITNK (NKp46⁺CD3⁻) but not in T (NKp46⁻CD3⁺) cell populations from DN1 and DN2 cultures. flox: conditional knockout allele; del: deletion allele. H₂O: no DNA template control. (E) No NKp46⁺ cells but T cells were obtained from untreated flox/flox thymocytes. (F) NKp46⁺TCRβ⁻ cells from OHT-treated DN1 and DN2 flox/flox thymocytes in the absence of IL-2 or IL-15 cultured on OP9 stromal cells. (G) NKp46⁺TCRβ⁻ cells were detected in OHT-treated DN3 flox/flox, but not flox/+, thymocytes in T cell media. (H) Reprogramming of Bcl11b-deficient DN3 thymocytes to NKp46⁺ cells in myeloid cell culture condition. (I) Reprogramming of Bcl11b-deficient DN3 thymocytes to NKp46⁺CD19⁻ cells in B cell culture condition. (J) Venn diagram comparison of the upregulated (>2-fold) genes between LAK vs DN3 (green) and ITNK vs DN3 (purple) shows a significant overlapping between the two gene lists. (K) ITNKs from DP flox/flox thymocytes treated with OHT and cultured on OP9-DL1 in the presence of IL-2. Untreated cells died rapidly under this condition. (L) ITNKs from splenic flox/flox CD8⁺ T cells treated with OHT cultured on OP9-DL1 in the presence of IL-2. All FACS data in this figure are representative of 2-4 experiments.

FIG. 9. Characterization of in vitro reprogrammed ITNK phenotype. (A) Experimental design for reprogramming of single DN3 thymocytes to ITNK. Whole thymocytes from flox/flox mice were treated with OHT (+OHT) or left untreated (−OHT) and 48-hours later single DN3 cells were sorted and seeded on OP9-DL1 stromal cells in 96-well plates for 10-14 days supplemented with IL-2. (a) Experimental design for analyzing single DN3 thymocytes conversion to iTNKs. DN3 thymocytes (either treated with OHT, or untreated) were sorted into individual wells of 96-well plates pre-seeded with OP9-DL1 stromal cells. Two weeks (with 112) or three weeks (without 112) later, the OHT-treated DN3 cells (Bcl11b-deficient) converted to iTNKs, confirmed by FACS analysis and genomic DNA PCR. (B-C) Expression of intracellular (TRAIL, perforin, IFNγ) and NK cell surface markers by the reprogrammed ITNK from DN3 thymocytes in vitro. (D) Expression of NK cell markers by ITNKs reprogrammed from Bcl11b-deficient DP thymocytes in vitro. (E) ITNKs did not express CD127 and thus were not thymic NK cells. (F) Analysis of CD27 and CD11b in bulk-cultured ITNKs reprogrammed from DN3 thymocytes. All FACS data are representative of three experiments.

FIG. 10. Analysis of in vivo reprogrammed ITNK cells in the flox/flox mouse. (A) Experimental design for the analysis of in vivo reprogrammed ITNK cells. flox/flox or flox/+ mice were treated with Tamoxifen by oral gavage on three consecutive days, and the thymi and spleens were analyzed 2-3 weeks later. We observed a 5-10 fold reduction in total thymocytes and about 2-fold reduction in splenocytes in the treated flox/flox mice compared to treated flox/+ control mice. (B) PCR of Bcl11b deletion in ITNK (NKp46⁺CD3⁺ and NKp46⁺CD3⁻) cell populations in flox/flox mice. flox: conditional knockout allele; del: deletion allele. H₂O: no DNA template control. All the NKp46⁺CD3⁺ and NKp46⁺CD3⁻ cells in the thymus were ITNKs. Analyzing ITNKs in the spleen was more complicated due to the presence of many NKp46⁺ conventional NK cells. However, most of the NKp46⁺CD3⁺ cells in the spleen had Bcl11b deficiency and thus were ITNKs. PCR data are representative of three experiments. (C) Flow cytometric analysis of CD4 and CD8 expression in NKp46⁺ ITNKs. Note that both CD4 and CD8 expression was down in ITNKs (CD4⁺NKp46⁺ or CD8⁺NKp46⁺) compared to CD4⁺NKp46⁻ or CD8⁺NKp46⁻ T cells. (D) Flow cytometric analysis of cells following ex vivo expansion of whole thymocytes or splenocytes from OHT treated mice. (E) Flow cytometric analysis of CD1d-restriced NKT cells in thymus and spleen. Total lymphocytes and CD19⁻ splenocytes were gated in the thymus and spleen, respectively. Note the reduction of NKT cells in the OHT-treated flox/flox mice. (F) Analysis of CD1d-restricted cells in the ex vivo-expanded ITNK culture. Numbers refer to percentages in lymphocyte gate. All FACS data in this figure are representative of 3-4 individual mice. (G) qRT-PCR analysis of several key T or NK cell-associated genes in CD8⁺ T cells, CD8⁺ ITNKs and LAKs. Bars are mean±SEM of 3 samples. The highest expression level for each gene was chosen as 1. (H) Splenocytes from flox/flox or flox/+ mice treated with Tamoxifen were stained with NKp46, NK1.1, CD8 and CD3 to confirm expression of CD3 on ITNKs. A separate aliquot was loaded with Indo-1, stained with antibodies to NKp46, NK1.1 and CD8 and analyzed for calcium flux by flow cytometry. Top panel: Phenotype of splenocytes from flox/flox or flox/+ mice indicating gated T cells (CD3⁺NKp46⁻) and ITNKs (CD3⁺NKp46⁺) cells. Numbers refer to percentages in gates of total lymphocytes. Lower panel: Calcium flux plots from the indicated cell subset. A baseline was established at the start of the assay, before acquisition was interrupted and anti-CD3 (145-2C11) was added (first arrow). CD3 was then cross-linked by addition of anti-hamster secondary antibody (second arrow). Ionomycin was added (third arrow) as a positive control. Numbers in gates refer to responders (upper gate) and non-responders (lower gates) after addition of anti-hamster antibody. Data below calcium plots show ratio of responders to non-responders. Data are representative of two mice.

FIG. 11. In vivo reprogrammed ITNKs from DP thymocytes prevented tumour metastasis. (A) Experimental design for the analysis of in vivo reprogramming of DP thymocytes to ITNKs. Whole thymocytes from flox/flox mice were treated with OHT (+OHT) or left untreated (−OHT) and 48-hours later DP cells were sorted and injected intravenously into Rag2^−/−Il2rg^−/− mice. Two weeks later, splenocytes, bone marrow (BM) and peripheral blood cells (PB) were analyzed by flow cytometry fro ITNKs. (B) Most ITNKs in the spleen were CD8⁺. Numbers in gates refer to percentages. Data are representative of three experiments. (C) ITNKs had complete Bcl11b deletion whereas donor derived NKp46 cells still retained at least one copy of the foxed allele. PCR data are representative of two individual experiments. (D) ITNKs were also found in bone marrow and peripheral blood. About 1.0% of bone marrow and 6-7% of peripheral white blood cells expressed NKp46 and thus ITNKs in the recipients injected with Bcl11b-deficient DP thymocytes. (E) Expression of additional NK cell surface markers on the in vivo reprogrammed ITNKs. The in vivo iTNKs expressed more NK-specific receptors such as Ly49C/I and Ly49G2. (F) ITNKs prevented tumour metastasis. Rag2^−/−Il2rg^−/− recipients were transplanted with treated (+OHT) or untreated (−OHT) flox/flox DP thymocytes or PBS. Recipients were subsequently injected intravenously with 5×10⁴B16F10 melanoma cells. Lung tumour colonies were enumerated two weeks after tumour challenge. Experiment was performed twice. (G) Plot shows inverse correlation between the percentage of ITNK cells (squares) obtained from recipient mice following in vivo reprogramming and tumor challenge and the number of lung colonies (circles) observed. Data are individual mice and are representative of two independent experiments, each with 5 mice per group. Chart shows that in vivo the percentages of ITNKs in spleen (squares) correlated with reduction of metastatic sites (+OHT circles) in the Rag2^−/−Il2γc^−/− mice after injection of OHT treated DP thymocytes. The −OHT squares and circles represent iTNKs and the metastatic sites respectively in recipient mice that were injected OHT untreated DP thymocytes. In mice injected with OHT-treated DP thymocytes, about 4% of splenocytes were iTNKs. FIG. 12. A working model showing that Bcl11b acts downstream of Notch signaling and promotes T cell development and maintains T cell identity.

FIG. 13. Bcl11b is expressed in early T cell precursors and is essential for T cell differentiation.

- a. Expression of Bcl11b in thymocytes from Bcl11b-lacZ knock-in mice using the fluorescent substrate FDG. Almost all of the DN2-DN4 thymocytes were stained positively for FDG. However a significant DN1 population did not express Bcl11b.
- b. Detection of Bcl11b expression in the five DN1 subpopulations. Approximately half of the DN1a and DN1b thymocytes, which were CD117⁺ and were thought to contain the true T cell progenitors, expressed Bcl11b.
- c. Top, acute loss of Bcl11b caused DN1 thymocytes to express NK-specific genes NK1.1 and NKp46 on OP9-DL1 stromal cells. Bottom, deleting Bcl11b in DN2 thymocytes gave rise to the same phenotype of losing T cell differentiation potential and converting to NK-like cells.

FIG. 14.

- a. Left panel: different double negative (DN) thymocyte populations defined by expression of CD25 and CD44. Right panel: five subpopulations of DN1 thymcoytes based on expression of CD24 and CD117 (c-Kit).
- b. Flow chart of analyzing Bcl11b-deficient DN1 thymocytes. The Bcl11b-deficient cells (+OHT) acquired NK properties while the untreated ones (−OHT) proliferated and differentiated into T cells on OP9-DL1 stromal cells.

FIG. 15.

- a. Double positive (DP) thymocytes expressed NKp46 after Bcl11b deletion. The untreated DP cells died in T cell media about one week after plated on OP9-DL1 stromal cells (not shown).
- b. Purified CD8 single positive cells (−OHT) proliferated on OP9-DL1 stromal cells. They did not express NKp46. Once Bcl11b was deleted, 38% of the cells now expressed NKp46 which killed the stromal cells. Note that these iTNKs still expressed Tcrβ and CD8.
- c. Purified CD4 single positive cells (−OHT) growing in T cell media (left). Bcl11b deletion (+OHT) caused these CD4 T cells to express NKp46. Note that most of the cells now did not express CD4 anymore.

Materials and Methods

1.1.1 Mice

The Bcl11b conditional knockout targeting vector was constructed using recombineering (Liu et al., 2003), and the mice (Bcl11b^flox/flox) were made according to a standard gene targeting approach in ES cells. The Bcl11b^flox/floxmice were crossed to Cre-ERT2 mice to generate Cre-ERT2; Bcl11b^flox/floxmice. Cre-ERT2; mice were a mixed C57BL/6J and 129S5 genetic background. A SA-lacZ cassette was targeted into the intron 3 of Bcl11b gene in Bcl11b-lacZ reporter mice (Song-Choon Lee and Pentao Liu, unpublished). All mice were NK1.1⁺ by flow cytometry, suggesting that they had inherited the C57BL/6 haplotype at the NK gene complex. Bcl11b tdTomato reporter mice were constructed by inserting the tdTomato cassette into the 3′ UTR of Bcl11b. Bcl11b tdTomato mice are on a C57BL/6 background. Rag2^−/−Il2rg^−/− are on a C57BL/6 background. Both C57BL/6 and 129S5 have the H-2^bhaplotype at the MHC. All animal experiments were performed in accordance with the UK 1986 Animals Scientific Procedure Act and local institute ethics committee regulations.

1.1.2 Reprogramming of T Cells to ITNKS In Vivo Flox/Flox

To test for the in vivo reprogramming of endogenous T cells to ITNK, Cre-ERT2; Bcl11b^flox/floxand Cre-ERT2; Bcl11b^flox/+ mice were given 3 doses of 1 mg Tamoxifen (indicated in the text as OHT) (Sigma) dissolved in sunflower oil by oral gavage on 3 consecutive days. Mice were analysed 2-3 weeks later. For the in vivo reprogramming of in vitro-treated thymocytes, thymocytes from Cre-ERT2; were treated with 4-hydroxytamoxifen (indicated in the text as OHT) (Sigma) or left untreated for 48 hours. 2−4×10⁶DP thymocytes were then sorted and injected intravenously into Rag2^−/−Il2rg^−/− recipient mice without irradiation. At various time points thereafter, blood, bone marrow and/or splenocytes were prepared for analysis.

1.1.3 PCR Genotyping and qRT-PCR

To extract genomic DNA, sorted cells were incubated in 400 μl of lysis buffer (50 mM Tris with pH 8.0, 100 mM NaCl, 25 mM EDTA with pH 8.0, 0.5% SDS, and 0.5 mg/ml Proteinase K) at 65° C. for 2 hrs. Genomic DNA was precipitated by adding 500 μl of isopropanol into cell lysis buffer. After centrifugation, DNA was washed once with 500 μl 70% ethanol and air dried before being re-suspended as template for PCR. The Bcl11b cko allele and the deletion after Cre-loxP recombination were detected by PCR with primers described in Table 4. PCR primers to detect TCRβ D-J and V-DJ are also listed in Table 4. For qRT-PCR, RNA was isolated using the RNAqueous Micro Kit (Ambion) from FACS sorted cells. After DNase I treatment, RNA was reverse transcribed to make cDNA with Super Script 11 (Invitrogen). qRT-PCR was performed with either SYBR (Invitrogen) or Taqman Master Mix (ABgene). cDNA input was standardized and PCR was performed for 40 cycles. Primers for qRT-PCR are listed in Table 4.

FDG Staining

For FDG staining, cells were first surface stained as above. Cells were then warmed at for 5 minutes before 20 μl pre-warmed FDG (Sigma) was added for a further 1 minute. The reaction was quenched by addition of 2.0 ml ice-cold PBS plus 1% BSA, and the cells were incubated on ice for a further 30 minutes. The cells were centrifuged and resuspended in PBS before analysis.

1.1.4 Flow Cytometry and Cell Sorting

Cells from spleen, thymus and bone marrow were mechanically disrupted and the red blood cells were removed with ACK lysis buffer (Lonza). Blood was collected into EDTA tubes (Sarstedt). In vitro-cultured cells were collected and washed with PBS/1% BSA prior to antibody labelling. For all cells, Fc receptors were blocked with anti-CD16 (2.4G2) prior to antibody labelling. Antibodies to the following antigens were used: CD3s (145-2C11), CD4 (L3T4), CD8a (53-6.7), CD25 (PC61), CD44 (IM7), CD122 (TM-131), CD27 (LG.3A10), CD11b (M1/70), CD45.2 (104), TCRβ (H57-597), CD117 (2B8), NK1.1 (PK136), CD49b (DX5), NKp46 (29A1.4), Ly49C/I (5E6), Ly49G2 (4D11), Ly49D (4E5). All antibodies were from BD Biosciences or eBioscience. Cells were incubated with antibody for 30 minutes at 4° C. before being washed. In some cases biotinylated antibodies were revealed by incubation with fluorochrome-conjugated streptavidin for a further 20 minutes at 4° C. CD1d-restricted NKT were detected by labelling cells with CD1d-mouse IgGl Fc fusion protein (BD Biosciences) loaded with α-galactosylceramide (Kirin), followed by fluorochrome-conjugated anti-mouse IgGl (BD Biosciences). Data acquisition was performed using a FACSCalibur (BD Biosciences), LSR II (BD Biosciences) or a FC 500 (Beckman Coulter) with dead cells excluded based on scatter profile or DAPI inclusion. Analysis was performed using FlowJo (Tree Star) software. Sorting was performed using a MoFlo (DAKO) or FACS Aria (BD Biosciences).

1.1.5 OP9 Stromal Cell Culture

OP9 stromal cells were cultured in alpha-MEM (Sigma) with 10% FCS (heat inactivated at 56° C. for 30 min), 1% penicillin/streptomycin, and 2 mM L-glutamine (Life Technologies). OP9-DL1 stromal cells were cultured in alpha-MEM (Sigma) with 20% FCS, 1% penicillin/streptomycin, and 2 mM L-glutamine (Life Technologies). Cells were passaged every 2 to 3 days by trypsinization (Invitrogen). A monolayer (70%-80% confluent) of OP9 or OP9-DL1 cells was prepared 24 hours prior to co-culture.

1.1.6 OHT Treatment In Vitro

Thymocytes or splenocytes from Cre-ERT2; Bcl11b^flox/floxmice were cultured in T cell medium with 1 μM 4-hydroxytamoxifen (indicated in the text as OHT) at 37° C. for 48 hrs. After this time, cells were washed and resuspended with fresh media. T cell media: RPMI-1640, 10% FCS, 1% penicillin/streptomycin, 2 mM L-glutamine, 5 ng/ml muFlt-3L, 5 ng/ml hulL-7. All cytokines used in this study were purchased from PeproTech.

1.1.7 Reprogramming of T Cells to ITNKS In Vitro

After OHT treatment, thymocytes were sorted by FACS and co-cultured with OP9-DL1 in T cell culture media (3,000 cells per well in 24-well plates). To promote ITNK proliferation, 20 ng/ml mulL-15 or 100 ng/ml hulL-2 was supplemented in T cell medium as indicated. Every three days, half of the media was replaced with fresh T cell media with IL-15 or IL-2 as indicated in text. Every seven days, cells were collected by vigorous pipetting, filtered through cell strainers and transferred to new tissue culture plates pre-seeded with fresh OP9-DL1 stromal cells. Fourteen days after OHT treatment, cells were collected and analyzed by FACS. For analysis of ITNK production in myeloid cell differentiation conditions, IMDM was used supplemented with 10% FCS, 1% penicillin/streptomycin, 2 mM L-gluatamine, 1 ng/ml hulL-7, 5 ng/ml muFlt-3L, 10 ng/ml hulL-3, hulL-6, stem cell factor (muSCF), and granulocyte/macrophage colony-stimulating factor (muGM-CSF). Cells were cultured on OP9 stromal cells. For analysis of ITNK production in B cell differentiation conditions, IMDM was used supplemented with 10% FCS, 1% penicillin/streptomycin, 2 mM L-gluatamine, 5 ng/ml hulL-7, 5 ng/ml muFlt-3L. Cells were cultured on OP9 stromal cells.

1.1.8 Reprogramming of Single Thymocyte to ITNKs

Thymocytes of Cre-ERT2; Bcl11b^flox/floxwere treated with OHT as above. Single DN3 thymocytes were sorted directly into individual wells of a 96-well plate pre-seeded with OP9-DL1 stromal cells in T cell medium supplemented with 100 ng/ml hulL-2. Medium was changed every three days. After 10-14 days cells were analyzed in flow cytometry. Genomic DNA was extracted for genotyping of the Bcl11b locus and for amplifying 6TCR rearrangement with PCR.

1.1.9 Tumour Cell Killing Assay

B16F10 melanoma (H-2^b), RMA lymphoma and RMA-S lymphoma (N-2^bTAP-1-deficient variant) were maintained in RPMI-1640, 5% FCS, 1% penicillin/streptomycin, 2 mM L-glutamine. For killing assays, target cells were washed and incubated with 0.1 μCi Na₂⁵¹CrO₄(Perkin Elmer) for 45 mins. at 37° C. The cells were then washed and added in triplicate to effector cells at the indicated E:T ratio. Plates were incubated for 4 hours at 37° C. before the supernatant was tested for chromium release in a scintillation counter. Percent specific lysis was calculated as (experimental release—spontaneous release)/(maximum release—spontaneous release)×100.

T Cells to iTNKs In Vivo

Thymocytes from Cre-ERT2; b^flox/floxwere treated with OHT as above. 2−4×10⁶DP thymocytes were sorted and injected intravenously into Rag2^−/−Il2γc^−/− recipient mice without irradiation. At various time points thereafter, blood and/or splenocytes were prepared for analysis.

1.1.10 ITNK Ex Vivo Expansion and LAK Culturing

For ex vivo expansion, splenic ITNK cells were enriched using the NK Isolation Kit (Miltenyi) and cultured for 6-9 days at 1×10⁶cells/ml in RPMI 1640 medium containing 10% FCS/50 μM 2-mercaptoethanol/2.0 mM L-glutamine and 1000 U/ml hIL-2 (Chiron). The cells were split every two days and supplemented with fresh IL-2. Purity was always >90%. For culturing reprogrammed ITNK cells ex vivo, whole splenocytes were cultured without pre-enrichment.

1.1.11 Tumour Experiments In Vivo

After OHT treatment, 2−4×10⁶DP T cells were sorted from Cre-ERT2; Bcl11b^flox/floxthymocytes and injected intravenously into each Rag2^−/−Il2rg^−/− recipient mouse without irradiation. Two weeks later, 5×10⁴B16F10 melanoma cells were injected intravenously and the lung colonies were enumerated 14 days after tumour inoculation.

Calcium Flux Experiments

flox/flox or flox/+ mice were treated with Tamoxifen to derive in vivo-reprogrammed ITNK as described above and splenocytes were analyzed 4-5 weeks later. Splenocytes were either stained directly with antibodies to NKp46, NK1.1, CD8 and CD3 for phenotyping, or loaded with 2 μM Indo-1 (Invitrogen), washed and stained with antibodies to NKp46, NK1.1 and CD8. Data was then acquired using an LSR II flow cytometer gating on lymphocytes, measuring Indo-1 (violet)/Indo-1 (blue) ratio against time. Unstimulated cells were run to establish the baseline Indo-1 (violet)/Indo-1 (blue) fluorescence before acquisition was interrupted, anti-CD3 (145-2C11; μg/ml) added and acquisition continued. Acquisition was interrupted again and cross-linking anti-hamster IgG secondary antibody was added before continuing. Ionomycin (1 μg/ml) was added at the end of the acquisiton to serve as a positive control.

1.1.12 Gene Expression Analysis

RNA was extracted using the RNAqueous Micro Kit (Ambion) from FACS sorted cells. Quality and quantity of RNA samples was tested with Bioanalyzer. Total RNA was amplified using the Illumina Total Prep RNA Amplification Kit (Ambion) according to the manufacture's instructions. The biotinlated cRNA (1500 ng per sample) was applied to Illumina Mouse-6 Expression BeadChips and hybridized overnight at 58° C. Chips were washed, detected and scanned according to the manufacture's instruction and the scanner output imported into BeadStudio software (Illumina).

Chromatin Immunoprecipitation

Chromatin immunoprecipitation was performed as previously described (38). Control IgG and the CSL antibody were purchased from Abcam. Genomic DNA was purified with Qiaquick PCR purification kit (QIAGEN) and specific genomic DNA regions were quantified by real-time quantitative PCR with Taqman (ABI) or SYBR Green (Invitrogen). Input DNA was used as a standard curve to quantify concentration of DNA recovered after IP. The amount of DNA recovered from each ChIP sample was presented as relative to the control IgG. Primers used in this assay are listed in table 4.

TABLE 1

ITNK vs. DN3

p-value
Ratio
Fold-Change

Column ID
(ITNK vs. DN3)
(ITNK vs. DN3)
(ITNK vs. DN3)

FCER1G
1.61E−08
38.85426542
38.8542

ROG
3.56E−09
38.51902069
38.519

UPP1
1.59E−11
27.95435613
27.9543

IFITM1
3.53E−06
27.42649015
27.4265

XCL1
1.21E−06
25.36912071
25.3691

SERPINA3G
7.75E−08
21.14875825
21.1487

SCIN
1.90E−08
20.78544021
20.7854

NKG7
1.76E−08
20.18204202
20.182

AQP9
1.71E−07
18.25220532
18.2522

KLRD1
7.30E−09
17.17811645
17.1781

LGALS3
7.17E−09
15.91702772
15.917

AVIL
9.13E−07
13.61854192
13.6185

IFITM3
1.54E−07
13.57143051
13.5714

TYROBP
1.57E−09
13.52446102
13.5245

GADD45G
4.52E−08
13.52446102
13.5245

CD160
3.07E−07
12.64065893
12.6407

IFITM2
4.83E−06
11.27456728
11.2746

CTSW
3.28E−06
9.798061943
9.79809

9130404D14RIK
6.12E−09
9.679979866
9.67995

LOC270152
2.41E−07
9.530162966
9.53016

BC025206
1.38E−08
9.063060777
9.06307

VIM
3.49E−08
8.891971439
8.89195

NFIL3
2.83E−06
8.426871608
8.42689

AMICA1
9.38E−08
8.267810932
8.26778

LTA
1.13E−11
8.210652501
8.21067

GLRX1
2.07E−06
8.027743883
8.02777

LITAF
1.96E−07
7.727497527
7.72749

CCR5
6.07E−09
7.323324789
7.32333

LMNA
5.83E−08
7.235052382
7.23503

BC049975
2.32E−08
6.988559728
6.98858

P2RY14
1.44E−07
6.797495803
6.79748

WBSCR5
8.76E−08
6.486766995
6.48677

LAG3
8.41E−08
6.190263953
6.19026

LY6A
1.40E−05
5.989781433
5.98977

E030006K04RIK
6.54E−09
5.948379959
5.94839

9130211I03RIK
4.39E−06
5.87668367
5.87667

1300002F13RIK
7.18E−06
5.866513355
5.8665

LOC381140
2.90E−07
5.84620961
5.8462

GPR114
2.07E−05
5.78573123
5.78573

2310067E08RIK
3.25E−07
5.725901114
5.72589

CDKN2B
6.18E−06
5.686341408
5.68634

IDB2
1.14E−08
5.637296353
5.63728

GOLPH2
4.35E−09
5.608053164
5.60805

PLCG2
2.95E−09
5.550036353
5.55005

1500031H04RIK
1.83E−07
5.492634377
5.49264

1110018K11RIK
7.30E−10
5.388947269
5.38893

CD9
9.16E−09
5.388947269
5.38893

LOC381319
6.47E−06
5.323963158
5.32396

SYTL2
3.83E−10
5.250666835
5.25066

SLC2A6
1.27E−07
5.223432317
5.22344

OSBPL3
3.09E−11
5.187367722
5.18736

2210411K11RIK
2.39E−06
5.080603779
5.0806

LRRK1
1.15E−07
5.045510505
5.04551

S100A6
3.80E−07
4.967438441
4.96743

KLRE1
8.01E−08
4.933107068
4.93312

PGLYRP1
6.39E−06
4.933107068
4.93312

GLRX
6.59E−06
4.873650608
4.87364

MYO1F
8.46E−09
4.839966507
4.83998

LOC269941
9.61E−10
4.831594764
4.8316

TRAF1
1.82E−06
4.69946896
4.69948

EMILIN2
0.000282473
4.691333699
4.69134

TNFRSF9
1.19E−07
4.67510367
4.67511

CD52
9.20E−05
4.618745641
4.61874

PLSCR1
2.75E−09
4.602758894
4.60276

BHLHB2
5.80E−06
4.570968863
4.57097

S100A1
8.07E−08
4.55514458
4.55515

LGALS1
4.96E−07
4.523699663
4.52369

2310046K01RIK
3.01E−06
4.4691539
4.46915

CAPG
5.03E−09
4.453688322
4.45369

C330008K14RIK
4.31E−07
4.430601277
4.43059

TNFRSF11B
0.000351472
4.384772562
4.38477

CCL4
8.44E−05
4.362031136
4.36203

SIAT10
6.50E−06
4.339411402
4.33941

HBA-A1
1.28E−06
4.301981923
4.30198

ROM1
1.73E−08
4.272262761
4.27226

1190002C06RIK
2.57E−07
4.198875541
4.19887

F2R
2.39E−05
4.184345527
4.18434

RGS1
5.76E−05
4.184345527
4.18434

CD69
2.26E−05
4.177091992
4.17709

CISH
1.63E−06
4.155429692
4.15544

DAPK2
9.71E−09
4.133888377
4.13389

SH3BP2
3.58E−08
4.098226288
4.09823

GCNT1
2.49E−08
4.069921247
4.06992

HAVCR2
5.18E−05
4.062877086
4.06287

DUSP6
3.27E−09
4.041808467
4.04181

CTNNA1
4.08E−08
3.993068034
3.99307

BC024955
6.91E−10
3.917681673
3.91768

ITGB7
0.000152314
3.917681673
3.91768

MLKL
7.92E−08
3.877156183
3.87716

SERPINE2
2.49E−05
3.870448353
3.87045

LY6G5B
7.00E−06
3.863748764
3.86375

PPP3CC
5.92E−09
3.850374449
3.85038

LOC218482
1.61E−08
3.837062959
3.83706

A430006M23RIK
3.38E−06
3.7776762
3.77768

2410008K03RIK
3.43E−08
3.732137059
3.73213

FURIN
1.27E−06
3.732137059
3.73213

F2RL2
9.14E−05
3.732137059
3.73213

GPR18
2.97E−06
3.712779387
3.71278

HGFAC
7.05E−05
3.70635306
3.70635

S100A10
4.21E−06
3.693525988
3.69353

APOB48R
4.85E−06
3.68074675
3.68075

OSM
1.62E−05
3.68074675
3.68075

AIM1L
2.96E−06
3.674376734
3.67438

IL18R1
3.56E−07
3.661662395
3.66167

NT5E
2.59E−08
3.655331484
3.65533

IFNG
3.30E−06
3.63637686
3.63637

H2-Q8
0.000127421
3.630080297
3.63008

FXYD4
1.04E−07
3.617513104
3.61752

PILRB
3.51E−05
3.592534713
3.59253

PLP2
3.87E−08
3.580097522
3.5801

MT1
0.000577989
3.580097522
3.5801

DOK2
7.55E−08
3.567707962
3.56771

0610037M15RIK
7.75E−06
3.549220591
3.54922

2310047C17RIK
4.43E−05
3.549220591
3.54922

S100A11
2.00E−07
3.530811628
3.53081

FES
1.98E−08
3.518599316
3.5186

BC029169
3.49E−07
3.500346534
3.50035

TNF
5.66E−10
3.482197266
3.4822

LRP12
7.21E−05
3.482197266
3.4822

IER3
0.000215344
3.476169123
3.47617

NAPSA
2.95E−05
3.470149772
3.47015

ANXA2
3.41E−06
3.434266424
3.43426

PRSS19
1.77E−05
3.428320671
3.42832

OSTF1
5.84E−09
3.381119827
3.38112

GVIN1
0.000422456
3.36941982
3.36942

1110007C02RIK
2.44E−08
3.340348064
3.34035

MAPKAPK3
1.77E−07
3.311532412
3.31153

CD244
7.87E−08
3.277280906
3.27728

F630022B06RIK
4.89E−06
3.271608977
3.27161

ID2
1.03E−07
3.265945981
3.26594

GPR68
6.05E−06
3.265945981
3.26594

GLIPR1
4.48E−07
3.260291927
3.26029

PDGFA
1.76E−06
3.254646822
3.25464

PKP3
1.71E−08
3.254646822
3.25464

D10BWG1379E
5.49E−08
3.254646822
3.25464

SLC39A4
4.73E−08
3.215403067
3.2154

TES
1.92E−07
3.182149415
3.18215

EGR1
0.0012963
3.182149415
3.18215

1110004P15RIK
5.91E−05
3.176640258
3.17664

B4GALNT4
4.86E−07
3.149229384
3.14923

CDKN1A
0.000553492
3.149229384
3.14923

D2ERTD217E
7.91E−08
3.079083172
3.07908

NCF4
5.37E−06
3.073754745
3.07375

LOC381924
1.33E−06
3.047229002
3.04723

170002G04RIK
2.02E−06
3.041954638
3.04196

AA467197
0.00100238
3.03668928
3.03669

SGK
5.37E−08
3.020947248
3.02095

BC021614
5.58E−05
3.015717922
3.01571

LOC385953
1.27E−10
3.005277267
3.00528

CDKN2A
8.87E−06
2.989697502
2.9897

2610009E16RIK
2.28E−05
2.953651304
2.95365

HIST1H1C
0.00103646
2.928171942
2.92817

DCI
1.02E−08
2.912989018
2.91299

NFKB1
1.21E−07
2.902909296
2.90291

TPST2
2.16E−06
2.902909296
2.90291

TRF
2.76E−06
2.89788715
2.89788

HK2
3.64E−06
2.887861452
2.88786

PDZK1
5.44E−10
2.88285795
2.88286

GNG2
6.86E−09
2.872886274
2.87288

S100A4
8.62E−08
2.848102167
2.8481

ZFP608
8.21E−08
2.838248233
2.83825

2210008N01RIK
6.64E−08
2.83333475
2.83333

SH3BGRL3
4.55E−05
2.828430249
2.82843

MYO1G
2.01E−06
2.823526754
2.82353

1110019C08RIK
1.34E−05
2.818640232
2.81864

S100A13
8.29E−05
2.818640232
2.81864

PPAP2C
3.95E−06
2.813762676
2.81376

MYO1E
7.25E−08
2.808886192
2.80889

IFI30
1.07E−06
2.799168087
2.79917

LTB4R1
0.000868775
2.784654327
2.78466

TMEM126A
3.48E−08
2.775025877
2.77502

1110020C13RIK
7.24E−08
2.775025877
2.77502

CD7
7.40E−06
2.775025877
2.77502

4933439K08RIK
1.06E−08
2.751084615
2.75108

E030003N15RIK
2.16E−05
2.732083678
2.73208

CCL5
0.0117888
2.732083678
2.73208

7420404O03RIK
1.10E−05
2.722629407
2.72263

4930486L24RIK
1.95E−06
2.717915685
2.71791

BC022224
9.36E−07
2.713210895
2.71321

5330403J18RIK
5.98E−06
2.708507694
2.70851

FXYD5
4.01E−06
2.703820769
2.70382

A430038C16RIK
2.01E−06
2.699142752
2.69914

GPC1
0.00472373
2.675857345
2.67586

AHNAK
7.45E−07
2.666595557
2.6666

EMP1
0.000268268
2.666595557
2.6666

CX3CR1
3.25E−09
2.661981579
2.66198

2810032E02RIK
4.29E−07
2.661981579
2.66198

LOC327957
0.000272484
2.657369417
2.65737

FCGR3
3.00E−05
2.625333417
2.62533

CLNK
1.01E−08
2.620785449
2.62079

HVCN1
0.000222656
2.620785449
2.62079

BSCL2
5.79E−06
2.616246367
2.61625

LGALS3BP
6.49E−06
2.616246367
2.61625

CYP51
7.38E−07
2.607194815
2.6072

PIM3
6.98E−05
2.593677134
2.59368

BC03881
7.63E−09
2.584707321
2.58471

LOC383981
0.00132323
2.584707321
2.58471

PDLIM7
5.33E−07
2.580232324
2.58023

SLC24A3
6.09E−05
2.580232324
2.58023

HHEX
5.95E−07
2.575766162
2.57576

D930046M13RIK
1.74E−05
2.575766162
2.57576

BC087945
7.68E−07
2.57130221
2.5713

9830144J08RIK
1.24E−07
2.566853705
2.56685

DP1
1.29E−06
2.566853705
2.56685

E130012A19RIK
1.31E−05
2.562407433
2.56241

4631423F02RIK
7.66E−05
2.540301889
2.5403

4930555L03RIK
4.40E−08
2.535902033
2.5359

FOSL2
6.92E−06
2.535902033
2.5359

ZFP296
2.68E−09
2.518384205
2.51839

F730045P10RIK
0.000293728
2.518384205
2.51839

PEA15
5.59E−08
2.509674796
2.50967

ITGAE
0.000534865
2.50533009
2.50533

A530050E01RIK
1.01E−05
2.50533009
2.50533

SCL0001419.1_32
1.67E−07
2.500994145
2.50099

SPP1
0.00129868
2.496660716
2.49666

ASB2
2.54E−05
2.492336067
2.49234

CCNG1
4.25E−05
2.492336067
2.49234

TUBA6
1.47E−06
2.483713052
2.48372

SDF2L1
7.56E−05
2.483713052
2.48372

RHOF
9.16E−06
2.47941466
2.47942

1110030J09RIK
2.72E−10
2.45802311
2.45803

EGR3
0.00128127
2.45802311
2.45803

CXCR3
5.82E−05
2.453770955
2.45377

ALDOA
0.000483723
2.449521486
2.44952

GCNT2
3.22E−08
2.44528073
2.44528

MVP
4.37E−07
2.44528073
2.44528

C130027E04RIK
5.53E−07
2.44528073
2.44528

SEC61B
1.47E−09
2.436819366
2.43682

E430036I04RIK
2.87E−07
2.43259877
2.4326

AI481100
9.30E−05
2.424183656
2.42419

CD63
0.000436295
2.41998911
2.41999

DEGS
4.85E−07
2.415797382
2.4158

LOC385699
1.78E−05
2.415797382
2.4158

EG331493
3.45E−06
2.415797382
2.4158

A930008A22RIK
6.04E−07
2.411614335
2.41162

4930504E06RIK
0.000436715
2.411614335
2.41162

LOC212399
1.73E−06
2.407439952
2.40744

AI850995
3.62E−06
2.407439952
2.40744

CTGF
0.000743838
2.407439952
2.40744

KIRL2
3.13E−06
2.394957178
2.39496

1700017I11RIK
6.32E−08
2.390811633
2.39081

2310037P21RIK
2.51E−06
2.386669022
2.38667

UAP1L1
3.29E−06
2.382540741
2.38254

D14ERTD449E
0.000867516
2.382540741
2.38254

BC023892
4.42E−09
2.378415405
2.37841

AA175286
0.000409847
2.361983405
2.36199

PPIB
5.05E−09
2.357895531
2.3579

GPR34
1.68E−07
2.357895531
2.3579

IRAK2
7.34E−05
2.357895531
2.3579

SH2D1B1
0.000605208
2.353810702
2.35381

HSD11B1
6.66E−05
2.353810702
2.35381

LOC328703
3.58E−06
2.349740001
2.34974

BC004728
5.49E−07
2.349740001
2.34974

LOC215405
4.47E−05
2.349740001
2.34974

RAB3D
1.16E−05
2.337551835
2.33755

SULT2B1
0.000164735
2.337551835
2.33755

EVI2A
2.55E−07
2.333509902
2.33351

TSPO
2.22E−06
2.333509902
2.33351

TNFRSF18
2.31E−05
2.333509902
2.33351

EG630499
0.000147665
2.329465644
2.32947

SERPINB6A
0.00133703
2.32543538
2.32543

SYPL
6.57E−07
2.321408259
2.32141

8030402P03RIK
0.000483123
2.321408259
2.32141

HAAO
3.04E−05
2.317389692
2.31739

NRGN
5.54E−05
2.313374311
2.31338

TAF9B
1.33E−06
2.309372821
2.30937

9930117H01RIK
6.56E−05
2.309372821
2.30937

GBP2
0.000466109
2.309372821
2.30937

AW212394
7.13E−06
2.30537452
2.30537

KIT
0.000138895
2.301379447
2.30138

A630086H07RIK
3.07E−05
2.297398197
2.2974

ANK
1.14E−07
2.293420178
2.29342

BATF
6.24E−06
2.293420178
2.29342

TIAM1
1.13E−07
2.289450669
2.28945

TCRD-V1
6.14E−06
2.285484431
2.28548

ARL6IP5
2.09E−07
2.285484431
2.28548

EHD4
3.43E−05
2.285484431
2.28548

N4WBP5-PENDING
9.39E−08
2.281526706
2.28153

B3GNT8
1.44E−07
2.281526706
2.28153

BLR1
8.56E−06
2.281526706
2.28153

NDFIP1
2.11E−06
2.261844715
2.26184

PRDX4
1.09E−05
2.261844715
2.26184

SNAG1
4.20E−07
2.250118694
2.25012

B4GALNT2
0.000956597
2.250118694
2.25012

TRPM6
9.96E−08
2.238448487
2.23845

CXCL9
0.00820156
2.230703854
2.23071

0610009O03RIK
8.23E−09
2.222987918
2.22299

PRR7
1.26E−06
2.207632226
2.20763

A630077B13RIK
3.14E−05
2.207632226
2.20763

SLC19A2
4.63E−05
2.207632226
2.20763

2810440J20RIK
7.67E−07
2.192381474
2.19238

MED10
3.04E−06
2.192381474
2.19238

COMT
8.15E−09
2.188586086
2.18859

PLTP
6.19E−07
2.188586086
2.18859

2310010I15RIK
1.21E−08
2.181015568
2.18102

0610039P13RIK
0.000646447
2.181015568
2.18102

VPS29
1.09E−07
2.177240435
2.17724

AI847670
1.60E−06
2.16595082
2.16595

ASAH1
3.80E−08
2.162199562
2.1622

B830021E24RIK
1.42E−05
2.162199562
2.1622

SRGAP2
4.27E−06
2.158456617
2.15846

IQGAP2
0.000141764
2.158456617
2.15846

LASP1
2.73E−06
2.154717323
2.15472

CORO1C
7.53E−07
2.150990962
2.15099

H2-Q6
3.02E−06
2.150990962
2.15099

9130604K18RIK
4.57E−06
2.147263635
2.14726

FNBP1
2.88E−08
2.143549203
2.14355

TMPIT
3.47E−06
2.139833863
2.13984

H2-Q7
5.84E−05
2.136131381
2.13613

0610007H07RIK
2.48E−06
2.132432594
2.13243

CCND2
3.28E−06
2.125055251
2.12505

SERTAD1
0.000141861
2.121376689
2.12138

RAB19
7.99E−06
2.114035292
2.11404

BAG3
0.00480518
2.110376934
2.11038

VTI1B
1.30E−05
2.10672234
2.10672

CAPN2
2.23E−06
2.103075959
2.10307

2310057H16RIK
6.11E−05
2.099433363
2.09943

STX11
0.000109261
2.095798971
2.0958

FTL1
0.00023523
2.095798971
2.0958

ARF6
3.80E−07
2.092168386
2.09217

2900026A02RIK
2.85E−07
2.088545996
2.08855

CSTB
3.81E−05
2.088545996
2.08855

LOC383189
5.48E−07
2.084931781
2.08493

CCL3
0.00511685
2.084931781
2.08493

GLIPR2
6.01E−05
2.084931781
2.08493

C330023F11RIK
1.81E−06
2.081321389
2.08132

SIRT3
2.69E−06
2.081321389
2.08132

CAPNS1
3.44E−08
2.077719163
2.07772

RBMS1
1.72E−07
2.077719163
2.07772

1110008P14RIK
2.41E−07
2.077719163
2.07772

MINA
1.96E−08
2.07412078
2.07412

CCDC132
2.24E−06
2.07412078
2.07412

LOC234582
1.09E−06
2.066944188
2.06695

KCTD10
9.73E−05
2.066944188
2.06695

LOC240672
5.49E−06
2.056229656
2.05623

A230057G18RIK
9.28E−07
2.056229656
2.05623

ELOVL1
3.81E−06
2.056229656
2.05623

STX7
5.97E−06
2.052667339
2.05267

BC017612
5.16E−06
2.052667339
2.05267

ZBTB32
3.61E−05
2.052667339
2.05267

H47
3.72E−06
2.049113144
2.04911

TNFRSF22
7.52E−05
2.049113144
2.04911

AI115600
1.65E−06
2.045567052
2.04557

MYL6
1.28E−07
2.045567052
2.04557

H2-GS17
0.000428176
2.042024872
2.04202

CAPZB
5.20E−07
2.038490783
2.03849

SC4MOL
3.38E−06
2.038490783
2.03849

FHL2
6.33E−07
2.034960624
2.03496

3010031K01RIK
4.37E−08
2.034960624
2.03496

A330042I21RIK
1.18E−06
2.034960624
2.03496

D15MGI27
2.70E−05
2.034960624
2.03496

RAB4A
4.71E−08
2.024406242
2.02441

DCXR
0.000151877
2.024406242
2.02441

AIM1
0.000114024
2.024406242
2.02441

SEMA4A
7.97E−05
2.017402111
2.0174

XBP1
0.000154152
2.017402111
2.0174

LOC383099
0.000417017
2.013912105
2.01391

JUNB
0.00325781
2.013912105
2.01391

HRMT1L1
7.88E−06
2.01042607
2.01042

GPR97
0.000165473
2.01042607
2.01042

COTL1
0.000539041
2.01042607
2.01042

2310061N23RIK
0.025362
2.006944026
2.00694

9130227C08RIK
4.47E−06
2.00347001
2.00347

AI840980
0.000521047
2.00347001
2.00347

DYRK3
0.000241993
2.00347001
2.00347

CASP1
0.00573828
2.00347001
2.00347

TRBV11_AE000663_T_CELL_RECEPTOR_BETA_VARIABLE_11_—
0.000105474
0.5
−2

C920004C08RIK
0.0113298
0.5
−2

A930023F05RIK
1.93E−06
0.499134003
−2.00347

PLEKHG2
1.52E−05
0.499134003
−2.00347

ABHD8
7.47E−05
0.499134003
−2.00347

3110013H01RIK
0.00068445
0.499134003
−2.00347

E030007N04RIK
3.23E−07
0.496546519
−2.01391

PRKCD
1.24E−06
0.495687519
−2.0174

9130430L19RIK
1.04E−05
0.494829037
−2.0209

6030443O07RIK
1.78E−05
0.494829037
−2.0209

A130062D16RIK
1.86E−06
0.493971083
−2.02441

5930416I19RIK
2.26E−06
0.493971083
−2.02441

FYB
9.40E−06
0.493971083
−2.02441

AA408556
0.000447224
0.491410151
−2.03496

TRBV31_X03277_T_CELL_RECEPTOR_BETA_VARIABLE_31_33
0.00870127
0.491410151
−2.03496

A130038J17RIK
8.99E−05
0.490559188
−2.03849

AJ237586
1.68E−05
0.490559188
−2.03849

ZFP260
3.69E−06
0.490559188
−2.03849

0710008K08RIK
9.80E−06
0.489711168
−2.04202

ANP32E
0.000183335
0.485486385
−2.05979

4921518A06RIK
8.35E−06
0.484644053
−2.06337

4933421G18RIK
1.04E−05
0.484644053
−2.06337

3110018A08RIK
0.00338526
0.484644053
−2.06337

C730009F21RIK
1.18E−07
0.48380464
−2.06695

OLFML3
1.61E−05
0.482132181
−2.07412

A330103N21RIK
9.39E−05
0.481296806
−2.07772

H2-T9
0.000542267
0.481296806
−2.07772

LOC386360
0.00269607
0.481296806
−2.07772

ILVBL
4.13E−05
0.478801082
−2.08855

SBK
5.91E−07
0.477972631
−2.09217

6330403M23RIK
6.88E−08
0.476319763
−2.09943

C230075L19RIK
4.42E−07
0.475495347
−2.10307

MSH6
0.00037102
0.475495347
−2.10307

CXCL12
0.00745872
0.475495347
−2.10307

BC035291
1.11E−05
0.474671527
−2.10672

MMP2
0.000824671
0.473848312
−2.11038

GM525
0.000141229
0.473027946
−2.11404

STK4
2.73E−05
0.472210417
−2.1177

A130093I21RIK
7.45E−05
0.471391264
−2.12138

A230013K13RIK
3.45E−06
0.471391264
−2.12138

0610041G09RIK
0.00831324
0.471391264
−2.12138

TRIM28
3.45E−05
0.470577163
−2.12505

B230342M21RIK
7.67E−06
0.46894857
−2.13243

1190002H23RIK
5.23E−05
0.464902208
−2.15099

TSPAN32
9.87E−06
0.462491906
−2.1622

2610019F03RIK
3.67E−05
0.462491906
−2.1622

H2-OB
1.61E−05
0.461691175
−2.16595

4732481H14RIK
1.72E−05
0.460891087
−2.16971

COL5A1
0.00288136
0.460891087
−2.16971

LDH2
8.49E−05
0.459297092
−2.17724

6720418B01RIK
7.62E−06
0.458501068
−2.18102

6430510M02RIK
1.62E−06
0.458501068
−2.18102

TNFRSF7
0.000102955
0.458501068
−2.18102

CRYL1
8.89E−07
0.458501068
−2.18102

B230345P09RIK
5.94E−05
0.458501068
−2.18102

CTLA4
0.000629703
0.456915183
−2.18859

RGL2
2.61E−06
0.45533401
−2.19619

1810015C11RIK
3.91E−09
0.452974457
−2.20763

F730003H07RIK
0.00244488
0.452974457
−2.20763

CD97
0.000238915
0.452189956
−2.21146

LLGL1
1.90E−06
0.450625017
−2.21914

LOX
0.000702165
0.450625017
−2.21914

PDXP
3.27E−05
0.449844579
−2.22299

TRIB2
4.12E−06
0.449066839
−2.22684

2210008I11RIK
0.000393721
0.449066839
−2.22684

H2-AB1
0.00426068
0.449066839
−2.22684

SLC29A1
8.67E−06
0.448287765
−2.23071

ITPR2
5.30E−06
0.446737698
−2.23845

TPST1
9.65E−06
0.445964689
−2.24233

RPS6KL1
9.39E−07
0.442883526
−2.25793

RIL-PENDING
1.64E−06
0.441351064
−2.26577

TTC3
8.83E−09
0.439825301
−2.27363

MAPK1
2.50E−07
0.439062514
−2.27758

H2-EB1
0.00320736
0.438302367
−2.28153

CD3D
0.000186864
0.435274658
−2.2974

TRBV8_AE000663_T_CELL_RECEPTOR_BETA_VARIABLE_8_27
0.000766092
0.435274658
−2.2974

PPP1R1C
6.89E−07
0.434521896
−2.30138

PITPNM2
5.21E−07
0.432267937
−2.31338

2210419D22RIK
1.12E−07
0.431519943
−2.31739

RAPGEF3
1.11E−07
0.430772677
−2.32141

SATB1
5.66E−06
0.427056598
−2.34161

C530015C18
9.54E−09
0.426317427
−2.34567

5830496L11RIK
1.02E−06
0.424843127
−2.35381

BCL7A
3.37E−07
0.424843127
−2.35381

GLDC
0.000448781
0.424106196
−2.3579

CD27
0.000101099
0.423371818
−2.36199

A830080H07RIK
1.99E−05
0.423371818
−2.36199

ART4
9.75E−06
0.422639978
−2.36608

SCL000121.1_106
6.92E−06
0.419720131
−2.38254

4932414K18RIK
2.36E−06
0.418993828
−2.38667

ACVR2B
4.27E−08
0.417543508
−2.39496

AI481316
4.04E−07
0.417543508
−2.39496

POU6F1
8.86E−07
0.417543508
−2.39496

NCK2
0.000270785
0.412508921
−2.42419

1110046J11RIK
5.70E−05
0.411082792
−2.4326

ETS2
1.91E−06
0.408243248
−2.44952

FBP1
0.00205479
0.408243248
−2.44952

TPCN1
3.56E−08
0.407536159
−2.45377

TBXA2R
3.11E−06
0.405422937
−2.46656

5430417L22RIK
5.18E−07
0.404020815
−2.47512

PPARGC1B
1.79E−07
0.404020815
−2.47512

TCF7
1.40E−05
0.398458762
−2.50967

DNTT
0.00027177
0.397767728
−2.51403

LOC386545
0.00608272
0.394337316
−2.5359

SOX4
1.85E−07
0.390257609
−2.56241

GPR83
1.40E−05
0.388908334
−2.5713

HIBADH
2.18E−08
0.387562349
−2.58023

IGH-6
2.87E−05
0.386221173
−2.58919

LOC381739
1.61E−06
0.382889437
−2.61172

DAP3
2.30E−08
0.380904496
−2.62533

DGKA
8.26E−05
0.378928542
−2.63902

SNAI3
4.51E−07
0.376964456
−2.65277

SLC5A9
2.33E−05
0.376964456
−2.65277

2410008J05RIK
1.24E−06
0.376311917
−2.65737

NAV1
2.19E−06
0.376311917
−2.65737

HDAC7A
2.22E−06
0.376311917
−2.65737

A130092J06RIK
6.62E−06
0.375660223
−2.66198

SLA
2.73E−05
0.373064727
−2.6805

MTF2
2.39E−06
0.371130501
−2.69447

C230098O21RIK
6.12E−05
0.370488378
−2.69914

GFI1
2.05E−06
0.368567122
−2.71321

EPHX1
2.39E−08
0.36792977
−2.71791

BRD3
2.92E−06
0.36792977
−2.71791

AQP11
4.62E−07
0.364754502
−2.74157

IL17RB
2.04E−07
0.362236156
−2.76063

RAMP1
0.000135079
0.361608725
−2.76542

NISCH
3.31E−07
0.361608725
−2.76542

BGN
0.0025721
0.36098216
−2.77022

TXNIP
0.000742272
0.359732934
−2.77984

COL6A1
0.00166573
0.359110268
−2.78466

CCL9
0.000264226
0.356012517
−2.80889

DPP4
1.63E−06
0.354166593
−2.82353

MLL
1.54E−07
0.352329781
−2.83825

C3
0.0001376
0.350503323
−2.85304

MARCKS
4.55E−06
0.349896256
−2.85799

TRBV1_AE000663_T_CELL_RECEPTOR_BETA_VARIABLE_1_20
0.00160689
0.34808276
−2.87288

3830612M24
2.00E−06
0.344481916
−2.90291

PP11R
0.000186213
0.344481916
−2.90291

2510015F01RIK
0.000255295
0.342695782
−2.91804

PARD6G
1.26E−07
0.327598181
−3.05252

NOTCH3
3.24E−06
0.327598181
−3.05252

H2-T10
0.000761011
0.327598181
−3.05252

LMAN2L
1.87E−07
0.327031438
−3.05781

DTX1
2.93E−07
0.324772335
−3.07908

TMEM108
2.25E−05
0.324210062
−3.08442

ETS1
5.90E−07
0.322528624
−3.1005

SH2D1A
2.22E−06
0.320856301
−3.11666

9626100_15
0.00161165
0.314252942
−3.18215

CD8B
3.35E−05
0.313166457
−3.19319

ACAS2L
7.23E−06
0.30992664
−3.22657

LOC434197
1.33E−06
0.305659904
−3.27161

9626100_224
0.00113435
0.305131084
−3.27728

FRAT2
2.01E−06
0.302498639
−3.3058

NRP
8.11E−07
0.299888741
−3.33457

G22P1
8.46E−08
0.296273472
−3.37526

RNPEPL1
4.01E−09
0.295759985
−3.38112

9626958_317
0.00188407
0.293717673
−3.40463

H19
0.000268847
0.283221131
−3.53081

ACTN1
2.58E−07
0.278838137
−3.58631

SLC16A5
3.62E−09
0.275476023
−3.63008

CD2
1.50E−06
0.274999519
−3.63637

PRKCB
2.18E−07
0.272154758
−3.67438

ST6GAL1
7.38E−08
0.268873581
−3.71922

PRELP
1.65E−05
0.268408098
−3.72567

CDCA7
4.57E−05
0.267943507
−3.73213

PDLIM4
4.93E−06
0.26701628
−3.74509

CD6
1.61E−09
0.264712734
−3.77768

ALDH2
7.40E−07
0.248703632
−4.02085

CD81
6.28E−06
0.247413906
−4.04181

9430068D06RIK
3.23E−10
0.239816205
−4.16986

H2-BL
9.51E−06
0.232854352
−4.29453

AI132321
1.19E−06
0.20877219
−4.78991

LY6D
2.50E−08
0.184923553
−5.40764

COX6A2
0.000397718
0.158219587
−6.32033

BCL11B
1.64E−08
0.150204503
−6.65759

LOC382896
6.81E−09
0.112266584
−8.90737

LAK vs. DN3

p-value
Ratio
Fold-Change

Column ID
(LAK vs. DN3)
(LAK vs. DN3)
(LAK vs. DN3)

GZMD
2.97E−07
79.3411511
79.3413

FCER1G
8.83E−09
50.3005458
50.3005

ROG
3.50E−09
38.7869009
38.787

CCL4
4.38E−08
38.5859038
38.5858

KLRE1
6.58E−11
35.1998648
35.1999

MT1
1.56E−07
35.0174387
35.0174

SPP1
2.96E−08
32.5029903
32.503

AVIL
8.90E−08
30.6432634
30.6433

TYROBP
1.95E−10
26.8620793
26.8621

GZME
6.58E−10
26.1728718
26.1729

XCL1
1.12E−06
26.1275337
26.1276

ASB2
4.61E−10
25.8125134
25.8125

KLRA7
9.89E−11
22.7059601
22.706

PRF1
9.89E−08
22.2386788
22.2387

KLRD1
4.72E−09
19.8353271
19.8353

LGALS3
4.28E−09
18.7979112
18.7979

GZMG
6.92E−08
18.0634207
18.0634

KLRA18
6.87E−08
16.6794542
16.6795

SERPINA3G
1.62E−07
16.5069346
16.5069

NKG7
3.26E−08
16.4783466
16.4784

LTB4R1
3.28E−07
16.1392364
16.1392

GADD45G
2.57E−08
16.0834021
16.0834

CTSG
5.88E−06
15.9446402
15.9446

CCL3
2.43E−07
15.5624826
15.5625

NFIL3
3.46E−07
15.3482283
15.3482

AQP9
3.11E−07
15.0323722
15.0324

1300002F13RIK
2.22E−07
14.4450222
14.445

KLRA4
9.74E−08
14.2461507
14.2461

LITAF
2.42E−08
13.4543507
13.4543

KLRA3
5.93E−09
13.2691108
13.2691

LRRK1
1.90E−09
13.2232104
13.2232

KLRG1
1.44E−07
13.2003094
13.2003

EMILIN2
5.64E−06
12.8393439
12.8393

1110007C02RIK
3.19E−11
12.7727787
12.7728

TNFRSF11B
5.09E−06
12.7727787
12.7728

LOC381140
1.29E−08
12.4666362
12.4666

HAVCR2
4.62E−07
12.0211235
12.0211

LOC327957
1.39E−07
12.0003072
12.0003

PDGFA
2.78E−09
11.9174171
11.9174

SCIN
1.18E−07
11.7329992
11.733

BC049975
2.96E−09
11.6519561
11.652

PGLYRP1
1.73E−07
11.5114671
11.5115

IFITM1
4.19E−05
11.4716392
11.4716

SPEER3
4.84E−05
10.7405848
10.7406

1810044J04RIK
3.59E−09
10.6848908
10.6849

CCR5
1.38E−09
10.519539
10.5195

WBSCR5
1.21E−08
10.4287901
10.4288

DAF1
3.14E−07
10.3388239
10.3388

2210411K11RIK
1.18E−07
10.091021
10.091

P2RY14
3.12E−08
9.81508382
9.81508

BCL2A1B
2.31E−08
9.781099
9.78112

F2R
5.12E−07
9.71354748
9.71356

LOC268288
1.40E−08
9.67997987
9.67995

RGS1
1.42E−06
9.54672166
9.54669

2310057H16RIK
5.56E−09
9.30171989
9.30174

BHLHB2
2.26E−07
9.30171989
9.30174

CTSW
4.35E−06
9.07877654
9.07879

5330403J18RIK
6.45E−09
9.06306078
9.06307

SH2D1B1
3.54E−07
8.89197144
8.69195

PLSCR1
1.15E−10
8.86116329
8.86119

1110018K11RIK
7.85E−11
8.69391339
8.69388

ICSBP1
4.16E−07
8.58907298
8.58906

SPEER1-PS1
0.000100291
8.29648312
8.29648

RGS16
6.08E−08
8.22490171
8.22491

EG433016
0.000986707
8.2106525
8.21067

DHRS6
2.02E−06
8.19645258
8.19646

2310067E08RIK
7.56E−08
7.88985759
7.88986

KLRA13
3.45E−10
7.87618635
7.8762

TNFSF6
7.56E−09
7.70077855
7.70076

CCL5
0.000147502
7.38705198
7.38706

E030006K04RIK
2.60E−09
7.23505238
7.23503

CAR2
7.88E−11
6.90431312
6.90432

SERPINE2
1.33E−06
6.90431312
6.90432

IER3
7.22E−06
6.84476752
6.84476

TNFRSF9
1.74E−08
6.83288806
6.83291

SIAT10
6.93E−07
6.7974958
6.79748

GLRX1
4.58E−06
6.64606387
6.64606

DAPK2
7.72E−10
6.61161396
6.6116

F2RL2
5.36E−06
6.55458329
6.55456

IFITM3
2.68E−06
6.46433304
6.46433

MLKL
5.34E−09
6.30938711
6.30939

SYTL2
1.70E−10
6.14749059
6.1475

TMEM119
0.000650503
6.1368518
6.13686

2810025M15RIK
1.47E−08
6.0418945
6.04189

SEPN1
8.40E−07
6.0418945
6.04189

TCRD-V1
7.87E−09
6.01055453
6.01056

OSBPL3
1.46E−11
5.98978143
5.98977

APOB48R
3.32E−07
5.98978143
5.98977

CD52
2.73E−05
5.979395
5.9794

MYO1F
2.88E−09
5.93810123
5.93809

SH3BP2
4.75E−09
5.8970609
5.89706

RASD2
1.15E−06
5.85634388
5.85634

LOC269941
3.60E−10
5.80581859
5.80582

IFITM2
6.30E−05
5.80581859
5.80582

GPR87
1.21E−06
5.77570622
5.77572

LOC270152
2.10E−06
5.74577255
5.74577

HIST1H1C
2.95E−05
5.71598418
5.71598

AA467197
3.72E−05
5.70606897
5.70608

KLRA1
3.83E−07
5.63729635
5.63728

IDB2
1.24E−08
5.55003635
5.55005

LY6A
2.01E−05
5.54044246
5.54044

FCGR3
2.61E−07
5.51170687
5.51171

GVIN1
3.28E−05
5.51170687
5.51171

A430038C16RIK
2.04E−08
5.42640707
5.42642

ID2
4.68E−09
5.37961235
5.3796

S100A1
3.25E−08
5.37961235
5.3796

CAPG
1.77E−09
5.37028087
5.37029

PPP3CC
8.68E−10
5.33319111
5.33319

1500031H04RIK
2.19E−07
5.30554642
5.30554

PLCG2
3.91E−09
5.25978056
5.25977

NCF4
1.99E−07
5.24158464
5.24157

AI115600
1.22E−09
5.14260442
5.14261

DUSP6
8.20E−10
5.11595308
5.11594

SERPINB6A
9.97E−06
5.09824315
5.09824

BCL2A1D
1.37E−08
4.89905497
4.89904

UPP1
1.21E−08
4.85677374
4.85678

PIM3
1.14E−06
4.85677374
4.85678

AMICA1
1.15E−06
4.84836731
4.84837

SLC2A3
4.16E−06
4.8148915
4.81488

5031436O0RIK
2.13E−06
4.77333804
4.77334

CD160
1.84E−05
4.73215976
4.73216

A430084P05RIK
2.72E−07
4.6913337
4.69134

GOLPH2
1.25E−08
4.61874564
4.61874

CD244
8.49E−09
4.61874564
4.61874

DMWD
1.23E−06
4.57890134
4.5789

AHNAK
1.67E−08
4.57096886
4.57097

TRAF1
2.12E−06
4.57096886
4.57097

TES
1.82E−08
4.55514458
4.55515

CDKN2B
1.88E−05
4.55514458
4.55515

1110004P15RIK
6.74E−06
4.55514458
4.55515

SRGAP2
1.33E−08
4.50803783
4.50804

SULF2
3.60E−09
4.4691539
4.46915

HHEX
1.23E−08
4.38477256
4.38477

LAG3
5.75E−07
4.30198192
4.30198

ALDOA
1.30E−05
4.22074496
4.22075

AI850995
5.34E−08
4.2134358
4.21344

PTER
3.14E−06
4.20615192
4.20615

LOC218482
9.08E−09
4.19887554
4.19887

1190002C06RIK
2.68E−07
4.16986356
4.16986

CTNNA1
3.18E−08
4.15542969
4.15544

CDKN1A
0.000116956
4.14106111
4.14106

2310016C16RIK
9.43E−06
4.12674045
4.12673

A530050E01RIK
2.79E−07
4.08404974
4.08405

LMNA
1.14E−06
4.06287709
4.06287

GLIPR1
1.07E−07
4.03481236
4.03481

9830144J08RIK
4.06E−09
4.02084406
4.02085

PPAP2C
3.54E−07
3.95862446
3.95863

SAT1
1.91E−05
3.95862446
3.95863

SLC2A1
5.83E−05
3.95862446
3.95863

HAK
4.36E−08
3.93809318
3.9381

SCL0003187.1_40
6.01E−07
3.91768167
3.91768

2310046K01RIK
7.02E−06
3.86374876
3.86375

RHOF
3.34E−07
3.84371457
3.84371

LOC212399
4.55E−08
3.82378471
3.82378

CD69
3.83E−05
3.81055447
3.51055

B230343A10RIK
1.49E−08
3.80395916
3.80395

BC022224
7.94E−08
3.7776762
3.77768

KLRB1C
9.87E−09
3.74509393
3.74509

MYO1E
8.79E−09
3.7192142
3.71922

KLRA33
3.61E−06
3.69352599
3.69353

WDFY1
5.95E−08
3.68074675
3.68075

GCNT1
5.12E−08
3.64268203
3.64268

C80638
3.80E−05
3.64268203
3.64268

HGFAC
7.89E−05
3.63637686
3.63637

RASL12
4.28E−07
3.61125556
3.61125

GNG2
1.28E−09
3.58009752
3.5801

SH2D2A
1.04E−06
3.56770796
3.56771

GPR141
1.94E−05
3.56770796
3.56771

DCI
2.41E−09
3.51859932
3.5186

GLRX
4.20E−05
3.51859932
3.5186

LOC385699
9.48E−07
3.51250628
3.5125

CD72
8.84E−05
3.48824635
3.48824

EGR1
0.000778832
3.48824635
3.48824

2210008N01RIK
1.43E−08
3.45814948
3.45815

ADORA2B
6.64E−07
3.44618438
3.44618

KLRA10
4.19E−08
3.43426642
3.43426

S100A11
2.42E−07
3.43426642
3.43426

F730045P10RIK
3.27E−05
3.43426642
3.43426

PILRB
4.70E−05
3.42832067
3.42832

LGALS1
2.90E−06
3.4046378
3.40463

S100A10
7.33E−06
3.39285533
3.39286

NQO2
4.88E−08
3.38698315
3.38698

CD9
1.56E−07
3.38111983
3.38112

PLP2
5.92E−08
3.36941982
3.36942

SLC39A4
3.40E−08
3.35194782
3.35195

7420404O03RIK
2.40E−06
3.33456712
3.33457

NEDD9
3.25E−07
3.31153241
3.31153

BSPRY
1.11E−06
3.28866234
3.28866

STX11
2.41E−06
3.28866234
3.28866

NFKBIZ
5.41E−07
3.27728091
3.27728

ADAM8
1.09E−06
3.27728091
3.27728

PPP1R3B
1.39E−06
3.27728091
3.27728

SEC61B
1.17E−10
3.26594598
3.26594

S100A6
4.92E−06
3.26594598
3.26594

ANXA2
4.93E−06
3.25464682
3.25464

HK2
1.70E−06
3.19319466
3.19319

PDLIM7
9.37E−08
3.18766755
3.18766

TRIO
4.54E−07
3.17664026
3.17664

ENO1
2.54E−07
3.16016673
3.16017

LOC383981
0.000359161
3.15469355
3.15469

N4WBP5-PENDING
5.19E−09
3.14922938
3.14923

LOC328703
2.98E−07
3.14378411
3.14378

HIC1
5.07E−06
3.11666293
3.11666

AOAH
2.89E−09
3.11126183
3.11126

1110020C13R1K
2.86E−08
3.11126183
3.11126

MAPKAPK3
2.85E−07
3.10587943
3.10588

FTL1
8.72E−06
3.09513198
3.09513

BC046404
6.62E−07
3.07908317
3.07908

E030003N15RIK
8.70E−06
3.07908317
3.07908

VIM
1.02E−05
3.0684259
3.06843

CISH
1.19E−05
3.0684259
3.06843

C230043G09RIK
1.82E−07
3.05252169
3.05252

GPR97
4.15E−06
3.04195464
3.04196

LASP1
1.17E−07
3.03668928
3.03669

9930117H01RIK
6.85E−06
3.03668928
3.03669

CASP1
0.000296727
3.000075
3.00008

D10ERTD438E
3.40E−07
2.97935604
2.97935

TBC1D2B
7.23E−09
2.97419883
2.9742

1700129I15RIK
2.83E−05
2.96390263
2.96391

NDFIP1
1.87E−07
2.95877246
2.95878

5730469M10RIK
9.16E−05
2.95877246
2.95878

JUNB
0.000168611
2.95877246
2.95878

PGK1
2.49E−06
2.9536513
2.95365

LOC240672
1.77E−07
2.94853915
2.94854

BSCL2
2.21E−06
2.94343599
2.94343

IFNG
1.45E−05
2.9383332
2.93834

S100A13
6.20E−05
2.93324807
2.93325

CHN2
1.49E−07
2.92817194
2.92817

CST7
1.31E−07
2.9231048
2.9231

POLD4
1.30E−05
2.91298902
2.91299

BC087945
2.69E−07
2.90794888
2.90794

4631423F02RIK
2.76E−05
2.89286561
2.89287

2310061N23RIK
0.00286637
2.87288627
2.87288

GIPC2
3.20E−05
2.86294236
2.86295

AA175286
9.62E−05
2.84810217
2.8481

TUBA6
4.58E−07
2.83824823
2.83825

KLRB1D
7.14E−11
2.83333475
2.83333

HRMT1L1
2.71E−07
2.83333475
2.83333

TMEM126A
2.96E−08
2.82843025
2.82843

TRF
3.44E−06
2.81864023
2.81864

LOC383189
2.82E−08
2.81376268
2.81376

AW536289
4.33E−07
2.81376268
2.81376

CCNG1
1.58E−05
2.81376268
2.81376

HAAO
5.90E−06
2.79916809
2.79917

6720467C03RIK
2.29E−11
2.7943264
2.79433

SLC24A3
3.28E−05
2.7948587
2.78949

PSTPIP1
5.32E−06
2.77502588
2.77502

TBX21
4.36E−06
2.76542554
2.76542

PRDX5
7.28E−05
2.76542554
2.76542

EGR3
0.000564399
2.76063119
2.76063

TFF1
0.00864278
2.76063119
2.76063

E130012A19RIK
7.32E−06
2.74631719
2.74632

BCAP29
8.13E−06
2.74631719
2.74632

IRAK2
2.06E−05
2.74156626
2.74157

2310016C08RIK
0.000897768
2.74156626
2.74157

E430036I04RIK
9.77E−08
2.73681675
2.73682

FES
1.41E−07
2.73208368
2.73208

C330023F11RIK
1.27E−07
2.71791568
2.71791

FKBP11
0.000163935
2.7132109
2.71321

GP49A
8.69E−07
2.70850769
2.70851

FHL2
3.43E−08
2.70382077
2.70382

PRMT2
5.08E−07
2.69446637
2.69447

3300005D01RIK
5.96E−05
2.68514764
2.68514

RPS6KA1
5.06E−08
2.66659556
2.6666

GPD2
2.77E−08
2.66198158
2.66198

SNAG1
8.24E−08
2.66198158
2.66198

CLN3
2.04E−05
2.65277321
2.65277

TMPIT
4.29E−07
2.64817685
2.64816

1810011E08RIK
4.07E−06
2.64817885
2.64818

BIN1
6.15E−08
2.64359338
2.64359

PEA15
3.62E−08
2.62988336
2.62989

SDF2L1
4.71E−05
2.62988336
2.62989

LOC383099
3.70E−05
2.62988336
2.62989

4930486L24RIK
2.78E−06
2.60719481
2.6072

CAMK2N1
2.06E−06
2.60268232
2.60266

IFI30
2.03E−06
2.59817868
2.59818

4930513E20RIK
0.000992688
2.59367713
2.59368

0610037M15RIK
7.56E−05
2.58919116
2.58919

PFKP
1.61E−05
2.58470732
2.58471

A630024B12RIK
2.56E−06
2.58023232
2.58023

SPIN2
4.24E−07
2.57576616
2.57576

MMD
1.33E−06
2.57576616
2.57576

MGC18837
1.51E−06
2.56240743
2.56241

C130027E04RIK
3.69E−07
2.55354138
2.55354

IL18R1
5.54E−06
2.55354138
2.55354

GALGT1
1.55E−05
2.55354138
2.55354

STK39
3.36E−08
2.54912157
2.54912

OBFC2A
8.44E−06
2.54912157
2.54912

D930046M13RIK
1.92E−05
2.54471056
2.54471

ALDOC
0.00134083
2.54471056
2.54471

IL2RB
4.01E−07
2.54030189
2.5403

LOC381319
0.000586562
2.53151098
2.53151

BAG3
0.00125353
2.53151098
2.53151

DOK2
1.17E−06
2.52275525
2.52275

UGCG
8.13E−05
2.52275525
2.52275

ARRDC4
0.000487201
2.5183842
2.51839

ATF4
0.00225306
2.5183842
2.51839

IL12RB1
6.73E−07
2.51402828
2.51403

9130211I03RIK
0.000664109
2.51402828
2.51403

1810061M12RIK
7.55E−08
2.50533009
2.50533

KLRA21
1.66E−06
2.50099415
2.50099

MVP
3.59E−07
2.49666072
2.49666

CYBA
1.03E−06
2.49233607
2.49234

BATF
2.83E−06
2.49233607
2.49234

NENF
1.16E−05
2.49233607
2.49234

TNFSF13
2.10E−08
2.48802018
2.48802

EHD4
1.57E−05
2.48802018
2.48802

CSDA
3.96E−08
2.47941466
2.47942

CRELD2
1.86E−05
2.47512499
2.47512

LOC238943
2.92E−06
2.47083794
2.47084

1110019C08RIK
3.97E−05
2.47083794
2.47084

OSTF1
8.10E−08
2.46655962
2.46656

2510048K03RIK
4.28E−07
2.46655962
2.46656

1700025G04RIK
1.15E−05
2.46229003
2.46229

PADI2
1.20E−08
2.45802311
2.45803

A530060O05RIK
9.89E−07
2.45802311
2.45803

FXYD5
9.20E−06
2.45802311
2.45803

MYO1G
6.72E−06
2.45377096
2.45377

ECH1
1.99E−05
2.45377096
2.45377

GABARAPL1
5.14E−09
2.44528073
2.44528

AI847670
4.70E−07
2.44104867
2.44105

PPIB
3.62E−09
2.43681937
2.43682

FOSL2
1.02E−05
2.42839277
2.42839

BC023892
3.65E−09
2.42418366
2.42419

STX7
1.05E−06
2.42418366
2.42419

NUCB1
1.57E−05
2.42418366
2.42419

KLF7
1.89E−05
2.42418366
2.42419

TAF9B
8.39E−07
2.41998911
2.41999

LOC381683
4.83E−07
2.41998911
2.41999

LOC386405
0.00419371
2.41998911
2.41999

H47
6.51E−07
2.41579738
2.4158

GPR34
1.34E−07
2.41161433
2.41162

1110006I15RIK
1.08E−07
2.40743995
2.40744

MYL6
2.17E−08
2.40327422
2.40327

SOAT2
1.66E−05
2.40327422
2.40327

HIP1
1.04E−10
2.39911137
2.39911

DEGS
5.19E−07
2.39911137
2.39911

2810032E02RIK
1.12E−06
2.39911137
2.39911

SH3BGRL3
0.000169298
2.39911137
2.39911

DTR
4.75E−06
2.38666902
2.38667

SLK
1.18E−08
2.38254074
2.38254

EOMES
8.89E−07
2.3784154
2.37841

GMDS
2.61E−06
2.3784154
2.37841

DCXR
3.22E−05
2.37018497
2.37019

H2-Q8
0.00206753
2.37018497
2.37019

HIP-1
1.62E−10
2.36607988
2.36608

DAB2IP
5.55E−08
2.36607988
2.36608

KLRK1
3.04E−08
2.36607988
2.36608

OLFM1
7.39E−08
2.3619834
2.36199

CABLES1
2.23E−06
2.3619834
2.36199

AI840980
0.000110731
2.3619834
2.36199

BC024955
4.20E−08
2.35789553
2.3579

MINA
4.70E−09
2.35789553
2.3579

A530090P03RIK
6.45E−06
2.35789553
2.3579

NRGN
4.73E−05
2.3538107
2.35381

ZFP52
0.000839487
2.3538107
2.35381

TSPO
2.07E−06
2.34974
2.34974

TDRD7
2.88E−06
2.34567235
2.34567

TMEM38B
6.08E−08
2.34160779
2.34161

SAMSN1
1.16E−06
2.34160779
2.34161

IAN4
0.000116004
2.34160779
2.34161

IMPA2
0.000464842
2.33755184
2.33755

2310056P07RIK
0.000374894
2.33755184
2.33755

ETFB
8.23E−07
2.3335099
2.33351

GZMK
0.000514646
2.32140826
2.32141

5730438N18RIK
1.67E−05
2.31738969
2.31739

LOC215678
4.32E−05
2.31337431
2.31338

LOC269355
1.94E−05
2.30937282
2.30937

STK32C
4.69E−07
2.30537452
2.30537

SLAMF7
1.11E−06
2.30537452
2.30537

ABCB1B
1.12E−06
2.30137945
2.30138

4930539E08RIK
3.24E−05
2.30137945
2.30138

CLNK
3.67E−08
2.2973982
2.2974

TEX9
1.16E−06
2.2973982
2.2974

LOC218617
6.06E−08
2.29342018
2.29342

PALD
8.48E−07
2.29342018
2.29342

GPR114
0.00411637
2.29342018
2.29342

GOLGA7
1.77E−06
2.28945067
2.28945

GPR160
5.41E−06
2.28945067
2.28945

KIT
0.00014555
2.28945067
2.28945

SQSTM1
0.000722465
2.28945067
2.28945

KLRA16
3.18E−07
2.28548443
2.28548

ZBTB32
1.19E−05
2.28548443
2.28548

1110030J09RIK
5.87E−10
2.28152671
2.28153

CYP51
2.62E−06
2.28152671
2.28153

3110054C06RIK
1.87E−06
2.28152671
2.28153

C730026J16
4.19E−05
2.26969929
2.2697

SERTAD1
7.39E−05
2.26969929
2.2697

2310004N11RIK
8.08E−05
2.26969929
2.2697

VEGFC
1.79E−07
2.26576519
2.26577

LOC114601
7.89E−06
2.26576519
2.26577

A930008A22RIK
1.15E−06
2.26184472
2.26184

GFOD1
6.22E−07
2.26184472
2.26184

STK2
7.89E−09
2.25011869
2.25012

BC036961
4.29E−06
2.25011869
2.25012

1810006K23RIK
4.62E−06
2.25011869
2.25012

2310047C17RIK
0.00113428
2.25011869
2.25012

CAI
2.16E−07
2.23844849
2.23845

CAPNS1
1.49E−08
2.23457196
2.23457

RPL36
3.70E−05
2.23457196
2.23457

2310043N10RIK
7.41E−05
2.23070385
2.23071

SYPL
1.01E−06
2.22684416
2.22684

GZMN
0.00220233
2.22684416
2.22684

COMT
6.85E−09
2.22298792
2.22299

SCL000416.1_19
1.60E−08
2.22298792
2.22299

LOC382127
0.000238522
2.22298792
2.22299

2610009E16RIK
0.000238786
2.21914008
2.21914

GPC1
0.0145298
2.21914008
2.21914

ASAH1
2.95E−08
2.21145978
2.21146

HADH2
7.87E−06
2.21145978
2.21146

XDH
1.37E−06
2.20763223
2.20763

DP1
5.63E−06
2.20380818
2.20381

TGFBR2
3.17E−07
2.19999252
2.19999

SC4MOL
1.44E−06
2.19999252
2.19999

XAB1
1.99E−06
2.19999252
2.19999

PTPN8
3.84E−06
2.19238147
2.19238

DIP3B
3.01E−06
2.18479904
2.1848

VTI1B
8.79E−06
2.18479904
2.1848

STK17B
1.46E−05
2.18479904
2.1848

GNS
0.000892576
2.18479904
2.1848

MTMR9
0.00171537
2.18479904
2.1848

ALAD
2.13E−08
2.18101557
2.18102

ARPC1B
3.71E−07
2.18101557
2.18102

2600010E01RIK
1.17E−05
2.18101557
2.18102

LOC237361
1.67E−05
2.15845662
2.15846

MPP6
2.77E−06
2.15471732
2.15472

PDCD1LG2
8.02E−06
2.15471732
2.15472

SERPINB6B
2.58E−05
2.15471732
2.15472

CAPN2
1.74E−06
2.15099096
2.15099

ELOVL1
2.29E−06
2.15099096
2.15099

RAB3D
2.65E−05
2.15099096
2.15099

HINT2
0.000350321
2.14726363
2.14726

FIGF
4.55E−05
2.1435492
2.14355

OSM
0.000887713
2.1435492
2.14355

ACATE3
1.20E−06
2.13983386
2.13984

5430427O19RIK
3.35E−06
2.13983386
2.13984

EG630499
0.00033688
2.12874206
2.12874

KLK1B11
0.00366553
2.12874206
2.12874

FNBP1
3.25E−08
2.12137669
2.12138

SRI
2.09E−08
2.12137669
2.12138

2610529H08RIK
2.11E−07
2.12137669
2.12138

ANXA5
0.000191309
2.12137669
2.12138

RPIA
2.24E−08
2.11403529
2.11404

LCP1
1.15E−05
2.11403529
2.11404

LOC23352
3.13E−05
2.11037693
2.11038

UGALT2
1.37E−05
2.10672234
2.10672

ANXA3
0.00588432
2.10307596
2.10307

JAM4
2.28E−07
2.09943336
2.09943

CHST12
6.51E−07
2.09943336
2.09943

SCL0001297.1_42
3.97E−05
2.09943336
2.09943

PYGL
9.38E−05
2.09943336
2.09943

MREG
1.19E−07
2.09579897
2.0958

1810003N24RIK
2.62E−06
2.09579897
2.0958

HRC
0.000100933
2.09579897
2.0958

DDIT4
0.0101176
2.09216839
2.09217

FBXO4
2.51E−07
2.08493178
2.08493

201007E07RIK
5.45E−06
2.08493178
2.08493

ZFP296
2.06E−08
2.08132139
2.08132

0610039D01RIK
7.86E−07
2.08132139
2.08132

COTL1
0.000383267
2.08132139
2.08132

KLRI1
1.30E−08
2.07771916
2.07772

UBL4
2.81E−07
2.07771916
2.07772

ARHGAP18
3.33E−07
2.07771916
2.07772

SNX9
1.75E−07
2.07412078
2.07412

PFN1
3.03E−07
2.07412078
2.07412

9130227C08RIK
2.98E−06
2.07412078
2.07412

DAP
2.54E−05
2.07412078
2.07412

9030611O19RIK
7.73E−05
2.07412078
2.07412

D8ERTD354E
0.000213556
2.07412078
2.07412

2900026A02RIK
3.15E−07
2.07053055
2.07053

M6PR
1.54E−06
2.07053055
2.07053

MRPS6
1.82E−05
2.07053055
2.07053

0610009O03RIK
1.91E−08
2.06694419
2.06695

CAPZB
4.50E−07
2.06336597
2.06337

ARRB2
1.28E−06
2.06336597
2.06337

A430093B03RIK
4.59E−07
2.05979587
2.05979

NIBAN
3.10E−05
2.05979587
2.05979

2610036L11RIK
0.00147553
2.05979587
2.05979

DHRS7
1.18E−08
2.05622966
2.05623

LOC241621
3.38E−06
2.05266734
2.05267

COX7A1
0.000599505
2.05266734
2.05267

RBMS1
2.03E−07
2.04911314
2.04911

CDKN2A
0.000238387
2.04911314
2.04911

BB220380
1.50E−07
2.03849078
2.03849

CMKBR2
3.22E−06
2.03849078
2.03849

AW212394
2.59E−05
2.03849078
2.03849

1110030C22RIK
0.000169536
2.03496062
2.03496

SCL00319622.1_241
8.06E−06
2.03143442
2.03144

LCN4
0.00307845
2.03143442
2.03144

KDELR2
1.28E−06
2.02792041
2.02792

CD59A
0.00082023
2.02440624
2.02441

CAPN5
0.00123881
2.01740211
2.0174

ZFP608
2.50E−06
2.0139121
2.01391

SLC2A6
0.000147228
2.0139121
2.01391

CORO1C
1.61E−06
2.0139121
2.01391

GNPDA1
7.70E−07
2.0139121
2.01391

CARD4
8.05E−06
2.01042607
2.01042

09-Sep
2.79E−06
2.00694403
2.00694

2410012H22RIK
8.81E−07
2.00347001
2.00347

SKAP2
1.02E−06
2.00347001
2.00347

IAN3
0.0043871
2.00347001
2.00347

TPI1
0.00037172
2.00347001
2.00347

9130422G05RIK
8.52E−08
2
2

2810004N20RIK
1.13E−06
2
2

B4GALNT2
0.00262346
2
2

DNMT3B
1.05E−06
0.5
−2

SCL000548.1_6
2.96E−06
0.5
−2

LYT-2
2.32E−05
0.5
−2

LBR
1.18E−05
0.499134
−2.00347

6330406L22RIK
5.22E−05
0.499134
−2.00347

MIER1
1.68E−08
0.498271
−2.00694

1810020D17RIK
2.21E−07
0.498271
−2.00694

SLC9A9
4.02E−05
0.498271
−2.00694

TCRG-V5
0.00106772
0.498271
−2.00694

BC035295
8.94E−08
0.4974085
−2.01042

LOC386192
0.0045284
0.4974085
−2.01042

ARHGEF11
2.19E−08
0.49654652
−2.01391

B230114J08RIK
6.30E−07
0.49654652
−2.01391

ARID1A
2.03E−06
0.49654652
−2.01391

C030002B11RIK
3.75E−05
0.49654652
−2.01391

E430013K19RIK
3.08E−06
0.49568752
−2.0174

A130022A09RIK
4.53E−06
0.49568752
−2.0174

LOC269401
5.58E−06
0.49568752
−2.0174

2700007B13RIK
3.11E−05
0.49568752
−2.0174

DDAH1
0.000139319
0.49568752
−2.0174

HP
0.00181193
0.49568752
−2.0174

DNCHC1
5.02E−06
0.49482904
−2.0209

SPEC1
1.46E−05
0.49397108
−2.02441

KCNH3
5.03E−08
0.49226165
−2.03144

LRMP
3.72E−06
0.49226165
−2.03144

CAMK4
5.35E−08
0.49141015
−2.03496

1110001P04RIK
2.50E−06
0.49141015
−2.03496

COL15A1
7.73E−05
0.49055919
−2.03849

E130307M08RIK
3.42E−05
0.48971117
−2.04202

NFE2
3.74E−05
0.48971117
−2.04202

ASB13
4.76E−07
0.48717037
−2.05267

XLR4A
0.000619052
0.48717037
−2.05267

LOC382020
0.0011986
0.48717037
−2.05267

3110018A08RIK
0.00353392
0.48717037
−2.05267

BC020108
0.000419903
0.48632692
−2.05623

SOX9
0.00119698
0.48632692
−2.05623

CD5
2.42E−06
0.48548638
−2.05979

ZFP96
2.60E−05
0.48548638
−2.05979

AKAP8L
8.62E−07
0.48464405
−2.06337

5530400P07RIK
1.11E−06
0.48380464
−2.06695

A430107D22RIK
2.94E−06
0.48380464
−2.06695

0610012D17RIK
7.81E−08
0.48296813
−2.07053

GALNT2
5.48E−07
0.48296813
−2.07053

EPPB9
2.51E−05
0.48296813
−2.07053

NSG2
8.56E−08
0.48213218
−2.07412

DUSP10
4.51E−08
0.48129681
−2.07772

9430080K19RIK
3.61E−08
0.48129681
−2.07772

RNASEN
2.84E−06
0.48129681
−2.07772

GAS6
0.000310919
0.48129681
−2.07772

1810015C11RIK
7.75E−09
0.48046432
−2.08132

SLITL2
0.000449972
0.48046432
−2.08132

LOC386330
0.00291956
0.48046432
−2.08132

FKBP9
0.000504748
0.47963241
−2.08493

ZFPN1A1
8.59E−07
0.47880108
−2.08855

DDX6
3.03E−07
0.47797263
−2.09217

BACH1
1.88E−06
0.47797263
−2.09217

TNNT1
0.000226617
0.47797263
−2.09217

BLK
2.09E−08
0.47714477
−2.0958

MSCP
3.61E−07
0.47714477
−2.0958

2900060B14RIK
0.0188022
0.47714477
−2.0958

CNN3
2.16E−06
0.47549535
−2.10307

REEP1
3.13E−08
0.47467153
−2.10672

SDH1
4.36E−08
0.47302795
−2.11404

PPARGC1B
9.21E−07
0.47302795
−2.11404

TLK1
5.15E−07
0.47302795
−2.11404

A630097D09RIK
3.46E−05
0.47221042
−2.1177

3110078M01RIK
8.46E−07
0.47139126
−2.12138

1110015K06RIK
2.05E−06
0.47057716
−2.12505

EXT1
5.14E−06
0.46894857
−2.13243

FBLN2
1.48E−07
0.4681363
−2.13613

1810018P12RIK
4.02E−05
0.46651583
−2.14355

DCAMKL2
1.02E−06
0.46570979
−2.14726

PPT1
1.66E−06
0.46570979
−2.14726

2810036L13RIK
3.90E−05
0.46570979
−2.14726

H2-EB1
0.00495563
0.46490221
−2.15099

2810470K03RIK
6.54E−06
0.46409742
−2.15472

RAG1
2.91E−05
0.46329327
−2.15846

2610020H15RIK
1.83E−07
0.46249191
−2.1622

D10UCLA1
4.00E−05
0.46169117
−2.16595

1110003A17RIK
7.74E−07
0.46089109
−2.16971

KCTD2
2.52E−05
0.46089109
−2.16971

G630024G08RIK
3.72E−08
0.45929709
−2.17724

1700026B20RIK
3.84E−06
0.45850107
−2.18102

FAS
7.68E−09
0.4577078
−2.1848

4933424M23RIK
1.76E−07
0.4577078
−2.1848

4921518A06RIK
4.47E−06
0.4577078
−2.1848

IGTP
0.00151097
0.4577078
−2.1848

9430068D06RIK
6.51E−08
0.45691518
−2.18859

A930005H10RIK
1.37E−07
0.45691518
−2.18859

ABCA3
2.04E−07
0.45533401
−2.19619

5330403D14RIK
3.86E−07
0.45533401
−2.19619

4631427C17RIK
9.36E−07
0.45533401
−2.19619

TRIM28
2.42E−05
0.45454752
−2.19999

CERK
1.15E−05
0.45454752
−2.19999

CRYL1
7.94E−07
0.45375963
−2.20381

IL7R
1.25E−08
0.45297446
−2.20763

IHPK1
4.52E−07
0.45297446
−2.20763

RENBP
1.68E−08
0.45218996
−2.21146

TPST1
1.05E−05
0.44984458
−2.22299

9430029L20RIK
9.20E−09
0.44828776
−2.23071

5730593F17RIK
3.51E−06
0.44828776
−2.23071

C530015C18
1.57E−08
0.4467377
−2.23845

HMGN2
0.00010418
0.4467377
−2.23845

TRAF4
8.39E−08
0.44596469
−2.24233

AXIN2
2.23E−09
0.44519237
−2.24622

BCL7A
5.46E−07
0.44519237
−2.24622

A630082K20RIK
7.65E−08
0.44442074
−2.25012

TNRC6C
1.44E−07
0.44442074
−2.25012

PCOLCE
0.000259954
0.44365179
−2.25402

PRICKLE1
6.89E−07
0.44288353
−2.25793

BCL6
1.24E−05
0.44288353
−2.25793

COL2A1
3.25E−05
0.44211792
−2.26184

MRPL14
5.42E−07
0.44135106
−2.26577

ZFP148
8.82E−07
0.44058686
−2.2697

CNOT2
1.44E−07
0.4398253
−2.27363

C230075L19RIK
1.84E−07
0.43906251
−2.27758

2700083E18RIK
2.52E−07
0.43906251
−2.27758

CCND1
1.92E−05
0.43830237
−2.28153

CUTL1
8.66E−08
0.43754485
−2.28548

AI467606
5.41E−07
0.43603003
−2.29342

GMFG
7.47E−05
0.43603003
−2.29342

GLDC
0.000564796
0.43603003
−2.29342

CNP1
1.01E−07
0.43527466
−2.2974

RBM38
2.02E−08
0.4345219
−2.30138

BC039093
2.61E−06
0.4345219
−2.30138

6.33E+19
2.02E−05
0.4345219
−2.30138

SCARA3
5.87E−05
0.4345219
−2.30138

FKBP5
3.81E−07
0.43376985
−2.30537

BC063749
1.34E−09
0.43301853
−2.30937

LOC226135
0.000314737
0.43301853
−2.30937

AFF1
5.19E−07
0.43226794
−2.31338

COL4A1
3.11E−05
0.43002799
−2.32543

COL6A3
0.00167802
0.43002799
−2.32543

VAMP4
4.17E−07
0.4292822
−2.32947

NUP210
0.000100262
0.42853898
−2.33351

ADCY6
1.78E−07
0.42779834
−2.33755

UHRF1
0.000151815
0.42779834
−2.33755

PTPRS
3.83E−07
0.4270566
−2.34161

LBH
5.98E−05
0.42631743
−2.34567

SCML4
1.21E−07
0.425579
−2.34974

1700095N21RIK
2.00E−07
0.425579
−2.34974

5930416I19RIK
4.45E−07
0.425579
−2.34974

SEMA4B
1.98E−06
0.425579
−2.34974

SCA2
6.05E−07
0.42484313
−2.35381

5830431A10RIK
2.60E−06
0.42484313
−2.35381

MSH6
0.000132082
0.42484313
−2.35381

TTC3
5.71E−09
0.4219071
−2.37019

KCTD1
1.30E−06
0.4219071
−2.37019

BC028975
1.19E−08
0.42117677
−2.3743

GPSM1
2.16E−06
0.42117677
−2.3743

ERICH1
3.26E−08
0.42044896
−2.37841

GATA3
6.03E−07
0.42044896
−2.37841

TTYH3
4.09E−06
0.42044896
−2.37841

H2-OB
6.36E−06
0.42044896
−2.37841

BHLHB9
1.72E−05
0.42044896
−2.37841

AW046396
0.00133505
0.42044896
−2.37841

4632417D23
5.33E−07
0.41754351
−2.39496

PDLIM1
8.32E−07
0.41754351
−2.39496

1810010N17RIK
1.37E−06
0.41682124
−2.39911

CHRNA9
1.78E−09
0.41609973
−2.40327

GSTM2
0.000400316
0.41537899
−2.40744

SCL000121.1_106
6.07E−06
0.41394155
−2.4158

MNS1
0.000531845
0.41394155
−2.4158

GABABRBP
3.45E−05
0.41250892
−2.42419

ANP32E
3.77E−05
0.41108279
−2.4326

SMARCD2
6.84E−06
0.40402082
−2.47512

CASP6
6.84E−07
0.40332013
−2.47942

LTAP
3.13E−08
0.40053511
−2.49666

EFEMP2
8.18E−05
0.39984166
−2.50099

4932408F19RIK
3.03E−07
0.39845876
−2.50967

CD1D1
9.54E−07
0.39845876
−2.50967

SH2D1A
1.27E−05
0.39845876
−2.50967

ADRB2
1.53E−08
0.39776773
−2.51403

6330403E01RIK
2.81E−07
0.39707909
−2.51839

C230082I21RIK
1.32E−09
0.39639283
−2.52275

FBXL12
3.35E−06
0.39502115
−2.53151

SMO
3.82E−09
0.39433732
−2.5359

6720469N11RIK
1.55E−07
0.39365429
−2.5403

ZFPN1A2
1.20E−07
0.39297209
−2.54471

PHF2
2.68E−07
0.39297209
−2.54471

LIP1
8.34E−07
0.39297209
−2.54471

IFNGR1
1.21E−07
0.39229224
−2.54912

SPATA13
1.64E−07
0.39161321
−2.55354

NEDD4L
4.00E−09
0.390935
−2.55797

SLA
4.00E−05
0.390935
−2.55797

ARHGEF18
2.56E−05
0.390935
−2.55797

RASGRP1
7.85E−08
0.39025761
−2.56241

NOTCH1
2.02E−08
0.38823493
−2.57576

2900016B01RIK
6.34E−09
0.38756235
−2.58023

PITPNM2
1.79E−07
0.3868906
−2.58471

SMAD3
2.00E−06
0.38555257
−2.59368

CHDH
1.11E−07
0.38355324
−2.6072

C920011N12RIK
5.34E−06
0.38355324
−2.6072

NISCH
5.43E−07
0.38288944
−2.61172

2310007G05RIK
1.06E−06
0.38288944
−2.61172

SIT1
3.76E−09
0.38222647
−2.61625

SLC29A3
7.22E−07
0.3809045
−2.62533

AEBP1
0.000162054
0.3809045
−2.62533

C730009F21RIK
9.48E−09
0.37958587
−2.63445

PLEKHG2
9.65E−07
0.37958587
−2.63445

MBP
2.65E−08
0.37827348
−2.64359

D8ERTD325E
9.64E−06
0.37827348
−2.64359

VPS54
4.16E−08
0.37761784
−2.64818

MLL
2.80E−07
0.37761784
−2.64818

LOC386144
4.21E−05
0.37761784
−2.64818

LOC386360
0.000403683
0.37761784
−2.64818

BACH2
0.000136247
0.37696446
−2.65277

BDH
2.28E−10
0.37500938
−2.6666

KLHL6
2.22E−08
0.37500938
−2.6666

DAP3
1.93E−08
0.37371163
−2.67586

TCRB-V8.2
8.04E−08
0.37371163
−2.67586

A130062D16RIK
1.07E−07
0.37371163
−2.67586

SSBP3
1.03E−07
0.37371163
−2.67586

MAPK1
5.13E−08
0.37242006
−2.68514

FRMD6
3.31E−08
0.37177485
−2.6898

TNFRSF13B
6.85E−08
0.37177485
−2.6898

MMP2
0.000109136
0.37177485
−2.6898

ECM1
1.84E−07
0.3711305
−2.69447

CUL7
2.80E−07
0.36602149
−2.73208

NOTCH3
7.70E−06
0.36602149
−2.73208

D930015E06RIK
2.19E−07
0.36538757
−2.73682

A430106G13RIK
1.00E−06
0.3647545
−2.74157

5830468F06RIK
5.09E−06
0.36412363
−2.74632

HIBADH
1.23E−08
0.36349361
−2.75108

TMEM9
4.73E−07
0.36223616
−2.76063

TSPAN32
8.74E−07
0.35662957
−2.80403

H2-T9
4.22E−05
0.35662957
−2.80403

ESM1
0.00132143
0.35601252
−2.80889

ALOX5AP
1.28E−07
0.35539634
−2.81376

RFX2
1.13E−06
0.35539634
−2.81376

2610019F03RIK
3.33E−06
0.35478103
−2.81864

WHRN
4.89E−07
0.35355303
−2.82843

5830496L11RIK
1.90E−07
0.35294159
−2.83333

GSTP1
7.09E−07
0.35294159
−2.83333

3100002J23RIK
3.91E−07
0.35232978
−2.83825

YPEL3
0.0001326
0.35111127
−2.8481

A130092J06RIK
3.76E−06
0.35050332
−2.85304

IGSF3
5.35E−07
0.34929007
−2.86295

HDAC7A
1.07E−06
0.34448192
−2.90291

IDB3
1.63E−07
0.34388605
−2.90794

OLFML3
6.69E−07
0.34328988
−2.91299

6430510M02RIK
1.07E−07
0.34269578
−2.91804

TRBV13-
1.50E−05
0.34151023
−2.92817

1_M15618_T_CELL_RECEPTOR_BETA_VARIABLE_13-

CD97
2.26E−05
0.33973969
−2.94343

MTF2
1.12E−06
0.33797714
−2.95878

PLA2G12A
1.72E−08
0.33622487
−2.9742

D15WSU75E
2.04E−06
0.33622487
−2.9742

ETHE1
1.66E−08
0.33448172
−2.9897

HIVEP3
3.41E−09
0.33390319
−2.99488

CYB5
3.77E−08
0.33390319
−2.99488

CTSE
0.149884
0.33332444
−3.00008

ZFP219
2.26E−07
0.32987732
−3.03143

ABHD8
1.69E−06
0.32987732
−3.03143

4732481H14RIK
8.63E−07
0.32930592
−3.03669

PRNP
3.14E−05
0.32873542
−3.04196

A630038E17RIK
0.000216397
0.32873542
−3.04196

A930013B10RIK
8.09E−08
0.3253355
−3.07375

ETS1
5.90E−07
0.32252862
−3.1005

LOC385086
0.00104356
0.32252862
−3.1005

RNPEPL1
7.60E−09
0.32196994
−3.10588

KLF13
6.51E−07
0.32141319
−3.11126

KCNN4
1.98E−06
0.3208563
−3.11666

NIPSNAP1
1.39E−06
0.3208563
−3.11666

C920004C08RIK
0.00056251
0.3208563
−3.11666

TRBV7_AE000663_T_CELL_RECEPTOR_BETA_VARIABLE_7_29
6.95E−05
0.31974625
−3.12748

SLC43A1
3.64E−08
0.31643867
−3.16017

STK4
7.89E−07
0.31589089
−3.16565

WISP2
1.27E−05
0.31262407
−3.19873

ACTN2
3.36E−07
0.31046452
−3.22098

EPB4.1L4B
4.42E−07
0.31046452
−3.22098

RNF144
2.74E−09
0.30885455
−3.23777

SCL0001849.1_2273
1.73E−05
0.30778606
−3.24901

ART4
6.83E−07
0.30619056
−3.26594

18S_RRNA_X00686_301
0.00445027
0.29988874
−3.33457

A330103N21RIK
1.65E−06
0.29936983
−3.34035

TPCN1
2.60E−09
0.29885092
−3.34615

AJ237586
1.97E−07
0.29833381
−3.35195

BC026370
6.26E−09
0.29575998
−3.38112

EPHX1
4.14E−09
0.29524827
−3.38698

COL5A1
0.000124246
0.29371767
−3.40463

SNN
1.47E−08
0.29270149
−3.41645

BCL9L
1.00E−08
0.29219432
−3.42238

0710008K08RIK
9.65E−08
0.29168806
−3.42832

F730003H07RIK
0.000110195
0.28967371
−3.45216

AI504432
2.49E−08
0.28717477
−3.4822

OACT1
9.74E−09
0.28667752
−3.48824

A130093I21RIK
1.16E−06
0.28519116
−3.50642

RGS10
2.26E−05
0.28469751
−3.5125

1110046J11RIK
3.46E−06
0.28420394
−3.5186

E2F2
2.29E−06
0.27835537
−3.59253

BRD3
3.58E−07
0.27787349
−3.59876

AA408556
4.12E−06
0.27262665
−3.66802

SATB1
1.49E−07
0.27074371
−3.69353

ILVBL
3.59E−07
0.27074371
−3.69353

TIAM1
1.97E−09
0.26887358
−3.71922

POU6F1
2.59E−08
0.26887358
−3.71922

MAGED1
1.17E−05
0.26887358
−3.71922

1810055G02RIK
1.69E−09
0.26655436
−3.75158

0710001E13RIK
6.72E−10
0.26471273
−3.77768

LMAN2L
4.14E−08
0.26471273
−3.77768

SLC29A1
1.20E−07
0.26425455
−3.78423

3830612M24
2.98E−07
0.26379726
−3.79079

H2-T10
0.000216723
0.26379726
−3.79079

CXCR4
4.41E−08
0.26334015
−3.79737

RIL-PENDING
2.35E−08
0.26242931
−3.81055

SERPINH1
3.41E−05
0.26242931
−3.81055

PALM
2.59E−09
0.26197487
−3.81716

CXCL12
0.000158114
0.26106866
−3.83041

5430417L22RIK
1.68E−08
0.2606162
−3.83706

TRIB2
4.82E−08
0.26016531
−3.84371

ETS2
5.78E−08
0.26016531
−3.84371

ALDH2
9.48E−07
0.25881592
−3.86375

HMGN1
3.20E−05
0.25702844
−3.89062

TRP53INP1
4.08E−08
0.25569562
−3.9109

ITPR2
5.70E−08
0.25392956
−3.9381

TCRB
8.36E−06
0.25348992
−3.94493

A930023F05RIK
4.58E−09
0.24784315
−4.03481

SLC5A9
1.20E−06
0.24698554
−4.04882

ICAM2
2.53E−07
0.24655805
−4.05584

H2-DMA
1.25E−05
0.24655805
−4.05584

4932414K18RIK
3.75E−08
0.24443125
−4.09113

TAP2
9.85E−09
0.24358518
−4.10534

TRBV12-
3.25E−05
0.24358518
−4.10534

2_M15613_T_CELL_RECEPTOR_BETA_VARIABLE_12-

PRELP
8.99E−06
0.24232261
−4.12673

TRBV12-
1.47E−06
0.24106609
−4.14824

1_M15614_T_CELL_RECEPTOR_BETA_VARIABLE_12-

LOX
8.44E−06
0.24023216
−4.16264

1500004A08RIK
1.03E−07
0.2398162
−4.16986

6720418B01RIK
4.27E−08
0.23857238
−4.1916

4930572J05RIK
4.75E−07
0.23692472
−4.22075

SCL0001032.1_178
7.57E−06
0.23692472
−4.22075

PSAP
4.23E−08
0.23610521
−4.2354

ASS1
5.82E−08
0.23569619
−4.24275

PARD6G
1.30E−08
0.23528803
−4.25011

1500009L16RIK
9.73E−07
0.2324511
−4.30198

GM2A
2.89E−06
0.23124595
−4.3244

LAT
5.46E−08
0.23084559
−4.3319

C3
9.90E−06
0.23084559
−4.3319

PPAP2B
1.69E−05
0.23044607
−4.33941

CTLA4
5.06E−06
0.23044607
−4.33941

FBP1
5.98E−05
0.22845811
−4.37717

B3BP
1.28E−10
0.22453292
−4.45369

PRKCB
6.59E−08
0.22453292
−4.45369

PPP1R1C
3.85E−09
0.22067553
−4.53154

RAPGEF3
6.21E−10
0.21953174
−4.55515

BAMBI-PS1
7.87E−08
0.21915118
−4.56306

1700012H17RIK
5.26E−09
0.21839306
−4.5789

ACVR2B
3.12E−10
0.21801502
−4.58684

18S_RRNA_X00686_849
0.000602805
0.21538634
−4.64282

SERPINF1
1.43E−05
0.21464156
−4.65893

NAV1
4.40E−08
0.21426995
−4.66701

TBXA2R
3.11E−08
0.2135283
−4.68322

SCL0001132.1_96
1.63E−05
0.20732994
−4.82323

SOX4
1.93E−09
0.2055414
−4.8652

E430021E22RIK
3.57E−07
0.20447556
−4.89056

LOC381739
2.02E−08
0.203063
−4.92458

CD6
3.02E−10
0.20096261
−4.97605

H2-OA
1.09E−07
0.19888426
−5.02805

LOC384370
7.41E−06
0.1978529
−5.05426

ZDHHC8
3.73E−09
0.19751019
−5.06303

AI481316
1.75E−09
0.19682715
−5.0806

H2-BL
3.80E−06
0.19614612
−5.09824

AA407270
7.17E−09
0.1954675
−5.11594

ITGAE
6.39E−06
0.1951288
−5.12482

GPR83
1.37E−07
0.19479128
−5.1337

SBK
4.54E−10
0.18783011
−5.32396

RPS6KL1
1.75E−09
0.18750504
−5.33319

TCF7
8.08E−08
0.18396455
−5.43583

NRP
4.24E−08
0.18364596
−5.44526

DNAJC6
3.58E−11
0.18269378
−5.47364

SCL0001090.1_202
3.66E−09
0.18269378
−5.47364

SCL0001131.1_227
1.31E−06
0.17924456
−5.57897

IL17RB
1.94E−09
0.17800626
−5.61778

ACAS2L
2.63E−07
0.17647048
−5.66667

AKR1C12
8.40E−09
0.1761652
−5.67649

COL6A1
3.56E−05
0.17464534
−5.72589

SOCS3
3.74E−08
0.17283904
−5.78573

LDH2
1.01E−07
0.17283904
−5.78573

DGKA
6.54E−07
0.17283904
−5.78573

GM525
1.28E−07
0.17253963
−5.79577

TIMP2
2.00E−08
0.17224096
−5.80582

AQP11
3.40E−09
0.17045939
−5.8665

TRBV6_AE000663_T_CELL_RECEPTOR_BETA_VARIABLE_6_11
5.66E−08
0.16695132
−5.98977

TNFRSF7
9.20E−08
0.1615441
−6.19026

CD2
7.22E−08
0.15932015
−6.27667

DTX1
3.94E−09
0.15876897
−6.29846

AI875142
2.49E−07
0.15712672
−6.36429

IGFBP4
1.57E−08
0.15577125
−6.41967

SH3KBP1
2.13E−10
0.15496356
−6.45313

2510015F01RIK
3.11E−06
0.15362659
−6.50929

TRBV11_AE000663_T_CELL_RECEPTOR_BETA_VARIABLE_11_—
2.50E−08
0.15151148
−6.60016

SLC16A5
1.12E−10
0.14865092
−6.72717

TUBB2B
4.82E−08
0.14813652
−6.75053

DNTT
7.39E−07
0.14533954
−6.88044

SYTL1
6.70E−08
0.14309058
−6.98858

2410008J05RIK
2.59E−09
0.13726158
−7.28536

0610041G09RIK
9.40E−06
0.13702403
−7.29799

2210408F11RIK
2.70E−08
0.13513751
−7.39987

LOC386545
2.77E−05
0.13121465
−7.6211

TRBV31_X03277_T_CELL_RECEPTOR_BETA_VARIABLE_31_33
4.97E−06
0.12918411
−7.74089

TCRB-V8.3
1.15E−08
0.12829181
−7.79473

KLF2
3.06E−06
0.125434
−7.97232

TCRB-V13
5.74E−08
0.12435182
−8.0417

A130038J17RIK
8.77E−09
0.11784809
−8.4855

LOC381738
1.87E−09
0.11723701
−8.52973

G22P1
5.44E−10
0.11582345
−8.63383

CD27
4.55E−08
0.11383376
−8.78474

TMEM108
8.61E−08
0.11265625
−8.87656

ACTN1
2.20E−09
0.11091295
−9.01608

ST6GAL1
6.69E−10
0.10657936
−9.38268

9626100_15
1.11E−05
0.10348549
−9.66319

9626100_224
8.13E−06
0.10118027
−9.88335

C030046M14RIK
1.45E−12
0.1010051
−9.90049

SELL
6.85E−09
0.09944213
−10.0561

COX6A2
7.37E−05
0.0987547
−10.1261

FRAT2
6.49E−09
0.09841357
−10.1612

LY6D
1.48E−09
0.09790292
−10.2142

9130430L19RIK
2.71E−10
0.09278245
−10.7779

CDCA7
3.37E−07
0.09214212
−10.8528

LOC382896
3.09E−09
0.09150554
−10.9283

CD8B
7.37E−08
0.08993372
−11.1193

E430002D04RIK
2.09E−10
0.08931203
−11.1967

TRGV2_M12831_T_CELL_RECEPTOR_GAMMA_VARIABLE_2_3
2.57E−11
0.08656959
−11.5514

PP11R
2.12E−07
0.08641922
−11.5715

CD81
4.58E−08
0.08318706
−12.0211

IGH-6
8.08E−09
0.08289881
−12.0629

AI132321
2.09E−08
0.08204186
−12.1889

9626958_317
8.03E−06
0.07721352
−12.9511

TRBV8_AE000663_T_CELL_RECEPTOR_BETA_VARIABLE_8_27
9.31E−08
0.07694675
−12.996

MGST2
3.44E−09
0.07419664
−13.4777

RAMP1
5.23E−08
0.072043
−13.8806

NCK2
3.02E−08
0.06863088
−14.5707

MARCKS
1.18E−09
0.06572116
−15.2158

DPP4
2.19E−10
0.05642577
−17.7224

TRBV1_AE000663_T_CELL_RECEPTOR_BETA_VARIABLE_1_20
6.96E−07
0.05603622
−17.8456

H19
3.39E−07
0.0557454
−17.9387

TCRG-V4
3.97E−09
0.05129626
−19.4946

1190002H23RIK
4.58E−10
0.05024065
−19.9042

BCL11B
2.77E−10
0.0495491
−20.182

CD3G
8.20E−11
0.04647186
−21.5184

BGN
3.71E−07
0.03969215
−25.1939

CD3D
1.53E−09
0.0349758
−28.5912

CD3E
7.19E−10
0.03190668
−31.3414

LOC434197
5.40E−11
0.02356029
−42.4443

MYLC2PL
8.10E−10
0.01703917
−58.6883

PDLIM4
8.76E−11
0.00978646
−102.182

TABLE 2

Comparison of cell surface receptor repertoires of ITNKs and LAKs.

Cell Type
Ly49C/I
Ly49D
Ly49G2
NK1.1
NKp46
NKG2A/C/E
NKG2D
CD3

DN3-
−
−
−
+
+
+
−
−

reprogrammed

ITNK (in vitro)

DP-
−
−
−
+
+
+
ND
+

reprogrammed

ITNK (in vitro)

DP-
+
−
+
+
+
+
+
low

reprogrammed

ITNK (in vivo)

LAK
+
+
+
+
+
+
+
−

Note:

N.D., not determined. +, present;. −, absent; low, low levels.

TABLE 3

Fold-Change

Column ID
(+OHT vs. −OHT)

24 hours +OHT vs. −OHT

TCRB-V13
−14894.3

TCRB-V13
−412.694

HIST1H2AO
−35.3085

MTDNA_CYTB
−13.7646

IFITM1
−12.6519

CDCA7
−12.2615

PDLIM4
−10.206

CD3D
−9.81142

RPS29
−9.27924

MTDNA_COXIII
−8.38116

RPS14
−6.99771

MYLC2PL
−6.76599

CD3D
−5.74896

IFITM2
−5.49522

RPL13
−5.33629

RPS17
−5.30823

RPL41
−5.29871

18S_RRNA_X00686_301
−5.08974

HIST2H2AC
−4.87534

IFITM3
−4.86541

MTDNA_ND4
−4.61459

HIST1H2AI
−4.60413

MT-CYTB
−4.34

MYLC2PL
−4.32543

RPS11
−4.20813

ITGB7
−4.05052

RPL39
−3.69839

RPS27L
−3.63035

CD3G
−3.49152

EG668668
−3.46858

CD160
−3.44386

RPL23
−3.38327

CD3E
−3.30996

HIST1H2AO
−3.29719

EMP3
−3.22525

PDLIM4
−3.15574

TBCA
−3.1281

THY1
−3.11285

CD8B
−3.01933

PRKACB
−2.90749

HIST1H2AF
−2.78505

LOC226574
−2.74888

G22P1
−2.74634

HIST1H2AG
−2.64205

AI481316
−2.62333

IGH-6
−2.5636

A130092J06RIK
−2.55682

TCRG-V4
−2.54552

UBB
−2.45105

LOC434197
−2.4265

RPS17
−2.41723

MTDNA_ATP6
−2.41506

TXNIP
−2.40294

LOC381808
−2.39227

PPIA
−2.37637

LOC382896
−2.35265

HMGCS1
−2.30359

TCRB-V8.2
−2.2788

HMGN2
−2.2528

UPP1
−2.22963

CSTB
−2.21395

PSAP
−2.20351

TRBV1_AE000663_T_CELL_RECEPTOR_BETA_VARIABLE_1_207
−2.17733

HIBADH
−2.16873

E430002D04RIK
−2.15383

CDCA7
−2.15035

RPS27
−2.14844

RPL8
−2.11346

RNPEPL1
−2.11344

COX6A2
−2.10037

CD27
−2.07584

MARCKS
−2.05494

VIM
−2.04688

AA408556
−2.03945

4932414K18RIK
−2.01727

BCL11B
−2.011

RPS14
−2.0086

TCRB-V8.2
−2.00629

COX7C
2.11831

LOC270037
2.16383

RPA1
2.26914

LAPTM5
2.8707

UBL5
3.35851

ATP5G3
3.54832

1300002F13RIK
3.6466

CD52
3.81745

LDH1
3.82439

CYBA
6.431

FCER1G
7.97969

HMGCS1
27.1719

48 hours +OHT vs. −OHT

ROG
11.7941537

FCER1G
11.6317801

UPP1
9.00046788

IFITM1
8.6938789

SCIN
8.6938789

SERPINA3G
8.51496146

XCL1
7.62110398

AQP9
7.4127045

NKG7
7.01284577

IFITM2
6.40855902

IFITM3
6.36429187

9130404D14RIK
5.69620078

GADD45G
5.6177795

LGALS3
5.46416103

CD160
5.31474326

KLRD1
5.27803164

VIM
4.9933222

TYROBP
4.89056111

LITAF
4.82323131

BC025206
4.78991482

AVIL
4.72397065

LMNA
4.72397065

GLRX1
4.40762046

NFIL3
4.40762046

LTA
4.1410597

CCR5
4.0278222

WBSCR5
4

P2RY14
3.91768119

1300002F13RIK
3.83705648

AMICA1
3.73213197

LOC270152
3.70635225

9130211I03RIK
3.6553258

CDKN2B
3.6553258

PLCG2
3.55537072

CTSW
3.53081199

BC049975
3.50642289

LOC381140
3.36358566

LGALS1
3.34035168

MT1
3.27160823

SYTL2
3.27160823

GPR114
3.24900959

S100A1
3.24900959

2310067E08RIK
3.20427951

LRRK1
3.20427951

TNFRSF11B
3.18214594

IDB2
3.16016525

CCL4
3.11665832

E030006K04RIK
3.11665832

OSBPL3
3.11665832

LY6A
3.09512999

TNFRSF9
3.09512999

S100A6
3.07375036

1500031H04RIK
3.05251842

2210411K11RIK
3.05251842

CTNNA1
3.03143313

LOC381319
3.03143313

EMILIN2
3.01049349

1110018K11RIK
2.9896985

ANXA2
2.9896985

SIAT10
2.96904714

2310046K01RIK
2.94853843

CISH
2.92817139

1110004P15RIK
2.90794503

GOLPH2
2.88785839

HAVCR2
2.88785839

PLSCR1
2.88785839

SLC2A6
2.8679105

CAPG
2.84810039

LAG3
2.84810039

F2R
2.82842712

LOC269941
2.82842712

1190002C06RIK
2.80888975

CD9
2.78948733

S100A11
2.78948733

GCNT1
2.75108364

CDKN1A
2.73208051

KLRE1
2.73208051

GPC1
2.71320865

SERPINE2
2.69446715

LRP12
2.67585511

MLKL
2.67585511

BC024955
2.65737163

BHLHB2
2.65737163

C330008K14RIK
2.65737163

F2RL2
2.63901582

GLRX
2.63901582

IFNG
2.62078681

PGLYRP1
2.62078681

1110007C02RIK
2.60268371

BC029169
2.60268371

TRAF1
2.60268371

CDKN2A
2.58470566

DUSP6
2.58470566

LY6G5B
2.58470566

RGS1
2.5668518

MYO1F
2.54912125

HBA-A1
2.53151319

2310047C17RIK
2.51402675

AIM1L
2.51402675

PILRB
2.4966611

2410008K03RIK
2.4794154

APOB48R
2.4794154

PDGFA
2.4794154

FURIN
2.46228883

SPP1
2.46228883

ROM1
2.44528056

SH3BP2
2.44528056

PPP3CC
2.42838977

B4GALNT4
2.41161566

IER3
2.41161566

OSM
2.41161566

DAPK2
2.39495741

LOC218482
2.39495741

MAPKAPK3
2.39495741

PLP2
2.37841423

BAG3
2.36198532

OSTF1
2.36198532

SERPINB6A
2.3456699

FXYD4
2.32946717

LOC327957
2.32946717

AHNAK
2.29739671

CD69
2.28152743

HK2
2.28152743

FES
2.26576777

IL18R1
2.26576777

PPAP2C
2.26576777

SLC39A4
2.25011697

TES
2.25011697

TNF
2.25011697

HGFAC
2.23457428

CD244
2.21913894

6330414G02RIK
2.20381023

CD63
2.20381023

LOC383981
2.1885874

NAPSA
2.1885874

PKP3
2.1885874

EMP1
2.17346973

FOSL2
2.17346973

GLIPR1
2.17346973

NT5E
2.17346973

SLC24A3
2.17346973

2610009E16RIK
2.15845647

1110020C13RIK
2.14354693

D10BWG1379E
2.14354693

ID2
2.14354693

DOK2
2.12874036

LOC381924
2.12874036

2210008N01RIK
2.11403608

5330403J18RIK
2.11403608

HIST1H1C
2.09943337

0610037M15RIK
2.08493152

7420404O03RIK
2.08493152

A430006M23RIK
2.07052985

D930046M13RIK
2.07052985

GNG2
2.07052985

GPR68
2.07052985

H2-Q8
2.07052985

IFI30
2.07052985

ZFP608
2.07052985

DCI
2.05622765

NFKB1
2.05622765

PIM3
2.05622765

SGK
2.05622765

CCNG1
2.04202425

CYP51
2.04202425

LOC385953
2.04202425

EGR1
2.02791896

HHEX
2.02791896

MYO1E
2.02791896

TMEM126A
2.02791896

NCF4
2.0139111

PDLIM7
2.0139111

CXCL9
2

GPR18
2

MVP
2

PRSS19
2

A130038J17RIK
−2.0139111

A130093I21RIK
−2.0139111

EPHX1
−2.0139111

NOTCH3
−2.0139111

MTF2
−2.027919

TNFRSF7
−2.027919

4932414K18RIK
−2.0420243

GFI1
−2.0420243

2410008J05RIK
−2.0562277

2610019F03RIK
−2.0705298

H2-OB
−2.0705298

SATB1
−2.0705298

TCF7
−2.0705298

2900060B14RIK
−2.0849315

TBXA2R
−2.0849315

NISCH
−2.0994334

LOC434197
−2.1140361

PARD6G
−2.1140361

DPP4
−2.1435469

H2-AB1
−2.1435469

LMAN2L
−2.1435469

BRD3
−2.1584565

CD27
−2.1584565

LOC386192
−2.1584565

H2-EB1
−2.1734697

NCK2
−2.1734697

RAMP1
−2.1734697

1110046J11RIK
−2.1885874

AQP11
−2.2345743

SLA
−2.2345743

MARCKS
−2.250117

IGH-6
−2.2657678

SH2D1A
−2.2657678

F730003H07RIK
−2.2973967

H2-T10
−2.2973967

DGKA
−2.3133764

DNTT
−2.3133764

ETS1
−2.3294672

LOC268393
−2.3294672

LOC386360
−2.3294672

TMEM108
−2.3294672

C230098O21RIK
−2.3619853

RNPEPL1
−2.3619853

G22P1
−2.3784142

TRBV31_X03277_T_CELL_RECEPTOR_BETA_VARIABLE_31_33
−2.3784142

ALDH2
−2.4283898

CDCA7
−2.4622888

NRP
−2.4622888

TXNIP
−2.4622888

SLC16A5
−2.4966611

ACAS2L
−2.5140267

FRAT2
−2.5491213

CD81
−2.6390158

PRKCB
−2.6573716

PDLIM4
−2.6758551

H2-BL
−2.7132087

PP11R
−2.7320805

ACTN1
−2.7510836

CD6
−2.7510836

CD2
−2.7894873

ST6GAL1
−2.8088898

TRBV1_AE000663_T_CELL_RECEPTOR_BETA_VARIABLE_1_20
−2.8088898

CD8B
−2.8481004

9430068D06RIK
−2.8679105

AI132321
−3.0737504

H19
−3.1601652

LY6D
−3.3869812

CTSE
−3.5064229

BCL11B
−3.5801003

LOC382896
−4.5947934

COX6A2
−6.2766728

TABLE 4

The list of primers in this study.

Genotyping PCR

Size of PCR

primers
Primer sequences (5′-3′)
SEQ ID NO.
products (bp)

Genotyping primers.

Bcl11b-cko-FW
TGAGTCAATAAACCTGGGCGAC
1
243 (wild type);

Bcl11b-cko-RV
GGAATCCTTGGAGTCACTTGTGC
2
345 (flox);

Bcl11b-cko-DEL
TCCTGGTAACACACAATTGC
3
450 (del)

qPCR primers
Primer sequences (5′-3′)
SEQ ID NO.

qRT-PCR primers.

Notch1-Fwd
CCCTTGCTCTGCCTAACGC
4

Notch1-Rev
GGAGTCCTGGCATCGTTGG
5

Etsi-Fwd
TTAGGAAAGGCTCGTTTGCTC
6

Ets1-Rev
CCAAAGCACAAGCATAGTTTGC
7

Hes1-Fwd
CCAGCCAGTGTCAACACGA
8

Hes1-Rev
AATGCCGGGAGCTATCTTTCT
9

Gata3-Fwd
CTCGGCCATTCGTACATGGAA
10

Gata3-Rev
GGATACCTCTGCACCGTAGC
11

Deltax1-Fwd
TGTTCAGGCTATACACGCATCAA
12

Deltax1-Rev
CCACCGCCCACTTTCAAG
13

Tcf1-Fwd
ATGGGCGGCAACTCTTTGAT
14

Tcf1-Rev
CGTAGCCGGGCTGATTCAT
15

Cdkn1c-Fwd
CGAGGAGCAGGACGAGAATC
16

Cdkn1c-Rev
GAAGAAGTCGTTCGCATTGGC
17

Id2-Fwd
ATGAAAGCCTTCAGTCCGGTG
18

Id2-Rev
AGCAGACTCATCGGGTCGT
19

Il2rb-Fwd
TGGAGCCTGTCCCTCTACG
20

Il2rb-Rev
TCCACATGCAAGAGACATTGG
21

Zfp105-Fwd
GGCATCCAGCCAACAAGTGTA
22

Zfp105-Rev
CATTTCCTGACCCTTTTCCTCAT
23

Traf1-Fwd
GGAGGCATCCTTTGATGGT A
24

Traf1-Rev
AGGGACAGGTGGGTCTTCTT
25

Zbtb32-Fwd
GCTCTGAGAGAGGACTTGGGA
26

Zbtb32-Rev
TGCTTTATGCTTGTGTGACATCT
27

PCR primers
Primer sequences (5′-3′)
SEQ ID NO.

Tcrb rearrangement PCR primers.

TCRB_Dβ2-Fwd
GTAGGCACCTGTGGGGAAGAAACT
28

TCRB_Vβ2-Fwd
GGGTCACTGATACGGAGCTG
29

TCRB_Jβ2-Rev
TGAGAGCTGTCTCCTACTATCGATT
30

List of primers for ChIP assay qPCR.

BS1-Fwd
CCGCTACGAGGCACCCTCCTTT
31

BS1-Rev
AGTCTCCTTGGGAAGCACGCGCTA
32

Bs2-Fwd
GCTTGCTTGTTTTTAATTCAGTTTATGGG
33

BS2-Rev
TTGAATGTCTGTGTTGGTGTGTAATCAC
34

BS3-Fwd
GTGAAAAAAAGGGGGTAGGCCCTC
35

BS3-Rev
CAGCCCAAAGTCAAAAGGCAAGATG
36

CTL-Fwd
GTTCCTTAACTGAGAGTTCCTCCTCCC
37

CTL-Rev
TCACTCTGGGCCGGAGTCAGTT
38

Number	Date	Country	Kind
0912287.0	Jul 2009	GB	national
1006649.6	Apr 2010	GB	national

CELLS, COMPOSITIONS AND METHODS

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