Patent Application
20220252594
γδ T cells function at the interface between the innate and adaptive immune systems and have well-demonstrated roles in response to infection, autoimmunity, and tumors. A common characteristic of these seemingly disparate conditions may be cellular stress. Very few verified ligands for γδ T cells have been identified and these have been largely intact self-proteins with no obvious common structure. In addition, no traditional MHC-restricted recognition of ligands has been demonstrated for γδ T cells. Therefore, full understanding of γδ T cell biology has been handicapped by ignorance of the ligands for most TCR-γδ. To date no systematic process has been reported for determining the spectrum of human TCR-γδ ligands.
The disclosure, in some aspects, relates to a method of detecting ligands for γδ T cells in vitro, the method comprising contacting a sample with a soluble human γδ T cell receptor (sTCR-γδ) tetramer, wherein the sTCR-γδ produces a detectable signal in response to engagement with a γδ T cell surface ligand and detecting the measurable signal of the sTCR-γδ tetramer, wherein the detectable signal indicates the presence of the γδ T cell surface ligand in the sample.
In some embodiments, the detectable signal is a fluorescent, chemiluminescent, or absorbance signal. In one embodiment, the sTCR-γδ tetramer is biotinylated and the detectable signal is streptavidin-PE. In some embodiments, the staining is detected via flow cytometry.
In some embodiments, the sTCR-γδ binds to the γδ T cell surface ligand of a Vδ1 T cell. In some embodiments, the sample comprises primary cells or a tumor cell line. In some embodiments, the sample is from a primary tissue, a tumor, inflamed synovium, or intestinal epithelium.
In some embodiments, the method further comprises identifying the γδ T cell surface ligand. In some embodiments, the γδ T cell surface ligand is identified using RNA-seq and bioinformatics and/or mass spectrometry, and/or a transfection-based genetic screen.
The disclosure, in another aspect, provides a human synovial soluble TCR-γδ. In some embodiments, the human synovial soluble TCR-γδ is formulated as a tetramer using, for example, streptavidin-PE or avidin-conjugated magnetic beads.
The disclosure, in a further aspect, provides a single vector comprising a T cell receptor (TCR) γ chain sequence and a TCR δ chain sequence, and further comprising two promoters, a tag, and a binding partner sequence. In some embodiments, the tag is a hexa-His tag. In some embodiments, the binding partner sequence is a biotinylation sequence. In some embodiments, the two promoters comprise p10 and polyhedron.
In one embodiment, the disclosure provides a method of making the human synovial soluble TCR-γδ, the method comprising transfecting a cell with a vector described herein.
The disclosure, in another aspect, provides an anti-cancer therapeutic composition, comprising a unique TCR-γδ ligand, wherein the unique TCR-γδ ligand is a protein or a functional fragment thereof of Table 1 and a pharmaceutically acceptable carrier for administration to a subject to stimulate a γδ T cell subpopulation.
In a further aspect, the disclosure provides a method for stimulating a γδ T cell subpopulation in vivo, the method comprising: administering to a subject a unique TCR-γδ ligand, wherein the unique TCR-γδ ligand is a protein or a functional fragment thereof of Table 1 and a pharmaceutically acceptable carrier in an effective amount to stimulate a γδ T cell subpopulation.
Each of the limitations of the invention can encompass various embodiments of the invention. It is, therefore, anticipated that each of the limitations of the invention involving any one element or combinations of elements can be included in each aspect of the invention. This invention is not limited in its application to the details of construction and the arrangement of components set forth in the following description or illustrated in the drawings. The invention is capable of other embodiments and of being practiced or of being carried out in various ways. The details of one or more embodiments of the invention are set forth in the accompanying Detailed Description, Examples, Claims, and Figures. Other features, objects, and advantages of the invention will be apparent from the description and from the claims.
The accompanying drawings are not intended to be drawn to scale. In the drawings, each identical or nearly identical component that is illustrated in various figures is represented by a like numeral. For purposes of clarity, not every component may be labeled in every drawing. In the drawings:
Described herein are methods of identifying candidate ligands for human γδ T cells using a soluble human TCR-γδ molecule and related products.
A detectable form of human soluble TCR-γδ (sTCR-γδ) was produced from a synovial Vδ1 γδ T cell clone of a Lyme arthritis patient. As described herein, the tetramerized sTCR-γδ was used in flow cytometry to identify various cell types that expressed candidate ligands. Initial analysis of 24 tumor cell lines identified a set of 8 ligand-positive tumors, enriched for those of epithelial and fibroblast origin, and 16 ligand-negative tumors, largely of hematopoietic origin. In addition, ligand was not expressed by primary monocytes or T cells, although each could be induced to express ligand following their activation. Ligand expression was sensitive to trypsin digestion, revealing the protein nature of the ligands, and was also reduced by inhibition of glycolysis. These findings provide a framework and strategy for the identification of individual ligands for human synovial γδ T cells.
γδ T cells reside at mucosal and epithelial barriers, and often accumulate at sites of inflammation with autoimmunity, infections, or tumors (1). Evidence suggests that γδ T cells provide protection against infections with bacteria, viruses, and protozoans, and are generally beneficial in autoimmunity (1-17). In addition, a role for γδ T cells in the immune response against tumors in humans is evident from a seminal study reporting that intratumoral γδ T cells are the most favorable prognostic immune population across 39 cancer types in humans (18). γδ T cells are often highly lytic against transformed proliferative cells, infected cells, as well as infiltrating CD4+ T cells in inflammatory arthritis (9, 17, 19). They can produce a variety of cytokines including IFN-γ, TNF-α, and IL-17 (20), as well as insulin-like growth factor-1 (IGF1) and keratinocyte growth factor (KGF) that promote epithelial wound repair (21). These collective studies indicate that a principal function of γδ T cells is in response to tissue injury of various causes. It is, thus, not surprising that γδ T cells are often suggested to react to host components that are upregulated or exposed during proliferation or cell injury (22). As such, γδ T cells may function in tissue homeostasis and immunoregulation as much as in protection from infection. Yet in the vast majority of cases, little if anything is known regarding the nature of these self-components, or whether they actually engage the TCR-78.
Whereas αβ T cells recognize proteins that are processed into peptides and presented on MHC molecules, the few proposed ligands for γδ T cells suggest that they recognize mostly intact proteins directly, without MHC restriction. This makes them highly attractive for immunotherapy. Despite the elaborate mechanisms that αβ T cells and B cells use to prevent autoreactivity, γδ T cells have been frequently reported to respond to autologous proteins. Furthermore, in contrast to other lymphocytes that maximize the potential diversity of their receptors, γδ T cells frequently show limitations in their diversity. Thus, human γδ T cells comprise subset of Vδ2 T cells, the predominant γδ in peripheral blood that respond to prenyl phosphates and certain alkyl amines (23-25), and Vδ1 T cells that do not respond to these compounds and often accumulate at epithelial barriers and sites of inflammation (1). A similar limited repertoire occurs in the mouse in which Vγ5Vδ1 cells colonize the epidermis, and a Vγ6Vδ1 subset colonizes the tongue, lung, and female reproductive tract (21, 26). This restricted repertoire implies that TCR-γδ ligands may also be limited. This may provide for a more rapid response, and perhaps explain why, in contrast to αβ T cells and B cells, it is difficult to generate antigen-specific γδ T cells by immunization with a defined antigen.
Various ligands for γδ T cells have been proposed, although only a few have been confirmed to bind to TCR-γδ, and these lack any obvious similarity in structure. γδ T cells for which ligands have been identified include the murine γδ T cell clone G8, which recognizes the MHC class I-like molecules T10 and T22 (27), γδ T cells from mice infected with herpes simplex virus that recognize herpes glycoprotein gL (28), a subset of murine and human γδ T cells that bind the algae protein phycoerythrin (20), a human γδ T cell clone G115 that recognizes ATP synthase complexed with ApoA-1 (28), a human γδ T cell clone (Vγ4Vδ5) from a CMV-infected transplant patient that recognizes endothelial protein C receptor (EPCR) (29), and some human Vδ1 T cells that recognize CD1d-sulfatide antigens (30). However, to date no systematic process has been reported for determining the spectrum of human TCR-γδ ligands.
Therefore, in some aspects, the disclosure provides a method of systemically identifying human TCR-γδ ligands, such as those that interact with Vδ1 γδ T cells. In some embodiments, the human TCR-γδ ligands are identified with the use of a soluble TCR-γδ tetramer linked directly or indirectly to a detectable molecule.
As used herein, a “soluble TCR-γδ” refers to a T cell receptor consisting of the chains of a full-length (e.g., membrane bound) receptor, except that, minimally, the transmembrane regions of the receptor chains are deleted or mutated so that the receptor, when expressed by a cell, will not associate with the membrane. Most typically, a soluble receptor will consist of only the extracellular domains of the chains of the wild-type receptor (i.e., lacks the transmembrane and cytoplasmic domains). TCR-γδ molecules comprise a heterodimer of a γ chain and a δ chain. Multiple different functional murine γ chains, murine δ chains, human γ chains, and human δ chains are known in the art. Various specific combinations of γ and δ chains are preferred for use in the sTCR-γδs described herein, and particularly those corresponding to γδ T cell subsets that are known to exist in vivo, but it is to be understood that sTCR-γδs having virtually any combination of γ and δ chains are also contemplated for use herein. Preferably, sTCR-γδs comprise γ and δ chains derived from the same animal species (e.g., murine, human). In some embodiments, the sTCR-γδ comprises human γ chains and human δ chains. A sTCR-γδ described herein may comprise a heterodimer comprising a γ chain and a δ chain. In some embodiments, the sTCR-γδ described herein is a multimer (e.g., tetramer) comprising four of the same γδ heterodimers. In some embodiments, the sTCR-γδ comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, or more γδ heterodimers. As described above, in some embodiments, γ and δ chains from the same species of mammal (e.g., murine, human) are combined to form a γδ heterodimer.
The heterodimers may be linked or conjugated by any method known in the art, for example, by streptavidin tetramerization. In one embodiment, the heterodimers are linked via a linker radical comprising a polyalkylene glycol polymer or a peptidic sequence. In some embodiments, the linker radical should be capable of attachment to defined positions on the sTCR-γδs, so that the structural diversity of the multimers formed is minimized. In one embodiment, the polymer chain or peptidic linker sequence extends between amino acid residues of each sTCR-γδ which are not located in a variable region sequence of the sTCR-γδ thereof.
Methods of linking the heterodimer chains are known in the art, and two examples are provided below. In some embodiments, the mulitmer (e.g., tetramer) described herein is linked by a polyalkylene glycol chain. In some embodiments, the polyalkylene glycol chain comprises hydrophilic polymers. Examples of polyalkylene glycols include, but are not limited to those based on polyethylene glycol or PEG, as well as those based on other suitable, optionally substituted, polyalkylene glycols, such as polypropylene glycol, and copolymers of ethylene glycol and propylene glycol. In other embodiments, the multimer (e.g., tetramer) is multimerized using a non-PEG-based polymer, such as moieties comprising maleimide termini linked by aliphatic chains such as BMH and BMOE can be used.
In some embodiments, the multimerization is accomplished through the use of one or more peptidic linkers. These linkers are comprised of chains of amino acids, and function to produce simple linkers or multimerization domains onto which sTCR-γδs can be attached. As noted above, the biotin/streptavidin system has previously been used to produce tetramers of murine TCR-γδs (see WO 99/60119) for in vitro binding studies.
There are a number of human proteins that contain a multimerization domain that could be used in the production of sTCR-γδs. For example, the tetramerization domain of p53 which has been utilized to produce tetramers of scFv antibody fragments which exhibited increased serum persistence and significantly reduced off-rate compared to the monomeric scFV fragment may be used. (Willuda et al. (2001) J. Biol. Chem. 276 (17) 14385-14392) Likewise, hemoglobin also has a tetramerization domain that could potentially be used.
A multimer (e.g., tetramer) complex comprising at least two sTCR-γδs wherein at least one of said sTCR-γδs is a sTCR-γδ described herein provides another embodiment of the disclosure.
The sTCR-γδ produces a detectable signal in response to engagement with a γδ T cell surface ligand. A detectable signal may be produced once a ligand interacts with sTCR-γδ and induces a change that enables detection of a signal. The signal may be in the form of a detectable molecule.
The detectable molecule may be any agent known in the art, for example, an agent capable of generating a fluorescent, chemiluminescent, or absorbance signal. A suitable label may be chosen from a variety of known detectable labels. Exemplary labels include fluorescent, photoactivatable, enzymatic, epitope, magnetic and particle (e.g. gold) labels. In some embodiments, the detectable molecule comprises one or more fluorescent labels, such as FITC. For example, in tetrameric sTCR-γδ formed using biotinylated heterodimers, fluorescent streptavidin (commercially available) can be used to provide a detectable label. A fluorescently labeled tetramer will be suitable for use in FACS analysis, for example to detect one or more γδ T cell ligands.
In some embodiments, the detectable agent is directly conjugated to the sTCR-γδ. In other embodiments, the detectable agent is indirectly conjugated to the sTCR-γδ. For example, the sTCR-γδ may be labeled either directly with a fluorescent tag, or with a hapten such as biotin, followed by treatment with a fluorescently labeled second moiety such as streptavidin (or both). The latter technique may be particularly advantageous to “amplify” the fluorogenicity of the target (sTCR-γδ), thus allowing smaller amounts of target to be used and/or detected. Suitable fluorescent labels include, but are not limited to, fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosin, coumarin, methyl-coumarins, pyrene, Malacite green, stilbene, Lucifer Yellow, Cascade Blue™, and Texas Red. In addition, suitable optical dyes are described in the 1996 Molecular Probes Handbook by Richard P. Haugland.
In one embodiment, the fluorescent label is functionalized to facilitate covalent attachment of the label to the sTCR-γδ. A wide variety of fluorescent labels are commercially available which contain functional groups, including, but not limited to, isothiocyanate groups, amino groups, haloacetyl groups, maleimides, succinimidyl esters, and sulfonyl halides, all of which may be used to covalently attach the fluorescent label to the sTCR-γδ. The choice of the functional group of the fluorescent label will depend on the site of attachment to either a linker, as described below, or directly to the sTCR-γδ.
The covalent attachment of the fluorescent label may be either direct or via a linker. In one embodiment, the linker is a relatively short coupling moiety. A coupling moiety may be synthesized directly onto a sTCR-γδ molecule, for example, and may contain at least one functional group to facilitate attachment of the fluorescent label. Alternatively, the coupling moiety may have at least two functional groups, which are used to attach a functionalized candidate agent to a functionalized fluorescent label, for example. In an additional embodiment, the linker is a polymer. In this embodiment, covalent attachment is accomplished either directly, or through the use of coupling moieties from the agent or label to the polymer. In some embodiments, the covalent attachment is direct, that is, no linker is used. In this embodiment, the candidate agent preferably contains a functional group, such as a carboxylic acid, which is used for direct attachment to the functionalized fluorescent label. Thus, for example, for direct linkage to a carboxylic acid group of a candidate agent, amino modified or hydrazine modified fluorescent labels will be used for coupling via carbodiimide chemistry, for example using 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) as is known in the art (see Set 9 and Set 11 of the Molecular Probes Catalog, supra; see also the Pierce 1994 Catalog and Handbook, pages T-155 to T-200). In one embodiment, the carbodiimide is first attached to the fluorescent label, such as is commercially available.
In other embodiments, the labeling may be accomplished through the use of a binding pair, that is, a first binding moiety directly attached to the sTCR-γδ, and a second binding moiety comprising a detectable signal (e.g., a fluorescent molecule) and is capable of binding to the binding pair agent attached to the sTCR-γδ.
Suitable binding pairs include, but are not limited to, antigens/antibodies (e.g., anti-γδ TCR antibodies), including digoxigenin/antibody, dinitrophenyl (DNP)/anti-DNP, dansyl-X/anti-dansyl, fluorescein/anti-fluorescein, lucifer yellow/anti-lucifer yellow, rhodamine/anti-rhodamine; and biotin/avidin (or biotin/strepavidin). Preferred binding pairs (i.e., first and second labeling moieties) generally have high affinities for each other, and in some embodiments, are able to withstand the shear forces during FACS sorting.
The measurable/detectable signal may be identified using any method known in the art for the type of detectable signal used. In some embodiments, the analysis is carried out using flow cytometry (FACS). In other embodiments, signal-specific assays, such as ELISAs are used. In other embodiments, fluorescence imaging may be used.
The level of the detectable signal and/or the existence of a detectable signal may indicate the presence of one or more γδ T cell surface ligands. In further embodiments, the one or more γδ T cell surface ligands are identified using any method known in the art. For example, RNA sequencing (whole transcriptome shotgun sequencing, RNAseq), bioinformatics, and/or genetic screening (transfection-based genetic screens) may be used to identify the one or more γδ T cell surface ligands. A non-limiting exemplary list of γδ T cell surface ligands identified using any of the methods disclosed herein is provided in Table 1.
Samples may be screened for the presence of γδ T cell surface ligands. Such samples include, without limitation, plasma, serum, cell, or tissue samples. In some embodiments, a primary tissue sample is used (e.g., tissue from the gut mucosa (intestinal epithelium), synovium, skin, lungs, uterus, etc.). In one embodiment, the sample is from inflamed synovium. In another embodiment, a cell line, such as a tumor cell line is used. Tumor cell lines are known in the art and include, for example, CRF-CEM, HL-60(TB), K-562, MOLT-4, RPMI-8226, SR, A549/ATCC, EKVX, HOP-62, HOP-92, NCI-H226, NCI-H23, NCI-H322M, NCI-H460, NCI-H522, COLO 205, HCC-2998, HCT116, HCT-15, HT-29, KM12, SW-620, SF-268, SF-295, SF-539, SNB-19, SNB-75, U251, LOX IMVI, MALME-3M, M14, MDA-MB-435, SK-MEL-2, SK-MEL-28, SK-MEL-5, UACC-257, UACC-62, IGR-OV1, OVCAR-3, OVCAR-4, OVCAR-5, OVCAR-8, NCI/ADR-RES (previously, MCF-7/ADR-RES), SK-OV-3, 786-O, A498, ACHN, CAKI-1, RXF 393, SN12C, TK-10, UO-31, PC-3, DU-145, MCF7, MDA-MB-231/ATCC, MDA-MB-468, HS 578T, MDA-N, BT-549, T-47D, LXFL 529, DMS 114, SHP-77, DLD-1, KM20L2, SNB-78, XF 498, RPMI-7951, M19-MEL, RXF-631, SN12K1, P388, and P388/ADR. Other tumor lines include 2fTGH, HEK 293 T, Hep3B, HT-29, IMR-90, and TE671. Samples may be obtained by any means known in the art, for example, through commercial sources or through biopsies or blood draws.
The sample, in some embodiments, comes from a subject. A subject shall mean a human or vertebrate animal including but not limited to a dog, cat, horse, cow, pig, sheep, goat, turkey, chicken, primate, e.g., monkey, and fish (aquaculture species), e.g. salmon. In one embodiment, the subject is a human.
Without wishing to be bound by theory, it is thought that the γδ T cell surface ligands identified, for example, using any of the methods disclosed herein, will be useful in a wide variety of applications, such as cancer immunotherapy (Pauza et al., Frontiers in Immunology, 2018, 9(1305):1-11). For example, administration of synthetic γδ T cell surface ligands may activate γδ T cells in vivo, leading to enhanced antitumor effects. Activated γδ T cells, as noted above, produce a variety of chemokines and cytokines, regulate other immune and non-immune cells, and present antigen (e.g., may induce primary CD4+ and CD8+ T cell responses to antigens). The γδ T cells are also able to aid B helper cells and therefore play a regulatory role in humoral immunity. They can also activate immature dendritic cells. Taken together, activating ligands may yield significant immunotherapy benefits.
Therefore, in one embodiment, the disclosure provides an anti-cancer therapeutic composition. The anti-cancer therapeutic composition may comprise a unique TCR-γδ ligand (e.g., a protein or a functional fragment thereof of Table 1) and a pharmaceutically acceptable carrier (excipient) for administration to a subject to stimulate a γδ T cell subpopulation.
“Acceptable” means that the carrier must be compatible with the active ingredient of the composition (and preferably, capable of stabilizing the active ingredient) and not deleterious to the subject to be treated. Pharmaceutically acceptable excipients (carriers) include buffers, which are well known in the art. See, e.g., Remington: The Science and Practice of Pharmacy 20th Ed. (2000) Lippincott Williams and Wilkins, Ed. K. E. Hoover.
The pharmaceutical compositions to be used in the present methods can comprise pharmaceutically acceptable carriers, excipients, or stabilizers in the form of lyophilized formulations or aqueous solutions. (Remington: The Science and Practice of Pharmacy 20th Ed. (2000) Lippincott Williams and Wilkins, Ed. K. E. Hoover). Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations used, and may comprise buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrans; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).
The pharmaceutical compositions to be used for in vivo administration must be sterile. This is readily accomplished by, for example, filtration through sterile filtration membranes. Therapeutic antibody compositions are generally placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.
The pharmaceutical compositions described herein can be in unit dosage forms such as tablets, pills, capsules, powders, granules, solutions or suspensions, or suppositories, for oral, parenteral or rectal administration, or administration by inhalation or insufflation.
To practice the method disclosed herein, an effective amount of the pharmaceutical composition described herein can be administered to a subject (e.g., a human) in need of the treatment via a suitable route, such as intravenous administration, e.g., as a bolus or by continuous infusion over a period of time, by intramuscular, intraperitoneal, intracerebrospinal, subcutaneous, intra-articular, intrasynovial, intrathecal, oral, inhalation or topical routes. Commercially available nebulizers for liquid formulations, including jet nebulizers and ultrasonic nebulizers are useful for administration. Liquid formulations can be directly nebulized and lyophilized powder can be nebulized after reconstitution. Alternatively, the TCR-γδ ligands as described herein can be aerosolized using a fluorocarbon formulation and a metered dose inhaler, or inhaled as a lyophilized and milled powder.
The subject to be treated by the methods described herein can be a mammal, more preferably a human. Mammals include, but are not limited to, farm animals, sport animals, pets, primates, horses, dogs, cats, mice and rats. A human subject who needs the treatment may be a human patient having, at risk for, or suspected of having a target disease/disorder, such as a cancer.
A subject suspected of having any of such target disease/disorder (e.g., cancer) might show one or more symptoms of the disease/disorder. A subject at risk for the disease/disorder can be a subject having one or more of the risk factors for that disease/disorder.
Without further elaboration, it is believed that one skilled in the art can, based on the above description, utilize the present invention to its fullest extent. The following specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. All publications cited herein are incorporated by reference for the purposes or subject matter referenced herein.
In order that the invention described herein may be more fully understood, the following examples are set forth. The examples described in this application are offered to illustrate the compounds, pharmaceutical compositions, and methods provided herein and are not to be construed in any way as limiting their scope.
Human synovial γδ T cell clones from a Lyme arthritis patient were produced as previously described (9, 31). One of these clones, Bb15, was chosen for production of the sTCR-γδ using modification of a previously reported procedure (32, 33). Both TCR chains were produced as a single transcript in a baculovirus vector. The pBACp10 pH vector used contains two back-to-back promoters, p10 and polyhedrin (
Two additional rounds of viral amplification—P2 and P3—were completed using mid-log phase Sf21 cells (˜1.6×106 cells/mL) allowed to adhere for 1 hour (h) before infecting at a MOI of 0.01 or 0.1 with P1 and P2 stock, respectively. After 72 h of infection, culture media was clarified by centrifugation (1000×g for 10 minutes) and filtration (VacuCap 90PF 0.8/0.2 μm Supor membrane filter units; PALL Corporation, Westborough, Mass.) before being stored in the dark at 4° C. until use. Protein production occurred in 12 L batches of the mid-log phase (˜1.6×106 cells/mL) Hi5 cells were grown in suspension (0.5 L of culture in 1 L spinner flasks) and infected with P3 stock at a 1:50 dilution. Following 72 h of infection, cells were removed by centrifugation and filtration as described above. The filtered supernatant (approximately 12 L) containing secreted sTCR-γδ was concentrated to approximately 100 mL. The supernatant was then dialyzed against 1 L of nickel column loading buffer (20 mM NaPhosphate buffer pH 7.4, 20 mM imidazole, 0.5 M NaCl) using a Pellicon diafiltration system with two 10K MWCO membranes (Millipore, Burlington, Mass.) back down to a volume of approximately 100 mL. After system flushing, the final sample volume was approximately 200 mL. It was then loaded onto loading-buffer-equilibrated His-Trap HP columns (GE Healthcare, Little Chalfont, UK) at 100 mL per 2×5 mL columns. Columns were washed with at least 10 column volumes of loading buffer until baseline absorption was achieved. Bound proteins were eluted using a gradient from 20 mM to 500 mM imidiazole over 20 column volumes. Elution was monitored by absorbance at 280 nM and 1 mL fractions were collected. Fractions containing the target protein were identified using SDS-PAGE gel analysis using Coomassie Blue. High purity (>95%) sTCR-γδ fractions were pooled, dialyzed against PBS pH 7.4, and frozen at −80° C. until used in future studies. Yields were typically approximately 1.0 to 2.5 mg/L of culture.
Purified sTCR-γδ was then biotinylated using a biotin-protein ligase system (Avidity, Inc.) and tetramerized with streptavidin-PE (BioLegend) for FACS staining. Verification of TCR-γδ protein was confirmed by SDS-PAGE gel analysis using Coomassie Blue as well as immunoblot using antibodies to Vδ1 or Cγ (Endogen).
Human monocytes were purified from human peripheral blood mononuclear cells (PBMC) using CD14 labeled magnetic beads, followed by column purification (Miltenyi) and then cultured in RPMI complete with 10% FCS in the absence or presence of either a Borrelia burgdorferi sonicate (10 μg/ml) or LPS (1 μg/ml; Sigma) for 18 h. To some cultures, TNFα (10 ng/ml) (Biolegend), anti-TNFα (10 μg/ml) (Biolegend), IL-1β (10 pg/ml) (Invitrogen), or anti-IL-1β (5 μg/ml) (R&D Systems) were added. Cells were then stained with the sTCR-γδ tetramer.
T cells from PBMC were used either fresh or were activated with anti-CD3/anti-CD28 (each 10 μg/ml; BioLegend)+IL-2 (50 U/ml; Cetus) and propagated for three days. Cells were then stained with the sTCR-γδ-tetramer. Tumor cell lines were obtained from ATCC. CHO cells deficient for glycosaminoglycans were derived as previously described (34).
Inhibition of glycolysis was performed using the 2-deoxyglucose (2-DG, 5 mM; Sigma) for 48 h. Transcription and translation were inhibited using, respectively, Actinomycin D (5 ug/ml; ICN) or Cycloheximide (1 ug/ml; Millipore) for 18 h. ER-Golgi transport was blocked using Brefeldin A (1:1000) or Monensin (1:1400) (BD Bioscience) for 18 h. Cell surface protein digestion was performed using trypsin (Invitrogen) (1×; 5-10 minutes, 37° C.). Glycosaminoglycans were removed from cells by treatment with heparinases I-III (2 μU/ml) for 30 min in RPMI with no serum. The reaction was then stopped by the addition of PBS-BSA.
Cells were stained with either sTCR-γδ-PE (10 μg/ml) or negative controls that included Streptavidin-PE (10 ug/ml), IgG-PE (10 μg/ml) (BioLegend), or a sTCRαβ-PE (a kind gift of Dr. Mark Davis). Additional surface staining of T cells consisted of CD4, CD8, CD19, and CD25 (BioLegend). Live-Dead staining (BD Bioscience) was used to eliminate dead cells from analysis. Samples were run on an LSRII flow cytometer (Becton-Dickinson).
Expression profiling (35) based on Illumina RNA sequencing technology (36) was used to characterize the transcriptomes of 22 of the 24 tumor cell lines examined (excluding bronchoepithelial cell line and 2fTGH). Expression data for all known genes (37) was generated, and those genes whose representation in tetramer-positive cell lines was significantly higher than in negative cell lines were considered as candidate ligands.
Biotinylated sTCR-γδ was bound to avidin-magnetic beads and then incubated with cell lysates from monocytes activated with B. burgdorferi sonicate. Magnetic beads alone served as a negative control. After 4 h, beads were washed 5 times and bound proteins were then separated on polyacrylamide gels. Gel lanes for each sample type were cut into 12 identical regions and diced into 1 mm cubes. In-gel tryptic digestion was conducted on each region as previously described (38). Extracted peptides were subjected to liquid chromatography tandem mass spectrometry (38) except that the analysis was performed using a LTQ linear ion trap mass spectrometer (Thermo Fisher Scientific, Waltham, Mass.). Tandem mass spectra were searched against the forward and reverse concatenated human IPI database using SEQUEST, requiring fully tryptic peptides, allowing a mass tolerance of 2 Da and mass additions of 16 Da for the oxidation of methionine and 71 Da for the addition of acrylamide to cysteine. SEQUEST matches in the first position were then filtered by XCorr scores of 1.8, 2, and 2.7 for singly, doubly, and triply charged ions, respectively. Protein matches made with more than two unique peptides were further considered. This list had a peptide false discovery rate of less than 0.01%.
The following statistical tests were used: unpaired Student's t-test when comparing two conditions, and a one-way ANOVA with Sidak test for correction for multiple comparisons when comparing multiple variables across multiple conditions.
Production of a Human Synovial Soluble TCR-γδ(sTCR-γδ) A panel of synovial Vδ1 γδ T cells was previously produced from Lyme arthritis patients (9, 31). A representative clone, Bb15 (Vδ1Vγ9), was selected from which to clone its TCR-γδ. The pBACp10 pH vector has been used previously to produce murine sTCR-γδ tetramers (33). It contains two back-to-back promoters, p10 and polyhedrin, in which the p10 promoter is followed by multiple cloning sites for inserting the γ-chain, and the polyhedrin promoter is followed by multiple cloning sites for inserting the 6-chain (
Expression of sTCR-γδ Candidate Ligand(s) Varies Among Cell Lines
The sTCR-γδ tetramer was initially used to screen a panel of 24 cell lines from a variety of cell sources. None of the cell lines stained with the negative controls (IgG-PE, avidin-PE, or sTCR-αβ tetramer-PE), but the sTCR-γδ tetramer gave a spectrum of staining in which eight cell lines were strongly positive and the other cell lines manifested low to undetectable surface staining (
Candidate sTCR-γδ Ligands are Sensitive to Trypsin, and Reduced by Inhibition of Transcription, Translation, ER-Golgi Transport, or Removal of Glycosaminoglycans
The positively staining cell lines were treated with trypsin and a complete disappearance of surface staining was noted, as exemplified for bronchoepithelial cells in
It was further determined that surface TCR-γδ ligand expression was reduced by inhibition of protein translation or transcription with, respectively, cycloheximide or actinomycin D (
sTCR-γδ Ligands are Expressed by Activated Monocytes
In considering what primary cells might express ligand(s) for the sTCR-γδ, fresh monocytes were first examined, as it had previously been observed that following their activation with Borrelia burgdorferi or LPS, monocytes could activate the synovial γδ T cell clones (31). Consistent with these earlier findings, it was observed that the sTCR-γδ-tetramer did not stain freshly isolated human monocytes, but following 24 hours activation with a sonicate of B. burgdorferi or LPS there was a robust upregulation of sTCR-γδ tetramer staining (
Given the induction of sTCR-γδ ligand expression by activated monocytes, lysates from Borrelia-activated monocytes were prepared. The biotinylated sTCR-γδ complexed with avidin-magnetic beads was then used as a bait. Following incubation with the monocyte lysates, the sTCR-γδ was isolated by magnetic purification, washed five times, and bound proteins were separated on polyacrylamide gels. Gel slices were subjected to trypsin digestion and analyzed by mass spectrometry. Avidin-magnetic beads alone incubated with monocyte lysates served as a negative control. This analysis yielded 291 unique proteins (shown in Table 3). When compared to the list produced by the RNAseq bioinformatics approach of the tumor lines, 16 proteins were found in common. Fourteen unique protein TCR-γδ ligands identified as described herein are listed in Table 1.
sTCR-γδ Ligands are Expressed by Activated T Cells
Freshly isolated PBL from three individuals of various ages were further analyzed (28-66). This consistently revealed that fresh CD8+ T cells exhibited negligible sTCR-γδ staining, whereas a subset of fresh CD4+ T cells manifested modest levels of sTCR-γδ staining (
The finding that fresh monocytes and T lymphocytes expressed low to negligible levels of sTCR-γδ ligand(s), but upregulated expression following activation, raised the possibility that this might reflect the known induction of glycolysis following activation of T cells, monocytes, or dendritic cells (36, 37), and the resultant synthetic capacity promoted by glycolysis (38). This notion is supported by the fact that ligand-expressing Treg are also highly glycolytic (35). This question was thus examined in two ways. First, activated T cells were exposed to 2-deoxyglucose (2-DG), an inhibitor of glycolysis. This reduced expression of both CD25 and sTCR-γδ ligand (
The current findings provide the first unbiased characterization of the spectrum of ligand expression for human synovial Vδ1 γδ T cells. The range of ligand expression may reflect the various locations and seemingly diverse functions attributed to γδ T cells. For example, ligand induction by B. burgdorferi- or LPS-activated monocytes parallels their known ability to activate synovial γδ T cell clones (9, 31). In addition, ligand expression by fresh CD4+ but not CD8+ T cells also correlates with previous observations that Lyme arthritis synovial γδ T cells suppress by cytolysis the expansion of synovial CD4+ but not CD8+ T cells in response to B. burgdorferi (9). Finally, defining the spectrum of tumor cell types that express TCR-Vδ1 ligands may help explain which tumors contain Vδ1 γδ T cells, and impact their effectiveness as immunotherapy. The collective findings are also most consistent with the view that γδ T cells respond to self-proteins as much or possibly more than foreign proteins. Although these results were obtained using a sTCR-γδ tetramer from a single synovial γδ T cell clone, the fact that it shares a common Vδ1 chain found on most synovial γδ T cells (9), as well as γδ T cells found in intestinal epithelium (1, 10, 21), several tumors (18), and cells expanded in PBL following certain infections such as HIV (39, 40) and CMV (29), suggests the possibility that Vδ1 γδ T cells from these other sources may share a common physiology of ligand expression.
Previous studies of ligands for murine and human γδ T cells have come largely from the identification of individual molecules that activate a specific γδ T cell clone (27-30). Whereas this has been successful in some instances, the current study applied a broader approach of using a soluble TCR-γδ tetramer in an unbiased fashion to identify the spectrum of ligand expression and how they are regulated. This approach also provided two independent methods by which to identify candidate ligands. One method used RNAseq transcriptome analysis from 22 tumor cell lines to match genes increased in positively staining tumors and decreased in negatively staining tumors. The second approach used the sTCR-γδ tetramer as a bait to bind ligands from lysates of activated monocytes, and then identified the bound proteins by mass spectrometry. It is of considerable interest that among these two sets of candidate ligands were 16 in common, two of which, Annexin A2 and heat shock protein 70, have been previously proposed as ligands for γδ T cells (39-41). On the other hand, surface sTCR-γδ tetramer binding was eliminated by treatment with trypsin or removal of GAGs, and also suppressed by inhibition of ER-Golgi transport, suggesting the involvement of a combination of protein and GAGs in tetramer binding.
Although the findings thus far have not determined whether this represents one or several TCR-γδ ligands, they do provide a framework for understanding the distribution and regulation of ligand expression, which is critical for better understanding of γδ T cell biology. For example, γδ T cells have been implicated in the defense against a variety of infections (2-7), which is consistent with the finding that various TLR agonists induce TCR-γδ ligand expression on monocytes. Similar studies using a murine soluble TCR-76 also found ligands induced with bacterial infection (21). In addition, γδ T cells have been found to generally ameliorate various autoimmune models (12-15), which may be consistent with the expression of ligand by a subset of activated CD4+ T cells.
The induction of TCR-γδ ligand expression by activation of primary monocytes or T cells, as well as ligand expression by a variety of highly proliferative tumor cell lines, suggested that the metabolic state of cells may influence their ability to express TCR-γδ ligands. Activation of monocytes and T cells is known to induce a metabolic switch to glycolysis to provide the synthetic capacity for proliferation (36, 37). In addition, Treg, which are known to be glycolytic in vivo (35), spontaneously expressed ligand. Moreover, most tumors are highly glycolytic, and the inhibition of glycolysis in these cells also reduced ligand expression. Collectively, these findings suggest that some γδ T cells may function to survey and regulate highly proliferative cells.
It is of some interest that the cell lines bearing high levels of TCR-γδ ligand expression were enriched for those of epithelial and fibroblast origin, since Vδ1 γδ T cells are typically found at epithelial barriers, such as skin, intestinal epithelium, and in inflamed synovium, which is rich in fibroblasts (41). By contrast, sTCR-γδ ligand expression was noticeably absent from most tumor lines of hematopoietic origin. The spectrum of tumor staining with the human synovial sTCR-γδ also bears considerable similarity to results using a murine sTCR-γδ, which strongly stained epithelial and fibroblast tumors, and less well tumors of hematopoietic origin (33). These same murine sTCR-γδ also stained macrophages activated by TLR2 or TLR4 stimuli, similar to the findings with monocytes activated by Borrelia or LPS (42). Furthermore, staining of macrophages by the murine sTCR-γδ was also not affected by the absence of P2-microgloublin, suggesting little or no contribution of ligand by classical or non-classical MHC class I molecules. This agrees with the findings that the human synovial sTCR-γδ tetramer staining was not affected by the presence or absence of CD1 or MICA/B molecules.
The expression of ligand(s) by transformed cell lines suggests routes to identification of the TCR-γδ ligand for synovial Vδ1 T cells. The variation in ligand expression by the various tumor cell lines from negligible to high lends itself to an RNA-seq and bioinformatics approach to match expression levels of genes with the ligand expression as detected by the sTCR-γδ tetramer. This may provide a powerful tool by which to identify candidate TCR-γδ ligands in an unbiased fashion. This could be followed by CRISPR/Cas9 deletion of candidates to identify the ligand(s) as well as their regulatory pathways of synthesis and transport (43).
The findings in this study were made using primary cells or tumor cell lines. Future studies will attempt to extend these results to analyses of sTCR-γδ tetramer histologic staining of primary tissues as well as tumors and inflamed synovium to determine the spectrum of TCR-γδ ligand expression at these sites. Screening primary tumors for binding of TCR-γδ tetramer may also help identify tumors that may benefit from immunotherapy with Vδ1 γδ T cells. In addition, identifying the ligands in inflamed synovium or intestinal epithelium will provide therapeutic strategies for manipulating the function of infiltrating γδ T cells.
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In the claims articles such as “a,” “an,” and “the” may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include “or” between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The invention includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The invention includes embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process.
Furthermore, the invention encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, and descriptive terms from one or more of the listed claims is introduced into another claim. For example, any claim that is dependent on another claim can be modified to include one or more limitations found in any other claim that is dependent on the same base claim. Where elements are presented as lists, e.g., in Markush group format, each subgroup of the elements is also disclosed, and any element(s) can be removed from the group. It should it be understood that, in general, where the invention, or aspects of the invention, is/are referred to as comprising particular elements and/or features, certain embodiments of the invention or aspects of the invention consist, or consist essentially of, such elements and/or features. For purposes of simplicity, those embodiments have not been specifically set forth in haec verba herein. It is also noted that the terms “comprising” and “containing” are intended to be open and permits the inclusion of additional elements or steps. Where ranges are given, endpoints are included. Furthermore, unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or sub-range within the stated ranges in different embodiments of the invention, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.
This application refers to various issued patents, published patent applications, journal articles, and other publications, all of which are incorporated herein by reference. If there is a conflict between any of the incorporated references and the instant specification, the specification shall control. In addition, any particular embodiment of the present invention that falls within the prior art may be explicitly excluded from any one or more of the claims. Because such embodiments are deemed to be known to one of ordinary skill in the art, they may be excluded even if the exclusion is not set forth explicitly herein. Any particular embodiment of the invention can be excluded from any claim, for any reason, whether or not related to the existence of prior art.
Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation many equivalents to the specific embodiments described herein. The scope of the present embodiments described herein is not intended to be limited to the above Description, but rather is as set forth in the appended claims. Those of ordinary skill in the art will appreciate that various changes and modifications to this description may be made without departing from the spirit or scope of the present invention, as defined in the following claims.
This application claims the benefit under 35 U.S.C. § 119(e) of U.S. provisional application No. 62/853,314, filed May 28, 2019, and U.S. provisional application No. 62/879,999, filed Jul. 29, 2019, the entire disclosure of each of which is incorporated herein by reference in its entirety.
This invention was made with government support under R01 Grant AR43520, R21, Grant AI 107298, and P30 Grant GM118228, awarded by the National Institutes of Health. Accordingly, the government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US20/34678 | 5/27/2020 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 62853314 | May 2019 | US | |
| 62879999 | Jul 2019 | US |
