Tissue rejection

Abstract

This document relates to methods and materials involved in detecting tissue rejection (e.g., organ rejection). For example, this document relates to methods and materials involved in the early detection of kidney tissue rejection.

Description

BACKGROUND

1. Technical Field

This document relates to methods and materials involved in tissue rejection (e.g., organ rejection) and detecting tissue rejection.

2. Background Information

The transplantation of tissue from one mammal to another has been used for years to save lives and to improve the quality of lives. For example, the first successful kidney transplant was performed in the mid-1950s between identical twin brothers. Since then, donors have grown to include not only close relatives but also distant relatives, friends, and total strangers. In some cases, the recipient may reject the transplanted tissue. Thus, tissue rejection is a concern for any recipient of transplanted tissue. If a doctor is able to recognize early signs of tissue rejection, anti-rejection medication often can be used to reverse tissue rejection.

SUMMARY

This document relates to methods and materials involved in detecting tissue rejection (e.g., organ rejection). More particularly, this document relates to methods and materials involved in the early detection of tissue rejection (e.g., kidney rejection) and the assessment of a mammal's probability of rejecting tissue such as a transplanted organ. For example, this document provides nucleic acid arrays that can be used to diagnose tissue rejection in a mammal. Such arrays can allow clinicians to diagnose tissue rejection early based on a determination of the expression levels of nucleic acids that are differentially expressed in tissue being rejected as compared to control tissue not being rejected. The differential expression of such nucleic acids can be detected in tissue being rejected prior to the emergence of visually-observable, histological signs of tissue rejection. Early diagnosis of patients rejecting transplanted tissue (e.g., a kidney) can help clinicians determine appropriate treatments for those patients. For example, a clinician who diagnoses a patient as rejecting transplanted tissue can treat that patient with medication that suppresses tissue rejection (e.g., immunosuppressants).

The description provided herein is based, in part, on the discovery of nucleic acids that are differentially expressed in tissue being rejected as compared to control tissue that is not being rejected. Such nucleic acids can be nucleic acids expressed by, for example, cytotoxic T lymphocytes (CTL). The term “CTL associated transcripts” or “CATs” as used herein refers to transcripts that are expressed by activated CTL in culture at a level greater than the level of expression in normal kidney tissue. The description provided herein also is based, in part, on the discovery that the expression levels of CATs can be used to distinguish transplanted tissue that is being rejected from transplanted tissue that is not being rejected. For example, the expression levels of the nucleic acids listed in Table 4 or Table 5 can be assessed in transplanted tissue to determine whether or not that transplanted tissue is being rejected. In addition, the description provided herein is based, in part, on the discovery that the expression levels of CATs can be used to distinguish transplanted tissue that is being rejected from transplanted tissue that is not being rejected at a time point prior to the emergence of any visually-observable, histological sign of tissue rejection (e.g., tubulitis for kidney rejection).

In general, this description features a method for detecting tissue rejection. The method includes determining whether or not tissue transplanted into a mammal contains cells that express at least two of the nucleic acids listed in Table 4 or Table 5, wherein the presence of the cells indicates that the tissue is being rejected. The mammal can be a human. The tissue can be kidney tissue. The tissue can be a kidney. The method can include determining whether or not the tissue contains cells that express at least five of the nucleic acids. The method can include determining whether or not the tissue contains cells that express at least ten of the nucleic acids. The method can include determining whether or not the tissue contains cells that express at least twenty of the nucleic acids. The determining step can include measuring the level of mRNA expressed from the at least two nucleic acids. The determining step can include measuring the level of polypeptide expressed from the at least two nucleic acids. The method can include determining whether or not the tissue contains cells that express at least two of the nucleic acids at a level greater than the average level of expression exhibited in cells from control tissue that has not been transplanted.

In another embodiment, the description features a method for detecting tissue rejection. The method includes determining whether or not a sample contains cells that express at least two of the nucleic acids listed in Table 4 or Table 5, wherein the sample contains cells, was obtained from tissue that was transplanted into a mammal, and was obtained from the tissue within fifteen days of the tissue being transplanted into the mammal, and wherein the presence of the cells indicates that the tissue is being rejected. The mammal can be a human. The tissue can be kidney tissue. The tissue can be a kidney. The method can include determining whether or not the sample contains cells that express at least five of the nucleic acids. The method can include determining whether or not the sample contains cells that express at least ten of the nucleic acids. The method can include determining whether or not the sample contains cells that express at least twenty of the nucleic acids. The determining step can include measuring the level of mRNA expressed from the at least two nucleic acids. The determining step can include measuring the level of polypeptide expressed from the at least two nucleic acids. The sample can be a sample obtained from the tissue within ten days of the tissue being transplanted into the mammal. The sample can be a sample obtained from the tissue within five days of the tissue being transplanted into the mammal. The method can include determining whether or not the sample contains cells that express at least two of the nucleic acids at a level greater than the average level of expression exhibited in cells from control tissue that has not been transplanted.

In another embodiment, this description features a nucleic acid array containing at least 20 nucleic acid molecules, wherein each of the at least 20 nucleic acid molecules has a different nucleic acid sequence, and wherein at least 50 percent of the nucleic acid molecules of the array comprise a sequence from nucleic acid selected from the group consisting of the nucleic acids listed in Table 4 and Table 5. The array can contain at least 50 nucleic acid molecules, wherein each of the at least 50 nucleic acid molecules has a different nucleic acid sequence. The array can contain at least 100 nucleic acid molecules, wherein each of the at least 100 nucleic acid molecules has a different nucleic acid sequence. Each of the nucleic acid molecules that comprise a sequence from nucleic acid selected from the group can contain no more than three mismatches. At least 75 percent of the nucleic acid molecules of the array can contain a sequence from nucleic acid selected from the group. At least 95 percent of the nucleic acid molecules of the array can contain a sequence from nucleic acid selected from the group. The array can contain glass. The at least 20 nucleic acid molecules can contain a sequence present in a human.

In another embodiment, this description features a computer-readable storage medium having instructions stored thereon for causing a programmable processor to determine whether one or more nucleic acids listed in Table 4 or Table 5 are detected in a sample, wherein the sample is from a transplanted tissue. The computer-readable storage medium can further comprise instructions stored thereon for causing a programmable processor to determine whether one or more of the nucleic acids listed in Table 4 or Table 5 is expressed at a greater level in the sample than in a control sample of non-transplanted tissue.

This description also features an apparatus for determining whether a transplanted tissue is being rejected. The apparatus can include one or more collectors for obtaining signals representative of the presence of one or more nucleic acids listed in Table 4 or Table 5 in a sample from the transplanted tissue and a processor for analyzing the signals and determining whether the tissue is being rejected. The one or more collectors can be adapted to obtain further signals representative of the presence of the one or more nucleic acids in a control sample from non-transplanted tissue.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1 is a diagram of a process for determining whether a transcript is classified as a CAT.

FIG. 2 contains photographs of the histopathology of rejecting mouse allografts using PAS staining (magnification 40×). Panel A: isograft (CBA into CBA) at day 5 with normal histology. Panel B: rejecting kidney allograft (CBA into B6) at day 5 with periarterial mononuclear interstitial infiltration. Panel C: rejecting kidney allograft at D7 (CBA into B6) with mononuclear interstitial infiltration and mild tubulitis. Panel D: kidney transplant (CBA into B6) at day 21 with heavy tubulitis.

FIG. 3 is a graph plotting the reproducibility of gene expression analysis. Gene expression values (n=22,690) from two biological replicates of pools of three kidneys rejecting in wild-type hosts at D5 (WTD5) demonstrate good reproducibility of microarray data (r=0.92).

FIG. 4 contains graphs plotting the correlation of gene expression analysis for 12 selected genes using microarrays versus real-time RT-PCR. The time course of gene expression in kidneys rejecting at day 5, 7, and 21 post transplant in selected genes (fold change versus normal kidney (NCBA)) for RT-PCR data (left) and microarrays (right).

FIG. 5 is a diagram of unsupervised hierarchical clustering of experimental groups. Unsupervised clustering of all genes, based on distance, demonstrates three main groups with a good separation between (1) isografts (ISO), (2) allografts rejecting in wild-type hosts (WT) or B cell deficient hosts (IghKO), and (3) lymphocyte cultures (MLR=mixed lymphocyte culture; CTL=CTL clone).

FIG. 6 is a graph plotting the expression level of CATs in isografts and WT allografts. CATs were absent in normal kidney, low in isografts, but highly expressed in rejecting kidneys at day 5. The expression of this set of CATs persisted throughout the rejection process.

FIG. 7 is a bar graph plotting the expression of CATs for K-means clusters in d4MLR and WT allografts. Based on their expression in a CTL clone, CATs (n=287) cluster in 5 groups. Expression in MLR and WT allografts in clusters 1-5 is shown as the percent of expression in the CTL clone. The boxplots represent the median and quartiles of expression of CATs for each time point. The CATs of cluster 1 (n=140) had low expression in MLR, but stable expression in all allografts. The CATs of cluster 2 (n=23) were more highly expressed in MLR than CTL and exhibited relatively strongly increased expression in day 5 rejecting kidneys, further increasing expression at D14. The CATs of cluster 3 (n=74) had relatively high expression in MLR versus CTL but lower expression in rejecting kidney, fluctuating somewhat among the different times while increasing between D5 and D7. The CATs of cluster 4 (n=46) had less expression in MLR than CTL, increased expression between D5 and D14, and decreased expression thereafter. The CATs of cluster 5 (n=4) were as highly expressed in rejecting grafts as in the CTL clone and MLR.

FIG. 8 is a bar graph plotting the expression of CATs for K-means clusters in kidneys rejecting in wild-type hosts and B cell deficient hosts at D7 and D21. Cluster analysis of CATs was based on expression in WT allografts (FIG. 7). Expression for each cluster is shown for WT and IghKO D7 and D21 as the percent of expression in the CTL clone. The boxplots represent the median and quartiles of expression of CATs for each time point. Expression of CATs was slightly higher in IghKO compared to WT at D7 but exhibited some attenuation in IghKO compared to their wild-type counterparts at D21.

DETAILED DESCRIPTION

This description provides methods and materials involved in detecting tissue rejection (e.g., organ rejection). For example, this description provides methods and materials that can be used to diagnose a mammal (e.g., a human) as having transplanted tissue that is being rejected. A mammal can be diagnosed as having transplanted tissue that is being rejected if it is determined that the tissue contains cells that express one or more CATs or that express one or more of the nucleic acids listed in Table 4 or Table 5.

The methods and materials provided herein can be used to detect tissue rejection in any mammal such as a human, monkey, horse, dog, cat, cow, pig, mouse, or rat. In addition, the methods and materials provided herein can be used to detect rejection of any type of transplanted tissue including, without limitation, kidney, heart, liver, pancreas, and lung tissue. For example, the methods and materials provided herein can be used to determine whether or not a human who received a kidney transplant is rejecting that transplanted kidney.

Any type of sample containing cells can be used to determine whether or not transplanted tissue contains cells that express one or more CATs or that express one or more of the nucleic acids listed in Table 4 or Table 5. For example, biopsy (e.g., punch biopsy, aspiration biopsy, excision biopsy, needle biopsy, or shave biopsy), tissue section, lymph fluid, blood, and synovial fluid samples can be used. In some embodiments, a tissue biopsy sample can be obtained directly from the transplanted tissue. In some embodiments, a lymph fluid sample can be obtained from one or more lymph vessels that drain from the transplanted tissue. A sample can contain any type of cell including, without limitation, cytotoxic T lymphocytes, CD4⁺ T cells, B cells, peripheral blood mononuclear cells, macrophages, kidney cells, lymph node cells, or endothelial cells.

As explained herein, a CAT refers to a transcript that is expressed by activated CTL in culture at a level greater than the level of expression in normal kidney tissue. Examples of CATs include, without limitation, the nucleic acids listed in Table 4 and/or Table 5. Additional examples of CATs can be identified using the procedures described herein. For example, the procedures described in Example 1 and Example 3 can be used to identify CATs other than those listed in Tables 4 and 5.

Any suitable process can be used to determine whether a particular transcript is classified as a CAT. In some embodiments, for example, a process can include determining whether a transcript is expressed in CTL and/or MLR at a level that is at least three (e.g., at least four, at least five, at least six, or at least seven) times higher than the level at which the transcript is expressed in normal kidney cells. FIG. 1 is a diagram of another embodiment of a process for determining whether a particular transcript is classified as a CAT. With reference to FIG. 1, process 100 can include step 102 for determining whether the transcript has a signal less than 50 in normal kidney (e.g., in kidney tissue from mouse strains such as CBA, B6, and Balbc), step 104 for determining whether expression of the transcript is at least five times higher in CTL as compared to expression in normal kidney, determining whether expression is at least five times higher in CD8 cells as compared to expression in normal kidney, and determining whether expression is at least five times higher in MLR and is significantly higher (p (fdr)<0.01, where “fdr” is the false discovery rate) as compared to expression in normal kidney, and step 106 for determining whether the transcript is expressed at a level that is at least two times increased in wild type allografts (CBA into B6) at day 5 and is significant (p (fdr)<0.01) as compared to expression in normal kidney. If the answer to each of these steps is “yes,” then the transcript can be classified as a CAT. If the answer to any of the steps is “no,” then the transcript is classified as not a CAT. The steps depicted in FIG. 1 can be carried out in any suitable order. Further, the steps depicted in FIG. 1 can be further divided into separate steps (e.g., step 104 can be separated into four steps, for determining (a) whether expression of the transcript is at least five times higher in CTL as compared to normal kidney, (b) whether expression is at least five times higher in CD8 cells as compared to normal kidney, (c) whether expression is at least five times higher in MLR as compared to normal kidney, and (d) whether expression in MLR is significantly higher (p (fdr)<0.01) than expression in normal kidney. Similarly, step 106 can be divided into two separate steps.

The expression of any number of CATs or nucleic acids listed in Table 4 or Table 5 can be evaluated to determine whether or not transplanted tissue is being or is likely to be rejected. For example, the expression of one or more than one (e.g., two, three, four, five, six, seven, eight, nine, ten, 15, 20, 25, 30, 40, 50, 75, 100, or more than 100) of the nucleic acids listed in Table 4 or Table 5 can be used. In some embodiments, determining that a nucleic acid listed in Table 4 or Table 5 is expressed in a sample at a detectable level can indicate that the transplanted tissue will be rejected. In some embodiments, transplanted tissue can be evaluated by determining whether or not the tissue contains cells that express a nucleic acid listed in Table 4 or Table 5 at a level that is greater than the average expression level observed in control cells obtained from tissue that has not been transplanted. Typically, a nucleic acid can be classified as being expressed at a level that is greater than the average level observed in control cells if the expression levels differ by at least 1-fold (e.g., 1.5-fold, 2-fold, 3-fold, or more than 3-fold). Control cells typically are the same type of cells as those being evaluated. In some cases, the control cells can be isolated from kidney tissue that has not been transplanted into a mammal. Any number of tissues can be used to obtain control cells. For example, control cells can be obtained from one or more tissue samples (e.g., at least 5, 6, 7, 8, 9, 10, or more tissue samples) obtained from one or more healthy mammals (e.g., at least 5, 6, 7, 8, 9, 10, or more healthy mammals).

Any suitable process can be used to determine whether a transplanted tissue is being or is likely to be rejected. In some embodiments, for example, a process can include determining whether a pre-determined number (e.g., one, two, three, four, five, six, seven, eight, nine, ten, 15, 20, 25, 30, 40, 50, 75, 100, or more than 100) of the nucleic acids listed in Table 4 or Table 5 is expressed in a sample (e.g., a sample of transplanted tissue) at a detectable level. If the number of nucleic acids that are expressed in the sample is equal to or exceeds the pre-determined number, the transplanted tissue can be predicted to be rejected. If the number of nucleic acids that are expressed in the sample is less than the pre-determined number, the transplanted tissue can be predicted to not be rejected. The steps of this process (e.g., the detection, or non-detection, of each of the nucleic acids listed in Table 4 or Table 5) can be carried out in any suitable order. In some embodiments, a process can include determining whether a predetermined number of the nucleic acids listed in Table 4 or Table 5 is expressed in a sample at a level that is greater than the average level observed in control cells (e.g., cells obtained from tissue that has not been transplanted. If the number of nucleic acids having increased levels of expression in the sample is equal to or exceeds the pre-determined number, the transplanted tissue can be predicted to be rejected. If the number of nucleic acids having increased expression levels in the sample is less than the pre-determined number, the transplanted tissue can be predicted to not be rejected. Again, the steps of this process can be carried out in any suitable order.

Any suitable method can be used to determine whether or not a particular nucleic acid is expressed at a detectable level or at a level that is greater than the average level of expression observed in control cells. For example, expression of a particular nucleic acid can be measured by assessing mRNA expression. mRNA expression can be evaluated using, for example, northern blotting, slot blotting, quantitative reverse transcriptase polymerase chain reaction (RT-PCR), real-time RT-PCR, or chip hybridization techniques. Methods for chip hybridization assays include, without limitation, those described herein. Such methods can be used to determine simultaneously the relative expression levels of multiple mRNAs. Alternatively, expression of a particular nucleic acid can be measured by assessing polypeptide levels. For example, polypeptide levels can be measured using any method such as immuno-based assays (e.g., ELISA), western blotting, or silver staining.

The methods and materials provided herein can be used at any time following a tissue transplantation to determine whether or not the transplanted tissue is being or is likely to be rejected. For example, a sample obtained from transplanted tissue at any time following the tissue transplantation can be assessed for the presence of cells expressing a nucleic acid listed in Table 4. In some cases, a sample can be obtained from transplanted tissue 1, 2, 3, 4, 5, 6, 7, 8, or more hours after the transplanted tissue was transplanted. In some cases, a sample can be obtained from transplanted tissue one or more days (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, or more days) after the transplanted tissue was transplanted. Typically, a sample can be obtained from transplanted tissue 2 to 7 days (e.g., 5 to 7 days) after transplantation and assessed for the presence of cells expressing one or more CATs or expressing one or more nucleic acids listed in Table 4.

This description also provides nucleic acid arrays. The arrays provided herein can be two-dimensional arrays, and can contain at least 10 different nucleic acid molecules (e.g., at least 20, at least 30, at least 50, at least 100, or at least 200 different nucleic acid molecules). Each nucleic acid molecule can have any length. For example, each nucleic acid molecule can be between 10 and 250 nucleotides (e.g., between 12 and 200, 14 and 175, 15 and 150, 16 and 125, 18 and 100, 20 and 75, or 25 and 50 nucleotides) in length. In addition, each nucleic acid molecule can have any sequence. For example, the nucleic acid molecules of the arrays provided herein can contain sequences that are present within the nucleic acids listed in Table 4. For the purpose of this document, a sequence is considered present within a nucleic acid listed in Table 4 when the sequence is present within either the coding or non-coding strand. For example, both sense and anti-sense oligonucleotides designed to human CD2 nucleic acid are considered present within CD2 nucleic acid.

Typically, at least 25% (e.g., at least 30%, at least 40%, at least 50%, at least 60%, at least 75%, at least 80%, at least 90%, at least 95%, or 100%) of the nucleic acid molecules of an array provided herein contain a sequence that is (1) at least 10 nucleotides (e.g., at least 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, or more nucleotides) in length and (2) at least about 95 percent (e.g., at least about 96, 97, 98, 99, or 100) percent identical, over that length, to a sequence present within a nucleic acid listed in Table 4.

For example, an array can contain 100 nucleic acid molecules located in known positions, where each of the 100 nucleic acid molecules is 100 nucleotides in length while containing a sequence that is (1) 30 nucleotides in length, and (2) 100 percent identical, over that 30 nucleotide length, to a sequence of one of the nucleic acids listed in Table 4.

A nucleic acid molecule of an array provided herein can contain a sequence present within a nucleic acid listed in Table 4, where that sequence contains one or more (e.g., one, two, three, four, or more) mismatches. Similarly, an array can contain 100 nucleic acid molecules located in known positions, where each of the 100 nucleic acid molecules is 100 nucleotides in length while containing a sequence that is (1) 30 nucleotides in length, and (2) 100 percent identical, over that 30 nucleotide length, to a sequence of one of the nucleic acids listed in Table 5. A nucleic acid molecule of an array provided herein can contain a sequence present within a nucleic acid listed in Table 5, where that sequence contains one or more (e.g., one, two, three, four, or more) mismatches.

The nucleic acid arrays provided herein can contain nucleic acid molecules attached to any suitable surface (e.g., plastic or glass). In addition, any method can be use to make a nucleic acid array. For example, spotting techniques and in situ synthesis techniques can be used to make nucleic acid arrays. Further, the methods disclosed in U.S. Pat. Nos. 5,744,305 and 5,143,854 can be used to make nucleic acid arrays.

Computer-Readable Medium and an Apparatus for Predicting Rejection

This disclosure further provides a computer-readable storage medium configured with instructions for causing a programmable processor to determine whether a transplanted tissue is being or is likely to be rejected. The determination of whether a transplanted tissue is being or will be rejected can be carried out as described herein; that is, by determining whether one or more of the nucleic acids listed in Table 4 or Table 5 is detected in a sample (e.g., a sample of the tissue), or is expressed at a level that is greater than the level of expression in a corresponding tissue that is not transplanted. The processor also can be designed to perform functions such as removing baseline noise from detection signals.

Instructions carried on a computer-readable storage medium (e.g., for detecting signals) can be implemented in a high level procedural or object oriented programming language to communicate with a computer system. Alternatively, such instructions can be implemented in assembly or machine language. The language further can be compiled or interpreted language.

The nucleic acid detection signals can be obtained using an apparatus (e.g., a chip reader) and a determination of tissue rejection can be generated using a separate processor (e.g., a computer). Alternatively, a single apparatus having a programmable processor can both obtain the detection signals and process the signals to generate a determination of whether rejection is occurring or is likely to occur. In addition, the processing step can be performed simultaneously with the step of collecting the detection signals (e.g., “real-time”).

Also provided herein, therefore, is an apparatus for determining whether a transplanted tissue is being or is likely to be rejected. An apparatus for determining whether tissue rejection will occur can include one or more collectors for obtaining signals from a sample (e.g., a sample of nucleic acids hybridized to nucleic acid probes on a substrate such as a chip) and a processor for analyzing the signals and determining whether rejection will occur. By way of example, the collectors can include collection optics for collecting signals (e.g., fluorescence) emitted from the surface of the substrate, separation optics for separating the signal from background focusing the signal, and a recorder responsive to the signal, for recording the amount of signal. The collector can obtain signals representative of the presence of one or more nucleic acids listed in Table 4 or Table 5 (e.g., in samples from transplanted and/or non-transplanted tissue). The apparatus further can generate a visual or graphical display of the signals, such as a digitized representation. The apparatus further can include a display. In some embodiments, the apparatus can be portable.

The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.

EXAMPLES

Example 1

Early Diagnosis of Organ Rejection

Kidney rejection is mediated by infiltration of cytotoxic T lymphocytes (CTL) and diagnosed by histologic Banff lesions such as tubulitis. Using Affymetrix microarrays, the relationship between the evolution of pathologic lesions and the transcriptome in normal mouse kidneys, CBA isografts, CBA into C57B1/6 allografts at days 5 to 42, and kidneys rejecting in B cell deficient hosts was evaluated. Histology was dominated by early infiltrate of mononuclear cells and slower evolution of severe tubulitis. A set of CATs was identified as having high expression in a CTL clone and day 4 mixed lymphocyte culture, while being absent in normal kidney. This set of CATs was fully expressed in rejecting kidneys at day 5, representing about 14 to 20 percent of the transcriptome of rejecting kidney. The expression persisted through day 42. Lack of mature B cells had little effect on expression of the set of CATs. In addition, expression of the identified set of CATs was established before diagnostic Banff lesions were observed and remained consistent through day 42 despite massive alterations in the pathology. Thus, the expression of the identified set of CATs in rejecting organs indicates the state of effector T cell infiltration, and can establish the diagnosis of T cell mediated rejection earlier and more securely than pathologic criteria.

Materials and Methods

Mice

Male CBA/J (CBA), C57B1/6 (B6), B6.129P2-Igh-J^tmlCgn (Igh-j), and B6.129S2-Igh-6^tm1,Cgn (Igh-6) mice were obtained from Jackson Laboratory (Bar Harbor, Me.) and maintained in the Health Sciences Laboratory Animal Services at the University of Alberta. All maintenance and experiments conformed to approved animal care protocols. CBA (H-2K, I-A^k) into C57B1/6 (B6; H-2K^bD^b, I-A^b) mice strain combinations were studied across full MHC and non-MHC disparities. To ensure robust findings, two different types of IghKO mice, which were previously shown to have similar phenotypes as hosts for allografts (Jabs et al., Am. J. Transplant, 3(12):1501-1509 (2003)), were used.

Renal Transplantation

Donor mice of 9-11 weeks of age were anaesthetized, and the right kidney was removed through a midline abdominal incision and preserved in cold lactate Ringer's solution. Host mice were similarly anaesthetized, and the right native kidney excised. The donor kidney was anastomosed heterotopically to the aorta, inferior vena cava, and bladder on the right side, without removing the host's left kidney (non life-supporting kidney transplantation). Recovered mice were killed at day 5, 7, 14, 21, or 42 post-transplant, following anaesthesia and cervical dislocation. Kidneys were removed, snap frozen in liquid nitrogen, and stored at −70° C. No mice received immunosuppressive therapy. Kidneys with technical complications or infection at the time of harvesting were removed from the study.

Mixed Leukocyte Reaction (MLR)

CTL effectors were generated by co-culturing C57BL/61 responder splenocytes with mitomycin C-treated (5 μg/mL, Sigma Chemicals, St. Louis, Mo.) CBA splenocytes in complete RPMI 1640 medium (10% FCS, 1% antibiotic-antimycotic; Life Technologies, Grand Island, N.Y.), 1% nonessential amino acids, 1% sodium pyruvate (Flow Laboratories, McLean, Va.), and 50 μM β-ME at a concentration of 3×10⁶ cells/mL. Cultures were kept at 37° C., 5% CO₂ in 25 cm² cell culture flasks standing upright for 4 days. Cytolytic activity was confirmed by a ⁵¹Cr release assay.

CTL Culture

A CTL clone, C57/B6 anti C3H, was generated by co-culturing C57B1/6 splenocytes with irradiated (2500 rads) C3H splenocytes at a 1:1 ratio for 3 days in RPMI 1640 medium (same composition as for the 4-day MLR). CTLs were purified using Ficoll gradient and cultured for another 4 days. Re-stimulation was performed at a 1:14 ratio for 3 days. After purification, cells were used for RNA extraction. Cytolytic activity was confirmed by a ⁵¹Cr release assay.

RNA Preparation

Total RNA was extracted from individual kidneys by the guanidinium-caesium chloride method (transplants) or by Trizol extraction (4-day MLR and CTL cultures), and RNA yields were measured by UV absorbance. Quality was assessed by the absorbance ratio, by agarose gel electrophoresis, and, in select samples, by Affymetrix T3 Test arrays (Affymetrix, Santa Clara, Calif.). For microarray analysis, equal amounts of RNA from 3 mice (20-25 μg each) were pooled and purified using the RNeasy Mini Kit (Quiagen, Ont. Canada). dsDNA and cRNA synthesis, hybridization to MOE 430A oligonucleotide arrays (Affymetrix), washing, and staining were carried out according to the manufacturer's manual. See, e.g., Affymetrix Technical Manual, 2003 version downloaded from Affymetrix's website.

Real-Time RT-PCR

To confirm the microarray results, expression of selected genes was assessed by TaqMan real-time RT-PCR. Two micrograms of RNA were transcribed using M-MLV reverse transcriptase and random primers. All TaqMan probe/primer combinations were designed using Primer Express software version 1.5 or purchased as Assay on demand (PE Applied Biosystems). cDNA was amplified in a multiplex system using murine hypoxanthine phosphoribosyltransferase (HPRT) cDNA as the control. Quantification of gene expression was performed utilizing the ABI prism 7700 Sequence Detection System (PE Applied Biosystems) as described elsewhere (Heid et al., Genome Research, 6(10):986-994 (1996)). Fold change over control kidney was determined using the ΔCt or ΔΔCt methods as described by the manufacturer.

Sample Designation and Analysis

Normal control kidneys were from CBA mice (NCBA). Allografts rejecting in wild-type hosts (B6) at day 5, 7, 14, 21, and 42 post transplant were designated WT D5, WT D7, WT D14, WT D21, and WT D42, respectively. Corresponding isografts were designated Iso D5, Iso D7, and Iso D21. Allografts rejecting in mature B cell deficient B6 hosts studied at days 7 and 21 were designated IghKO D7 and IghKO D21. Mixed leukocyte reaction, day 4, was designated as d4MLR and CTL clone, day 4, was designated as CTL. Two biological replicates (each consisting of RNA pooled from 3 mice) were tested in the following groups: WT D7, WT D14, WT D21, WT D42, Iso D7, and IghKO D7. Biological triplicates were analyzed in NCBA, WT D5, IghKO D21 (2 arrays with RNA pooled from 3 Igh-6 hosts, and 1 array with RNA pooled from 3 Igh-j hosts), and a single analysis was done in Iso D5, Iso D21, d4MLR, and CTL. Before processing for mRNA studies, every kidney was examined histologically to exclude kidneys with infection or surgical complication (global early infarction).

Initial data analysis was performed using Microarray Suite Expression Analysis 5.0 software (Affymetrix). Software default conditions were used to flag transcripts as present, marginal, or absent and to calculate the absolute signal strength. Total fluorescence for each array was globally scaled to a target value of 500. GeneSpring™ software (Version 6.1, Silicon Genetics, Calif., USA) was used for further analyses. Following data importation, intensity values below 20 were adjusted to a value of 20, a per chip normalization was performed to the 50^th percentile, and a per gene normalization was performed using NCBA or CTL as control samples. Replicate samples were expressed as mean normalized value for further analysis. For unsupervised hierarchical cluster analysis, similarity measurements were based on distance and visualized by a tree diagram (Eisen et al., Proc. Natl. Acad. Sci., 95(25):14863-14868 (1998)). CATs were defined as CTL associated transcripts having a signal that was increased at least five-fold in CTL and MLR culture compared to the signal in normal kidney (significant by ANOVA; p<0.05), and that were “absent” (by Affymetrix GCOS software default conditions) in normal CBA kidney.

A second, more refined algorithm, used RMA (robust multichip analysis). In this process, CATs were identified based on (1) a signal less than 50 in normal kidneys in all three strains (CBA, B6, and Balb/c), (2) a signal at least 5 times higher in CTL, MLR, and CD8 as compared to normal kidneys, significantly higher (p(fdr)<0.01) in MLR vs. normal kidney, and at least 2 times higher in wild type allografts (CBA into B6) at day 5 and significant (p(fdr)<0.01) compared to normal kidney.

CATs were analyzed using a K-means cluster algorithm based on expression data normalized to the CTL clone.

Results

Pathological Lesions in Rejecting Kidneys

Histology of CBA kidney allografts in B6 hosts has been described elsewhere (Jabs et al., Am. J. Transplant., 3(12):1501-1509 (2003) and Halloran et al., Am. J. Transplant., 4(5):705-712 (2004)). Isografts at 5 (FIG. 2, panel A), 7, and 21 days post transplant appeared normal with no inflammation or acute tubular necrosis. Allografts exhibited an interstitial mononuclear infiltrate at day 5, which increased at day 7, and stabilized or regressed by day 21 (FIG. 2, panels B, C, and D, respectively). Tubulitis was absent at day 5, mild at day 7, and severe at days 14, 21, and 42. By immunostaining, the infiltrate in kidney allografts at days 5, 7, and 21 was comprised of 40-60 percent CD3⁺ T cells (mostly CD8⁺) and 35-50 percent CD68⁺ macrophages, with late appearance of about 5 percent CD 19⁺B cells at day 21. Hosts deficient in mature B cells (Igh6KO or IghJKO) exhibited similar infiltrate and tubulitis but less necrosis and hemorrhage at day 21 (Jabs et al., Am. J. Transplant., 3(12):1501-1509 (2003)), and 19 percent lower kidney weight (260±58 mg, n=8 versus 319±70 mg, n=6 in wild type hosts). Details of the histology of individual mice are found in Table 1 with the abbreviations being as follows: wt: weight; Tx: transplant; Nec: necrosis; PTC: peritubular capillary congestion; Glom: glomerulitis; Tub: tubulitis; Inf: interstitial infiltrate; Art: arteritis; AT: arterial thrombosis; Ven: venulitis; VT: venous thrombosis; NCBA: normal CBA kidney; iso: isograft; WT: wild-type allograft.

TABLE 1
Histology for individual mice.
				Mouse	Donor	Host	Tx
	Donor	Host	day	ID	wt	wt	wt	Nec	PTC	Glom	Tub	Inf	Art	AT	Ven	VT	Ed	Cast
NCBA	CBA	—	0	695		24	170	—	—	—	—	—	—	—	—	—	—	—
				627				—	—	—	—	—	—	—	—	—	—	—
				628				—	—	—	—	—	—	—	—	—	—	—
				696		25	165	—	—	—	—	—	—	—	—	—	—	—
				661				—	—	—	—	—	—	—	—	—	—	—
				662				—	—	—	—	—	—	—	—	—	—	—
				752		28	209	—	—	—	—	—	—	—	—	—	—	—
				755		20	133	—	—	—	—	—	—	—	—	—	—	—
				756		20	132	—	—	—	—	—	—	—	—	—	—	—
Iso	CBA	CBA	5	727	34	28	249	0	0	1	0	0	0	0	2	0	0	0
				728	27	26	226	0	0	0	0	0	0	0	1	0	0	0
				740	29	28	242	0	0	0	0	0	0	0	0	0	0	0
			7	520	25	23	207	0	0	1	0	5	0	0	0	0	0	0
				525	25	27	298	0	0	1	0	0	0	0	0	0	0	0
				528	24	25	229	0	10	1	0	0	1	0	1	0	0	0
				751	27	26	193	0	5	1	0	0	0	0	0	0	0	0
				744	28	24	231	0	0	1	0	0	0	0	2	0	0	0
				745	25	27	192	0	0	1	0	0	0	0	0	0	0	0
			21	513	27	27	193	0	0	1	0	0	0	0	1	0	0	0
				518	34	25	182	0	10	1	0	5	1	0	1	0	0	0
				531	26	27	204	0	0	1	0	0	0	0	0	0	0	0
WT	CBA	B6	5	495	30	26	253	0	0	1	10	30	0	0	0	0	0	0
				496	29	26	303	0	0	1	10	40	0	0	4	0	0	0
				499	33	23	279	0	0	0	10	20	0	0	4	0	0	0
				510	20	28	215	0	5	1	10	10	0	0	1	0	0	0
				511	23	26	309	0	0	1	20	50	3	0	1	0	0	0
				694	25	20	168	15	0	2	40	50	0	0	1	0	0	0
				831		24	299	0	0	1	10	20	0	0	4	0	0	0
				874	30	24	270	0	0	1	15	20	0	0	2	0	0	0
			7	447		31	423	0	0	2	20	40	0	0	5	0	0	0
				455		26	239	0	0	2	20	40	0	0	6	0	0	0
				471		26	338	0	0	2	20	60	0	0	8	0	0	0
				350		27	290	0	0	2	15	40	0	0	2	0	0	0
				351		28	267	0	0	2	10	30	0	0	3	0	0	0
				352		28	299	0	0	2	10	40	0	0	0	0	0	0
			14	404	24	27	349	0	10	3	50	60	2	0	3	0	10	5
				405	23	27	389	0	15	3	50	40	4	1	3	0	15	5
				406	24	28	228	0	15	3	60	50	3	2	2	0	10	5
				403	24	25	321	0	10	3	50	60	1	1	4	0	0	0
				787	26	27	347	0	0	1	30	50	0	0	2	0	0	0
				859	27	25	358	5	0	2	50	30	0	0	2	0	0	0
			21	470		26	371	5	0	3	50	60	1	0	2	0	0	0
				346		30	363	5	10	3	40	50	0	0	1	0	20	0
				456		32	398	10	0	3	60	70	1	0	3	0	20	10
				436		27	297	0	0	3	80	50	3	0	2	0	0	0
				438		28	264	5	0	3	70	50	4	0	3	0	10	10
				445		26	219	10	0	3	60	60	1	0	3	0	0	10
			42	287		25	285	0	0	1	40	40	0	0	2	1	0	0
				288		29	224	0	0	2	70	75	0	0	0	0	0	0
				566		25	348	30	0	2	80	60	2	1	0	0	80	0
				289		29	627	75	0	2	30	30	0	2	1	0	75	0
				291		27	499	20	40	2	80	70	1	1	2	0	40	0
				297		29	392	50	60	2	80	70	2	2	3	0	70	0
IghKO	CBA	Igh-6	7	116		25	294	0	0	1	50	60	0	0	0	0	0	0
				265		22	330	20	0	1	30	70	5	1	5	0	0	5
				274		17	160	0	0	1	40	50	0	0	2	0	0	0
				275		21	232	0	0	1	30	60	3	0	4	0	0	0
				276		19	262	5	0	1	30	60	2	0	3	0	0	0
				277		20	286	0	0	1	30	70	0	0	3	0	0	0
		Igh-6	21	155		28	283	0	0	1	30	0	0	0	0	0	0	0
				244		25	223	0	0	1	20	20	0	0	1	0	0	0
				259		24	214	0	0	0	20	20	1	0	0	0	0	0
				156		28	208	10	10	3	75	75	0	0	5	0	0	0
				264		25	216	0	10	2	75	75	1	1	0	0	0	0
		Igh-J		490		21	373	0	0	1	5	30	0	0	2	0	5	0
				491		26	260	0	0	1	20	30	1	0	2	0	0	0
				492		30	306	0	5	1	30	20	3	0	2	0	0	0

Affymetrix Microarray Analysis and Validation

The global gene expression correlated well in biological replicates from two independent pools of three kidneys (NCBA: r=0.96; Iso D7: r=0.96; WT D5: r=0.92; WT D7: r=0.96; WT D14: r=0.98; WT D21: r=0.86; WT D42: r=0.90). The results for WT D5 transplants are presented in FIG. 3. Correlation between the d4MLR and a CTL clone was r=0.82. Microarray results were compared with real-time RT-PCR for a set of 35 genes encoding cytokines, chemokines, CD markers, and other factors involved in inflammation and cytolysis. Results from twelve selected genes are presented in FIG. 4. RT-PCR results revealed a 10 fold higher increase in gene expression when compared to the results obtained from microarrays, but the patterns of gene expression were similar for microarray and RT-PCR (r=0.87).

Hierarchical Clustering of the Global Gene Expression in Rejecting Kidneys, Isografts, CTL, and d4 MLR

Unsupervised hierarchical cluster analysis was used to compare overall gene expression between control kidneys, isografts, allografts rejecting in WT and IghKO hosts, d4MLR, and the allostimulated CTL clone. The resulting dendrogram (FIG. 5) revealed that the transcriptomes cluster into three groups. One group included normal kidneys and isografts at days 5, 7, and 21, with Iso D21 being more similar to NCBA than Iso D5 or Iso D7. The allografts clustered in a second group, with WT D5, WT D7, IghKO D7, and IghKO D21 in one sub-cluster and WT D 14, WT D21, and WT D42 in a second sub-cluster. d4MLR and CTL formed a distinct third cluster.

CD Antigen Transcript Expression

Expression of CD gene transcripts as a reflection of cellular infiltration was analyzed. Transcripts were selected by searching a master table for “CD antigen.” Genes having an expression level that was increased greater than two fold at least at one time point during rejection in allografts were chosen and compared to other samples.

The expression of thirty-three CD transcripts was increased at least two fold in wild-type allografts as compared to the expression levels observed in NCBA kidney (Table 2). Twenty-one of these had high expression in d4MLR and CTL (increased more than 5 fold as compared to NCBA). High expression of these transcripts in rejecting kidney is consistent with CTL infiltration at D5, which increases at D7 and stabilizes thereafter. CD2f10 and CD14 were increased in rejecting allografts with no expression in d4MLR or CTL, suggesting that they represent infiltrating activated macrophages, which are poorly represented in d4MLR and absent in CTL. The relatively high CD68 expression in all rejecting grafts supports this view. The B cell specific transcripts CD79a and CD79b appeared late in rejection at days 14, 21, and 42 in wild-type but not in IghKO hosts, consistent with late recruitment of antibody-producing cells to the graft. The analysis of CD transcripts is consistent with an early and sustained CTL/macrophage infiltrate in wild-type and IghKO hosts, and with late B cell infiltration in wild-type hosts.

TABLE 2
Changes in CD antigen transcripts in isografts and kidneys rejecting in wild-type
hosts and in B cell deficient hosts.
				IghKO
				Allografts
	NCBA	Isografts	WT Allografts	Fold	Lymphocytes
	Signal	Fold Change	Fold Change	Change	Fold Change
Symbol	NCBA	D5	D7	D21	D5	D7	D14	D21	D42	D7	D21	CTL	MLR
Cd1d1	48	—	—	—	—	—	5.2	3.1	4.1	2.6	2.7	9.4	5.3
Cd2	15	—	—	—	10.4	13.6	15.8	12.7	9.3	10.2	9.9	269.5	166.0
Cd2f10-	112	—	—	—	4.2	5.5	10.8	7.9	7.4	3.9	4.8	—	—
pending
Cd3d	8	—	—	—	42.1	59.0	60.8	48.9	31.8	66.9	38.2	812.4	910.7
Cd3e	60	—	—	—	9.3	16.3	11.2	16.1	6.3	19.9	13.8	64.8	81.1
Cd3g	43	—	—	—	22.9	35.9	41.4	42.4	23.4	37.6	28.0	252.4	174.9
Cd3z	39	—	—	—	6.9	9.1	8.1	8.6	4.3	9.5	6.6	54.8	64.5
Cd5	112	—	—	—	2.9	4.1	2.4	3.2	—	3.8	2.7	14.6	17.1
Cd6	54	—	—	—	6.8	6.6	—	7.2	—	7.2	6.9	18.4	16.2
Cd7	24	—	—	—	—	—	—	—	—	—	9.6	—	—
Cd8a	97	—	—	—	9.3	18.8	17.6	11.6	8.8	27.9	10.9	39.7	32.8
Cd8b	22	—	—	—	26.9	39.6	50.3	40.0	24.8	47.1	29.1	251.6	111.8
Cd14	424	—	3.6	—	7.3	2.8	5.4	4.2	4.6	5.5	3.1	—	—
Cd22	153	—	—	—	—	—	2.4	3.1	3.2	—	2.6	—	14.4
Cd28	41	—	—	—	8.6	8.1	10.7	5.2	6.1	7.9	5.1	76.2	45.8
Cd38	343	—	—	—	—	—	3.1	2.3	2.9	3.1	2.0	—	3.2
Cd44	65	2.1	3.7	—	9.8	14.3	29.6	28.9	25.0	15.6	16.8	43.0	25.6
Cd47	990	—	—	—	3.2	2.9	4.0	3.7	2.9	3.8	3.1	13.3	9.5
Cd48	20	—	4.0	—	23.6	29.6	45.8	31.2	33.5	32.1	22.3	269.6	63.1
Cd52	287	—	2.1	—	15.1	19.1	30.6	19.8	19.7	21.6	15.3	71.0	58.8
Cd53	134	—	2.8	—	11.4	17.3	22.6	18.2	19.6	18.0	13.0	73.9	71.7
Cd68	161	—	—	—	4.8	6.7	13.4	10.6	18.3	9.8	8.9	2.5	2.7
Cd72	41	—	—	—	7.8	13.4	27.7	14.9	20.2	9.4	14.4	13.6	30.9
Cd79a	85	—	—	—	—	—	2.0	2.9	2.7	—	—	—	35.6
Cd79b	67	—	—	—	—	—	3.0	3.8	3.9	—	—	—	35.6
Cd80	54	—	—	—	—	2.0	3.0	2.3	2.4	—	—	6.3	3.1
Cd83	81	—	—	—	3.8	4.7	9.3	12.2	12.7	4.1	9.2	—	35.6
Cd84	71	—	—	—	2.9	3.8	11.6	10.5	12.7	6.9	8.9	20.8	12.3
Cd86	82	—	—	—	2.9	3.4	8.4	5.8	6.8	3.6	4.2	2.7	5.9
Cd97	272	—	—	—	2.1	2.8	2.8	3.6	3.1	2.8	2.9	14.2	8.9
Ptprc	187	—	2.5	—	20.1	21.3	28.0	24.3	16.7	23.8	18.9	88.2	72.9
(CD45)
Sdc1	247	—	—	—	—	—	3.3	3.6	3.8	3.1	2.8	—	—
(CD138)
Thy1	132	—	—	—	9.2	10.6	7.6	11.2	5.7	12.0	11.6	71.9	91.6
(CD90)

The table contains the signal strength for controls and fold changes for the transplants. (−) indicates that a given gene was not upregulated; bolded signal values indicate that a transcript was classified as present. In case of multiple probe sets querying the same gene, data obtained from probe sets with suffixes _s_at and _x_at were not considered, and a probe set displaying the most robust signal was selected.

Eighteen CD transcripts were present in normal kidney, perhaps reflecting immature dendritic cells in the interstitium (Austyn et al., J. Immunol., 152:2401-2410 (1994)). Expression of CD transcripts was similar between CTL and d4MLR. In addition, d4MLR contained the B cell specific transcripts CD79a and CD79b. Macrophage transcript CD14 was not expressed in CTL or d4MLR, while macrophage transcript CD68 was expressed at a low level in both.

Expression of CA Ts in Rejecting Mouse Kidney Allografts

CATs were defined by high expression in both the CTL clone and in d4MLR but rated as “absent” in normal kidney. This algorithm identified 287 CATs. Expression of CATs was lower in d4MLR than in the CTL clone (mean 91±59 percent, median 87 percent). Compared to NCBA and isografts, the CATs were strongly expressed in rejecting WT allografts (FIG. 6). At day 5 post-transplant, the signal for CATs was increased 6.4 fold compared to NCBA and 14 percent (median) of that observed with the CTL clone (mean 20±28 percent). These results indicate that the CTL infiltrating the kidney are diluted about 5-6 fold compared to the CTL clone or the d4MLR. To confirm this interpretation, RNA from d4MLR was diluted with kidney RNA in a ratio 1:4. The resulting signal was similar to the signal in all rejecting kidneys (mean 20±7 percent, median 20 percent of the d4MLR and mean 18±11 percent, median 15 percent of expression in the CTL clone). Thus, at day 5, about one fifth to one sixth of the transcriptome of rejecting kidney is attributable to CATs. After day 5, mean expression of CATs was stable as a percent of the CTL signal (D7, 23.2±28 percent; D14, 27.3±45 percent; D21, 26.2±34 percent; and D42, 22.5±38 percent) and the median was also consistent (D5, 14 percent; D7, 16 percent; D14, 16 percent; D21, 16 percent; and D42, 12 percent).

To determine whether the pattern of CAT expression is consistent in vivo, the consistency of expression of individual CATs in various experimental conditions was analyzed. By non-parametric regression analysis, the expression of CATs correlated in all conditions, indicating robust maintenance of CAT expression in vivo and in vitro (Table 3). The d4MLR correlated well with the diluted MLR (r=0.91), despite the 80 percent decrease in signal, and slightly less well with the CTL clone (r=0.81; p<0.001). In rejecting transplants, the CAT signals exhibited a striking correlation among all days in wild-type hosts (r=0.90-0.96), indicating that most CATs displayed predictable and stable levels of expression in vivo in all rejecting kidneys. The correlations of d4MLR with the rejecting transplants at all days were considerably less (r=0.70-0.78; p<0.001), indicating significant differences between the relative transcript levels in vivo and in vitro. Expression in the CTL clone correlated least with that in the transplants (r=0.66-0.74; p=n.s.). Thus, the relative level of expression of individual CATs was similar in vitro between CTL and d4MLR, and was similar in vivo under all conditions in rejecting transplants, but was somewhat different in vivo compared to in vitro.

TABLE 3
Spearman rank correlations for CATs in lymphocytes from d4MLR and a CTL clone
(CTL), MLR diluted with kidney RNA 1:4 (MLRdil), and kidneys rejecting
in wild-type (WT) and B-cell deficient (IghKO) hosts at
days 5-42 post transplant.
									IghKO	IghKO
	MLR	CTL	MLRdil	WTD5	WTD7	WTD14	WTD21	WTD42	D7	D21
MLR	1	.81	.91	.78	.79	.74	.74	.70	.80	.77
CTL	.81	1	.78	.74	.74	.74	.69	.37	.73	.69
MLRdil	.91	.78	1	.84	.82	.76	.75	.73	.81	.79
WTD5	.78	.74	.84	1	.96	.92	.90	.90	.96	.92
WTD7	.79	.74	.82	.96	1	.95	.96	.93	.98	.96
WTD14	.74	.74	.76	.92	.95	1	.96	.97	.95	.96
WTD21	.76	.69	.75	.90	.96	.96	1	.94	.96	.98
WTD42	.70	.66	.73	.90	.93	.97	.94	1	.93	.95
IghKO	.80	.73	.81	.96	.98	.95	.96	.93	1	.97
D7
IghKO	.77	.69	.79	.92	.96	.96	.98	.95	.97	1
D21

To further investigate expression patterns of individual CATs, a k-means cluster analysis of CATs was performed based on their expression level in wild-type allografts relative to the CTL clone. The 287 CATs were clustered into five clusters (FIG. 7). Cluster 1 has 140 transcripts (e.g., CD2, CD3g, GzmB, Tcrb, EOMES, and several genes related to the cell cycle) and was characterized by lower expression in d4MLR than CTL but relatively stable expression in all allografts (FIG. 7). The expression level for individual CATs are provided in Table 4. The mean expression was 6.1 fold increased versus NCBA at day 5, and remained unchanged thereafter. Cluster 2 has 23 transcripts (Table 4). The cluster 2 CATs were more highly expressed in d4MLR than CTL and relatively strongly increased in day 5 rejecting kidneys (6.7 fold; FIG. 7). A further 2.4 fold increase was observed from day 5 to day 14, and expression levels were stable thereafter. Cluster 3 has 74 transcripts, and the expression was also relatively high in d4MLR versus CTL, but lower in rejecting kidney, fluctuating somewhat among the different times (FIG. 7 and Table 4). Cluster 4 has 46 transcripts, and the CATs of this cluster were less expressed in d4MLR than CTL, exhibited a 2.2 fold increase in expression from day 5 to day 14, and exhibited a decreased expression thereafter by 1.4 fold. Cluster 5 has four transcripts, and the CATs of this cluster were as highly expressed in rejecting grafts as in the CTL clone and d4MLR (FIG. 7 and Table 4). Expression of CATs in cluster 2 and cluster 5 is higher than in clusters 1, 3, and 4, which contained the great majority of the CATs.

TABLE 4
CATs of clusters 1 through 5
		GenBank			IghKO
		Accession			Allografts
	GenBank	Number for	NCBA	WT Allografts	Fold	Lymphocytes
	Accession	Human	Signal	Fold Change	Change	Fold Change
Symbol	Gene Title	Number	Ortholog	NCBA	D5	D7	D14	D21	D42	D7	D21	CTL	MLRD4
CLUSTER 1
Adam19	a disintegrin and metalloproteinase domain 19 (meltrin beta)	D50410	NM_023038	34	8.3	8.9	7.8	11.1	6.7	10.1	6.9	29.8	18.9
			NM_033274
Adam19	a disintegrin and metalloproteinase domain 19 (meltrin beta)	NM_009616	NM_023038	12	3.5	5.1	4.5	5.2	3.6	5.4	3.2	15.2	11.4
			NM_033274
Ask-	activator of S phase kinase	NM_013726	NM_006716	72	3.6	3.5	3.8	3.2	2.8	3.4	2.1	22.3	18.5
pending
Aqp9	aquaporin 9	BC024105	NM_020980	17	2.5	1.2	2.7	2.7	2.9	1.1	2.5	20.9	14.5
Abcb9	ATP-binding cassette,	AK020749	NM_019624	8	1.6	1.3	1.1	1.0	0.8	1.7	1.8	19.6	16.0
	sub-family B	BC017348	NM_019625
	(MDR/TAP), member 9		NM_203444
			NM_203445
			BC017348
Brdg1-	BCR downstream	NM_019992	NM_012108	130	1.5	1.6	2.2	1.5	1.6	1.6	1.4	10.5	8.6
pending	signaling 1		BC014958
Brca1	breast cancer 1	U31625	AF005068	25	1.6	1.0	1.6	0.7	1.7	1.0	0.6	13.1	8.5
			NM_007295
Bub1	budding uninhibited by benzimidazoles 1 homolog (S. cerevisiae)	AF002823	AF043294	9	5.7	5.6	6.9	4.8	4.8	7.0	4.1	77.7	55.9
			AK023540
Bub1b	budding uninhibited by benzimidazoles 1 homolog, beta (S. cerevisiae)	NM_009773	NM_001211	19	11.0	8.9	8.8	6.9	5.9	13.7	8.1	80.2	65.2
Calmbp1	calmodulin binding	BB648052	AK001380	24	2.2	1.7	2.8	1.6	2.1	2.5	1.4	16.5	10.1
	protein 1
MGC38321	CasL interacting molecule	BB209438	NM_022765	10	5.3	6.5	6.7	8.4	5.7	6.4	8.1	62.6	44.4
	MICAL
Ctsw	cathepsin W	NM_009985	NM_001335	17	32.2	41.5	47.1	43.4	23.3	54.9	45.1	476.9	257.3
Cd2	CD2 antigen	NM_013486	NM_001767	15	10.4	13.6	15.8	12.7	9.3	10.2	9.9	269.5	166.0
Siva-	Cd27 binding protein	NM_013929	AF033111	14	2.1	1.2	3.6	3.2	3.5	4.5	3.3	33.7	35.1
pending	(Hindu God of		NM_006427
	destruction)		AW024335
Cd3g	CD3 antigen, gamma	M58149	NM_000073	43	22.9	35.9	41.4	42.4	23.4	37.6	28.0	252.4	174.9
	polypeptide
Cd53	CD53 antigen	NM_007651	NM_000560	134	11.4	17.3	22.6	18.2	19.6	18.0	13.0	73.9	71.7
BC003314	cDNA sequence	NM_030255	NM_004900	86	8.6	9.8	8.6	9.5	7.9	12.5	8.5	44.4	35.1
	BC003314		NM_145298
			NM_021822
Cdc6	cell division cycle 6	NM_011799	NM_001254	11	5.6	3.0	5.2	3.5	3.2	4.7	2.4	44.0	19.0
	homolog (S. cerevisiae)		U77949
Cenpa	centromere autoantigen A	AV132173	NM_001809	22	10.5	10.4	10.0	8.3	6.0	10.2	6.0	180.3	166.7
Chl12-	Chl12 homolog (yeast)	BM233289	AK024476	5	0.9	0.8	0.8	0.8	0.8	0.9	0.9	12.4	10.5
pending
Hcapg-	chromosome condensation	AV277326	NM_022346	5	4.4	3.0	4.7	2.7	3.8	3.5	1.6	73.1	22.0
pending	protein G
Coro1a	coronin, actin binding	BB740218	NM_007074	9	2.6	3.6	3.2	2.6	2.2	2.5	2.7	30.8	23.9
	protein 1A
Ccna2	cyclin A2	NM_009828	NM_001237	214	2.6	2.4	1.9	1.8	1.7	2.2	1.8	17.2	9.0
Ccnb1	cyclin B1	AU015121	NM_031966	15	12.4	11.0	12.1	7.2	4.1	8.5	6.0	109.3	51.0
Ccnb2	cyclin B2	AK013312	NM_004701	69	6.3	5.2	5.6	3.9	4.1	4.6	3.6	78.2	36.0
			BF509102
			AK023404
			AU134430
Ccnd2	cyclin D2	BM118679	NM_001759	4	1.0	1.7	1.4	1.5	1.8	1.5	2.0	7.1	5.5
Ccnd2	cyclin D2	BB840359	NM_001759	9	2.0	1.4	3.0	1.2	2.6	1.1	1.2	8.1	7.1
Ccne1	cyclin E1	NM_007633	NM_001238	78	1.6	1.4	1.4	1.3	1.1	1.8	1.4	6.7	5.4
			NM_057182
Cst7	cystatin F (leukocystatin)	NM_009977	AF031824	15	15.3	23.5	28.7	25.9	22.4	31.9	23.0	223.5	199.5
Diap3	diaphanous homolog 3	NM_019670	NM_030932	6	1.7	2.0	2.7	1.1	2.3	2.1	1.0	20.8	9.8
	(Drosophila)		AL354829
Dnmt1	DNA methyltransferase	BB165431	NM_001379	163	2.9	3.1	2.7	2.5	2.4	2.8	2.5	14.3	10.1
	(cytosine-5) 1
D2Ertd750e	DNA segment, Chr 2,	AK012148	NM_033286	28	2.2	2.4	2.3	1.4	0.8	2.2	1.1	33.9	15.1
	ERATO Doi 750,
	expressed
Emb	embigin	BG064842	NM_198449	428	1.8	1.9	1.9	1.9	1.7	2.7	1.6	14.9	10.7
Eomes	eomesodermin homolog (Xenopus laevis)	AB031037	NM_005442	9	1.1	3.2	0.8	5.2	0.8	1.7	2.4	82.4	24.3
	ESTs, Moderately similar to hypothetical protein FLJ23311 [Homo sapiens]	BM247465	NM_024680	23	3.6	5.8	3.3	3.2	3.6	4.1	2.5	20.6	18.0
	[H. sapiens]
Eef1b2	eukaryotic translation	C77437	NM_001008396	124	2.0	1.8	1.7	1.6	1.4	1.6	1.2	7.4	5.6
	elongation factor 1 beta 2		NCBI
			NM_007086
AA408511	expressed sequence	AU018569	AB040957	2	3.8	3.4	3.7	2.3	1.7	3.2	1.4	45.8	16.3
	AA408511		NM_020890
AA675320	expressed sequence	BC025223	NM_144595	100	1.4	1.5	1.9	1.4	1.2	1.6	1.4	8.5	6.9
	AA675320
AI173001	expressed sequence	BC024727	NM_014800	149	2.2	2.7	2.2	2.5	2.0	2.1	2.0	9.0	8.1
	AI173001		NM_130442
Fignl1	fidgetin-like 1	NM_021891	NM_022116	10	9.6	9.8	8.4	7.4	3.8	10.6	5.4	77.4	53.0
			AK023411
Gtse1	G two S phase expressed	NM_013882	NM_016426	20	2.0	2.5	3.6	1.5	1.9	1.8	1.7	26.8	22.8
	protein 1		BC006325
			BF973178
			AI218393
			AI340239
Glipr2	GLI pathogenesis-related 2	BM208214	NM_022343	69	3.8	4.0	5.0	6.8	4.0	5.8	6.1	21.7	18.2
Gzmb	granzyme B	NM_013542	J03189	43	38.6	44.8	58.8	23.0	24.2	65.1	30.3	703.2	476.8
Hemgn	hemogen	NM_053149	AF130060	5	1.3	2.4	0.8	0.8	0.9	1.0	1.5	22.3	7.2
			AF322875
Hmmr	hyaluronan mediated	AF079222	U29343	79	1.6	1.3	1.5	0.6	1.4	1.3	0.9	8.7	4.9
	motility receptor		NM_012485
	(RHAMM)		BC035392
			BC002966
			BM449961
MGC37568	hypothetical protein	BB327418	BC006107	22	20.0	22.5	26.2	24.6	23.3	25.1	24.4	136.5	107.9
	MGC37568
Icos	inducible T-cell co-	AB023132	AB023135	12	10.6	14.8	12.6	9.2	7.4	17.5	12.6	71.1	57.7
	stimulator
Incenp	inner centromere protein	BQ175667	NM_020238	60	4.2	4.4	3.3	4.0	3.0	4.6	3.0	18.7	14.6
Incenp	inner centromere protein	BB418702	NM_020238	85	5.1	5.3	5.0	5.5	4.0	5.1	3.5	59.0	19.6
Il2ra	interleukin 2 receptor,	AF054581	K03122	24	2.3	2.1	2.2	1.3	1.3	1.9	2.0	64.2	34.9
	alpha chain		NM_000417
Il2rb	interleukin 2 receptor, beta	NM_008368	NM_000878	24	23.8	30.9	32.9	39.5	26.2	32.4	30.2	168.8	119.7
	chain
Il7r	interleukin 7 receptor	AI573431	NM_002185	5	2.7	3.9	7.2	7.9	7.0	5.5	5.5	106.0	84.8
			BE217880
Kif10	kinesin family member 10	BG068387	NM_001813	14	1.8	2.1	1.9	1.5	2.0	1.0	0.9	11.6	6.0
Kif11	kinesin family member 11	BM234447	NM_004523	46	3.8	3.0	2.9	2.2	2.2	2.6	1.7	39.1	13.4
Kif11	kinesin family member 11	BB827235	NM_004523	60	2.4	1.9	2.3	1.2	1.5	1.8	0.9	25.4	7.0
Kif22-ps	kinesin family member 22,	BC003427	NM_007317	7	3.6	2.4	3.2	2.5	1.9	4.2	1.2	37.2	15.6
	pseudogene
Kif22-ps	kinesin family member 22,	BC003427	NM_007317	14	4.0	3.6	5.4	2.7	3.9	4.1	2.5	65.2	24.7
	pseudogene
Kif23	kinesin family member 23	BG082989	NM_004856	3	1.4	1.0	1.3	0.8	1.6	0.9	0.9	16.5	10.4
			NM_138555
Kif23	kinesin family member 23	AW986176	NM_004856	80	3.1	2.4	2.6	2.2	2.3	2.1	2.0	18.4	10.5
			NM_138555
Lmnb1	lamin B1	BG064054	NM_005573	70	3.7	3.6	3.3	2.9	2.6	3.5	2.2	23.4	8.2
Lek1	leucine, glutamic acid,	BB049243	NM_016343	152	1.1	1.5	1.4	1.3	0.7	1.5	1.2	11.5	7.4
	lysine family 1 protein
Melk	maternal embryonic	NM_010790	NM_014791	6	3.6	3.7	3.5	3.5	2.0	3.7	2.2	52.5	19.3
	leucine zipper kinase
Ms4a4b	membrane-spanning 4-	BB199001	n/a	6	71.4	97.6	89.2	77.4	53.6	87.0	54.7	1238.4	519.1
	domains, subfamily A,
	member 4B
Mcmd6	mini chromosome	NM_008567	NM_005915	285	3.1	3.2	3.4	2.9	2.3	3.4	2.5	18.7	13.2
	maintenance deficient 6
	(S. cerevisiae)
	Mus musculus adult male	BB014626	n/a	10	12.9	21.2	24.3	29.4	12.3	17.6	16.9	158.1	102.9
	testis cDNA, RIKEN full-
	length enriched library,
	clone: 4930483L24
	product: weakly similar to
	AT-HOOK PROTEIN
	AKNA [Homo sapiens],
	full insert sequence.
	Mus musculus, Similar to	BC026773	AL832450	26	2.7	4.1	3.2	4.9	2.5	4.3	3.3	29.4	14.1
	expressed sequence
	AI481279, clone
	MGC: 25733
	IMAGE: 3982549, mRNA,
	complete cds
Myb	myeloblastosis oncogene	BC011513	NM_005375	6	2.4	1.7	1.4	1.1	1.5	2.0	0.9	10.2	7.8
Myb	myeloblastosis oncogene	NM_033597	NM_005375	10	4.5	2.5	1.3	2.1	1.6	3.1	1.4	26.2	20.1
Ncf4	neutrophil cytosolic factor 4	NM_008677	NM_000631	122	5.6	6.1	7.8	6.2	6.9	7.0	6.0	17.3	14.7
			NM_013416
Np95	nuclear protein 95	NM_010931	NM_013282	40	8.0	7.9	6.6	6.9	4.6	8.1	4.7	55.7	27.6
Np95	nuclear protein 95	BB702754	NM_013282	10	5.7	6.3	3.8	4.7	2.4	4.8	2.0	44.4	29.6
Odf2	outer dense fiber of sperm	AF000968	AF053970	15	2.0	1.6	3.2	2.1	0.8	2.1	1.4	10.7	9.1
	tails 2		AL138382
Pvt1	plasmacytoma variant	BE956863	n/a	31	0.6	0.4	0.5	0.6	2.0	0.8	0.8	8.9	9.1
	translocation 1
Plk	polo-like kinase homolog,	NM_011121	NM_005030	102	2.9	2.8	2.5	1.9	2.0	2.5	1.8	19.7	14.7
	(Drosophila)
Pole	polymerase (DNA	NM_011132	NM_006231	7	2.8	4.5	4.2	2.6	1.2	4.1	3.6	53.4	36.5
	directed), epsilon
Kcnn4	potassium	NM_008433	NM_002250	4	8.4	8.8	14.0	12.0	14.2	8.9	8.9	49.5	53.7
	intermediate/small
	conductance calcium-
	activated channel,
	subfamily N, member 4
Kcnn4	potassium	BG865910	NM_002250	18	19.8	19.7	32.4	26.3	31.5	23.8	25.4	123.4	97.3
	intermediate/small
	conductance calcium-
	activated channel,
	subfamily N, member 4
Pstpip1	proline-serine-threonine	U87814	AF038602	44	5.9	7.1	8.8	8.8	7.4	9.1	9.1	51.1	40.7
	phosphatase-interacting
	protein 1
Prss19	protease, serine, 19	NM_008940	NM_007196	159	1.4	1.6	1.6	1.6	2.1	1.7	1.4	11.1	8.2
	(neuropsin)		NM_144505
			NM_144506
			NM_144507
Prkcq	protein kinase C, theta	AB062122	L01087	103	5.3	4.1	3.0	4.1	1.7	4.5	4.0	25.5	15.8
			AK024876
			AK024876
			AL137145
LOC233406	protein regulator of	BC005475	NM_003981	97	3.7	4.3	5.3	3.3	3.2	4.3	3.5	21.5	18.4
	cytokinesis 1-like		NM_199413
			NM_199414
Ptpn8	protein tyrosine	NM_008979	NM_015967	163	4.4	4.6	6.0	5.2	4.3	5.6	4.2	43.9	24.6
	phosphatase, non-receptor		NM_012411
	type 8		AW665758
Pycs	pyrroline-5-carboxylate	NM_019698	NM_002860	87	3.3	2.7	3.9	3.5	4.0	3.3	3.1	8.0	8.7
	synthetase (glutamate		U76542
	gamma-semialdehyde
	synthetase)
Pycs	pyrroline-5-carboxylate	BB833010	NM_002860	13	4.4	5.6	8.1	4.4	7.2	3.3	4.9	15.0	14.5
	synthetase (glutamate
	gamma-semialdehyde
	synthetase)
Racgap1	Rac GTPase-activating	NM_012025	AU153848	13	5.5	6.2	5.2	6.6	3.1	6.1	4.0	77.1	26.8
	protein 1
Racgap1	Rac GTPase-activating	AF212320	AU153848	164	2.1	2.2	2.1	1.7	1.5	2.3	1.8	21.2	7.9
	protein 1
Rad51ap1	RAD51 associated protein 1	NM_009013	NM_006479	3	1.0	1.1	1.1	0.8	1.6	1.1	0.9	10.6	6.3
Rad51	RAD51 homolog (S. cerevisiae)	NM_011234	D14134	8	14.6	12.8	12.3	9.7	8.8	12.4	7.5	96.8	51.6
			NM_002875
Rad541	RAD54 like (S. cerevisiae)	AV310220	NM_003579	45	3.6	4.0	4.0	3.3	3.2	4.2	2.9	17.8	13.9
Rassf5	Ras association	NM_018750	AY062002	93	4.0	4.8	5.8	5.9	4.5	5.2	4.0	39.2	20.4
	(RalGDS/AF-6) domain		BC004270
	family 5
Rgs1	regulator of G-protein	NM_015811	S59049	28	6.9	8.0	11.9	11.2	9.6	8.4	9.1	82.1	73.1
	signaling 1		NM_002922
Rrm2	ribonucleotide reductase	BF119714	NM_001034	164	4.5	3.6	4.0	3.0	2.8	3.9	2.8	22.5	13.2
	M2
1700054N08Rik	RIKEN cDNA	BC024705	n/a	15	2.5	1.2	3.6	2.8	0.8	4.6	3.0	14.0	10.4
	1700054N08 gene
2310009O17Rik	RIKEN cDNA	BB799833	NM_017447	28	4.0	3.7	7.1	3.3	5.3	3.8	3.1	55.6	30.4
	2310009O17 gene
2310009O17Rik	RIKEN cDNA	BC019957	NM_017447	214	1.6	1.8	2.3	2.3	1.8	1.9	2.0	8.3	6.2
	2310009O17 gene
2310035M22Rik	RIKEN cDNA	NM_025863	NM_173084	33	5.1	5.3	7.1	6.0	5.4	5.2	2.8	46.8	28.3
	2310035M22 gene
2410003C07Rik	RIKEN cDNA	AK010351	n/a	16	10.9	10.1	11.2	6.7	5.3	9.6	6.1	91.6	40.6
	2410003C07 gene
2410005L11Rik	RIKEN cDNA	BC022648	NM_031423	17	3.2	2.6	2.0	2.5	2.1	2.4	2.1	25.3	16.7
	2410005L11 gene		NM_145697
2600001J17Rik	RIKEN cDNA	BC006674	n/a	18	8.8	6.4	8.3	4.1	4.2	7.2	4.2	37.2	22.8
	2600001J17 gene
2610020P18Rik	RIKEN cDNA	NM_023294	NM_006101	47	1.9	1.8	2.3	1.7	1.8	2.0	1.3	19.2	6.9
	2610020P18 gene
2610036L13Rik	RIKEN cDNA	NM_026410	NM_080668	103	3.7	2.9	3.5	2.3	2.4	3.5	2.5	34.9	14.5
	2610036L13 gene
2610201A12Rik	RIKEN cDNA	NM_133851	NM_016359	103	3.4	3.3	3.3	2.5	2.3	3.4	2.3	51.0	26.5
	2610201A12 gene		NM_018454
			NM_002157
2610307O08Rik	RIKEN cDNA	AK012006	NM_198282	183	6.8	6.4	6.0	6.4	7.3	6.2	5.9	6.8	6.8
	2610307O08 gene
2610510J17Rik	RIKEN cDNA	BM230253	NM_018455	52	3.5	3.2	3.5	2.6	2.5	3.3	2.7	14.0	9.5
	2610510J17 gene
2810038K19Rik	RIKEN cDNA	NM_023684	NM_017806	198	2.0	2.1	1.9	2.0	1.2	1.9	1.7	22.5	8.4
	2810038K19 gene
3300001M08Rik	RIKEN cDNA	NM_028232	NM_001012409	16	4.6	2.9	4.3	3.7	1.2	5.0	1.4	53.0	29.7
	3300001M08 gene		NM_138484
			NM_001012413
5730403J10Rik	RIKEN cDNA	BC004617	n/a	228	1.8	1.5	1.9	1.2	1.6	1.5	1.3	11.2	5.0
	5730403J10 gene
A430107P09Rik	RIKEN cDNA	X01134	n/a	448	6.2	8.5	8.3	8.0	4.3	9.9	6.1	45.4	30.0
	A430107P09 gene
E430034C16Rik	RIKEN cDNA	NM_134163	NM_018388	22	3.2	3.6	3.0	2.5	1.6	3.2	2.4	44.9	10.9
	E430034C16 gene		NM_133486
Slfn1	schlafen 1	NM_011407	n/a	15	20.4	29.0	23.2	18.7	15.1	34.0	19.5	89.8	62.1
6-Sep	septin 6	NM_019942	D50918	27	3.0	4.1	3.5	2.8	2.7	3.8	2.0	30.9	23.2
			D50918
			AF403061
			AK026589
			T91323
			AW150913
			AI968130
			AL568374
Stk12	serine/threonine kinase 12	BC003261	AB011446	18	5.4	5.2	5.3	3.6	4.1	4.0	2.9	48.3	18.8
Stk18	serine/threonine kinase 18	BB706079	NM_014264	35	1.8	1.6	2.3	1.5	1.2	1.6	1.0	11.4	6.2
Stk4	serine/threonine kinase 4	NM_021420	Z25430	12	4.7	3.3	6.4	3.3	4.4	2.7	3.2	20.7	13.6
			NM_006282
			BC039023
			BC005231
			BE222274
			BF433725
			AI763206
Stk6	serine/threonine kinase 6	U80932	NM_003600	115	2.2	2.3	2.2	1.6	1.8	2.0	1.5	14.0	10.0
Sh2d1a	SH2 domain protein 1A	NM_011364	AF072930	2	2.6	3.9	4.1	4.4	1.6	2.9	2.0	119.9	64.7
			AF100540
			AF100539
			AF100542
Sh2d2a	SH2 domain protein 2A	NM_021309	NM_003975	22	14.4	20.0	18.9	21.1	10.1	21.9	14.0	109.9	68.7
Sh3kbp1	SH3-domain kinase	AK007283	AF230904	39	5.6	8.5	10.7	7.9	9.4	8.9	5.8	68.6	56.9
	binding protein 1		AF542051
Sh3kbp1	SH3-domain kinase	AK018032	AF230904	70	3.1	3.9	5.8	3.6	4.4	4.2	2.5	26.2	16.7
	binding protein 1		AF542051
Slc28a2	solute carrier family 28	NM_172980	NM_004212	8	10.4	15.8	21.3	16.0	16.0	17.9	11.7	64.3	56.1
	(sodium-coupled
	nucleoside transporter),
	member 2
Satb1	special AT-rich sequence	AV172776	NM_002971	28	3.2	2.8	3.3	3.0	1.9	4.2	2.9	111.1	85.1
	binding protein 1
Satb1	special AT-rich sequence	BG092481	NM_002971	26	0.8	0.7	0.7	0.9	0.6	0.8	0.8	30.5	17.7
	binding protein 1
Tcrb-V13	T-cell receptor beta,	M16120	n/a	173	12.7	15.2	19.1	13.0	8.8	15.6	9.4	122.8	88.3
	variable 13
Tcrb-V13	T-cell receptor beta,	U07661	n/a	94	20.7	24.0	22.8	19.3	13.5	21.3	15.6	142.0	97.1
	variable 13
Tcrb-V13	T-cell receptor beta,	U46841	n/a	67	2.1	2.3	2.7	1.7	1.8	2.0	1.5	44.5	12.3
	variable 13
Tcrb-V13	T-cell receptor beta,	X14388	n/a	9	14.4	15.5	16.5	13.8	8.5	15.9	8.5	301.5	71.4
	variable 13
Tcrb-	T-cell receptor beta,	BF658725	n/a	96	1.5	1.7	1.6	1.2	1.6	1.8	1.7	7.3	7.1
V8.2	variable 8.2
Tk1	thymidine kinase 1	NM_009387	NM_003258	168	2.0	2.0	1.9	1.8	1.3	2.3	2.0	10.9	9.7
			BC007986
Tyms	thymidylate synthase	BM068975	NM_001071	17	0.9	1.0	1.1	1.0	1.5	0.7	0.7	8.3	4.7
Trip13	thyroid hormone receptor	AK010336	NM_004237	18	3.2	2.8	2.5	2.0	2.4	2.7	2.1	18.1	15.1
	interactor 13
Traip	TRAF-interacting protein	NM_011634	NM_005879	4	1.9	1.6	1.2	1.6	1.2	1.0	1.1	24.1	9.1
Tacc3	transforming, acidic	NM_011524	NM_006342	6	3.8	3.6	4.0	2.8	2.9	3.9	2.4	43.6	24.2
	coiled-coil containing		AF289576
	protein 3
Tpp2	tripeptidyl peptidase II	BB484264	NM_003291	12	1.0	1.3	2.3	1.6	0.9	1.3	1.5	11.0	6.7
Tnfrsf7	tumor necrosis factor	L24495	n/a	9	6.5	12.2	6.4	7.6	5.3	12.8	7.9	51.8	42.1
	receptor superfamily,
	member 7
Ubl5	ubiquitin-like 5	AV210814	NM_017703	10	1.0	0.8	0.8	1.3	1.3	1.1	1.3	14.0	5.4
			AI479104
Xlr4	X-linked lymphocyte-	NM_021365	N/a	62	7.6	9.5	10.3	10.3	5.2	8.9	7.0	110.7	62.9
	regulated 4
Zap70	zeta-chain (TCR)	NM_009539	AB083211	33	13.9	20.2	16.3	18.0	10.6	19.7	13.4	90.0	60.0
	associated protein kinase
Znfn1a1	zinc finger protein,	NM_009578	S80876	30	5.7	7.1	5.6	4.2	3.7	8.0	3.4	39.2	26.4
	subfamily 1A, 1 (Ikaros)		NM_006060
		NM_053213	NM_031300	30	1.5	1.6	1.3	1.7	0.7	1.7	1.5	24.4	10.4
		AV126179	NM_018131	8	2.0	0.8	1.9	0.8	0.8	1.1	1.2	19.7	12.1
CLUSTER 2
Bcl2a1a	B-cell leukemia/lymphoma 2 related protein A1a	L16462	NM_004049	73	15.1	25.1	36.0	39.3	26.7	23.4	22.6	59.7	73.4
Ccl3	chemokine (C—C motif)	NM_011337	NM_002983	6	16.1	25.1	54.7	33.9	51.1	32.6	31.7	43.9	28.9
	ligand 3
Cd44	CD44 antigen	X66083	AF098641	5	16.3	16.0	53.4	31.8	41.0	23.6	16.5	61.3	48.2
			M24915
			NM_000610
			BC004372
Gadd45b	growth arrest and DNA-	AK010420	AF087853	108	3.8	2.8	4.6	5.6	4.9	4.9	3.4	6.8	11.6
	damage-inducible 45 beta		AF078077
			NM_015675
			AV658684
Ikbke	inhibitor of kappaB kinase	NM_019777	NM_014002	10	5.1	12.7	10.9	20.2	15.3	16.9	18.5	18.6	92.1
	epsilon		AW340333
Il10ra	interleukin 10 receptor,	NM_008348	NM_001558	10	8.8	13.2	19.1	20.4	18.1	12.9	13.7	28.0	26.7
	alpha
Il16	interleukin 16	BB167822	NM_004513	42	0.4	1.4	2.3	2.2	1.3	2.1	1.2	6.7	11.8
Il21r	interleukin 21 receptor	AB049137	AF269133	16	7.0	9.5	8.5	10.8	8.1	8.7	8.8	21.8	70.4
			NM_021798
			AK093371
Map3k8	mitogen activated protein	NM_007746	NM_005204	14	6.4	6.1	10.5	11.3	9.7	7.6	7.9	20.9	27.8
	kinase 8
Pglyrp	peptidoglycan recognition	NM_009402	NM_005091	9	6.1	12.3	9.4	13.2	9.9	17.3	18.1	22.8	78.9
	protein
Pim1	proviral integration site 1	AI323550	n/a	90	8.7	9.0	9.3	10.0	9.2	8.5	7.3	13.8	25.2
Plek	pleckstrin	NM_019549	NM_002664	41	17.8	29.9	28.7	30.7	28.0	33.6	25.5	40.5	40.6
Runx1	runt related transcription	NM_009821	U19601	5	3.5	4.1	9.2	11.8	9.3	4.5	4.3	7.5	8.9
	factor 1		D89788
			L34598
			NM_001754
			S76346
			D43968
			D43967
Tap1	transporter 1, ATP-	BC024897	n/a	257	10.3	11.4	10.5	13.4	9.3	12.6	11.5	12.3	23.1
	binding cassette, sub-
	family B (MDR/TAP)
Trim30	tripartite motif protein 30	BG068242	n/a	116	3.9	4.0	4.5	4.6	4.9	4.3	3.7	5.4	6.0
1300004C08Rik	RIKEN cDNA	AK004894	L13852	61	4.6	4.7	9.1	6.8	7.3	6.0	5.4	7.8	10.7
	1300004C08 gene		NM_003335
2610043M05Rik	RIKEN cDNA	BM247370	NM_002719	20	0.9	2.5	6.2	7.3	7.3	3.8	4.5	14.3	11.6
	2610043M05 gene		NCBI
			NM_178586
			NCBI
			NM_178587
			NCBI
			NM_178588
9030412M04Rik	RIKEN cDNA	AK018504	NM_014737	38	3.5	5.1	5.3	6.6	6.7	4.9	5.0	7.6	13.3
	9030412M04 gene		NM_170773
			NCBI
			NM_170774
E430025L02Rik	RIKEN cDNA	BC027411	NM_198565	120	4.2	6.4	5.8	6.3	6.5	7.7	7.6	8.4	11.8
	E430025L02 gene
MGC41320	hypothetical protein	BC006817	NM_025079	31	1.9	2.3	3.3	3.4	3.1	2.7	2.4	5.3	5.6
	MGC41320
		BC003855	n/a	174	1.2	0.9	3.7	2.3	2.2	1.6	2.2	4.5	5.9
		BC003855	n/a	5	3.4	4.9	10.1	15.8	8.7	5.9	9.9	10.9	16.6
		BC003855	n/a	20	5.0	8.8	15.4	18.6	11.4	5.8	13.1	12.3	27.7
CLUSTER 3
Abca7	ATP-binding cassette, sub-family A (ABC1), member 7	NM_013850	NM_019112	109	1.9	2.6	1.9	2.7	1.9	2.6	2.5	8.6	10.7
Apbblip-	amyloid beta (A4)	BC023110	NM_019043	21	6.5	8.9	5.1	8.8	4.9	9.6	9.3	21.1	23.3
pending	precursor protein-binding,		BC035636
	family B, member 1
	interacting protein
Batf	basic leucine zipper	NM_016767	NM_006399	31	10.0	11.6	9.6	11.3	7.2	8.3	8.7	20.4	23.5
	transcription factor, ATF-
	like
Bcl11b	B-cell	NM_021399	AB043584	17	6.0	8.6	5.4	9.1	3.2	7.0	8.3	101.8	100.7
	lymphoma/leukaemia 11B		NM_022898
			AA918317
			AU146285
Brca1	breast cancer 1	U36475	NM_007294	9	4.2	5.2	5.0	4.5	3.3	5.2	3.4	36.0	36.3
			NCBI
			NM_007295
			NCBI
			NM_007296
			NCBI
			NM_007297
			NCBI
			NM_007298
			NCBI
			NM_007299
			NCBI
			NM_007300
			NCBI
			NM_007301
			NCBI
			NM_007302
			NCBI
			NM_007303
			NCBI
			NM_007304
			NCBI
			NM_007305
			NCBI
			NM_007306
Brca1	breast cancer 1	U31625	AF005068	1	0.9	0.8	0.8	0.8	0.9	0.9	0.9	7.3	8.9
			NM_007295
Cd37	CD37 antigen	BC019402	NM_001774	21	9.9	15.8	14.4	21.3	11.0	18.5	11.9	106.1	127.4
Cd3d	CD3 antigen, delta	NM_013487	NM_000732	8	42.1	59.0	60.8	48.9	31.8	66.9	38.2	812.4	910.7
	polypeptide
Cd3z	CD3 antigen, zeta	X84237	J04132	4	4.1	4.7	5.7	6.0	2.2	5.8	3.8	56.8	70.3
	polypeptide
Cep2	centrosomal protein 2	NM_008383	NM_007186	11	1.1	2.5	0.8	3.8	0.8	2.9	3.3	11.9	17.8
Elmo1	engulfment and cell	BC006054	NM_014800	15	5.3	6.1	5.3	5.0	3.0	5.3	4.1	20.7	25.4
	motility 1, ced-12		NCBI
	homolog (C. elegans)		NM_130442
Fgf13	fibroblast growth factor 13	BC018238	NM_004114		2.7	3.1	1.6	2.9	1.1	4.2	2.1	15.4	12.9
			NM_033642
Foxm1	forkhead box M1	AK008037	NM_033642		1.4	1.8	1.1	1.3	1.2	1.4	1.4	6.4	6.2
Gfi1	growth factor independent 1	NM_010278	NM_005263	8	2.4	3.7	3.6	4.8	2.3	4.2	2.7	30.2	45.0
Gzmc	granzyme C	NM_010371	n/a	6	1.0	1.9	3.0	2.0	2.9	1.3	2.3	24.1	35.1
Ian4	immune associated	NM_031247	NM_018384	276	2.6	3.6	2.3	3.1	2.0	3.6	3.4	14.0	19.0
	nucleotide 4		AL080068
			AL080068
Il12rb2	interleukin 12 receptor,	NM_008354	NM_001559	56	1.3	1.6	1.8	1.2	1.0	1.7	1.1	8.1	14.0
	beta 2
Il2ra	interleukin 2 receptor,	M30856	NM_000417	76	1.8	1.6	1.1	1.2	1.1	1.7	1.0	11.2	22.0
	alpha chain
Il2rg	interleukin 2 receptor,	L20048	NM_000206	186	9.9	13.3	14.0	12.6	9.7	16.0	12.6	36.9	37.4
	gamma chain
Irf4	interferon regulatory	NM_013674	NM_002460	15	7.4	7.1	6.7	11.0	6.6	9.2	5.6	50.2	102.7
	factor 4
Itgal	integrin alpha L	AF065902	BC008777	67	3.7	5.6	4.7	5.6	2.8	6.4	4.4	28.2	24.9
Itgb7	integrin beta 7	NM_013566	NM_000889	30	14.0	21.1	16.0	22.6	11.4	23.1	20.3	48.5	96.2
			AI807169
Itk	IL2-inducible T-cell	NM_010583	D13720	8	10.7	17.1	15.5	20.3	8.2	15.4	13.7	152.5	152.4
	kinase
Itk	IL2-inducible T-cell	L10628	D13720	17	2.2	3.5	3.4	4.6	1.4	4.5	1.7	33.2	33.6
	kinase
Kcna3	potassium voltage-gated	NM_008418	NM_002232	48	1.3	1.6	1.2	1.4	1.4	1.6	1.2	4.7	6.4
	channel, shaker-related
	subfamily, member 3
Lat	linker for activation of T	AF036907	AF036905	18	32.1	35.5	25.0	31.5	17.9	43.8	34.9	205.2	179.7
	cells		AF036906
Lef1	lymphoid enhancer	NM_010703	AF294627	19	2.3	2.3	0.9	1.8	0.8	1.5	1.7	22.6	44.6
	binding factor 1		AF288571
			AW117601
			AI762816
Ltb	lymphotoxin B	NM_008518	NM_002341	8	41.1	66.4	52.2	70.2	30.3	65.5	59.8	354.8	366.2
Ly108	lymphocyte antigen 108	AF248636	NM_052931	61	5.4	5.4	6.0	4.7	3.3	6.4	3.9	7.7	8.9
Map4k1	mitogen activated protein	BB546619	NM_007181	7	11.5	13.1	11.0	15.0	7.5	12.2	10.4	56.3	81.1
	kinase 1
MGC37568	hypothetical protein	AU043488	BC006107	7	7.2	11.2	11.4	18.7	4.8	13.0	11.1	81.5	64.9
	MGC37568
MGC37914	hypothetical protein	BC021614	n/a	89	2.6	3.4	2.4	3.0	1.5	3.4	2.4	21.9	20.7
	MGC37914
Ms4a4c	membrane-spanning 4-	NM_029499	AF237912	136	8.2	8.3	7.0	4.0	3.4	8.5	4.4	16.7	24.7
	domains, subfamily A,		AF354928
	member 4C		NM_024021
Myb	myeloblastosis oncogene	NM_033597	NM_005375	5	9.2	7.9	4.1	5.8	2.2	6.0	3.2	59.7	50.0
Nfatc1	nuclear factor of activated	AK004810	NM_006162	150	2.9	4.3	3.8	3.8	2.7	4.5	3.3	7.9	13.5
	T-cells, cytoplasmic 1		NM_172387
			NM_172388
			NM_172389
			NM_172390
Pglyrpl-	peptidoglycan recognition	NM_021319	BE672390	3	1.4	2.9	5.6	5.6	1.3	4.1	3.2	47.0	34.1
pending	protein-like
Pik3cd	phosphatidylinositol 3-	NM_008840	U57843	10	6.8	10.5	15.1	12.5	9.0	12.8	7.5	111.8	154.1
	kinase catalytic delta		U86453
	polypeptide
Pik3cd	phosphatidylinositol 3-	BB700084	n/a	100	3.3	5.2	5.9	7.2	4.6	5.2	4.4	35.0	38.8
	kinase catalytic delta
	polypeptide
Plxnc1	plexin C1	BB765457	NM_005761	64	2.3	3.3	2.7	4.7	1.8	3.3	3.2	10.3	15.6
Pom121	nuclear pore membrane	C80273	AK022555	66	2.2	2.5	2.3	3.4	2.1	2.5	2.2	6.4	6.9
	protein 121
Prkcb	protein kinase C, beta	BF660388	NM_002738	6	5.3	7.3	6.5	11.3	4.1	7.2	5.6	13.2	18.9
			NM_212535
Rad51ap1	RAD51 associated protein 1	BC003738	NM_006479	71	2.1	2.0	1.3	1.5	1.3	2.2	1.7	10.2	11.3
Rgs10	regulator of G-protein	NM_026418	NM_002925	208	2.5	3.3	4.0	4.1	3.1	3.7	4.0	9.0	11.1
	signaling 10		AI744627
Rgs19	regulator of G-protein	BC003838	NM_005873	104	4.1	4.9	5.0	5.3	4.5	5.2	4.2	15.9	16.1
	signaling 19
Rog-	repressor of GATA	AK015881	NM_014383	11	10.9	9.5	6.8	3.8	5.3	11.0	2.1	49.3	143.0
pending
Selpl	selectin, platelet (p-	NM_009151	U02297	115	7.6	13.3	11.0	16.4	8.4	13.9	12.7	69.5	70.8
	selectin) ligand
Sema4d	sema domain,	NM_013660	NM_006378	149	2.3	2.8	2.6	2.4	1.4	3.5	2.5	8.0	10.4
	immunoglobulin domain
	(Ig), transmembrane
	domain (TM) and short
	cytoplasmic domain,
	(semaphorin) 4D
Sh3bp1	SH3-domain binding	NM_009164	NM_018957	48	4.3	7.4	4.1	7.6	2.1	7.2	7.4	10.8	14.3
	protein 1		AK024971
Slc1a7	solute carrier family 1,	NM_009201	AF105230	216	1.5	1.8	1.4	2.1	1.6	2.0	1.4	6.6	7.4
	member 7		BC000986
Slc2a3	solute carrier family 2	M75135	NM_006931	5	0.9	0.8	0.8	1.0	1.2	1.3	1.2	19.4	48.5
	(facilitated glucose		AL110298
	transporter), member 3
Stat4	signal transducer and	NM_011487	NM_003151	8	7.4	10.5	12.0	10.5	7.6	8.2	8.7	143.6	144.0
	activator of transcription 4
Stk10	serine/threonine kinase 10	NM_009288	AB015718	53	4.5	5.8	4.6	6.9	2.8	5.9	3.4	32.1	28.6
			NM_005990
			BE504180
			BE501281
			AF088069
Tacc3	transforming, acidic	BB787809	NM_006342	77	2.8	2.4	1.9	2.2	2.1	2.8	1.6	11.7	19.3
	coiled-coil containing		AF289576
	protein 3
Tcrb-V13	T-cell receptor beta,	M87849	n/a	15	6.5	6.4	4.5	5.7	3.9	7.1	4.2	19.2	27.3
	variable 13
Tcrb-	T-cell receptor beta,	BF318536	n/a	24	2.3	3.6	2.4	2.8	2.7	3.6	2.9	18.4	35.4
V8.2	variable 8.2
Trim34	tripartite motif protein 34	NM_030684	AB039904	94	2.8	3.7	4.4	3.7	3.9	3.6	2.5	7.7	8.4
			NM_021616
9-Sep	septin 9	NM_017380	AF142408	469	1.6	2.4	1.9	3.3	2.1	2.6	2.0	6.3	5.4
			AB023208
			NM_006640
2310021G01Rik	RIKEN cDNA	AK011289	AY029179	11	8.5	7.8	5.0	5.2	2.0	8.3	4.2	24.9	49.8
	2310021G01 gene
2700084L22Rik	RIKEN cDNA	NM_026024	AB032931	5	2.5	3.5	3.1	1.9	1.3	2.0	1.6	30.5	36.7
	2700084L22 gene
2810047L02Rik	RIKEN cDNA	AV270035	NM_016448	28	3.3	3.5	3.2	3.1	2.0	4.1	2.5	24.9	27.9
	2810047L02 gene
2810425K19Rik	RIKEN cDNA	BC025911	AF121856	6	2.1	4.6	1.5	3.5	3.2	1.3	0.9	9.4	11.0
	2810425K19 gene		NM_021249
3322402E17Rik	RIKEN cDNA	AK014382	AB006628	6	1.1	1.3	0.8	1.5	1.6	1.7	1.3	13.8	34.5
	3322402E17 gene
3322402E17Rik	RIKEN cDNA	BF730694	AB006628	14	6.0	8.6	6.5	8.6	2.8	9.5	9.2	38.3	80.9
	3322402E17 gene
5031419I10Rik	RIKEN cDNA	BB474868	NM_016573	39	2.9	3.7	2.3	3.6	2.7	3.9	3.8	8.7	13.1
	5031419I10 gene
5830400A04Rik	RIKEN cDNA	BM243660	NM_004310	14	5.7	8.7	10.5	11.5	9.6	9.1	7.4	43.0	102.2
	5830400A04 gene
9130017C17Rik	RIKEN cDNA	AF395844	AK055837	74	3.7	4.5	4.2	4.2	3.7	4.0	4.1	7.8	7.7
	9130017C17 gene		AW104269
			AI081246
			AA521424
			AL161979
A430104N18Rik	RIKEN cDNA	AA254104	n/a	25	4.0	5.5	6.6	7.0	6.0	3.9	4.8	72.9	125.6
	A430104N18 gene
AA409164	expressed sequence	BC006054	n/a	12	1.3	1.5	1.1	1.6	1.8	2.1	1.8	5.5	5.9
	AA409164
		AK004668	NM_012452	51	3.0	4.8	5.6	6.5	4.8	5.0	3.7	29.6	31.3
	Mus musculus BIC	AY096003	n/a	3	1.7	1.7	2.7	2.4	2.2	1.8	1.1	8.3	20.6
	noncoding mRNA,
	complete sequence.
		BG976607	n/a	75	2.9	2.3	2.4	2.2	1.1	2.4	1.3	9.4	12.6
	Mus musculus adult	AW557946	NM_016457	61	2.8	3.3	3.7	4.8	2.6	3.3	3.4	18.5	22.3
	female vagina cDNA,
	RIKEN full-length
	enriched library,
	clone: 9930101D06
	product: PROTEIN
	KINASE D2 homolog
	[Homo sapiens], full insert
	sequence.
	Mus musculus 9 days	AW552536	n/a	10	3.4	2.9	2.6	4.0	2.1	3.2	3.0	20.5	27.2
	embryo whole body
	cDNA, RIKEN full-length
	enriched library,
	clone: D030060F23
	product: Mus musculus
	U22 snoRNA host gene
	(UHG) gene, complete
	sequence, full insert
	sequence.
	Mus musculus adult male	BB014626	n/a	3	7.5	13.6	8.7	22.2	4.7	17.4	12.1	69.3	75.2
	testis cDNA, RIKEN full-
	length enriched library,
	clone: 4930483L24
	product: weakly similar to
	AT-HOOK PROTEIN
	AKNA [Homo sapiens],
	full insert sequence.
CLUSTER 4
Adcy7	adenylate cyclase 7	BB746807	NM_001114	73	8.2	12.6	20.1	16.9	12.5	14.2	12.7	53.2	34.4
AV278559	expressed sequence	BC026563	AA668763	83	7.0	8.7	9.1	9.2	7.9	9.7	6.7	69.9	24.8
	AV278559
C4st2-	chondroitin 4-	NM_021528	NM_018641	9	5.0	8.9	10.3	13.9	11.0	10.3	9.7	32.9	21.5
pending	sulfotransferase 2		BC002918
			BC029471
			BC029471
C79673	expressed sequence	BG066664	NM_031471	34	6.5	14.1	20.3	19.0	17.6	18.0	15.7	56.8	54.5
	C79673		NM_178443
Cd80	CD80 antigen	AA596883	NM_005191	31	1.5	1.0	1.9	2.3	1.7	1.2	1.6	9.8	5.2
Cd8a	CD8 antigen, alpha chain	AK017889	NM_001768	14	18.4	36.3	45.6	33.4	23.2	41.3	26.9	100.2	84.3
			NM_171827
Cd8b	CD8 antigen, beta chain	U34882	AW296309	22	26.9	39.6	50.3	40.0	24.8	47.1	29.1	251.6	111.8
			NM_172100
			NM_004931
Crmp1	collapsin response	AB006714	NM_001313	14	1.9	2.6	7.3	3.7	4.5	3.5	3.9	69.8	8.7
	mediator protein 1
Cxcr6	chemokine (C—X—C motif)	NM_030712	NM_006564	13	5.3	17.8	34.2	27.9	16.2	11.8	14.6	434.8	15.9
	receptor 6
Dock2	dedicator of cyto-kinesis 2	NM_033374	D86964	19	17.1	28.9	36.4	46.6	23.9	28.9	30.0	200.5	116.4
			BC016996
E430024D12	hypothetical protein	AV173260	AI342543	6	5.2	8.7	10.8	10.3	10.1	10.7	9.1	155.5	66.1
	E430024D12
Evi2	ecotropic viral integration	BB201368	NM_006495	19	14.2	24.5	27.0	24.5	19.9	20.9	16.9	107.4	55.5
	site 2
Flt3l	FMS-like tyrosine kinase	L23636	U03858	43	1.8	2.2	3.3	2.8	2.5	2.3	2.2	14.3	8.1
	3 ligand		NM_001459
Glipr2	GLI pathogenesis-related 2	AK017557	NM_022343	17	11.1	11.5	23.6	20.7	14.2	23.0	16.3	118.4	67.9
Gng2	guanine nucleotide	BC021599	NM_053064	14	10.3	16.5	28.0	15.7	24.0	17.7	14.1	182.8	45.9
	binding protein (G
	protein), gamma 2 subunit
Gpr34	G protein-coupled	NM_011823	NM_005300	5	1.0	1.3	2.1	2.5	2.3	0.9	2.0	16.5	6.9
	receptor 34
Hcls1	hematopoietic cell specific	NM_008225	NM_005335	8	31.0	44.1	51.3	48.2	33.3	44.8	36.1	175.3	75.3
	Lyn substrate 1
Hcst	hematopoietic cell signal	AF172930	AF285447	228	1.3	1.5	2.0	2.0	1.6	1.8	1.7	8.9	4.7
	transducer
Il18r1	interleukin 18 receptor 1	NM_008365	NM_003855	60	8.8	10.9	5.8	10.8	8.7	10.1	7.8	75.9	19.1
Klrc1	killer cell lectin-like	AF106008	NM_002260	5	8.7	16.6	33.2	20.3	13.3	20.3	10.1	331.4	11.3
	receptor subfamily C,		NM_002261
	member 1
Klrd1	killer cell lectin-like	NM_010654	U30610	27	14.4	19.4	28.3	22.5	27.9	22.9	18.8	489.9	94.6
	receptor, subfamily D,		AB009597
	member 1		NM_007334
Ly75	lymphocyte antigen 75	NM_013825	NM_002349	30	1.6	2.5	2.5	3.0	2.4	1.9	1.5	6.2	4.1
Ly9	lymphocyte antigen 9	NM_008534	NM_002348	7	8.2	13.3	22.9	19.4	19.7	18.4	16.5	82.2	42.8
Myolg	myosin IG	BB235320	NM_033054	98	7.2	8.4	10.6	11.0	6.3	8.7	8.1	70.3	29.0
Pik3cg	phosphoinositide-3-kinase,	BB205102	AF327656	20	2.7	3.9	6.3	5.8	3.9	3.7	3.1	19.8	11.3
	catalytic, gamma		NM_002649
	polypeptide
Plcl2	phospholipase C-like 2	BM207017	NM_015184	144	2.4	2.8	4.1	3.7	4.8	3.6	3.3	8.1	6.9
Plek	pleckstrin	AF303745	NM_002664	22	17.0	17.1	25.6	15.2	20.4	20.4	15.1	31.1	25.7
Rgs16	regulator of G-protein	U94828	U94829	3	6.5	13.3	12.2	16.3	14.5	16.5	13.9	17.1	8.7
	signaling 16
Ripk3	receptor-interacting	NM_019955	NM_006871	48	5.5	6.8	11.7	7.4	10.1	8.2	6.3	27.6	15.1
	serine-threonine kinase 3
Runx2	runt related transcription	D14636	L40992	52	3.1	4.6	4.0	7.1	4.0	3.9	3.2	46.1	16.8
	factor 2		NM_004348
			NM_004348
			AL353944
Sla	src-like adaptor	NM_009192	NM_006748	102	4.0	5.1	5.8	4.8	5.2	5.6	3.8	13.6	9.6
Sla2	Src-like-adaptor 2	AF287467	AF290986	24	1.5	8.1	6.3	5.9	2.9	6.6	3.1	23.5	15.7
Sp100	nuclear antigen Sp100	U83636	AF056322	89	2.5	2.9	3.5	3.5	2.5	3.3	2.7	28.1	10.0
			U36501
			U36501
			NM_003113
			NM_003113
Tcrb-V13	T-cell receptor beta,	U63547	n/a	5	1.5	3.0	3.4	2.3	1.2	2.2	1.4	26.6	7.8
	variable 13
Tcrg-V2	T-cell receptor gamma,	X03802	n/a	22	1.9	2.1	2.8	4.6	3.1	1.9	3.1	16.7	11.8
	variable 2
Tnfsf6	tumor necrosis factor	NM_010177	AF288573	73	1.8	2.4	4.2	4.2	2.8	2.8	3.0	57.7	20.3
	(ligand) superfamily,		D38122
	member 6
Tpm3	tropomyosin 3, gamma	NM_022314	AF362887	16	5.7	4.2	6.3	4.2	4.8	4.0	2.9	26.0	8.9
			AF362887
			AY004867
			BC000771
			X04201
Trex1	three prime repair	NM_011637	AJ243797	100	6.7	6.0	9.9	8.4	7.5	9.3	6.9	12.0	8.3
	exonuclease 1		NM_130384
			NM_016381
			BC002903
Trim12	tripartite motif protein 12	BM244351	n/a	3	2.0	3.7	7.1	5.4	3.1	4.0	3.1	30.6	17.2
Vav1	vav 1 oncogene	NM_011691	NM_005428	7	4.7	7.2	7.2	8.2	8.6	7.3	5.8	26.3	17.3
2410004L22Rik	RIKEN cDNA	NM_029621	NM_033417	23	4.5	7.0	7.6	7.3	5.4	6.3	6.3	24.8	16.8
	2410004L22 gene
2810433K01Rik	RIKEN cDNA	NM_025581	BF038461	2	1.0	1.4	2.3	1.2	1.0	1.1	0.9	33.2	9.2
	2810433K01 gene
4930422C14	hypothetical protein	BM241008	n/a	33	15.1	17.1	20.7	11.4	15.6	20.0	10.1	202.2	45.1
	4930422C14
9830126M18	hypothetical protein	BM224662	NM_019018	124	3.3	4.5	4.8	4.6	4.5	4.6	3.8	12.8	6.8
	9830126M18
		NM_011558	n/a	25	7.4	10.9	30.7	18.6	21.2	9.7	14.1	138.9	43.4
	Mus musculus adult female vagina cDNA, RIKEN full-length	BB204677	NM_016457	81	1.7	1.8	2.7	2.3	2.8	2.0	1.6	5.8	5.7
	enriched library, clone: 9930101D06 product: PROTEIN
	KINASE D2 homolog [Homo sapiens], full insert
	sequence.
CLUSTER 5
Pdcd1	programmed cell death 1	NM_008798	NM_005018	13	15.6	25.7	38.3	27.5	21.9	26.9	17.6	22.9	18.5
Socs1	suppressor of cytokine	AB000710	AB005043	46	10.5	9.3	7.5	9.3	7.1	13.3	8.6	7.9	26.9
	signaling 1		U88326
Stat1	signal transducer and activator of transcription 1	NM_009283	NM_007315	359	17.1	15.0	23.2	14.1	18.1	15.9	13.8	7.1	11.7
			NM_139266
		BC002065	n/a	95	45.0	42.6	81.1	50.9	68.8	56.8	48.5	13.7	33.4
Numbers indicate signal strength for NCBA and fold changes versus NCBA for allografts and lymphocyte cultures.
The abbreviations are as follows:
NCBA = normal CBA kidney;
WT allografts = CBA kidneys rejecting in wild-type B6 hosts;
IghKO allografts = CBA kidneys rejecting in B-cell deficient B6 hosts;
CTL = CTL clone;
MLRD4 = mixed lymphocyte culture day 4;
D5 = day 5 post transplant.

Expression of CA Ts in Allografts Rejecting in B-Cell Deficient Hosts

Whether the absence of B cells affects T-cell mediated rejection was analyzed by comparing CAT expression in kidneys rejecting in wild-type hosts to those rejecting in IghKO hosts at day 7 and day 21. The level of expression of CATs in grafts rejecting in IghKO hosts was highly correlated with that in wild-type hosts (D7: r=0.98; D21: r=0.98). The mean expression of the five clusters of CATs was also similar in IghKO versus wild-type hosts (FIG. 8), but was slightly higher in IghKO at day 7 (mean 23.2 percent in wild-type versus 25.3 percent in IghKO of the signal in the CTL clone) and lower in IghKO at day 21 (mean 26.2 percent in wild-type compared to the CTL clone versus 21.1 percent in IghKO).

In summary, the relationship between the pathologic Banff lesions of kidney rejection and the transcriptome, particularly in the CTL-associated transcripts, was studied. The interstitial infiltrate was established by day 5 and stable after day 7, whereas tubulitis and arteritis evolved slowly and progressively, being absent at day 5 and fully developed only after 14 days. The transcriptome changed markedly by day 5, with appearance of T cell and macrophage CD antigen transcripts. A set of CATs present in d4MLR and in a CTL clone but absent in normal kidney were identified. The CATs appeared in the transplants with a mean signal intensity about one fifth of that in the CTL clone, and was independent of B cells and alloantibody. In addition, CAT expression was essentially constant from day 5 through 42, despite massive changes in the histopathology. Thus, CTL transcripts appear early in rejecting kidneys, before the diagnostic Banff lesions, and persist for at least 6 weeks, providing a robust measurement of this aspect of rejection. This permits separation of the effectors of rejection, CTL, from the downstream consequences, parenchymal deterioration and pathologic lesions. In addition, CAT expression provides an approximation of the effector T cell burden and activity in rejecting kidneys. The interpretation of the CAT expression does not depend on the assumption that CATs are expressed exclusively in CTL, although it is likely that CTL account for most CAT expression.

The CD transcripts provide an overview of leukocyte population changes, and support the concept of a CTL and macrophage infiltrate with late B cell infiltration indicated by the histologic analysis. There is no real “gold standard” unbiased assessment of the composition of the infiltrate in rejecting transplants: both immunostaining of sections and cell isolation have potential for errors. Nevertheless, the arrays' estimates are fully compatible with estimates based on these methods. CD transcripts with high expression in CTL and d4MLR increased early during rejection and persisted throughout the time course, consistent with CTL infiltration and supporting the contention that CATs in the rejecting kidneys reflect transcripts in effector T cells. The macrophage markers CD14 and CD68 were present in rejecting kidneys, with low expression in CTL and d4MLR, consistent with macrophage infiltration. B cell markers CD79A and CD79B were present in d4MLR but not CTL, and appeared late in rejection, reflecting late B cell infiltration. There were few CD4⁺ cells in the infiltrate by immunostaining, and CD4 expression in the microarrays was low, in keeping with rejection being mainly driven by CD8⁺ CTL.

The constancy of CAT expression over weeks establishes a new concept of T cell mediated rejection, namely that CTL generated from secondary lymphoid organs create and maintain a constant state in which the parenchyma progressively changes, yielding the pathologic lesions. The surprising stability of CAT levels over time suggests that the CTLs in the graft are occupying a finite “space,” similar to other emerging concepts of space in the secondary lymphoid organs (Stockinger et al., Immunology, 111(3):241-247 (2004)). The differences in the regression coefficients indicate that relative expression of individual CATs was consistent over time in vivo, although somewhat altered relative to the patterns of expression in vitro in the d4MLR and CTL clone. The moderate differences in relative expression of transcripts in the in vivo grafts versus the in vitro conditions may reflect different stimuli for CTL in these conditions (e.g., CD44). Other cells may also be recruited to express selected CAT in vivo: transcripts in cluster 5 exhibited high expression in vivo, perhaps reflecting IFN-γ effects (e.g., STAT1). The algorithm defining CATs, however, may exclude most IFN-γ inducible genes.

B cells do appear late in kidney rejection in this model but have no critical role, either as antigen presenting cells or alloantibody producing cells. Grafts in IghKO hosts exhibited very similar CAT expression to those in wild-type hosts by regression analysis, with slightly higher mean CAT expression at day 7 and lower at day 21. The small decline in CAT expression at day 21 in B cell deficient hosts suggest a role of B cells as second line antigen presenting cells sustaining CTL generation in secondary lymphoid organs.

The sustained expression of transcripts associated with cytotoxicity (e.g., perforin, granzymes A and B) in rejecting grafts raises the question of the role of cytotoxic mechanisms. Typical lesions develop in mice lacking perforin or granzyme A plus granzyme B (Halloran et al., Am. J. Transplant., 4(5):705-712 (2004)). Fas ligand (Tnfsf6) is expressed in CTL and rejecting grafts, but is not necessary for organ rejection across MHC disparities (Larsen et al., Transplant, 60(3):221-224 (1995)). Thus, the alterations in the parenchyma could reflect non-cytotoxic CTL and macrophage products, acting either by direct engagement or by indirect actions, e.g., extracellular matrix alterations triggering secondary changes in the epithelium. On the other hand, the lytic mechanisms such as perforin, granzymes, and Fas ligand could contribute to homeostasis, through fratricide of T cells (Huang et al., Science, 286(5441):952-954 (1999)) or interactions with antigen presenting cells (Ludewig et al., Eur. J. Immunol., 31(6): 1772-1779 (2001)).

CAT expression can be used in estimating the burden of CTL in rejecting grafts, by analogy with viral load measurements in viral diseases. Moreover, although CD8⁺ CTL were used as the basis of the effector T cell signature, the definition of CATs probably includes most transcripts in CD4⁺ effector T cells. Less is known about effector CD4⁺ T cells in rejection, perhaps because CD8⁺ effectors develop more rapidly after short term stimulation (Seder and Ahmed, Nat. Immunol., 4(9):835-842 (2003)). CD4⁺ T cells may play a bigger role in human kidney allograft rejection than in mice, although in human rejection CD8⁺ T cells predominate (Hancock et al., Transplant, 35(5):458-463 (1983)). CD4⁺ effectors that home to inflammatory sites share many properties with CD8⁺ effectors, e.g., IFN-γ production, expression of P-selectin ligand and CXCR3, absence of CCR7 (Campbell et al., Nat. Immunol., 2(9):876-881 (2001)). Other transcript sets can be developed to reflect distinct events in a disease state, e.g., IFN-γ inducible transcripts or macrophage-associated transcripts.

Example 2

Kidney Rejection in Humans

Human Database and Comparison with Mouse Transcripts

Data obtained from the mouse model were compared to the gene expression data obtained from human kidney biopsies from nine living donor controls, seven recipients with histologically confirmed acute rejection, five recipients with renal dysfunction without rejection on biopsy, and 10 protocol biopsies carried out more than one year post-transplant in patients with good transplant function and normal histology. Microarray data from these biopsies were obtained from a database available on the World Wide Web at scrips.edu/services/dna_array/. Flechner et al., Halloran laboratory Reference Manager # 18134: Am. J. Transplant., 4(9):1475-1489 (2004)). Raw data were normalized as described herein for the mouse data, using the donor biopsies as controls. In GeneSpring, a homology database was created for the mouse and human data, and gene lists of interest were then used for supervised hierarchical clustering of the human biopsy samples.

CTL Gene Expression in Human Kidney Transplant Biopsies

The following was performed to determine whether or not the transcriptome pattern observed in mouse CTL and in rejecting mouse kidney reflects the rejection process in human transplant kidneys. A set of human kidney biopsies was analyzed based on the CTL signature identified in the mouse model. The database includes biopsies of normal kidneys (healthy donor biopsies), control biopsies of well functioning kidney transplants, rejecting transplants, and transplants with dysfunction but no rejection. The expression of CTL genes identified in mice in a published database of human renal transplants was examined. Of the 284 mouse CTL transcripts, 164 corresponding transcripts in the human database were identified. Supervised hierarchical cluster analysis based on the CTL transcripts separated the rejecting transplants from the other samples. In rejecting transplants, gene expression of CTL transcripts was increased compared to normal transplants with dysfunction but no rejection. Compared to donor biopsies, control biopsies of well functioning transplants had decreased expression of a subset of CTL transcripts, possibly due to immunosuppressive treatment. Another subset of transcripts exhibited increased expression in control biopsies, indicating some CTL activity in the transplant; however, expression levels were much lower than in rejecting kidneys. A class prediction model based on two classes (rejection—no rejection) identified 19 of the 21 samples correctly based on the expression of CTL transcripts in transplant biopsies (using the 100 best predictor genes (Fisher's Exact Test) and K-nearest neighbors (K=4)). The two samples that could not be classified were diagnosed as “borderline rejection” (AR5) and “tubular nephropathy” (NR5) based on histologic criteria.

In a first analysis of human kidney biopsies, the set of CTL genes identified in the mouse model exhibited striking upregulation in rejecting kidneys and permitted identification of samples from rejecting transplants without further refinement, indicating that the transcriptome patterns observed in rejecting mouse kidney reflect the rejection process in human transplant kidneys. Although this analysis includes only a limited number of human biopsies and may require verification and further refinement in a large patient population, this is a first indication that analysis of the CTL pattern in the transcriptome of kidney biopsies can be used as a diagnostic tool. Addition of other elements of the transcriptome to the CTL gene set may improve the diagnostic power, therefore allowing refinement of the gene set and reduction of the number of transcripts required for a diagnosis. The clinical application of this knowledge can involve either a microarray system using large numbers of genes or an RT-PCR system, depending on an evaluation of sensitivity, specificity, cost, and practicability. Based on the observation in the mouse model that transcriptome changes occur early before tubulitis develops, this approach can be more sensitive and quantitative than evaluation by histopathology and could be developed for use as an endpoint in clinical trials.

Example 3

CATs Identified Using a Second Algorithm

A second, more refined algorithm was used to identify CATs. This method involved RMA (robust multichip analysis). CATs were identified based on the following: a signal of less than 50 in normal kidneys in all three strains (CBA, B6, and Balbc); five times higher in CTL, MLR, and CD8 compared to normal kidneys; significantly (p (fdr)<0.01) higher in MLR versus normal kidney; two times increased in wild type allografts (CBA into B6) at day 5 compared to normal kidney; and significant in comparison to normal kidney (p(fdr)<0.01). This algorithm produced a list of 332 CATs, 91 of which were included in the original list of 287 CATs. The new list was checked for polymorphisms that would have been excluded if there had been any polymorphisms (5× difference between the strains or genes that are known to be highly polymorphic e.g., TCR, NKR, Ig, MHC). The list of 332 CATs is provided in Table 5.

TABLE 5
CATs identified using an RMA-based algorithm.
						Locus
Systematic	Symbol	Title	Genbank	Swissprot	Unigene	link
1424965_at	Lpxn	leupaxin	BC026563	0	Mm.313136	107321
1416016_at	Tap1	transporter 1, ATP-	AW048052	P21958,	Mm.207996	21354
		binding cassette, sub-		Q62427,
		family B		Q62428,
		(MDR/TAP)		Q62429,
				Q64333
1425226_x_at	Tcrb-V13	T-cell receptor beta,	M16120	0	Mm.333026	269846
		variable 13
1433935_at	AU020206	expressed sequence	BI151331	0	Mm.200422	101757
		AU020206
1419194_s_at	Gmfg	glia maturation	NM_022024	0	Mm.194536	63986
		factor, gamma
1451174_at	Lrrc33	leucine rich repeat	BC027411	0	Mm.33498	224109
		containing 33
1454169_a_at	Epstil	epithelial stromal	AK017174	0	Mm.68134	108670
		interaction 1 (breast)
1449127_at	Selpl	selectin, platelet (p-	NM_009151	Q62170	Mm.332590	20345
		selectin) ligand
1436199_at	Trim14	Tripartite motif-	AU042532	0	Mm.240252	74735
		containing 14
1436423_at	E430004N04Rik	RIKEN cDNA	BE628523	0	Mm.123021	210757
		E430004N04 gene
1439595_at	Tcra	T-cell receptor alpha	BM243643	0	Mm.213248	21473
		chain
1452352_at	Ctla2b	cytotoxic T	BG064656	0	0	13025
		lymphocyte-
		associated protein 2
		beta
1437886_at	Klhl6	kelch-like 6	BM247104	0	Mm.86699	239743
		(Drosophila)
1460245_at	Klrd1	killer cell lectin-like	NM_010654	O54707,	Mm.8186	16643
		receptor, subfamily		O54708
		D, member 1
1449925_at	Cxcr3	chemokine (C—X—C	NM_009910	O88410	Mm.12876	12766
		motif) receptor 3
1436212_at	AI661017	expressed sequence	AV173260	0	Mm.132299	213068
		AI661017
1444088_at		Similar to T-cell	BE447255	P04212	Mm.347827	381764
		receptor beta chain
		VNDNJC precursor
1440811_x_at	Cd8a	CD8 antigen, alpha	BB030365	P01731,	Mm.1858	12525
		chain		Q60965
1456064_at	AI504432	expressed sequence	AI323624	0	Mm.347584	229694
		AI504432
1448759_at	Il2rb	interleukin 2	M28052	P16297	Mm.35287	16185
		receptor, beta chain
1417597_at	Cd28	CD28 antigen	NM_007642	P31041	Mm.255003	12487
1429270_a_at	1700013H19Rik	RIKEN cDNA	AK005954	0	Mm.229128	71846
		1700013H19 gene
1426025_s_at	Laptm5	lysosomal-associated	U29539	Q61168,	Mm.271868	16792
		protein		Q60924
		transmembrane 5
1449220_at	Gimap3	GTPase, IMAP	NM_031247	0	Mm.333050	83408
		family member 3
1420876_a_at	6-Sep	septin 6	NM_019942	0	Mm.260036	56526
1456494_a_at	Trim30	tripartite motif	BG068242	P15533	Mm.295578	20128,
		protein 30				209387
1436570_at		Transcribed locus	BG143461	0	Mm.23897	0
1419178_at	Cd3g	CD3 antigen, gamma	M58149	P11942	Mm.335106	12502
		polypeptide
1434280_at			BG976607	0	0	0
1448713_at	Stat4	signal transducer and	NM_011487	P42228	Mm.1550	20849
		activator of
		transcription 4
1417171_at	Itk	IL2-inducible T-cell	NM_010583	Q03526	Mm.339927	16428
		kinase
1416118_at			NM_025863	0	0	0
1423760_at	Cd44	CD44 antigen	M27130	P15379	Mm.330428	12505
1434929_at	BC035044	cDNA sequence	BI076809	0	Mm.373829	232406
		BC035044
1454764_s_at		Transcribed locus	BF165681	0	Mm.376972	0
1416956_at	Kcnab2	potassium voltage-	U31908	P62482	Mm.302496	16498
		gated channel,
		shaker-related
		subfamily, beta
		member 2
1417546_at	Il2rb	interleukin 2	M28052	P16297	Mm.35287	16185
		receptor, beta chain
1419569_a_at	Isg20	interferon-stimulated	BC022751	0	Mm.322843	57444
		protein
1454850_at	Tbc1d10c	TBC1 domain family,	AV060417	0	Mm.288312	108995
		member 10c
1434380_at		Diabetic	BM241271	0	Mm.254851	0
		nephropathy-like
		protein (Dnr12)
		mRNA, partial
		sequence
1426396_at	Cd3z	CD3 antigen, zeta	AK017904	P29020,	Mm.217308	12503
		polypeptide		P24161
1443937_at	Il2rb	Interleukin 2	BE634648	P16297	Mm.35287	16185
		receptor, beta chain
1454893_at	1110013L07Rik	RIKEN cDNA	BB765852	0	Mm.274708	68521
		1110013L07 gene
1418842_at	Hcls1	hematopoietic cell	NM_008225	P49710	Mm.4091	15163
		specific Lyn substrate 1
1425396_a_at	Lck	lymphocyte protein	BC011474	P06240	Mm.293753	16818
		tyrosine kinase
1429197_s_at	Rabgap1l	RAB GTPase	BB431654	0	Mm.25833	29809
		activating protein 1-
		like
1436097_x_at	Arhgap9	Rho GTPase	BB327418	0	Mm.227198	216445
		activating protein 9
1438439_at	Gpr171	G protein-coupled	BB229616	0	Mm.123648	229323
		receptor 171
1431592_a_at	Sh3kbp1	SH3-domain kinase	AK007283	0	Mm.286495	58194
		binding protein 1
1455899_x_at	Socs3	suppressor of	BB241535	O35718	Mm.3468	12702
		cytokine signaling 3
1419193_a_at	Gmfg	glia maturation	NM_022024	0	Mm.194536	63986
		factor, gamma
1457725_at	0	Similar to membrane-	BB221406	0	Mm.233909	381214
		spanning 4-domains,
		subfamily A, member
		4C; membrane-
		spanning 4-domains,
		subfamily A, member 9
1434745_at	Ccnd2	cyclin D2	BQ175880	P30280	Mm.333406	12444
1423614_at	Lrrc8c	leucine rich repeat	BB329408	0	Mm.319847	100604
		containing 8 family,
		member C
1427539_a_at	Zwint	ZW10 interactor	BC013559	0	Mm.62876	52696
1454632_at	6330442E10Rik	RIKEN cDNA	AV328515	0	Mm.341747	268567
		6330442E10 gene
1424542_at	S100a4	S100 calcium binding	D00208	P07091	Mm.3925	20198
		protein A4
1435331_at	AI447904	expressed sequence	BM241008	0	Mm.360525	236312
		AI447904
1448441_at	Cks1b	CDC28 protein	NM_016904	P61025	Mm.3049	54124
		kinase 1b
1436171_at	Arhgap30	Rho GTPase	BM244999	0	Mm.251048	226652
		activating protein 30
1455576_at	5830482F20Rik	RIKEN cDNA	AW493583	0	Mm.74632	320435
		5830482F20 gene
1417104_at	Emp3	epithelial membrane	BC001999	O35912	Mm.20829	13732
		protein 3
1424727_at	Ccr5	chemokine (C—C	D83648	P51682	Mm.14302	12774
		motif) receptor 5
1419033_at	2610018G03Rik	RIKEN cDNA	AW556821	0	Mm.377135	70415
		2610018G03 gene
1416246_a_at	Coro1a	coronin, actin binding	BC002136	O89053	Mm.290482	12721
		protein 1A
1439956_at	0	Adult male aorta and	BE692425	0	Mm.123404	0
		vein cDNA, RIKEN
		full-length enriched
		library,
		clone: A530049N04
		product: unknown
		EST, full insert
		sequence
1433466_at	AI467606	expressed sequence	BB234337	0	Mm.284102	101602
		AI467606
1424560_at	Pstpip1	proline-serine-	U87814	P97814	Mm.2534	19200
		threonine
		phosphatase-
		interacting protein 1
1425947_at	Ifng	interferon gamma	K00083	P01580	Mm.240327	15978
1460338_a_at	Crlf3	cytokine receptor-like	BB161253	0	Mm.272093	54394
		factor 3
1450698_at	Dusp2	dual specificity	L11330	Q05922	Mm.4729	13537
		phosphatase 2
1438052_at	A130071D04Rik	RIKEN cDNA	BM239436	0	0	320791
		A130071D04 gene
1425335_at	Cd8a	CD8 antigen, alpha	M12825	P01731,	Mm.1858	12525
		chain		Q60965
1455898_x_at	Slc2a3	solute carrier family	BB414515	P32037,	Mm.269857	20527
		2 (facilitated glucose		Q61607
		transporter), member 3
1419135_at	Ltb	lymphotoxin B	NM_008518	P41155	Mm.1715	16994
1416022_at	Fabp5	fatty acid binding	BC002008	Q05816	Mm.741	16592
		protein 5, epidermal
1434873_a_at	Centb1	centaurin, beta 1	BB115902	0	Mm.288671	216859
1460419_a_at	Prkcb1	protein kinase C, beta 1	X59274	P68404	Mm.207496	18751
1441677_at	Smc4l1	SMC4 structural	BM244144	0	Mm.206841	70099
		maintenance of
		chromosomes 4-like
		1 (yeast)
1448500_a_at	Lime1	Lck interacting	NM_023684	0	Mm.272712	72699
		transmembrane
		adaptor 1
1447788_s_at	AW212607	expressed sequence	BB308532	0	Mm.277243	241732
		AW212607
1424927_at	Glipr1	GLI pathogenesis-	BC025083	0	Mm.173790	73690
		related 1 (glioma)
1455000_at	Gpr68	G protein-coupled	BB538372	0	Mm.32160	238377
		receptor 68
1439034_at	Spn	sialophorin	BB160586	0	Mm.283714	20737
1425854_x_at	Tcrb-V13	T-cell receptor beta,	U07661	0	Mm.333026	269846
		variable 13
1418126_at	Ccl5	chemokine (C—C	NM_013653	P30882	Mm.284248	20304
		motif) ligand 5
1437176_at	LOC434341	similar to nucleotide-	AV277444	0	0	434341
		binding
		oligomerization
		domains 27
1424278_a_at	Birc5	baculoviral IAP	BC004702	O70201	Mm.8552	11799
		repeat-containing 5
1424923_at	Serpina3g	serine (or cysteine)	BC002065	Q62259	Mm.264709	20715
		proteinase inhibitor,
		clade A, member 3G
1435529_at	0	Brain CRL-1443	BM245961	0	Mm.371956	0
		BC3H1 cDNA,
		RIKEN full-length
		enriched library,
		clone: G430091H17
		product: weakly
		similar to
		GLUCOCORTICOID-
		ATTENUATED
		RESPONSE GENE
		16 PRODUCT
		[Rattus norvegicus],
		full insert sequence
1416296_at	Il2rg	interleukin 2	L20048	P34902	Mm.2923	16186
		receptor, gamma
		chain
1424181_at	38966	septin 6	BC010489	0	Mm.260036	56526
1451099_at	Mbc2	membrane bound C2	BC011482	0	Mm.66056	23943
		domain containing
		protein
1426652_at	Mcm3	minichromosome	BI658327	P25206	Mm.4502	17215
		maintenance deficient
		3 (S. cerevisiae)
1416869_x_at	Lime1	Lck interacting	NM_023684	0	Mm.272712	72699
		transmembrane
		adaptor 1
1452954_at	Ube2c	ubiquitin-conjugating	AV162459	0	Mm.89830	68612
		enzyme E2C
1440196_at	0	3 days neonate	BB207611	0	Mm.1891	0
		thymus cDNA,
		RIKEN full-length
		enriched library,
		clone: A630020E03
		product: unknown
		EST, full insert
		sequence
1452117_a_at	Fyb	FYN binding protein	BB157866	O35601	Mm.170905	23880
1450842_a_at	Cenpa	centromere	AV132173	O35216	Mm.290563	12615
		autoantigen A
1427325_s_at	AI597013	expressed sequence	BB014626	0	Mm.258930	100182
		AI597013
1437432_a_at	Trim12	tripartite motif	BM244351	0	Mm.327033	76681
		protein 12
1418980_a_at	Cnp1	cyclic nucleotide	M58045	P16330	Mm.15711	12799
		phosphodiesterase 1
1427007_at	1200013B08Rik	RIKEN cDNA	AK004734	0	Mm.276131	74131
		1200013B08 gene
1435945_a_at	Kcnn4	potassium	BG865910	O89109	Mm.9911	16534
		intermediate/small
		conductance calcium-
		activated channel,
		subfamily N, member 4
1451910_a_at	Cd6	CD6 antigen	U12434	Q61003	Mm.290897	12511
1422808_s_at	Dock2	dedicator of cytokinesis 2	NM_033374	0	Mm.217288	94176
1423895_a_at	Cugbp2	CUG triplet repeat,	BB644164	0	Mm.147091	14007
		RNA binding protein 2
1418770_at	Cd2	CD2 antigen	NM_013486	P08920	Mm.22842	12481
1418465_at	Ncf4	neutrophil cytosolic	NM_008677	P97369	Mm.2068	17972
		factor 4
1418641_at	Lcp2	lymphocyte cytosolic	BC006948	Q60787	Mm.265350	16822
		protein 2
1448409_at	Lrmp	lymphoid-restricted	NM_008511	Q60664	Mm.843	16970
		membrane protein
1436953_at	Waspip	Wiskott-Aldrich	C76969	0	Mm.223504	215280
		syndrome protein
		interacting protein
1416619_at	4632428N05Rik	RIKEN cDNA	BC003967	0	Mm.273584	74048
		4632428N05 gene
1417898_a_at	Gzma	granzyme A	NM_010370	P11032	Mm.15510	14938
1449393_at	Sh2d1a	SH2 domain protein	NM_011364	O88890	Mm.235391	20400
		1A
1438577_at	0	Transcribed locus	BB376947	0	Mm.130040	0
1416759_at	Mical1	microtubule	NM_138315	0	Mm.290431	171580
		associated
		monoxygenase,
		calponin and LIM
		domain containing 1
1436905_x_at	Laptm5	lysosomal-associated	BB218107	Q61168,	Mm.271868	16792
		protein		Q60924
		transmembrane 5
1418396_at	Gpsm3	G-protein signalling	NM_134116	0	Mm.26584	106512
		modulator 3 (AGS3-
		like, C. elegans)
1424724_a_at	D16Ertd472e	DNA segment, Chr	BC019957	0	Mm.37332	67102
		16, ERATO Doi 472,
		expressed
1429947_a_at	Zbp1	Z-DNA binding	AK008179	0	Mm.116687	58203
		protein 1
1448748_at	Plek	pleckstrin	AF181829	0	Mm.98232	56193
1417620_at	Rac2	RAS-related C3	NM_009008	Q05144	Mm.1972	19354
		botulinum substrate 2
1427911_at	2610307O08Rik	RIKEN cDNA	AK012006	0	Mm.45995	72512
		2610307O08 gene
1451154_a_at	Cugbp2	CUG triplet repeat,	BB644164	0	Mm.147091	14007
		RNA binding protein 2
1416008_at	Satb1	special AT-rich	AV172776	Q60611	Mm.311655	20230
		sequence binding
		protein 1
1442700_at	Pde4b	phosphodiesterase	BG793493	0	Mm.20181	18578
		4B, cAMP specific
1437249_at	Scap1	src family associated	BG075562	0	Mm.340720	78473
		phosphoprotein 1
1438475_at	0	0	BM246462	0	0	0
1421931_at	Icos	inducible T-cell co-	AB023132	0	Mm.42044	54167
		stimulator
1419206_at	Cd37	CD37 antigen	BC019402	Q61470	Mm.3689	12493
1449175_at	Gpr65	G-protein coupled	NM_008152	Q61038	Mm.207528	14744
		receptor 65
1422701_at	Zap70	zeta-chain (TCR)	NM_009539	P43404,	Mm.8038	22637
		associated protein		P97455
		kinase
1450291_s_at	Ms4a4c	membrane-spanning	NM_022429	0	Mm.353643	64380
		4-domains, subfamily
		A, member 4C
1417601_at	Rgs1	regulator of G-protein	NM_015811	0	Mm.103701	50778
		signaling 1
1437072_at	Arhgap25	Rho GTPase	BM241218	0	Mm.119564	232201
		activating protein 25
1436847_s_at	Cdca8	cell division cycle	BB702047	0	Mm.28038	52276
		associated 8
1457404_at	Nfkbiz	nuclear factor of	BM240058	0	Mm.247272	80859
		kappa light
		polypeptide gene
		enhancer in B-cells
		inhibitor, zeta
1421173_at	Irf4	interferon regulatory	U34307	Q64287	Mm.4677	16364
		factor 4
1416295_a_at	Il2rg	interleukin 2	L20048	P34902	Mm.2923	16186
		receptor, gamma
		chain
1428242_at	6330406L22Rik	RIKEN cDNA	AK018130	0	Mm.243954	70719
		6330406L22 gene
1418392_a_at	Gbp4	guanylate nucleotide	NM_018734	Q61107	Mm.1909	55932
		binding protein 4
1437025_at	Cd28	CD28 antigen	AV313615	P31041	Mm.255003	12487
1422637_at	Rassf5	Ras association	NM_018750	O70407	Mm.248291	54354
		(RalGDS/AF-6)
		domain family 5
1439323_a_at	Map4k1	mitogen activated	BB546619	P70218	Mm.148278	26411
		protein kinase kinase
		kinase kinase 1
1424674_at	Slc39a6	solute carrier family	BB825002	0	Mm.21688	106957
		39 (metal ion
		transporter), member 6
1434920_a_at	Evl	Ena-vasodilator	AW553781	P70429	Mm.238841	14026
		stimulated
		phosphoprotein
1415850_at	Rasa3	RAS p21 protein	NM_009025	Q60790	Mm.18517	19414
		activator 3
1435560_at	0	0	BI554446	0	0	0
1428735_at	Cd69	CD69 antigen	AK017979	0	Mm.74745	12515
1434573_at	Traf3ip3	TRAF3 interacting	BE986588	0	Mm.261259	215243
		protein 3
1419060_at	Gzmb	granzyme B	NM_013542	P04187	Mm.14874	14939
1450241_a_at	Evi2a	ecotropic viral	NM_010161	P20934	Mm.164948	14017
		integration site 2a
1442219_at	Ms4a6b	Membrane-spanning	BB218965	0	Mm.278844	69774
		4-domains, subfamily
		A, member 6B
1460337_at	Sh3kbp1	SH3-domain kinase	BB326929	0	Mm.286495	58194
		binding protein 1
1425084_at	Gimap7	GTPase, IMAP	BC026200	0	Mm.30479	231932
		family member 7
1435343_at	Dock10	dedicator of	BF715043	0	Mm.133473	210293
		cytokinesis 10
1436598_at	Icos	inducible T-cell co-	AV313923	0	Mm.42044	54167
		stimulator
1422612_at	Hk2	hexokinase 2	NM_013820	O08528	Mm.255848	15277
1423135_at	Thy1	thymus cell antigen 1,	AV028402	P01831	Mm.3951	21838
		theta
1439436_x_at	Incenp	inner centromere	BB418702	0	Mm.29755	16319
		protein
1426505_at	Evi2b	ecotropic viral	AI122415	0	0	216984
		integration site 2b
1420515_a_at	Pglyrp2	peptidoglycan	NM_021319	0	Mm.86752	57757
		recognition protein 2
1448511_at	Ptprcap	protein tyrosine	NM_016933	Q64697	Mm.329686	19265
		phosphatase, receptor
		type, C polypeptide-
		associated protein
1442338_at	0	Transcribed locus	BB740904	0	Mm.35746	0
1417391_a_at	Il16	interleukin 16	BC026894	O54824	Mm.10137	16170
1434376_at	Cd44	CD44 antigen	AW146109	P15379	Mm.330428	12505
1433465_a_at	AI467606	expressed sequence	BB234337	0	Mm.284102	101602
		AI467606
1460253_at	Cklfsf7	chemokine-like factor	NM_133978	0	Mm.35600	102545
		super family 7
1429028_at	Dock11	dedicator of	AK017170	0	Mm.32873	75974
		cytokinesis 11
1428787_at	Nckap11	NCK associated	BM238906	0	Mm.30805	105855
		protein 1 like
1436576_at	A630077B13Rik	RIKEN cDNA	BB239429	0	Mm.34479	215900
		A630077B13 gene
1440481_at	0	0	BB229853	0	0	0
1418353_at	Cd5	CD5 antigen	NM_007650	P13379	Mm.779	12507
1427301_at	Cd48	CD48 antigen	BE634960	P18181	Mm.1738	12506
1417756_a_at	Lsp1	lymphocyte specific 1	NM_019391	P19973	Mm.234003	16985
1422812_at	Cxcr6	chemokine (C—X—C	NM_030712	0	Mm.124289	80901
		motif) receptor 6
1456307_s_at	Adcy7	Adenylate cyclase 7	BB746807	P51829	Mm.288206	11513
1418131_at	Samhd1	SAM domain and HD	NM_018851	Q60710	Mm.248478	56045
		domain, 1
1455132_at	A430107D22Rik	RIKEN cDNA	AV312663	0	Mm.122284	320484
		A430107D22 gene
1440275_at	Runx3	Runt related	AV233043	Q64131,	Mm.247493	12399
		transcription factor 3		O88674
1417786_a_at	Rgs19	regulator of G-protein	BC003838	0	Mm.274366	56470
		signaling 19
1448449_at	Ripk3	receptor-interacting	NM_019955	0	Mm.46612	56532
		serine-threonine
		kinase 3
1422632_at	Ctsw	cathepsin W	NM_009985	P56203	Mm.113590	13041
1454694_a_at	Top2a	topoisomerase	BM211413	Q01320	Mm.4237	21973
		(DNA) II alpha
1434940_x_at	Rgs19	regulator of G-protein	BB233670	0	Mm.274366	56470
		signaling 19
1449156_at	Ly9	lymphocyte antigen 9	NM_008534	Q01965	Mm.560	17085
1435084_at	C730049O14Rik	RIKEN cDNA	BB200607	0	Mm.209644	320117
		C730049O14 gene
1420819_at	Sla	src-like adaptor	NM_009192	Q60898	Mm.7601	20491
1434067_at	AI662270	expressed sequence	BE688410	0	Mm.295569	103814
		AI662270
1416007_at	Satb1	special AT-rich	AV172776	Q60611	Mm.311655	20230
		sequence binding
		protein 1
1452087_at	Epsti1	epithelial stromal	BF020640	0	Mm.68134	108670
		interaction 1 (breast)
1436649_at	Zfpn1a3	RIKEN cDNA	BB151746	O08900	Mm.133367	22780
		5830411O07 gene
1449235_at	Fasl	Fas ligand (TNF	NM_010177	P41047	Mm.3355	14103
		superfamily, member
		6)
1450639_at	Slc28a2	solute carrier family	NM_021520	O88627	Mm.29510	269346,
		28 (sodium-coupled				381417
		nucleoside
		transporter), member 2
1416076_at	Ccnb1-rs1	cyclin B1, related	NM_007629	P24860	Mm.260114	12429,
		sequence 1				268697,
						434175,
						545021
1421038_a_at	Kcnn4	potassium	NM_008433	O89109	Mm.9911	16534
		intermediate/small
		conductance calcium-
		activated channel,
		subfamily N, member 4
1447792_x_at	0	Adult male thymus	BB241847	0	Mm.179798	0
		cDNA, RIKEN full-
		length enriched
		library,
		clone: 5830404C02
		product: unknown
		EST, full insert
		sequence
1419598_at	Ms4a6d	membrane-spanning	NM_026835	0	Mm.290390	68774
		4-domains, subfamily
		A, member 6D
1426159_x_at	Tcrb-V13	T-cell receptor beta,	U46841	0	Mm.333026	269846
		variable 13
1456014_s_at	BC032204	cDNA sequence	BB113173	0	Mm.157591	108101
		BC032204
1443534_at	0	0	BM201095	0	0	0
1419226_at	Cd96	CD96 antigen	NM_032465	0	Mm.29204	84544
1428696_at	2310015N21Rik	RIKEN cDNA	AK009372	0	Mm.41854	76438
		2310015N21 gene
1448314_at	Cdc2a	cell division cycle 2	NM_007659	P11440	Mm.281367	12534
		homolog A (S. pombe)
1424443_at	Tm6sf1	transmembrane 6	AV378394	P58749	Mm.221412	107769
		superfamily member 1
1433826_at	AW212607	expressed sequence	AV325152	0	Mm.277243	241732
		AW212607
1455269_a_at	Coro1a	coronin, actin binding	BB740218	O89053	Mm.290482	12721
		protein 1A
1450106_a_at	Evl	Ena-vasodilator	NM_007965	P70429	Mm.238841	14026
		stimulated
		phosphoprotein
1434399_at	Galnt6	UDP-N-acetyl-alpha-	AV231866	0	Mm.22969	207839
		D-
		galactosamine: polypeptide
		N-
		acetylgalactosaminyltransferase 6
1419153_at	2810417H13Rik	RIKEN cDNA	AK017673	0	Mm.269025	68026
		2810417H13 gene
1426278_at	Ifi27	interferon, alpha-	AY090098	0	Mm.271275	76933
		inducible protein 27
1432459_a_at	MGI: 1891838	repressor of GATA	AK015881	0	Mm.116789	58206
1451860_a_at	Trim30	tripartite motif	AF220015	P15533	Mm.295578	20128
		protein 30
1452393_at	AI597013	expressed sequence	BB014626	0	Mm.258930	100182
		AI597013
1452205_x_at	Tcrb-V13	T-cell receptor beta,	X67128	0	Mm.333026	269846
		variable 13
1420394_s_at	Gp49a	glycoprotein 49 A	U05264	Q61450,	Mm.358601	14727,
				Q64281		14728
1427656_at	Tcrb-V13	T-cell receptor beta,	X14388	0	Mm.333026	269846
		variable 13
1430165_at	Stk17b	serine/threonine	AI661948	0	Mm.25559	98267
		kinase 17b
		(apoptosis-inducing)
1450997_at	Stk17b	serine/threonine	AV173139	0	Mm.25559	98267
		kinase 17b
		(apoptosis-inducing)
1415899_at	Junb	Jun-B oncogene	NM_008416	P10922,	Mm.1167	16477
				P09450
1449988_at	Gimap1	GTPase, IMAP	NM_008376	P70224	Mm.252599	16205
		family member 1
1431292_a_at	Ptk91	protein tyrosine	AK002699	0	Mm.274346	23999
		kinase 9-like (A6-
		related protein)
1447621_s_at	2610307O08Rik	RIKEN cDNA	AV300716	0	Mm.45995	72512
		2610307O08 gene
1434980_at	Pik3r5	phosphoinositide-3-	AV230647	0	Mm.244960	320207
		kinase, regulatory
		subunit 5, p101
1424953_at	BC021614	cDNA sequence	BC021614	0	Mm.26996	225884
		BC021614
1435144_at	0	Transcribed locus	BM243379	0	Mm.364092	0
1433963_a_at	BC032204	cDNA sequence	BG066664	0	Mm.157591	108101
		BC032204
1419599_s_at	Ms4a11	membrane-spanning	NM_026835	0	0	64382
		4-domains, subfamily
		A, member 11
1422303_a_at	Tnfrsf18	tumor necrosis factor	AF229434	O35714	Mm.3180	21936
		receptor superfamily,
		member 18
1450678_at	Itgb2	integrin beta 2	NM_008404	P11835	Mm.1137	16414
1427892_at	MyoIg	myosin IG	BB235320	0	Mm.239554	246177
1427511_at	B2m	Beta-2 microglobulin	AA170322	P01887	Mm.163	12010
1444177_at	0	Transcribed locus,	AI451538	0	Mm.31556	0
		moderately similar to
		XP_576460.1
		PREDICTED: similar
		to hypothetical
		protein
		PB402898.00.0
		[Rattus norvegicus]
1452539_a_at	Cd3z	CD3 antigen, zeta	X84237	P29020,	Mm.217308	12503
		polypeptide		P24161
1416882_at	Rgs10	regulator of G-protein	NM_026418	0	Mm.18635	67865
		signalling 10
1449361_at	Tbx21	T-box 21	NM_019507	0	Mm.94519	57765
1417065_at	Egr1	early growth response 1	NM_007913	P08046	Mm.181959	13653
1425860_x_at	Cklf	chemokine-like factor	AY046597	0	Mm.269219	75458
1419561_at	Ccl3	chemokine (C—C	NM_011337	P10855	Mm.1282	20302
		motif) ligand 3
1450753_at	Nkg7	natural killer cell	NM_024253	0	Mm.34613	72310
		group 7 sequence
1422875_at	Cd84	CD84 antigen	NM_013489	0	Mm.259115	12523
1426817_at	Mki67	antigen identified by	X82786	Q61769	Mm.4078	17345
		monoclonal antibody
		Ki 67
1418655_at	Galgt1	UDP-N-acetyl-alpha-	U18975	Q09200	Mm.1853	14421
		D-galactosamine: (N-
		acetylneuraminyl)-
		galactosylglucosylcer
		amide-beta-1,4-N-
		acetylgalactosaminyltransferase
1456439_x_at	Mical1	microtubule	BB209438	0	Mm.290431	171580
		associated
		monoxygenase,
		calponin and LIM
		domain containing 1
1452348_s_at	Mnda	myeloid cell nuclear	AI481797	0	0	381308
		differentiation
		antigen
1453228_at	Stx11	syntaxin 11	AK017897	0	Mm.248648	74732
1449347_a_at	Xlr4	X-linked	NM_021365	0	Mm.104764	27083,
		lymphocyte-regulated 4				434794
1416379_at	Panx1	pannexin 1	NM_019482	0	Mm.142253	55991
1416935_at	Trpv2	transient receptor	NM_011706	0	Mm.288064	22368
		potential cation
		channel, subfamily V,
		member 2
1450069_a_at	Cugbp2	CUG triplet repeat,	BB667096	0	Mm.147091	14007
		RNA binding protein 2
1458299_s_at	Nfkbie	nuclear factor of	BB820441	O54910	Mm.57043	18037
		kappa light
		polypeptide gene
		enhancer in B-cells
		inhibitor, epsilon
1415945_at	Mcm5	minichromosome	NM_008566	P49718	Mm.5048	17218
		maintenance deficient
		5, cell division cycle
		46 (S. cerevisiae)
1426170_a_at	Cd8b1	CD8 antigen, beta	U34882	P10300	Mm.333148	12526
		chain 1
1434388_at	Mobkl2a	MOB1, Mps One	BB023868	0	Mm.49309	208228
		Binder kinase
		activator-like 2A
		(yeast)
1428786_at	Nckap1l	NCK associated	BM238906	0	Mm.30805	105855
		protein 1 like
1429525_s_at	Myo1f	myosin IF	AK021181	0	Mm.42019	17916
1419004_s_at	Bcl2a1a	B-cell	L16462	Q07440,	Mm.244917	12044,
		leukemia/lymphoma		O55179		12045,
		2 related protein A1a				12047
1421317_x_at	Myb	myeloblastosis	NM_033597	P06876,	Mm.52109	17863
		oncogene		Q61927,
				Q61421,
				Q61926,
				Q61928
1443894_at	Evi2b	ecotropic viral	BB236216	0	0	216984
		integration site 2b
1433699_at	Tnfaip3	tumor necrosis factor,	BM241351	Q60769	Mm.116683	21929
		alpha-induced protein 3
1452389_at	Tnfrsf7	tumor necrosis factor	L24495	P41272	Mm.121	21940
		receptor superfamily,
		member 7
1418398_a_at	Phemx	pan hematopoietic	AF175771	0	Mm.28172	27027
		expression
1419186_a_at	St8sia4	ST8 alpha-N-acetyl-	NM_009183	Q64692	Mm.306228	20452
		neuraminide alpha-
		2,8-sialyltransferase 4
1438676_at	Mpa2l	macrophage	BM241485	0	Mm.275893	100702
		activation 2 like
1423182_at	0	0	AK004668	0	0	0
1421628_at	Il18r1	interleukin 18	NM_008365	Q61098	Mm.253664	16182
		receptor 1
1424906_at	E030024M05Rik	RIKEN cDNA	BC025220	0	Mm.5675	217430
		E030024M05 gene
1418612_at	Slfn1	schlafen 1	NM_011407	0	Mm.10948	20555
1418776_at	5830443L24Rik	RIKEN cDNA	NM_029509	0	Mm.301868	76074
		5830443L24 gene
1439440_x_at	Ptk9l	protein tyrosine	BB397672	0	Mm.274346	23999
		kinase 9-like (A6-
		related protein)
1434068_s_at	AI662270	expressed sequence	BE688410	0	Mm.295569	103814
		AI662270
1435458_at	0	0	AI323550	0	0	0
1453281_at	Pik3cd	Phosphatidylinositol	BB700084	O35904	Mm.229108	18707
		3-kinase catalytic
		delta polypeptide
1435710_at	AI661384	expressed sequence	BB034038	0	Mm.30743	106930
		AI661384
1451673_at	Cd8a	CD8 antigen, alpha	M12825	P01731,	Mm.1858	12525
		chain		Q60965
1452815_at	P2ry10	purinergic receptor	AK020001	0	Mm.74639	78826
		P2Y, G-protein
		coupled 10
1416811_s_at	Ctla2a	cytotoxic T	NM_007796	P12399,	Mm.358584	13024,
		lymphocyte-		P12400		13025
		associated protein 2
		alpha
1436329_at	Egr3	early growth response 3	AV346607	P43300	Mm.103737	13655
1416875_at	Parvg	parvin, gamma	NM_022321	0	Mm.251356	64099
1423467_at	Ms4a4b	membrane-spanning	BB199001	0	Mm.33957	60361
		4-domains, subfamily
		A, member 4B
1444078_at	Cd8a	CD8 antigen, alpha	BB154331	P01731,	Mm.1858	12525
		chain		Q60965
1436808_x_at	Mcm5	minichromosome	AI324988	P49718	Mm.5048	17218
		maintenance deficient
		5, cell division cycle
		46 (S. cerevisiae)
1416802_a_at	Cdca5	cell division cycle	NM_026410	0	Mm.23526	67849
		associated 5
1426239_s_at	0	0	BC016642	0	0	0
1416028_a_at	Hn1	hematological and	NM_008258	P97825	Mm.1775	15374
		neurological
		expressed sequence 1
1429524_at	Myo1f	myosin IF	AK021181	0	Mm.42019	17916
1419254_at	Mthfd2	methylenetetrahydrofolate	BG076333	P18155	Mm.443	17768
		dehydrogenase
		(NAD+ dependent),
		methenyltetrahydrofolate
		cyclohydrolase
1441317_x_at	MGI: 1923321	gamma-aminobutyric	BB316060	0	Mm.228812	76071
		acid (GABA-B)
		receptor binding
		protein
1438917_x_at	Nup62	nucleoporin 62	AW240611	Q63850	Mm.2565	18226
1429319_at	Rhoh	ras homolog gene	BM243660	0	Mm.358763	74734
		family, member H
1437636_at	LOC433377	similar to Interferon-	BB135602	0	0	433377
		activatable protein
		203 (Ifi-203)
		(Interferon-inducible
		protein p203)
1435330_at	AI447904	expressed sequence	BM241008	0	Mm.360525	236312,
		AI447904				545384
1416698_a_at	Cks1b	CDC28 protein	NM_016904	P61025	Mm.3049	54124
		kinase 1b
1460651_at	Lat	linker for activation	AF036907	O54957	Mm.10280	16797
		of T cells
1433964_s_at	BC032204	cDNA sequence	BG066664	0	Mm.157591	108101
		BC032204
1434295_at	Rasgrp1	RAS guanyl releasing	BE691356	0	Mm.42150	19419
		protein 1
1437325_x_at	Aldh18a1	aldehyde	BB251523	Q63739	Mm.233117	56454
		dehydrogenase 18
		family, member A1
1426772_x_at	Tcrb-J	T-cell receptor beta,	M11456	0	Mm.333026	21580,
		joining region				269846,
						381765
1451363_a_at	2010308M01Rik	RIKEN cDNA	BC008266	0	Mm.371646	72121
		2010308M01 gene
1439814_at	0	Transcribed locus	BM246630	0	Mm.315271	0
1448575_at	Il7r	interleukin 7 receptor	AI573431	P16872	Mm.389	16197
1422188_s_at	Tcrg	T-cell receptor	NM_011558	0	Mm.350873	110067,
		gamma chain				434531
1437760_at	Galnt12	UDP-N-acetyl-alpha-	AV376137	0	Mm.132246	230145
		D-
		galactosamine: polypeptide
		N-
		acetylgalactosaminyltransferase
		12
1428492_at	Glipr2	GLI pathogenesis-	BM208214	0	Mm.22213	384009
		related 2
1460437_at	Pscd4	pleckstrin homology,	AK010908	0	Mm.32911	72318
		Sec7 and coiled/coil
		domains 4
1437052_s_at	Slc2a3	solute carrier family	BB414515	P32037,	Mm.269857	20527
		2 (facilitated glucose		Q61607
		transporter), member 3
1422638_s_at	Rassf5	Ras association	NM_018750	O70407	Mm.248291	54354
		(RalGDS/AF-6)
		domain family 5
1418826_at	Ms4a6b	membrane-spanning	NM_027209	0	Mm.278844	69774
		4-domains, subfamily
		A, member 6B
1422828_at	Cd3d	CD3 antigen, delta	NM_013487	0	Mm.4527	12500
		polypeptide
1452948_at	Tnfaip8l2	tumor necrosis factor,	AK007540	0	Mm.34368	69769
		alpha-induced protein
		8-like 2
1422932_a_at	Vav1	vav 1 oncogene	NM_011691	P27870,	Mm.248172	22324
				O08526
1436312_at	Zfpn1a1	zinc finger protein,	AV317621	Q03267	Mm.103545	22778
		subfamily 1A, 1
		(Ikaros)
1418451_at	Gng2	guanine nucleotide	BB522409	P63213	Mm.41737	14702
		binding protein (G
		protein), gamma 2
		subunit
1418166_at	I112rb1	interleukin 12	NM_008353	Q60837	Mm.731	16161
		receptor, beta 1
1448749_at	Plek	pleckstrin	AF181829	0	Mm.98232	56193
1452483_a_at	Cd44	CD44 antigen	X66083	P15379	Mm.330428	12505
1448617_at	Cd53	CD53 antigen	NM_007651	Q61451	Mm.316861	12508
1425832_a_at	Cxcr6	chemokine (C—X—C	AF301018	0	Mm.124289	80901
		motif) receptor 6
1421855_at	Fgl2	fibrinogen-like	BF136544	P12804	Mm.292100	14190
		protein 2
1419202_at	Cst7	cystatin F	NM_009977	O89098	Mm.12965	13011
		(leukocystatin)
1423602_at	Traf1	Tnf receptor-	BG064103	P39428	Mm.239514	22029
		associated factor 1
1450905_at	Plxnc1	plexin C1	BB476707	0	Mm.256712	54712
1439141_at	Gpr18	G protein-coupled	BG145550	0	Mm.37405	110168
		receptor 18
1426324_at	H2-D1	histocompatibility 2,	M33151	P01899,	Mm.33263	14964
		D region locus 1		P01900,
				P01897,
				P01895,
				Q31116,
				Q31198,
				Q31168,
				O19467,
				O78207,
				Q31167,
				Q31209,
				Q31149,
				Q31169,
				Q31170,
				Q31188,
				Q61891,
				Q61892
1425086_a_at	Slamf6	SLAM family	AF248636	0	Mm.245727	30925
		member 6
1420671_x_at	Ms4a4c	membrane-spanning	NM_029499	0	Mm.353643	64380
		4-domains, subfamily
		A, member 4C
1422628_at	4632417K18Rik	RIKEN cDNA	NM_026640	0	Mm.1643	107373
		4632417K18 gene
1417164_at	Dusp10	dual specificity	NM_022019	0	Mm.266191	63953
		phosphatase 10
1452796_at	Def6	differentially	AK010356	0	Mm.204731	23853
		expressed in FDCP 6
1419631_at	Was	Wiskott-Aldrich	NM_009515	P70315,	Mm.4735	22376
		syndrome homolog		Q61078
		(human)
1421457_a_at	Samsn1	SAM domain, SH3	NM_023380	P57725	Mm.131406	67742
		domain and nuclear
		localisation signals, 1

Other Embodiments

It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

1. A method for detecting tissue rejection, wherein said method comprises determining whether or not tissue transplanted into a mammal contains cells that express at least two of the nucleic acids listed in Table 4 or Table 5, wherein the presence of said cells indicates that said tissue is being rejected.

2. The method of claim 1, wherein said mammal is a human.

3. The method of claim 1, wherein said tissue is kidney tissue.

4. The method of claim 1, wherein said tissue is a kidney.

5. The method of claim 1, wherein said method comprises determining whether or not said tissue contains cells that express at least five of said nucleic acids.

6. The method of claim 1, wherein said method comprises determining whether or not said tissue contains cells that express at least ten of said nucleic acids.

7. The method of claim 1, wherein said method comprises determining whether or not said tissue contains cells that express at least twenty of said nucleic acids.

8. The method of claim 1, wherein said determining step comprises measuring the level of mRNA expressed from said at least two nucleic acids.

9. The method of claim 1, wherein said determining step comprises measuring the level of polypeptide expressed from said at least two nucleic acids.

10. The method of claim 1, wherein said method comprises determining whether or not said tissue contains cells that express at least two of said nucleic acids at a level greater than the average level of expression exhibited in cells from control tissue that has not been transplanted.

11. A method for detecting tissue rejection, wherein said method comprises determining whether or not a sample contains cells that express at least two of the nucleic acids listed in Table 4 or Table 5, wherein said sample comprises cells, was obtained from tissue that was transplanted into a mammal, and was obtained from said tissue within fifteen days of said tissue being transplanted into said mammal, and wherein the presence of said cells indicates that said tissue is being rejected.

12. The method of claim 11, wherein said mammal is a human.

13. The method of claim 11, wherein said tissue is kidney tissue.

14. The method of claim 11, wherein said tissue is a kidney.

15. The method of claim 11, wherein said method comprises determining whether or not said sample contains cells that express at least five of said nucleic acids.

16. The method of claim 11, wherein said method comprises determining whether or not said sample contains cells that express at least ten of said nucleic acids.

17. The method of claim 11, wherein said method comprises determining whether or not said sample contains cells that express at least twenty of said nucleic acids.

18. The method of claim 11, wherein said determining step comprises measuring the level of mRNA expressed from said at least two nucleic acids.

19. The method of claim 11, wherein said determining step comprises measuring the level of polypeptide expressed from said at least two nucleic acids.

20. The method of claim 11, wherein said sample was obtained from said tissue within ten days of said tissue being transplanted into said mammal.

21. The method of claim 11, wherein said sample was obtained from said tissue within five days of said tissue being transplanted into said mammal.

22. The method of claim 11, wherein said method comprises determining whether or not said sample contains cells that express at least two of said nucleic acids at a level greater than the average level of expression exhibited in cells from control tissue that has not been transplanted.

23. A nucleic acid array comprising at least 20 nucleic acid molecules, wherein each of said at least 20 nucleic acid molecules has a different nucleic acid sequence, and wherein at least 50 percent of the nucleic acid molecules of said array comprise a sequence from nucleic acid selected from the group consisting of the nucleic acids listed in Table 4 and Table 5.

24. The array of claim 23, wherein said array comprises at least 50 nucleic acid molecules, wherein each of said at least 50 nucleic acid molecules has a different nucleic acid sequence.

25. The array of claim 23, wherein said array comprises at least 100 nucleic acid molecules, wherein each of said at least 100 nucleic acid molecules has a different nucleic acid sequence.

26. The array of claim 23, wherein each of said nucleic acid molecules that comprise a sequence from nucleic acid selected from said group comprises no more than three mismatches.

27. The array of claim 23, wherein at least 75 percent of the nucleic acid molecules of said array comprise a sequence from nucleic acid selected from said group.

28. The array of claim 23, wherein at least 95 percent of the nucleic acid molecules of said array comprise a sequence from nucleic acid selected from said group.

29. The array of claim 23, wherein said array comprises glass.

30. The array of claim 23, wherein said at least 20 nucleic acid molecules comprise a sequence present in a human.

31. A computer-readable storage medium having instructions stored thereon for causing a programmable processor to determine whether one or more nucleic acids listed in Table 4 or Table 5 are detected in a sample, wherein said sample is from a transplanted tissue.

32. The computer-readable storage medium of claim 31, further comprising instructions stored thereon for causing a programmable processor to determine whether one or more of the nucleic acids listed in Table 4 or Table 5 is expressed at a greater level in said sample than in a control sample of non-transplanted tissue.

33. An apparatus for determining whether a transplanted tissue is being rejected, said apparatus comprising: one or more collectors for obtaining signals representative of the presence of one or more nucleic acids listed in Table 4 or Table 5 in a sample from said transplanted tissue; and a processor for analyzing said signals and determining whether said tissue is being rejected.

34. The apparatus of claim 33, wherein said one or more collectors are configured to obtain further signals representative of the presence of said one or more nucleic acids in a control sample from non-transplanted tissue.