Molecular Profile of Tumor-Specific CD8+ T Cell Hypofunction in a Transplantable Murine Cancer Model

This information is current as Katherine A. Waugh, Sonia M. Leach, Brandon L. Moore, of September 25, 2021. Tullia C. Bruno, Jonathan D. Buhrman and Jill E. Slansky J Immunol published online 1 July 2016 http://www.jimmunol.org/content/early/2016/07/01/jimmun ol.1600589 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2016 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published July 1, 2016, doi:10.4049/jimmunol.1600589 The Journal of Immunology

Molecular Profile of Tumor-Specific CD8+ T Cell Hypofunction in a Transplantable Murine Cancer Model

Katherine A. Waugh,* Sonia M. Leach,† Brandon L. Moore,* Tullia C. Bruno,* Jonathan D. Buhrman,* and Jill E. Slansky*

Mechanisms of self-tolerance often result in CD8+ tumor-infiltrating lymphocytes (TIL) with a hypofunctional phenotype incapable of tumor clearance. Using a transplantable colon carcinoma model, we found that CD8+ T cells became tolerized in <24 h in an established tumor environment. To define the collective impact of pathways suppressing TIL function, we compared genome-wide mRNA expression of tumor-specific CD8+ T cells from the tumor and periphery. Notably, gene expression induced during TIL hypofunction more closely resembled self-tolerance than viral exhaustion. Differential gene expression was refined to identify a core set of genes that defined hypofunctional TIL; these data comprise the first molecular profile of tumor-specific TIL that are

naturally responding and represent a polyclonal repertoire. The molecular profile of TIL was further dissected to determine the Downloaded from extent of overlap and distinction between pathways that collectively restrict T cell functions. As suggested by the molecular profile

of TIL, protein expression of inhibitory receptor LAG-3 was differentially regulated throughout prolonged late-G1/early-S phase of the cell cycle. Our data may accelerate efficient identification of combination therapies to boost anti-tumor function of TIL specifically against tumor cells. The Journal of Immunology, 2016, 197: 000–000. http://www.jimmunol.org/ or some cancers, immunological data from tumor samples substantially altered by slight changes in the environment and Ag are better predictors of patient survival than those cur- encounter. In the context of cancer, tumor environments evolve to rently used to stage disease (1, 2). Compelling evidence suppress and evade CD8+ T cells (4); therefore, TIL typically have F + associates functional CD8 tumor-infiltrating lymphocytes (TIL) a hypofunctional phenotype incapable of mounting an effective with survival of patients with cancer. For example, patient survival attack against an established solid tumor (5). positively correlates with TIL that produce proinflammatory cy- Exhaustion, tolerance, anergy, senescence, and ignorance are tokines such as IFN-g, kill target cells, and progress through the states of CD8+ T cell hypofunction that have been tied to TIL (3). cell cycle. These data suggest that the immunological status of Of these programs, hypofunctional TIL are most commonly re-

TIL is paramount for patient response. ferred to as exhausted or tolerant. T cell exhaustion occurs during by guest on September 25, 2021 To acquire cytotoxic functions, the Ag receptor on naive T cells prolonged inflammation and/or TCR exposure to an Ag, such as must be engaged in a proinflammatory environment (3). Cell- during a chronic viral infection. Peripheral T cell tolerance occurs intrinsic pathways are then initiated to drive T cell proliferation more quickly when Ag is presented in the absence of immunos- and differentiation to cytotoxic T cells that kill cells expressing timulatory signals to avoid self-reactivity and autoimmunity. + the associated Ag. CD8 T cell activation and differentiation are Unlike naive or memory CD8+ T cells that, upon TCR and co- stimulation, enter the cell cycle to undergo clonal expansion into functional effector T cells, a functional response by exhausted and † *University of Colorado School of Medicine, Aurora, CO 80045; and Center for self-tolerant CD8+ T cells is constrained. Genes, Environment and Health, National Jewish Health, Denver, CO 80206 The promise of promoting functional TIL to treat patients with Received for publication April 5, 2016. Accepted for publication June 9, 2016. cancer has been developing for decades to produce a recent ex- This work was supported by American Cancer Society Grant RSG-08-184-01-L1B, plosion of clinical successes (6). Such therapies include blockade National Institutes of Health Grants 5T32AI007405 and 5R25GM083333, and a generous donation from Richard and Donna Hammel. of receptors, such as programmed cell death-1 (PD-1) (7), that The sequences presented in this article have been submitted to the National Center inhibit TIL function. However, a therapy that targets just one in- for Biotechnology Information Gene Expression Omnibus under accession number hibitory pathway is often ineffective against established solid tu- GSE79858. mors. Cotherapies targeting multiple pathways inhibiting TIL Address correspondence and reprint requests to Dr. Jill E. Slansky, University of function have recently translated from mouse models to the clinic Colorado School of Medicine, 12800 East 19th Avenue, Mail Stop 8333, Aurora, CO 80045. E-mail address: [email protected] (8). For instance, dual blockade of T cell inhibitory receptors PD-1 The online version of this article contains supplemental material. and TIM-3 was first shown to augment clearance of the CT26 Abbreviations used in this article: 7-AAD, 7-aminoactinomycin D; CU-SOM, Uni- solid tumor model in mice (9) and is now in clinical trials (trial versity of Colorado School of Medicine; EdU, 5-ethynyl-2’-deoxyuridine; FDR, false registration ID: NCT02608268). discovery rate; FOM, figure of merit; GSEA, gene set enrichment analysis; IPA, The transplantable colon carcinoma CT26 (10) reflects both the ingenuity pathway analysis; KMC, K-means clustering; NES, normalized enrichment score; PCA, principal component analysis; PD-1, programmed cell death-1; SAM, infiltrating immune cells and pathways of immune suppression significance analysis of microarray; SpTV, AH1-specific CD8+ T cell from spleens found in many solid human tumors that are immunogenic (11, 12). in CT26 tumor-bearing mice; SpV, AH1-specific CD8+ T cell from spleens in non–tumor-bearing mice; TASPR, tumor Ag-specific polyclonal repertoire; TIL, As with many Ags being pursued in patients with cancer (13, 14), + CD8+ tumor-infiltrating lymphocyte; TILN, tumor-specific CD8+ T cell isolated the immunodominant CD8 T cell response against CT26 is di- from lymph node metastases; TILV, tumor-infiltrating lymphocyte from vaccinated rected against a tumor/self-antigen, GP70 , also known as tumor-bearing mice. 423–431 AH1 (15, 16). CT26 TIL exhibit an exhausted phenotype similar + Copyright Ó 2016 by The American Association of Immunologists, Inc. 0022-1767/16/$30.00 to CD8 T cells from patients with melanoma that bear TCRs

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1600589 2 MOLECULAR PROFILE OF TIL specific for a tumor/self-antigen (9, 17). The majority of CT26 When indicated, Ficoll-Paque PLUS (GE Healthcare) was used accord- TIL express multiple inhibitory receptors and are hypofunctional ing to the manufacturer’s instructions to enrich for live lymphocytes. (9). Nevertheless, mobilizing a functional CD8+ T cell response is Splenocytes were harvested from mice, mechanically dissociated, treated for ∼2 min with ammonium chloride-potassium lysis buffer (ACK) (22), necessary for successful anti-CT26 immunotherapy (18). filtered through a 100-mm cell strainer, and washed in complete medium. In this study, we show within just 24 h of transfer into a CT26 Before FACS, spleens were processed into single-cell suspensions through tumor-bearing host, AH1-specific CD8+ T cells that were pro- Liberase digestion, as performed for tumors. Before FACS and adoptive + tective in a prophylactic setting (19) were markedly hypofunc- transfer experiments, CD8 T cells were enriched from splenocytes using the Mouse CD8 Negative Selection Kit (Invitrogen) according to the tional in the tumor compared with those in the periphery. To manufacturer’s instructions. Cells were used directly for staining, in vitro molecularly define the collective impact of pathways that suppress stimulation assays, or adoptive cell transfer. AH1-specific TIL function, we compared genome-wide mRNA expression of AH1-specific CD8+ T cells from the CT26 tumor Abs, staining reagents, and flow cytometry and periphery. Gene expression of TIL hypofunction more closely For flow cytometry analyses, 2 3 106 live cells were incubated in 30-ml resembled self-tolerance than viral exhaustion. Differential gene stains containing an Ab to block FcRs (2.4G2) and fluorochrome-conjugated expression identified a core set of genes, or molecular profile, that Abs for at least 45 min at room temperature while protected from light. All staining was done in flow cytometry wash buffer [FACS buffer, 13 PBS, 2% defined TIL hypofunction. Further scrutiny of the molecular FBS, 10 mmol HEPES (pH 7.5), and 0.1% (w/v) sodium azide, as described profile of TIL delineated overlapping and distinct pathways that (21)] for at least 45 min at room temperature. Stain time was increased to restrict function of polyclonal TIL to highlight key mechanisms 1.5 h when peptide-loaded H-2Ld-tetramer (23) was included. that are known, hypothesized, and potentially novel drivers of Fluorochrome-conjugated Abs purchased from BioLegend, unless oth- erwise noted, included the following: mouse CD8a (53-6.7), CD11a (M17/4), immune suppression and evasion. The T cells from the tumor were Downloaded from CD4 (RM4-5), B220 (RA3-6B2), MHC class II (I-A/I-E; M5/114.15.2), hypofunctional and expressed more inhibitory receptor protein CD279/PD-1 (RMP1-30), CD366/TIM-3 (B8.2C12), CD223/LAG-3 when compared with those in the periphery. Known restrictors of (C9B7W), CD244.1/2B4 (C9.1; BD Biosciences), and Thy1.1 (53-2.1; TIL function were predicted to converge at cell cycle inhibition. eBioscience). Other staining reagents included viability-discriminating Inhibitory receptor LAG-3 and cell cycle progression validated a agents 7-aminoactinomycin D (7-AAD; 1 mg/ml; Sigma-Aldrich) and Fixable Viability Dye eFluor 450 (eBioscience) that were used according simplified approach for pathway analysis of small gene clusters to the manufacturer’s instructions. CD4/B220/I-A/I-E Abs and viability- within the molecular profile of TIL. TIL progressed more slowly discriminating agents are collectively referred to as the dump. http://www.jimmunol.org/ 3 7 ∼ 3 through late-G1 and early S-phase, associated with expression of Cells were stained for sorts at 35 10 cells/ml, then diluted to 5 7 inhibitory receptor LAG-3. Our molecular dissection of over- 10 cells/ml sorting buffer (13 PBS, 1 mmol EDTA, 25 mmol HEPES lapping and distinct pathways that restrict TIL function may ac- [pH 7], and 1% FBS), and sorted by FACS into complete medium. Intra- cellular cytokines were stained as described (24) following peptide stim- celerate identification of efficient cotherapy combinations to boost ulations at 10 pg/ml, 10 ng/ml, and 10 mg/ml, but stains contained an Ab naturally responding TIL specifically against tumors. directed against Thy1.1 rather than CD11a. Samples were analyzed on a variety of flow cytometers, and data were analyzed using Kaluza (Beck- man Coulter) or FlowJo software (Tree Star). Materials and Methods Mice Microarray by guest on September 25, 2021 Female BALB/c (BALB/cAnNCr) mice were purchased from the Na- Single-cell suspensions were stained with Fixable Viability Dye, then with d tional Cancer Institute and Charles River Laboratories. Mice congenic for AH1–H-2L -tetramer (23), and fluorochrome-conjugated Abs against CD8a, + thymus cell Ag-1 (Thy1), a kind gift of Dr. Charles D. Surh of The Scripps CD11a, CD4, B220, and I-A/I-E. Tumor-specific live CD8 T cells were Research Institute, were bred onto the BALB/cAnCr background for six sorted by FACS to a purity of 90–100% from tumors on the MoFlo XDP100 generations. For all experiments, mice were 6–12 wk old unless other- (Beckman Coulter) and from spleensontheMoFloXDP70(Beckman wise indicated; all animal protocols were reviewed and approved by the Coulter) at the CU-SOM Cancer Center Flow Cytometry Shared Resource. Institutional Animal Care and Use Committee at National Jewish Health RNAwas immediately extracted using the RNeasy Mini Kit (Qiagen) and or the University of Colorado School of Medicine (CU-SOM). treated once with ∼30 Kunitz units of DNase I (Qiagen) for 15 min. RNA quality was determined by BioAnalyzer (Agilent Technologies) and Peptides, vaccine, and tumor challenge quantified by Qubit (Life Technologies). Samples were narrowed to those that had an RNA integrity number of 9.6-10 and a minimum of 5 ng total Peptides used were bgal (TPHPARIGL) (20), AH1 (SPSYVYHQF) (15), RNA (25) before submission to the CU-SOM Genomics and Microarray and a TCR agonist of AH1 (amino acid substitution in boldface), A5 Core to be amplified by the Ovation RNA Amplification System V2 (SPSYAYHQF) (21). Soluble synthetic peptides were $95% pure (Chi (NuGEN) (25) and hybridized to Mouse Gene1.0 ST Arrays (Affymetrix) Scientific), and stocks were diluted to 10 mg/ml in double distilled H2O. according to the manufacturer’s instructions. CD8+ T cells bearing TCRs specific for the immunodominant CT26 T cell epitope AH1 (15) were expanded in vivo through vaccination with peptide Cell cycle analysis A5, as described (18). Mice were challenged s.c. with 5 3 104 CT26 tumor cells on the hind flank (21). Tumors were measured with calipers, and mice Tumors or spleens were processed into single-cell suspensions, and were sacrificed when indicated or the tumor size reached 1 cm in the longest live lymphocytes were enriched from tumors using Ficoll-Paque PLUS (GE diameter. Spleens from five vaccinated mice were combined, enriched for Healthcare) according to the manufacturer’s instructions. Cells were plated 6 CD8+ T cells with a Mouse CD8 Negative Selection Kit (Invitrogen), and at 2 3 10 cells/ml complete medium in 24-well tissue culture plates and then divided equally among 10 recipient mice (∼1 3 107 cells/mouse) for stimulated by plate-bound CD3 and CD28, each at 2 mg/ml, and spun for i.v. injection into Thy1 congenic naive or tumor-bearing hosts. Spleens and 2 min at ∼340 3 g to synchronize cell cycle progression. After 48 h, cells tumors were harvested at the indicated time points and assayed for inhibitory were pulsed for 1 or 2 h with 5-ethynyl-2’-deoxyuridine (EdU; 10 mmol; + receptor expression and IFN-g production as specified below. Sigma-Aldrich), washed in complete medium, and stained for live CD8 T cells and EdU using the Click-iT Plus EdU Flow Cytometry Assay Kit Isolation of tumor-specific CD8+ T cells (Life Technologies) according to the manufacturer’s instructions. Briefly, cells were fixed, permeabilized, and EdU was detected with one-fifth the Tumors were collected from BALB/c mice, minced using a razor blade, and recommended Click-iT reaction to decrease background. To stain total treated for 45 min at 37˚C with 0.1 mg/ml Liberase (Research Grade, DNA, 7-AAD was diluted 1:400 in permeabilization buffer (Life Tech- Dispase Low) in serum-free RPMI 1640 medium according to the manu- nologies) and used in combination with RNaseA (0.2 mg/ml; Qiagen). Cell facturer’s instructions (Roche Life Science). Large clumps of tumor were cycle phase was determined for live CD8+ T cells by flow cytometry. broken up through an 18-gauge needle, filtered through a 100-mm cell strainer, and washed in complete medium [RPMI with L-glutamine, 10% Statistical analyses FBS, 100 U/ml each penicillin and streptomycin, 1 mmol sodium pyruvate, 10 mmol HEPES, 13 MEM nonessential amino acids, 2 mmol additional High-throughput microarray data were processed by Affymetrix Expression L-glutamine, and 0.1 mmol 2-ME (Sigma-Aldrich), as described (21)]. Console software (Affymetrix; affymetrix.com), performing robust multi- The Journal of Immunology 3 chip analysis background correction, quantile normalization, and median duction to distinguish between exhausted and tolerant TIL, as loss polish summarization. The gene-expression data described in this paper of IFN-g production should occur overtime during exhaustion and have been deposited in the National Center for Biotechnology Information immediately in tolerance (3). Gene Expression Omnibus under accession number GSE79858 (http:// + www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE79858). Data sets were Functional tumor-specific CD8 T cells were expanded in vivo by compared by one-way ANOVA to delineate significant differential gene a vaccine strategy that is protective against CT26 tumor challenge expression when p # 0.01, false discovery rate (FDR) #0.01 [Benjamini- (18). We transferred these cells into congenic hosts bearing an Hochberg method (26)], and |log2 (ratio)| $ (+2-fold). The molecular established CT26 tumor. Within 24 h of transfer, tumor-specific TIL profile of the tumor Ag-specific polyclonal repertoire (TASPR) TIL con- sists of differentially expressed genes that have been narrowed by multiple became markedly hypofunctional relative to peripheral counterparts statistical analysis as described above for ANOVA and significance anal- (Fig. 1A, 1B), a phenotype that became more pronounced over 1 wk ysis of microarray (SAM) (27, 28), where q #0.05 [Benjamini-Hochberg (Fig. 1C, 1D). Transferred T cells were similarly functional in the method, FDR #0.01 (26)] and |log2 (ratio)| $ (+2-fold). SAM was done in spleens of tumor-bearing and non–tumor-bearing mice (K.A. Waugh, MeV version 4.8 (29). Principal component analysis (PCA), ANOVA, and unpublished observations). IFN-g protein expression in response corresponding Volcano plots were done using Partek software (Partek, St. Louis, MO). The PCA was done on scaled data. to PMA/ionomycin stimulation (a means to bypass TCR signaling) All gene set enrichment analyses (GSEAs) were run simultaneously was also decreased, suggesting that functional defects of TIL were using the GSEA software from the Broad Institute (30) and used the fol- cell intrinsic. In addition, the immediate loss of function and accu- lowing parameters recommended for expression data sets that contain a mulation of multiple inhibitory receptors suggested that self- sample with fewer than seven replicates: all data were converted into gene symbols, redundant probe sets were collapsed using probe medians, a tolerance of TIL is established quickly in a solid tumor environment. Signal2Noise metric was used for ranking genes, the weighted enrichment Alternatively, differences among transferred T cells in the spleen statistic was employed, and 1000 gene set permutations were used. All and tumor may have been due to altered trafficking of differentially Downloaded from comparison populations and statistics used to generate gene sets have been activated polyclonal T cells after vaccination rather than a sup- published (17, 31–34). For gene sets corresponding to human data, gene pressive tumor environment. However, when the vaccination is symbols were converted to mouse gene symbols by the Database for + Annotation, Visualization and Integrated Discovery (DAVID) version 6.7 given prophylactically, functional CD8 T cells traffic to eliminate (35, 36), and gene symbols were manually verified to contain the gene CT26 tumors (18). Therefore, immediate differential expression of symbol identifier(s) corresponding to our microarray Probe Set identifi- proteins, such as IFN-g, in T cells from the spleen and tumor cation. A compiled list of all gene sets used for GSEA has been included strongly suggested that the tumor and spleen environments dif- http://www.jimmunol.org/ (Supplemental Table I). Leading edge comparisons were done in a Pan- gloss Venn diagram generator. ferentially affect the functionality of T cells. Figure of merit (FOM) (37) was used to estimate the appropriate number of clusters to divide these data for the K-means clustering (KMC) algo- Genome-wide mRNA expression of TIL and functional rithm (38, 39). The smallest cluster number was used for which FOM validation values did not continue to decrease. To verify the appropriate number of To ultimately define the collective impact of multiple pathways clusters to divide differentially expressed genes for pathway analysis, we analyzed these data divided into 5, 7, and 13 clusters by ingenuity pathway suppressing CT26 TIL function (44), a comparison of genome-wide analysis (IPA). Thirteen clusters were ultimately used, as it was the mRNA expression of tumor-specific CD8+ T cells from the tumor smallest number of clusters for which the FOM value leveled out (at 6) and was required (Fig. 2A). Tumor-bearing mice were vaccinated to had the lowest trend in overall p values associated with biofunctions that expand tumor-specific CD8+ T cells for detection in the periphery. by guest on September 25, 2021 were predicted to be enriched among clusters by pathway analysis. All pathway analyses were done through Qiagen’s IPA (Qiagen, Redwood Although this vaccination strategy is protective when given pro- City, CA). MeV was used for FOM, KMC, and to generate the histogram phylatically (18), under the therapeutic conditions used, vaccination (29). Expression profiles for clusters 1, 3, and 5 were made using custom R only slightly delayed tumor growth. Therefore, we hypothesized scripts. Gene type was determined in PANTHER through Gene Ontology that the TIL from vaccinated tumor-bearing mice (TILV) have a protein class (40); unidentified genes in PANTHER were manually hypofunctional phenotype similar to naturally responding TIL in assigned a gene type through a Gene Ontology molecular function backed with stringent evidence in mice by inference from a mutant phenotype or a comparison with a peripheral counterpart in vaccinated tumor- direct assay in the National Center for Biotechnology Information data- bearing mice (AH1-specific CD8+ T cells from spleens in CT26 base. All gene products that did not meet the criteria to merit a specified tumor-bearing mice [SpTV]). Because tumors have systemic effects gene type were listed as miscellaneous. TRANSFAC provided by the on the immune system, we also included a cohort of AH1-specific GATHER Web tool was used to analyze promoter regions of differentially + expressed genes for enrichment of transcription factor motifs (41). CD8 T cells from vaccinated spleens in non–tumor-bearing mice Unless otherwise indicated, all other statistical significance was analyzed (SpV) as a positive control for effective antitumor function. Rather using an unpaired two-tailed Student t test (Prism version 4.0; GraphPad). than transferred T cells, we defined the hypofunction of naturally A p value #0.05 was considered statistically significant, and error bars responding TIL after chronic exposure to a tumor environment. represent SEM. Statistical significance is denoted by an asterisk, in which Tumor-specific CD8+ T cells were tetramer sorted from tumors *p # 0.05, **p # 0.01, ***p # 0.001, and ****p # 0.0001. (TIL and TILV) and spleens (SpTVand SpV) (Supplemental Fig. 1) for genome-wide mRNA expression analysis. Results To identify genome-wide similarities and differences in the TIL rapidly lose function in the CT26 tumor environment mRNA of samples and determine the most appropriate grouping of We previously showed that vaccine strategies that are protective samples, we performed PCA (45). PCA is a statistical technique to against CT26 tumor growth do not work as well in a therapeutic reduce a large set of variables to a small set of principal compo- setting (19). These results led us to determine how quickly tumor- nents that contain most of the original variation. The two largest specific CD8+ T cells become hypofunctional in an established principal components, PC 1 and PC 2, accounted for 48.5% of tumor environment. We investigated the loss of the production of the variance between samples and divided tumor-specific CD8+ the anti-tumor cytokine, IFN-g (4). Although hypofunction of T cells into two distinct clusters, those from the spleen and the early exhausted CD8+ T cells often cannot be detected without tumor (Fig. 2B). Therefore, genome-wide mRNA expression of analysis of multiple functions, reduced capacity to produce IFN-g TIL and TILV was most similar to each other and diverged from generally occurs more slowly than loss of target cell lysis, pro- peripheral counterparts SpTV and SpV. liferative potential, as well as IL-2 and TNF-a production (42). We next determined if tumor-specific CD8+ T cells from the Deficient IFN-g production is also a hallmark of CD8+ T cell spleen and tumor also represented functionally distinct cohorts tolerance (43). We expected functional analysis of IFN-g pro- and expressed the expected inhibitory receptors. As determined by 4 MOLECULAR PROFILE OF TIL Downloaded from

FIGURE 1. Effector CD8+ T cells become hypofunctional within 24 h in a CT26 tumor environment. Transferred live CD8+ T cells, known to protect against tumor challenge, were adoptively transferred into a tumor-bearing host and monitored at the indicated time points from the tumor (Tum) and spleen (Sp). (A) One day after adoptive transfer into a tumor-bearing host, transferred (Thy1.1+) CD8+ T cells from the Tum and Sp were assayed for IFN-g protein in response to A5 peptide (10 mg/ml) stimulation ex vivo. Geometric mean fluorescence intensities (gMFIs) in representative dot plots from IFN-g+ endogenous (upper left quadrant, black) and transferred (upper right quadrant, red) live CD8+ T cells are shown. (B) Expression level of IFN-g in http://www.jimmunol.org/ transferred CD8+ T cells from the Tum and Sp was measured in response to A5 peptide (10 mg/ml) and PMA/ionomycin stimulation ex vivo 1 d after adoptive transfer into a tumor-bearing host. b2galactosidase (bgal; 10 mg/ml) is an H-2Ld–binding irrelevant peptide. (C) Coexpression of inhibitory receptors was monitored over time on transferred CD8+ T cells from the Tum and Sp. 0d represents immediately before transfer, and a frequency of “0” designates no dual PD-1+/TIM-3+ cells of interest. (D) Transferred CD8+ T cells from the Tum and Sp were monitored over time for IFN-g protein production following ex vivo PMA/ionomycin stimulation. A gMFI of “0” designates no IFN-g+ among cells of interest. Data represent at least two independent experiments, n = 2 to 3 biological replicates per group, and error bars indicate SD of the mean. **p # 0.01, ***p # 0.001, ****p # 0.0001.

IFN-g production, TIL from vaccinated mice were comparably TASPR TIL were most similar to a self-tolerant profile (33) hypofunctional to TIL from nonvaccinated mice, whereas function followed by the virally exhausted profile (31). Exhaustion occurs by guest on September 25, 2021 of tumor-specific CD8+ T cells from the spleens of vaccinated over time with chronic TCR Ag or inflammatory cytokine exposure tumor-bearing mice was similar to that of cells from vaccinated (47, 48), whereas self-tolerance generally occurs quickly in re- non–tumor-bearing mice (Fig. 2C). TIL and TILV had higher sponse to initial TCR exposure to self-antigen in the absence of protein expression of multiple inhibitory receptors than SpTV and immunostimulatory signals (3). As expected, similarities were also SpV (Fig. 2D); the expression data were comparable to those identified between TASPR TIL, deletional tolerance (32), and ef- obtained in Fig. 1 and previously published studies of naturally fector T cells (9, 31). We compared TASPR TIL with tumor-specific responding CD8+ TIL of CT26 tumors that did not consider Ag CD8+ T cells isolated from lymph node metastases (TILN) of specificity (9). These results suggested that our genome-wide previously vaccinated patients with melanoma (17) and total natu- mRNA expression data reflect differential gene expression re- rally responding CD8+ TIL from a mouse model of melanoma for sponsible for hypofunction of TIL in the tumor environment. For which Ag specificity was not restricted (34). Curiously, although the remaining analyses, the tumor Ag-specific polyclonal reper- genes highly expressed by vaccinated TILN (17) were enriched toire of CD8+ T cells from tumors (TIL and TILV) and spleens among TASPR TIL, genes highly expressed by the total naturally (SpTV and SpV) will collectively be referred to as TASPR T cells responding CD8+ TIL (34) were not. A likely explanation for such a from the CT26 tumor. large differential impact is the lack of TCR specificity for a tumor Ag in the total CD8+ TIL (34). For example, in the CT26 tumor Gene expression of heterogeneous TASPR TIL resembles model, protein expression of inhibitory receptors by total CD8+ TIL self-tolerance is much lower and more variable than by CD8+ TIL that have been GSEA is a statistical method commonly used to compare gene narrowed by tumor-specificity (K.A. Waugh, unpublished obser- expression results between data sets that are derived from inde- vations). Genes highly expressed by memory CD8+ T cells were in pendent experiments (17, 30, 46). We used GSEA to determine if direct opposition of TASPR TIL and similar to functional TASPR TASPR TIL had similar gene expression to other published gene T cells in the spleen. Unique enrichment of individual CD8+ Tcell expression profiles of hypofunctional T cells that have been as- programs among TASPR TIL (Fig. 3B, Supplemental Table II) and sociated with TIL. These included exhaustion, tolerance, anergy, rapid loss of T cell function in a tumor that became more pro- and other TIL profiles (Fig. 3A, Supplemental Table I) (17, 31– nounced over time (Fig. 1) strongly suggested that pathways do not 34). TASPR TIL were also compared by GSEA to other functional just overlap between exhausted and self-tolerant CD8+ Tcells,but T cell subsets that can infiltrate tumors, such as memory and ef- that TASPR TIL are a heterogeneous population that is largely self- fector T cells (31). Statistical significance, as reflected by the tolerant and exhausted. normalized enrichment scores (NES), p values, and familywise The gene set highly expressed by anergic T cells was ∼15 genes error rates, was analyzed for enrichment of genes associated with (32) and was too small (30) to obtain an accurate estimate of T cell subsets among TASPR T cells. enrichment among TASPR T cells (see statistics in Anergy The Journal of Immunology 5 Downloaded from http://www.jimmunol.org/

FIGURE 2. Genome-wide mRNA expression of tumor-specific CD8+ T cells segregates into two functionally distinct clusters. (A) Schematic of the origin of tumor Ag-specific T cells is shown. AH1-specific CD8+ T cells were tetramer-sorted from CT26 tumors of mice that had (TILV) or had not (TIL) been vaccinated and spleens of vaccinated mice that either harbored (SpTV) or did not harbor tumors (SpV). (B) mRNA from the groups described in (A) was interrogated by microarray. PCA divided genome-wide mRNA expression of the indicated cell populations into two distinct clusters: tumor-specific CD8+ T cells from the spleen or the tumor. Five to 10 mice were pooled per group, and each group was repeated three times. (C) AH1-specific CD8+ T cells described in (A) were stimulated ex vivo with increasing concentrations of the indicated peptide and assayed for IFN-g production. (D) AH1-specific CD8+ by guest on September 25, 2021 T cells described in (A) were analyzed for inhibitory receptor expression. The gray histogram represents naive CD8+ T cells from a spleen as a negative control for inhibitory receptor expression. (C) and (D) are representative data of at least two independent experiments, n = 1–4 biological replicates, and error bars indicate SD. **p # 0.01, ***p # 0.001. enrichment plot, Fig. 3A). The gene TOB1 consistently downreg- determining differential expression (27, 28). Both statistical ulated by anergic T cells was also downregulated by TASPR TIL analyses are commonly used to assess genome-wide mRNA ex- but could not be systematically compared with TASPR T cells by pression data: we reasoned dual criteria would increase confidence GSEA (30, 32) (Supplemental Table I). Although not available for that genes are not falsely included (51) in the molecular profile of systematic comparisons by GSEA, we also manually compared the TASPR TIL hypofunction. TASPR T cells to signature genes expressed by ignorant and Molecular profile of TIL highlights cell cycle inhibition senescent T cells. Genes such as KLRG1 and CD57, which are associated with senescent CD8+ T cells in a nonreversible state of To dissect the downstream impact of collective pathways suppressing cell cycle arrest (49), were not upregulated by TASPR TIL. Finally, CT26 TIL function, the 1164 genes comprising the molecular profile TASPR TIL were Ag experienced and did not downregulate the of TASPR TIL were sequestered into smaller gene clusters enriched TCR-CD3 complex and therefore did not correspond to T cells for biological pathways using KMC (Fig. 5A, Supplemental Table III) “ignorant” of the growing tumor (3, 50). (38, 39). IPA of individual clusters indicated that genes upregulated by TASPR T cells in the periphery were associated with lymphocyte Molecular profile of tumor-specific TIL proliferation (Fig. 5B, cluster 1), whereas those upregulated in the To delineate significant differential gene expression between tumor were broadly associated with regulation of cell cycle pro- TASPR T cells, genome-wide mRNA expression was compared by gression (Fig. 5A, cluster 2, p =2.793 10228). KMC further divided one-way ANOVA and narrowed by a 2-fold-change (Fig. 4A–C). cluster 2 into 13 smaller clusters by similar patterns of gene ex- There were no significant differences among TASPR TIL, re- pression that were enriched for more specialized biological pathways gardless of vaccination, or between TASPR T cells from the pe- (Fig. 5A, clusters 3–14). These clusters within the molecular profile riphery, regardless of tumor growth (Fig. 4A, 4B). However, when of TASPR TIL allow systematic focus of individual pathways asso- TASPR T cells from the tumor were compared with those from the ciated with TIL immunosuppression. For instance, cluster 5 was periphery, we identified a total of 1973 genes that were differen- enriched for many gene products associated with arrest in cell cycle tially expressed (Fig. 4C). We further narrowed the list to 1164 (Fig. 5B), and the top biofunction related to the cell cycle in cluster 26 differentially expressed genes by SAM (Fig. 4D, Supplemental 5wasG1/S-phase transition (p =2.123 10 ). Additionally, genes Table III) (27, 28). Unlike ANOVA, which assumes an underly- upregulated in the periphery were associated with TCR signaling and ing normal distribution, SAM is a nonparametric technique for CD28 costimulation (cluster 1, p =3.163 1027 and 1.37 3 1027, 6 MOLECULAR PROFILE OF TIL Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 3. Genome-wide mRNA expression of tumor-specific TIL from the CT26 tumor is similar to transcription profiles of other hypofunctional CD8+ Tcells; similarities are driven by distinct gene expression. Genome-wide mRNA expression of TASPR T cells, acquired as described in Fig. 2A, was simultaneously compared with other published CD8+ T cell transcriptional profiles for similarities by GSEA (30). (A) Plots show enrichment of genes associated with the indicated T cell profile or gene set. Vertical black lines show where gene sets match genes expressed by TASPR T cells from the tumor (red, TILTOT = TIL and TILV) and spleen (blue, SpTOT = SpTV and SpV). The NES reflect the degree to which a gene set is overrepresented at the extremes of the entire ranked list of gene expression differences between TASPR T cells from the tumor and spleen; an NES value farther from 0 indicates greater enrichment of the indicated profile genes among TASPR T cells from the tumor (positive NES) or spleen (negative NES). The green line is a visual representation of gene set enrichment along the ranked list. The leading edge, roughly shown by a dashed circle, includes the core genes from a gene set that contribute most to the deviation of an NES from 0. Profile gene sets were determined to significantly cluster among genes highly expressed by TASPR T cells from the tumor or spleen when p #0.001 (30) and familywise error rate (FWER) #0.05 (which suggests the probability of type 1 errors) and are delineated by green rather than gray labels. (B) The majority of leading edge genes differ between the hypofunctional CD8+ Tcell profiles. These core genes drive the NES away from 0 in the leading edge (circled in A) and are compared in the Venn diagram and Supplemental Table II. respectively), whereas genes induced in TASPR TIL included in- (cluster 2, p =4.93 10236). Suboptimal TCR and costimulation, hibitory receptors, such as PD-1 (PDCD1, cluster 12, Supplemental PD-1, and TGF-b are all known to restrict TIL function and to inhibit Table III), and reflected an environment of strong TGF-b signaling cell cycle progression (5, 52–54). The Journal of Immunology 7 Downloaded from http://www.jimmunol.org/

FIGURE 4. Molecular profile of tumor-specific TIL that are naturally responding and polyclonal. Genome-wide mRNA expression of TASPR T cells acquired in Fig. 2A (TILTOT = TIL and TILVand SpTOT = SpTV and SpV) were compared for differential gene expression. (A–C) Volcano plots comparing the indicated groups illustrate significant 2-fold differential gene-expression by one-way ANOVA in red. (D) Differentially expressed genes were narrowed by ANOVA, as in (C), and SAM. Genes common to all three groups were designated the molecular profile of TASPR TIL.

Interestingly, the majority of inhibitory receptors were distributed phases (68), and total DNA staining to set apart the G2/M phase between different clusters. Cluster 9 contained two surface receptors, (9, 69). Because many of the same gene products are associated TIM-3 (HAVCR2) and 2B4 (CD244), that can broadly inhibit function with cell cycle inhibition and progression depending on post- by guest on September 25, 2021 of CD8+ T cells (55, 56), whereas another such inhibitory receptor, translational modifications (66, 70), we determined cell cycle T cell Ig and ITIM domain (TIGIT) (57), was contained separately in progression of CD8+ T cells from the CT26 tumor relative to those cluster 13. PD-1 (PDCD1) (7) segregated into cluster 12 that included from the periphery (Fig. 6A). As expected (3, 71), TIL did not other surface receptors not usually expressed on CD8+ T cells, such as progress through the cell cycle as readily as peripheral counter- CD80, a molecule that is expressed on CD8+ T cells from HIV- parts in response to TCR and costimulation. Most stimulated and infected patients (58). Inhibitory receptor CTLA-4 and the contro- unstimulated TIL were in the late-G1/early-S phase. versial surface receptor IL-2RA (CD25), which are associated with Dual PD-1 and LAG-3–positive CD8+ T cells were the most negative and positive effects on antitumor T cell function (59–61), abundant among CT26 TIL (Fig. 6B). Although previous studies respectively, were grouped together in cluster 4 that was broadly as- have tied PD-1 more strongly to the cell cycle, specifically arrest 27 sociated with cell-to-cell signaling and interactions (p =2.13 10 ). in the G0/G1 phase (53), we were interested in analyzing both Clusters that contained inhibitory receptors were also enriched PD-1 and LAG-3 expression in TIL because TIL were blocked in for other gene products that have been independently tied to some late-G1/early S-phase, and LAG-3 surprisingly clustered with of the same biological pathways (Fig. 5A, 5B). For instance, in- many cell cycle–regulated genes. Stimulated TIL in G0/G1 phase hibitory receptor LAG-3 (62) was among the gene products were enriched among those that express PD-1, whereas stimulated grouped into cluster 3 that was associated with arrest in cell cycle, TIL in S phase were enriched among those that express LAG-3 including CDKN2C, CDKN3, CHN2, LYN, and PLK1. Besides and those that express both PD-1 and LAG-3 (Fig. 6C). Stimulated LAG-3, many gene products associated with cell cycle arrest in TIL also expressed higher levels of PD-1 and LAG-3 in the G0/G1 KMCs 3 and 5 have recently attracted attention for functions that and S phase, respectively (Fig. 6D). Although expected to peak in may ultimately regulate DNA replication and licensing cell cycle the S phase (72), LAG-3 protein expression has not previously + progression beyond the G1 and S phases (63–66). Because much been investigated on wild-type CD8 T cells throughout the cell of the downstream signaling that mediates the effect of these in- cycle. As suggested by cluster analysis of the molecular profile of hibitory receptors on TIL hypofunction is unknown, these clusters TASPR TIL, these data functionally tie control of LAG-3 ex- may provide a valuable and fluid tool to dissect the extent of pression with the cell cycle and corroborate that progression of distinction and overlap between such pathways. CT26 TIL through the G1/S-phase boundary is inhibited upon LAG-3 expression associated with cell cycle progression TCR- and costimulation. Hypofunctional TIL incapable of tumor clearance have decreased Discussion capacity to progress through the cell cycle (9, 48, 67). This The molecular profile of TASPR TIL from CT26 tumors identifies foundation was largely laid by studies that distinguish cycling many known, hypothesized, and potentially novel pathways that cells by staining for Ki-67 (9, 67), which groups noncycling cells underlie TIL hypofunction. As expected, CT26 TIL express high in the G0/G1 phase from cycling cells in the late-G1, S, and G2/M levels of multiple inhibitory receptors and are hypofunctional; 8 MOLECULAR PROFILE OF TIL Downloaded from http://www.jimmunol.org/

FIGURE 5. Molecular profile of tumor-specific TIL from the CT26 tumor highlights transcriptional regulation of cell cycle inhibition. The molecular profile of TASPR T cells acquired in Fig. 2A was analyzed in clusters for enrichment of biological pathways. (A) KMC divided differentially expressed by guest on September 25, 2021 genes into 13 clusters for focused pathway analyses. Heat map shows log2-transformed expression intensities mean centered at the probe level. (B) Pathway analysis of gene products from clusters 1, 3, and 5 revealed corresponding biofunctions enriched in TASPR T cells from the spleen and tumor. Gray dashed lines represent p = 0.05. however, immediate accumulation of inhibitory receptors and lack Although the profile of patient TILN expressed TCRs specific for of function in the tumor environment revealed a robust similarity a tumor/self-antigen, GSEA showed that TILN hypofunction most between the molecular profiles of TASPR TIL and self-tolerance. resembles murine viral exhaustion (17, 30, 46). The more recent Differential gene expression between additional hypofunctional genome-wide mRNA expression profile of total CD8+ TIL from a and functional CD8+ T cell subsets, as well as between pathways mouse model of melanoma also found TIL hypofunction similar that restrict T cell function, provide a map to navigate the intri- to viral exhaustion (34). Neither molecular profile was systemat- cacies of TIL hypofunction. ically compared with self-tolerance (17). In addition, others have The molecular profile of TASPR TIL may provide a novel re- previously observed that total CD8+ TIL that naturally respond to source for studies to characterize and boost the response of nat- CT26 tumors, regardless of TCR specificity, are comprised largely urally responding TIL against a tumor. The TASPR profile differs of exhausted T cells and a smaller fraction of effector T cells (9). from other relevant strategies as follows. Tumor-specific CD8+ Therefore, we expected the profile of TASPR TIL from CT26 T cells isolated from lymph node metastases of previously vac- tumors to reflect a hypofunctional T cell program most similar to cinated patients with melanoma, or TILN, likely express T cell exhausted CD8+ T cells during chronic viral infection (31). genes reflective of an anatomical location of the metastases in a TASPR TIL have gene expression and functional capacities most lymph node rather than a primary tumor tissue (73). Another similar to a recently published molecular profile of self-tolerance molecular profile corresponding to total CD8+ TIL is not restricted (33) followed by exhaustion (31). Functional tumor-specific CD8+ to tumor reactivity (34); both profiles were not as similar to T cells do not become gradually hypofunctional over weeks, but TASPR TIL as exhausted and self-tolerant T cells. Genome-wide become tolerized within 1 d of trafficking into this solid tumor mRNA expression of TASPR TIL reflect a molecular profile of model. Although both self-tolerance and viral exhaustion can be at T cells that bear TCRs of relatively low to intermediate affinity for least temporarily overridden for a functional T cell response (3), tumor Ag (24). Conversely, a molecular profile of tumor-specific peripheral self-tolerance is a T cell program acquired quickly in TIL that have been genetically modified to all express the same response to initial TCR exposure to self-antigen in the absence of tumor-specific TCR may not model inhibitory pathways, such as immunostimulatory signals (3), whereas viral-exhaustion is a suboptimal TCR stimulation, that are known to restrict function of T cell program acquired gradually during chronic non–self-TCR a naturally responding polyclonal repertoire of TIL. or immunostimulatory signals (47, 48). Distinctions between T cell The Journal of Immunology 9 Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 6. Slowed cell cycle progression of CD8+ T cells in the CT26 tumor corresponds to a novel pattern of inhibitory receptor expression predicted + by the molecular profile of TIL. Total CD8 T cells from CT26 tumors (TILTOT) and spleens (SpTOT) from timelines shown in Fig. 2A were untreated or treated with Abs to CD3 and CD28 for 48 h before a 1 h (or indicated time) pulse with the thymidine analog EdU. Flow cytometric analyses identified cell cycle phase and inhibitory receptor expression of live CD8+ T cells. (A) Cell cycle phase was resolved by staining for EdU and by the DNA dye 7-AAD.

Frequencies are shown in sub-G0/G1 (gray), G0/G1 (black), S (red), and G2/M (blue) phase. (B) Inhibitory receptor expression (PD-1 and LAG-3) was determined in the presence and absence of stimulation. (C) Inhibitory receptor expression on TILTOT during G0/G1 and S phase was determined. (D) + Representative histograms (left panel) of naive CD8 T cells from spleen (gray, negative control) and stimulated TILTOT cells (G0/G1 [black], S [red], and G2/M [blue] phase) are shown. Numbers indicate the geometric mean fluorescence intensity (gMFI). At this time point (48 h), frequency of synchronized T cells in G2/M phase (blue) was too small to confidently determine inhibitory receptor expression. All data (right panel) represent three independent experiments, n = 3–10 biological replicates per group, and error bars indicate SEM. *p # 0.05, **p # 0.01, ***p # 0.001, ****p # 0.0001. programs of self-tolerance and exhaustion are therefore not re- heterogeneously exhausted and self-tolerant or that TILN (5) and stricted semantics, but have widespread implications for devel- CT26 TIL hypofunction are T cell programs that are distinct yet opment of cancer immunotherapies that mobilize patient TIL overlapping with other T cell subsets. Both scenarios are in line against a mutated or nonmutated self-tumor Ag (13–16). with current literature; CD8+ T cell hypofunction varies by pa- We have since reanalyzed the patient TILN data and found tient, assaulting malignancy, and over time (5, 48). A shift in focus significant overlap between gene expression of TILN (5) and self- toward overlapping mechanisms known to underlie multiple tolerant murine T cells (33) that was also associated with control hypofunctional T cell subsets may enhance the functional re- of cell cycle progression (74). The line between self-tolerance and sponse in the majority of heterogeneous TILN and TIL (44). exhaustion gene sets among TILN and TASPR TIL is blurred, and The tumor environment varies in response to different Ags, in pathways may overlap. However, there is little overlap between different tumor models, and across species (75, 76). Nevertheless, the exhausted and self-tolerant gene sets that define hypofunction CT26 TIL have a hypofunctional phenotype (9) and molecular of TILN (4 out of 114 genes) and TASPR TIL from CT26 tumors profile similar to exhausted patient TILN (9, 17) and exhausted (12 out of 195 genes) as similar to viral exhaustion and self- TIL from an inducible model of melanoma (34). CT26 is therefore tolerance. These data suggest that TILN (5) and CT26 TIL are an immunogenic tumor model with widespread implications as 10 MOLECULAR PROFILE OF TIL to whether TIL tolerance can be overridden against tumor/self- phase (72), variations in LAG-3 expression on cycling wild-type antigens or if efforts should be focused on restoring function of CD8+ T cells has not been investigated prior to this study. exhausted TIL against mutated/foreign tumor Ags (11, 76, 77). We Although PD-1 and CTLA-4 specifically restrict cell cycle pro- + predict as molecular profiles of TIL specific for mutated tumor Ags gression of CD8 T cells beyond G0/G1 phase (87–89), LAG-3 has become available, comparison with the CT26 TIL that are self-specific been more broadly demonstrated to restrict T cell expansion in will identify a highly conserved gene expression signature among TIL. many inflammatory contexts in vivo (90). Although rare, CT26 TIL Transcriptional regulators interpret multiple extracellular sig- singularly positive for PD-1 were slightly enriched in G0/G1 phase; nals to determine T cell function and differentiation; such intra- cycling TIL did not express significantly higher levels of PD-1 in cellular targets represent promising avenues to simultaneously disrupt any phase. Although these data do not confirm in vitro experiments multiple inhibitory pathways that restrict antitumor function of regarding PD-1 regulation of cycling CD8+ T cells (53), IPA of the TIL (44). FOXP1, a transcription factor that has recently been report- cluster in which PD-1 was grouped predicts less association of ed as highly expressed in TIL and a key regulator of T cell hypo- PD-1 and similarly expressed gene products with the cell cycle function in many cancers (78), is downregulated in TASPR TIL (p =5.103 1027) than other biofunctions in TASPR TIL, such as from CT26 tumors. FOXP1 expression in TASPR TIL is similar to cell death and survival (p =6.623 1028) and immune cell traf- the genome-wide mRNA expression profiles of virally exhausted ficking (p = 6.45 3 1028) (49). As with cotherapies that target both T cells (79), hypofunctional TILN (17), and naturally responding PD-1 and CTLA-4 (8), deletion of both PD-1 and LAG-3 in tumor- total CD8+ TIL from a mouse melanoma model (34). In the model specific CD8+ T cells increases control of tumor burden more than melanoma system, MAF was the alternatively pursued transcrip- singularly targeting one of these inhibitory receptors (62, 91) and tional regulator of TIL hypofunction (34), but MAF is not dif- represents attractive therapeutic targets to restore TIL function (92). Downloaded from ferentially expressed between TASPR T cells from the tumor and Notably, deletion of both PD-1 and LAG-3 accounts for less au- periphery. Among gene products corresponding to transcriptional toimmunity relative to deletion of CTLA-4 (91, 93). regulators that are upregulated between TASPR T cells from the LAG-3 gene expression was among the few to overlap between tumor versus the periphery are HIF1A, E2F7, IRF8, IKZF2, and TASPR TIL hypofunction, self-tolerance, and viral exhaustion STAT3; conversely, among those downregulated in TASPR TIL (Supplemental Table II). Although the entire gene set upregulated + are TCF7, TOB1, FOS, and RNX1 (Supplemental Table III). by self-tolerant CD8 T cells (33) was associated with the cell http://www.jimmunol.org/ Differential expression of genes involved in regulating transcrip- cycle (p = 2.59 3 10218, Supplemental Table I), overlapping gene tion in TASPR TIL suggests an altered pattern of differentiation expression of TASPR TIL and self-tolerant CD8+ T cells (33) was among T cells from the tumor and spleen. further enriched for cell cycle gene products (p = 8.60 3 10220, E2F1, a transcription factor closely associated with late-G1/ Supplemental Table II). Genes associated with the cell cycle were early-S phase, was predicted to be a key mediator between ex- similarly noted among vaccinated TILN and virally exhausted tracellular stimuli and the decreased proliferative capacity of TIL, T cells when compared with their functional counterparts (17, 31). although increased expression did not meet the strict 2-fold These data are striking in that alterations in cell cycle progression/ change criterion used for true difference among TASPR T cells proliferation modify TIL susceptibility to apoptosis and memory (q-value = 0.00; 1.98 fold-change). Nevertheless, it is not uncom- T cell development (69, 94, 95). Genes highly expressed by by guest on September 25, 2021 mon for transcriptional regulators to be controlled posttransla- memory CD8+ T cells (31) were collectively downregulated by tionally and for subtle changes in expression of transcriptional TASPR TIL. Our analyses dissect the extent of overlap and dis- regulators to have global effects (44, 70). The top five transcription tinction between the pathways that cooperatively restrict T cell factor motifs enriched in promoter regions (41) of genes upregu- functions and may accelerate identification of effective cotherapy lated by TASPR TIL were all variants for E2F1 (p , 1 3 10210, combinations and intracellular targets to efficiently boost antitu- FDR , 1 3 1029, Fisher exact test). Additionally, E2F1 was mor function of TIL. among the top transcriptional regulators predicted by IPA to be a As evident by the lack of published studies, isolating a sufficient key upstream regulator of differential gene expression in TASPR number of tumor-specific TIL has been historically limiting for high- TIL (p =2.63 10221, cluster 2). E2F activity is extremely complex throughput analyses. Only differences between the most abundant and regulates some of the most basic cell functions, such as cell transcripts are reliably detectable after the necessary amplification division and apoptosis (80, 81). During late-G1/early-S phase, E2F1 methods to detect genome-wide mRNA expression (96). Subtleties activity determines if cells progress through the cell cycle or arrest that underlie genome-wide mRNA expression profiles of small and apoptose (66). E2F1 is also a potent regulator of central and numbers of patient TIL, such as differential expression of tran- peripheral tolerance (80, 82–85). Implications of these data are that scription factors, are difficult to biologically replicate due to het- E2F1 regulates self-tolerance (44) and is likely to compromise erogeneity (17, 96). We avoided these problems by tetramer sorting + function of CT26 TIL in late-G1/early-S phase. many tumor-specific CD8 Tcells($40,000 cells, 5 ng RNA) from Additionally, TIL hypofunction is downstream of many known pooled samples of genetically identical mice. Future mRNA ex- inhibitory pathways targeted individually in patients to restore pression profiles from relatively small numbers of patient TIL may antitumor function of T cells (86), which we analyzed by KMC, an be validated by comparison with the TASPR profile, as technologies unsupervised sequestering of differentially expressed genes into are rapidly advancing (96). We predict that genes with high ex- smaller clusters by similar patterns of gene expression (38). These pression differences between patient TIL and their functional clusters allow focus on gene products associated with specific counterparts will have similar expression to the TASPR profile, but biological pathways that have not yet been investigated together or that subtle yet significant differences among TASPR T cells will in TIL. The TASPR profile predicts that heterogeneous CT26 TIL elucidate of the intricacies of collective pathways that restrict an- receive many inhibitory signals through overlapping mechanisms titumor function of patient TIL. to restrict cell cycle progression. Cell cycle progression and LAG-3 protein expression confirm pathway analysis of the TASPR Acknowledgments TIL profile. We identified novel control of inhibitory receptor We thank the exceptional personnel of the CU-SOM Cancer Center Flow + LAG-3 expression on cycling CD8 T cells, which peaks in S Cytometry Shared Resource, especially Lester Acosta and Karen Helm. We phase. Although LAG-3 is highly expressed on NKT cells in S also thank Todd Woessner, Curtis J. Henry, Cydney Rios, Brian P. O’Connor, The Journal of Immunology 11

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Sort

Scatter Singlets Live CD8+ T cells Antigen-experience Tumor-specifcity c Blue f Tet PE Tet d SSC-H SSC-H AH1-L Dump Paci

FSC-H SSC-W CD8α FITC CD11a AF647

Post sort

67% 100%67% 100% 98% c Blue f Tet PE Tet d SSC-H SSC-H AH1-L SSC-Height Dump Paci FSC-H SSC-W CD8α FITC CD11a AF647 SSC-Height Supplemental Figure 1. Tumor-specific CD8+ T cells were isolated for microarray analysis from the timelines illustrated in Figure 2A. Representative FAC-sort of CD8+

7-AAD- CD4- B220- MHC-II- CD11a+ AH1-Ld Tet+ cells from a tumor sample is shown. The “Dump” collectively refers to 7-AAD+, CD4+, B220+, and MHC-II+. For each sample, βgal-Ld Tet was used as a negative control to set AH1-Ld Tet gates. After

FAC-sorts, all samples were 90-100% pure tumor-specific CD8+ T cells.

Supplemental Table 1. Transcriptional profiles of published T cell programs compared to tumor-specific TIL T cell program of interest Exhausted (overexpressed) Exhausted (underrexpressed) Memory (overexpressed) Memory (underexpressed) Effector (overexpressed) Effector (underexpressed)

Naïve virus-specific CD8+ T Naïve virus-specific CD8+ T Naïve virus-specific CD8+ Exhausted virus-specific CD8+ cells; also not in memory and Memory virus-specific CD8+ cells; also not in exhausted Effector virus-specific T cells; als not in memory, T cell programs compared for T cells vs. effector vs naïve virus-specific T cells vs. and effector vs naïve virus- CD8+ T cells vs. and exhausted vs naïve relative gene expression CD8+ T cells specific CD8+ T cells virus-specific CD8+ T cells Naïve virus-specific CD8+ T Naïve virus-specific CD8+ T Naïve virus-specific CD8+ T Naïve virus-specific CD8+ T Naïve virus-specific CD8+ Naïve virus-specific CD8+ cells cells cells cells T cells T cells Reference Wherry EJ et al. 2007 Wherry EJ et al. 2007 Wherry EJ et al. 2007 Wherry EJ et al. 2007 Wherry EJ et al. 2007 Wherry EJ et al. 2007 Gene set 1110067D22Rik 1110038D17Rik 6330512M04Rik 2610019F03Rik 1110008P14Rik 1190002H23Rik 1810035L17Rik/Nrp 1190002H23Rik Acot7 Actn1 2810417H13Rik 2610019F03Rik 2810417H13Rik 2610019F03Rik Ahnak Blk Acot7 5730403B10Rik/Hmox2 5730469M10Rik 2810407C02Rik Anxa1 Ccr7 Actb Abca1 6330403K07Rik 4833420G17Rik Anxa2 Cd24a Adam19 Acss1 Acot7 4922505G16Rik/Rpn2 Aqp9 Cd74 Ahnak Actn1 Adam19 A630038E17Rik AW112010 Cd79b Aim1 Amd-Ps6 Ahnak Abca1 Bag3 Cnn3 Anxa1 Amd1/Amd2 Alcam Abce1 Bhlhe40 Cxxc5 Anxa2 Amd1/Amd2 Anxa2 Ablim1 Casp1 Ddit4 Apobec2 Amd1/Amd2 Art3 Acadm Ccl5 Dntt Atp5l Apex1/Tmem55b Atf1/Gm1862 Acp5 Ccr2 Gsn Atp5l Bcl2 Atf1/Gm1862 Acss1 Ccr5/Ccr2 H2-Aa AW112010 Bzw2 AW112010 Actb Cebpb/Tmem189 H2-Ea-ps Bag3 Ccnd2 Bag3 Actn1 Crtam H2-Eb1 Bhlhe40 Ccr7 BC055004 Adcy7 Ctla2a Idh2 Birc5 Cd72/Tesk1 Bhlhe40 Add1 Ctla2b Igfbp4 Bub1 Clk1 Bub1 Adh5 Ctla4 Igk-V28 C330027C09Rik Clk4 C330007P06Rik Akap8 Cxcr3 Il6ra Capn2 Cnn3 C330027C09Rik Anapc5 Ddx6 Il6st Casp1 Ctsl Capzb Arhgap1 Dock5 Itgae Casp3 Ctss Car2 Arhgef1 Dusp1 Kif23 Ccdc50 Cxcr4 Casp1 Arl4c Ell2 Lef1 Ccl5 Cxxc5 Casp3 Ascc1 Emp1 Lyz2 Ccl9 Ddx3x Casp4 Atp13a3 Eomes Ms4a1 Ccnb1/Gm5593 Dgka Ccdc50 Atp6v0a2 Errfi1 Myb Ccnb2 Dnajb2 Ccl3 Atp6v0b F2r Pdk1 Ccr2 Dnajc7/Nkiras2 Bnip3l/LOC100044398/Gm104 Ccl4 Fasl Piga Ccr5/Ccr2 Dntt 80 Ccl5 Bzw1 Fcgr2b Ppic Cd48 Dph5 Ccnb1/Gm5593 Bzw2 Fos Rab3ip Cdc6 Dtx1/Gm19706 Ccnb2 Ccnd2 Fosb Ramp1 Cdk1 Dusp1 Ccr5/Ccr2 Ccr7 Gm6742 Scd1 Cks1b/Gm6340 Dusp6 Ccrl2 Cct3 Gm7278 Sfmbt2 Cks2 Eif4a2 Cd160 Cct4 Gp49a Smc4 Clic1 Eif4g1 Cd200 Cct5 Gzmb St6gal1 Cma1 Eif5/LOC100047658 Cd244/LOC677008 Cct8 Gzmk Tec Cox17 Elovl5 Cd7 Cd1d1 Gzmm Trim59 Crip1 Emb Cd84 Cd1d2 H2-Q10 Tspan32 Csda Ets2 Cd9 Cd24a Hiatl1 Ttc14/Ccdc39 Ctla2a Evl Cdk1 Cd72/Tesk1 Hopx Ctla2b Fam101b Chek1 Cdc42se2/LOC100045021 Id2 Ctla4 Fam134b Chl1 Cirh1a Ifng Ctsd Fkbp3/Prpf39 Cit Clk2 Il18r1 Cxcr3 Fos Ckmt2 Clk4 Il18rap Cyfip1 Gadd45a Cks2 Cmah Itgax Dennd5a Gart Clic4 Cnn3 Itgb1 Dnajc15 Gas5 Coch Crlf3 Junb Dock5 Gm1005/Hnrnpa Cox17 Ctsl Kcnj8 Dstn Gm10052/Hnrnpa Cpa3 Cxxc5 Klf4 E2f8 Gm5643/Hnrnpa1 Cpsf2 Cytip Klrc1 Ech1 Gm5803/Hnrnpa1 Cpt2 D10Wsu52e Klrg1 Ect2 Gm7862 Crygb Ddx39b Klrk1 Ell2 Gnl3 Cryl1 Ddx5 Lgals1 Emp1 H2-Aa Cst7 Dgka Lilrb4 Eomes H2-Ea-ps Ctla2a Dnajc7/Nkiras2 Ly6a Errfi1 H2-Ob Ctla2b Dntt Ly6c1/Ly6c2 Ezh2 Hmgn1/LOC100044391 Ctla4 Dph5 Ly6c1/Ly6c2 F2r Hnrnpa1L2/Loc6 Cxcl10 Dscaml1 Mdfic Fasl Hnrnph1 Cxcr3 Dtx1/Gm19706 Myo1f Fcgr2b Hspa9 Cyfip1 Eef2 Nrp1 Fgl2 Igfbp4 Dock5 Eif2s1 Null Fignl1 Il4ra Dock7 Eif4a2 Odc1 Fyn Il6ra E2f8 Eif4g1 Pglyrp1 Gapdh Il6st Ect2 Elovl5 Prf1 Gapdh Il7r Eea1 Emb Prr13 Gapdh Isoc1 Ell2 Eml5 Rn18s/Rn45s Gapdh/Gm2606 Itgae Entpd1 Eng Rnf138 Glrx Itm2a Eomes Etnk1 Runx2 Gm10124 Itpr2 Etf1 Ets1 S100a4 Gm10291 Kctd12/Mir5130 F2r Evl S100a6 Gm10291 Lef1 Fasl Fam101b St3gal6/Dcbld2 Gm11401 Mdm4 Fgl2 Fam102a Tnfaip3 Gm12070/Gapdh Mettl8 Fignl1 Fam134b Txndc5 Gm12070/Gapdh Mettl9 Fyn Fkbp4 Gm12070/Gm2606 Mll1 Gapdh Fos Gm12070/Gm4609 Morf4l2 Gapdh Frat2 Gm14148 Ms4a1 Gapdh Gadd45a Gm14407 Myb Gapdh Glt25d1 Gm2467 Myc Gapdh/Gm12070 Gm10845 Gm2606/Gm10291 Mycbp2 Gas2 Gm13777 Gm3671 Nedd4l Gcdh Gm14165 Gm3809 Nfkbiz Gdf3 Gm14407 Gm4804 Nrip1 Gdpd5 Gm2a Gm4929 Nsg2 Gem Gm4638/Gm4671 Gm5068 Null Glrx Gm4671 Gm5177 Nxf1 Gm10291 Gm4761 Gm5523/Gapdh/Gm12070 P2rx4 Gm10291/Gm1029 Gm4833 Gm6340 Parp8 Gm12070/Gapdh Gm4959 Gm6946 Pdk1 Gm12070/Gapdh Gm5148 Gm7123 Peli1 Gm12070/Gapdh Gm5191 Gm7183 Phf12 Gm14148 Gm6983 Gm7286 Piga Gm2467 Gm7246 Gm8055 Pkp4 Gm2606/Gm10291 Gm7290 Gm8174 Plekha1 Gm3671 Gm7862 Gm8513 Ppic Gm3809 Gm8231 Gm8681 Prkd3 Gm4804 Gm8624 Gm9028 Rab3ip Gm4833 Gm9514 Gm9034 Ramp1 Gm4929 Gnas Gm9081 Rere Gm5068 Gnb2l1 Gm9238/Loc6776 Rgs10 Gm5177 Gnl3 Gm9568 Satb1 Gm5523/Gapdh/Gm12070 Gsn Gp49a Scd1 Gm6946 Gtf2i Gzma Sell Gm7123 H2-Ke6 Gzmb Sema4b Gm7183 H2-Ob Gzmk Sfmbt2 Gm7286 Hba-a1/Hba-a2 H2-Q1 Sfrs7 Gm8055 Hba-a1/Hba-a2 H2-Q10 Slc12a7 Gm8174 Hbb-b1/Hbb-b2/Beta-s H2-Q2 Smad1 Gm8231 Hexa H2-Q6/H2-Q8/LOC68395 Smad7 Gm8513 Hmgcs1 H2-Q6/H2-Q8/LOC68395 Smarca2 H2-Q7/H2-Q9/H2-Q8/H2- Gm8681 Hmgn1/LOC100044391 Smc4 Q6/LOC68395 Gm9028 Hmha1 H2-T10/H2-T9 Snhg5 Gm9034 Hnrnpu H2afz Socs3 Gm9081 Hspa8 Hba-a1/Hba-a2 Spred2 Gm9568 Icam2 Hba-a1/Hba-a2 Ssbp2 Gp49a Idh2 Hbb-b1/Hbb-b2/Beta-s St6gal1 Gpd2 Ifnar1 Hiatl1 Tcf12 Gpr56 Igk-V28 Hist3h2a/Trim17 Tcf7 Gpr65 Ikbke Hmgb2 Tec Gzma Il17ra Hopx Tgtp1/Tgtp2/Gm12185 Gzmb Il4ra Id2 Tlr6 Gzmk Il6ra Ifng Tnfaip8 Hiatl1 Il6st Il18r1 Tspan13 Hist3h2a/Trim17 Il7r Il18rap Tsr1/Srr Hmgb2 Ilvbl Il1rl1 Ttc3 Id2 Impdh2 Itga4 Vcam1 Ifih1 Isoc1 Itgax Wdr92 Ifng Itgae Itgb1 Xlr3b/Xlr3c/Xlr3a Irf4 Itgb7 Itgb2 Xlr3b/Xlr3c/Xlr3a Irf8 Kcnn4 Kif11 Xlr3b/Xlr3c/Xlr3a Klra3/LOC100862437/LO Isg15 Kctd10 Zbtb20 C100862410/Klra9 Isg20 Kdm3a Klra9/Klra3/Klra10 Zfp281 Itga4 Klf13 Klrc1 Zmym6/Gm12942 Itgav Klf2 Klrg1 Itgb1 Klf3 Klrk1 Itih5 Klk8 Kpna2 Jak3/Insl3 Klrd1 Lgals1 Klf10 Kpnb1 Lgals3 Klk1/Klk1b5/Klk1b4 Lbr Lilrb4 Klra3/LOC100862437/LOC10 Lck Lonrf1 0862410/Klra9 Klra9/Klra3/Klra10 Lef1 Lxn Klrg1 Ltb Ly6a Kpna2 Lyz2 Ly6c1/Ly6c2 Lag3 Macf1 Ly6c1/Ly6c2 Lat2 Map4k4 Mad2l1/LOC100045924 Lclat1 Mapk8 Mdfic Lgals1 Mat2a Med12l Lgals3 Mettl8 Mki67 Lilrb4 Mettl9 Myo1f Litaf Mipol1/Prps1l3 Ndufa6 Lman2 Ms4a1 Nrp1 Lonrf1 Nedd4l Null Ly6a Nfe2l2 Nusap1/Oip5 Mad2l1/LOC100045924 Nfkbiz Pbx3 Mdfic Npc2 Pfkp Mki67 Nsg2 Pglyrp1 Mrpl46 Nsun2 Phf13 Mx1 Null Plscr1 Myh4 Numb Pqlc3 Ndfip1 Nxf1 Prc1 Nfatc1 Osbpl11 Prdm1 Nfil3 Pak2 Prf1 Npcd/Cbx6/Nptxr Pdha1/Map3k15 Prr13 Nr4a2 Pdk1 Ptgr1 Nrp1 Peli1 Racgap1 Nucb1 Piga Reep5 Null Pik3cd Rhoq/Pigf Rn18S ??=>?? 10445185 Nusap1/Oip5 Pitpnc1 Rn18s/Rn45s OR 10536170 Rn18s/Rn45s Pawr Plac8 Rora Pbx3 Pld3 Rpa2 Pdcd1 Ppp2R5a Rpl27a Penk Ppp2R5a Rpl27a Perp Prdx6/LOC677654 Rpl27a Pglyrp1 Prkd2 Rpl38 Plin2 Prkd3 Rrm2 Plk4 Prps1 Runx2 Plscr1 Prps1l1 S100a10 Pon2 Ptpn6 S100a11 Pqlc3 Ptprc S100a13 Prc1 Rabac1 S100a4 Prdm1 Ramp1 S100a6 Ptger2 Rap1gds1 Sec61g Ptger4 Rbm38 Serpinb9 Ptpn13 Rere Sh2d1a Rbm39 Rgs10 Sh3bgrl Rcn1 Rpl10 Slc4a7 Rgs16 Rpl10a Smc2 Rhoq/Pigf Rpl10l Snx10 Rnf11 Rpl13 St3gal6/Dcbld2 Romo1 Rpl22/LOC100504863 Stard10 Rpa2 Rpl27a Stmn1 Rpl38 Rpl27a Tacc3 Rps4X Rpl27a Tceb2 Rps4x Rpl28 Tmem49/Mir21 Rrm2 Rpl28 Top2a Rsad2 Rpl3 Trappc1 Runx2 Rpl8 Txn1 S100a11 Rplp0 Txndc5 S100a13 Rplp1 S100a4 Rps16/Gm10079 S100a6 Rps23 Scin Rps23/Gm5148/Gm8618 Sec61g Rps28 Sept4 Rps3 Serpinb6a Rps3a Serpinb9 Rps3a Sh2d2a Rps4x Sh3bgrl Rps4x Shkbp1 Rps7 Slc29a1 Rps8 Slc4a7 Rps8 Smc2 Rps8 Snrpb2 S1pr4 Snx10 Satb1 Spock2 Scd1 Spp1 Sdha Stmn1 Sell Sypl Sema4a Tacc3 Sfmbt2 Tank Sfpq Tbc1d22a Sgk1 Tcea2 Siah1a Tcrg-V3/Tcrg-V Skp1a Tcta Slc12a7 Tctn3 Slco3a1 Tfdp1 Smad1 Tmem109 Smarca2 Tnfrsf1a Snord15b/Rps3 Snord38a/LOC100862183/Gm Tnfrsf1b 15501/Rps8 Tnfrsf9 Snrpd3 Top2a Snx4 Tor3a Spnb2 Trim25 Srpk1 Trim47 Ss18 Ttc39b Ssbp2 Tubb2a St6gal1 Txn1 Stk38 Ube2t Supt4h1/Gm3258 Vamp7 Supt4h1/Gm3258 Vamp8 Supt5h Vmp1 Tcf7 Vps37a/Mtmr7 Tec Wbp5 Tgtp1/Tgtp2/Gm12185 Wnk1 Tlr6 Zfp91-Cntf/Zfp Tmc6 Tmed2 Tmem50b Tmem66 Trim28 Tsr1/Srr Ttc3 Tubb5 Twf2 Ubp1/Fbxl2 Use1 Wdr92 Xlr3b/Xlr3c/Xlr3a Xlr3b/Xlr3c/Xlr3a Xlr3b/Xlr3c/Xlr3a Zfp36 Znrf2

Self tolerant Deletional tolerance Deletional tolerance T cell program of interest Self tolerant (overexpressed) Anergy (overexpressed) Anergy (underexpressed) (underexpressed) (overexpressed) (underexpressed) Functionally rescued tolerant Tolerant self-specific CD8+ T CD8+ T cells during and memory self-specific CD8+ T cells undergoing cells (K means clusters 9 and immunity and lymphopenia- Various anergic T cells vs. Various anergic T cells vs. CD8+ T cells (K means clusters deletional tolerance vs. T cell programs compared for 13) vs. induced proliferation vs. relative gene expression 7, 8, 10, 11 and 14) vs. CD8+ T cells during Naïve and memory self-specific Tolerant self-specific CD8+ T CD8+ T cells undergoing immunity and lymphopenia- Various T cell programs Various T cell programs CD8+ T cells cells deletional tolerance induced proliferation Parish IA et al. 2009; Redmond WL et al. 2005; Davey GM et al. 2002; Parish IA et al. 2009; Reference Schietinger A et al. 2012 Schietinger A et al. 2012 Parish IA et al. 2009 Parish IA et al. 2009 Schwartz RH, 2003;Rocha Davey GM et al. 2002 B et al. 1995; Hernandez J et al. 2001 Gene set Cd81 1700025G04Rik 4921525O09Rik 6330409N04Rik Cblb Tob1 Clspn 4930486L24Rik 4930539E08Rik Axin2 Cd5 Cdc20 Abcb9 A830073O21Rik Dtx1/Gm19706 Cdkn1b Kif15 Abhd14b Cd20041 Fech Ctla4 Tcf19 Acpl2 Crem Gimap7 Dgka LOC237877 Acsbg1 Cyth3 Gzma Dgkz Lag3 Acy1 Dgkz H2-Aa Egr2 Tnfrsf9 Adssl1 Egr2 Hbb-bh1 Egr3 Aurka Ahnak Endod1 Il18rap Itch Bcat1 Aim1 Ephx1 Klf3 Nfatc2 Tfrc Alad Fcrl1 Ly6a Pag1 Icenp Alox5ap Galm Ly6c1/Ly6c2 Pdcd1 Mcm10 Amica1 Gpm6b Lyz1 Rnf128 H3f3b/Gm12657/Gm6749/H Hmgn3 Ank Lyz2 3f3c/H3f3a Snn/Txndc11 Anxa3 Hspa4l Nedd4 Espl1 Ap1g2/Thtpa Ikzf2 Psmb2 Hist1h3e Apobr/Cln3 Lpcat1 Rras2 Chtf18 Aqp1 Lrig1 Sike1 Hist1h2bn Aqp9 Lta Snca Kntc1 Arl4d Marcks Sytl1 Gtse1 Arsb Mdh1 Sgol1 Asb2 Nab2 Ecm1 Ass1/Gm5424 Nr4a1 Kif18b Axl Nr4a3 Hist1h3a Bag3 Nrip1 Kif2c Bbc3 Nrn1 Cdca2 Bbs9 Pacsin1 Fancd2 BC021614 Prmt2 Dctpp1 Bcl2 Ptprs Cdc6 Bhlhe40 Rasa1 Chaf1a/Ubxn6 Blvrb Reep3 Chst2 C1qb Rgs16 Spag5 C1qc Skil Hist1h2bm C1qtnf4 Soat1 Cldn10 C3 Ssh1 Igf2bp3 Camk2n1 Synj2 Nhedc2 Camp Tbc1d4 Apitd1 Car11 Tnfsf11 Ccdc99 Casp1 Tox Lig1 Casp4 Ypel2 Stmn1 Ccl4 Zranb3 Ccl5 Cdc45 Ccl9 Marcksl1 Ccr5/Ccr2 Rgs16 Cd163l1 Ptpla Cd177 Chst3 Cd22 Cenpm Cd44 Ptprs Cd63 Ncapd2 Cd7 Nrgn Cd74 Hist1h3c Cdc42ep3 Sox5 Cdh1 Hist1h3d Cdk5rap1 Sxb Cdkn2c Nrn1 Cebpb/Tmem189 Hist1h2bh Cebpe Rcc1 Chi3l1 Bub1b Chi3l3 Hist1h2bj Chi3l7 Hist1h2ab Cobll1 Tpi1 Csprs/Gm7609 Mcm5 Ctsa Nmral1 Ctsw Rad51 Cxcr3 Cdca7 Cxcr5 Spc25 Cxcr6 Ndrg1 Cyb5r4 Pdzd11/Kif4 D14Ertd668e Rilpl2 D8Ertd82e Pif1 Dapk2 Cenpa Dennd3 Rad54l Dkkl1 Hist1h3h Dmrta1 Cenpn Dstn Hist1h2bk Ear2 Hist1h2bf Elane Rad54b/Fsbp Emilin2 Cdc7 Emr1 Itm2a Eomes Fibcd1 Epb4.1l3 Xcl1 Epsti1 Sh3bp2 Eraf Top2a Errfi1 Hist1h2af F2r Ppic F2rl1 Hist1h2an Fam26f Cdk1 Fcgr4 Birc5 Fcgrt Hist1H2ag Fcna Hist2ab Fgf13 Litaf Fndc3b E2f2 Fpr3/Fpr2 Prc1 Gab3 Cdca3 Gas7 H2afx Gata3 Hist1h2ah Gatsl3 Nusap1/Oip5 Gbp3 Ppa1 Gimap7 Tyms/Tyms-ps Glrx LOC100047934 Gm12253 Ccnb1/Gm5593 Gm4814/Cmya5 Psat1/LOC100047252 Gng2 Pbk Gpc1 Hist1h2ak Gpr183 Mcm6 Gramd3 Hist1h2ad Gsto1 Tyms-ps Gtdc2 Uhrf1 Gvin1/Gm4070 Klrb1c Gzmm Adk H2-Ab1 Rrm1 H2-DMb2/H2-DMb1 Hist1h2ao H2-Eb1 Tnfrsf21 Hcst 2810417H13Rik Hdc Scamp1 Hebp1 Chn2/9130019P16Rik Hk3 Lmnb1 Hmox1 Anln Hp Rrm2 Hsd11b1 Pkm Ier3 Kif22 Ifi30 Prm2/Wdr19 Ifit3 Plk1 Ifitm2 Mad2l1/LOC100045924 Ifitm3 LOC100045304 Ifitm6 Ncaph Ifng E2f1 Ifngr1 Hist2H2ac Il10ra Tk1 Il12rb1 Fen1 Il18r1 Fignl1 Il18rap D17H6S56E-5 Il1b Ndrg1 Il7r Mcm2 Inppl1 Kif11 Insl6 Cdca5 Itgad Chaf1b Itgb1 Capg Kcnj8 Endod1 Kcnk6 Cenph Klra16 Galk1/Itgb4 Klra7 Swap70 Klra9/Klra3/Klra10 LOC100047651 Klre1 Geod1 Klrk1 Lat2 Lcn2 Lgals3bp Lmna Loc100046232 Loc100046608 Loc100047167 Loc100048346 Loc641240 Lpxn Lrg1 Lrrk1 Ltb4r1 Ltf Ly6a Ly6c1/Ly6c2 Ly6e Lyz1 Lyz2 Lyz2 Mapre2 Mdfic Med7 Mfsd10 Mgst1 Mlkl Mmp9 Mocos Mpo Mrc1 Mrpl52 Ms4a6d Mt1 Myadm Myl4 Myo1f Myo6 Naip2 Nans Nccrp1 Ncf4 Ngp Nkg7 Nod1 Nrarp Nt5e Nuak2 Nudt4 Oas1g/Oas1a Osbpl3 P2ry14 Pde2a Pfkp Phf11 Pkig/Gm6334 Plac8 Plcg2 Plek/Cnrip1 Plekhg3 Plscr1 Pou6f1 Pqlc3 Prf1 Prtn3 Ptpn12 Pygl Rab19 Rab31 Rap1gap2 Rassf4 Reck Retnlg Rgs1 Rnf138 Ryk S100a6 S100a8 S100a9 Samd3 Scly Scn2b Sdc3 Sdcbp2 Sepn1 Serpina3f/Serpina3g Serpina3f/Serpina3g Serpinb6b Sidt1 Sla Slamf7 Slc11a1 Slc38a5 Slc39a4 Slc40a1 Slc4a1 Slc52a3 Slpi Smad3 Sntb2 Snx10 Snx8 Soat2 Sorl1 Sort1 Spin2 St3gal6/Dcbld2 St6galnac4 Stard10 Stat4 Susd3 Swap70 Sytl2 Tbx21 Tdrd7 Tgfbi Tgm2 Tm6sf1/Hdgfrp3 Tmem51 Trem3 Trf/Srprb Tshz3 Ttc39b Txndc5 Tyrobp Uap1l1 Ublcp1 Unc93b1 Usp18 Vcam1 Vim/LOC100862060 Vkorc1 Wdr95 X99384 Zc3h6 Zcchc18 Zfhx2/Zfhx2as Zfp296 Zfp3 Zscan12

Patient TILN All CD8+ TIL All CD8+ TIL T cell program of interest Patient TILN (overexpressed) (underexpressed) (overexpressed) (underexpressed) 200 most differentailly 200 most differentially expressed genes of all CD8+ Tumor-specific CD8+ T cells Tumor-specific CD8+ T cells expressed genes of all naïve TIL (regardless of from a tumor vs. from the periphery vs. CD8+ T cells from the spleen T cell programs compared for specificity), from a mouse vs. relative gene expression model of melanoma vs. All CD8+ TIL (regardless of Tumor-specific CD8+ T cells Tumor-specific CD8+ T cells Naïve CD8+ T cells from the specificity), from a mouse from the periphery from a tumor spleen model of melanoma Reference Baitsch L et al. 2010 Baitsch L et al. 2010 Giordano M et al. 2015 Giordano M et al. 2015 Gene set 4931431C16Rik/Acox3 Abhd14b Acsbg1 Abcc5 Ak2 Anapc5 Actg1 Abcg3 Alg10b Atg16l1 Ahnak Acot2 Angptl6/A230050P20Rik Atg3 Ap2m1 Adar Apeh Atp2b1 Atp2b4 Akr1b3/Gm6644 Atf3 BC005561 Batf Ank Atf4 Blmh Bcl2a1c Ap1ar Becn1/Cntd1 Cacybp Bcl2a1d Arhgef18 Bst2 Casp8 Bcl2l1 Arid3b Calml4 Cd300c Calm2/Calm3/Calm1 Arpc1b/Gm5637 Cblb Clpx Calu Atp6ap2 Cct6a Cog4 Camk2n1 Atp6v0a1 Cd27 Commd2 Caml Bace1 Cklf Cox6b1 Casp1 Bbs9 Cltb Cul4a Casp8 Bcl9 Cotl1 Derl2/LOC100862452 Ccdc72 Brix1 Cpt1c E2f1 Ccl25 Carhsp1 Crtam Eif4g3 Ccl3 Ccdc101 Csnk2a2 Fbxo3 Ccl4 Ccdc114 Ctla4 Flna Ccl5 Ccdc30 Cxcl13 Fxyd5 Ccr5/Ccr2 Ccdc64 Dclre1c Fzr1 Cct5 Cd2ap Ddx11 G3bp1 Cdt1 Cept1/Dennd2d/Dram2 Dgke Gins4 Cerkl Chmp2a Dtnbp1 Gk5 Crem Chrnb1 Dusp16 Golga4 Csnk2a1 Cmah Dusp4 Inpp5d Ctla2a Col10a1 Dusp5 Itgam Ctla2b Cpeb4 Erbb2ip Jhdm1d Ctss Cyth1 Erf Kcnn1 Cul2 Dapl1 Exoc4 Lrrc47/1190007F08Rik Cxcl10 Ddx23 Exoc5/Mudeng Lyar Cyp51 Ddx39 Exoc7 Metap1 Cysltr2 Dlg1 Fam105b Mif4gd Dcaf5 Dph5 Fam53c Myo1f Ddx3x Drg1 Fos Ndufs8 Ddx54 Dtnb Galm Nf2 Dennd4a Ears2 Gapvd1 Nr3c1 Dhx58 Eif3j/Gm9781 Gbp1/Gbp5/LOC100047734 Pdcd2 Dnajb1 Entpd4/LOC100862375 Gcc2 Pik3r1 Dusp1 Eya2 Gem Pno1 Ecd Fam53b Gm10784 Polr1d Eif5al3-ps Fam65b Gm5472 Pom121/Nsun5 Emg1/Lpcat3 Fkbp15 Gm6560 Ppp2r5c Eml5 Fntb/LOC639541 Gpr19 Ppp3r1/Wdr92 Eomes Foxk1 Gramd1b Ppp4r1 Ergic2/Far2 Foxk1 Hdgf Psma2 F2r Fry Hdhd2 Pxn Fam46c Ggnbp1/Zbtb9 Hip1 Rbl2 Fam96a Ggt1 Hspd1 Rev3l Fbxo40 Gmps Icos Rpl18a Fcer1g Gon4l Ier3ip1/Hdhd2 Rpl18a Frrs1 H3f3a Ifng Samhd1 Fyn Haus7/LOC100048005 Ilf2/Snapin Scaf11 Gabarap Hs6st1 Irf4 Sh3bgrl3 Gatad2a Hspa14/LOC100861945 Itga1 Ssbp3 Gch1 Igf1r Itm2a Ssbp4 Glcci1 Igfbp4 Jmjd1c Thoc2 Gng2 Ikbke Jtb Tmub2 Golph3 Il6st Kif3b Tnfrsf25 Gp49a Ino80 Lasp1 Tpst2 Gramd1b Irgm2/Igtp Lix1l Traf3ip3/A130010J15Rik Gramd4 Isg20l2 LOC545845 Txnip Grina Kcnmb1 Mapre2 Ube2b Gzmb Kif9 Mbnl1 Uqcrq H2-Ab1 Klhl22 Mcl1 Vdac2 H2-Ea-ps Krtap5-4 Mcm6 Zbtb44 H2afy Larp1b Myo7a Zyx/Epha1 H2afz Las1l Ndfip1 Hba-a1/Hba-a2 Limd2/LOC632329 Ndufc2/LOC675851/LOC6350 Hip1 Lin52 87 Neil2 Hmbox1 Lrrc51 Nfat5 Hmgb2 Map2k5 Nod2 Hsp90aa1 Map3k7 Nploc4 Hspa1a Map4k2 Otud7b Hsph1 Mbp Parvb Icam1 Mdn1 Parvg Idh3a Med20 Pcbp2/Map3k12 Ier3 Mettl3/Tox4 Pes1 Ifi35 Mfsd4 Phlda1 Ifit3 Miip Pik3ip1 Igf2r Mpdu1 Pik3r1 Il18bp Msh3 Pkm Il2rb Mtch2 Polr1d Irgm1 Mthfd1 Pom121/Nsun5 Itgb1 Mthfd1l Ppp1cc Iws1 Myc Ppp1r2 Klf10 Mylip Ppp1r2/Ppp1r2-ps3 Klra23 N6amt1 Ppp1r2/Ppp1r2-ps3 Klrc1 Nadk Prdx5/Trmt112 Krr1/Glipr1 Nap1l4 Psenen Lag3 Npas2 Ptprj Lass2 Nsg2 Rab27a Lilrb4 Nsun2 Rala Lpcat1 Nucb2 Ranbp2 Lphn1 Nup188 Rdh10 Lsm3 Osbpl7 Rgs1 Lsm6 Pak1ip1 Rgs3 Lsr Parp1 Rnf24 Ly6c1/Ly6c2 Pcsk1 Sardh Lzic Pdcd11 Sat1 Mad2l1/LOC100045924 Pdzk1/Gpr89 Sema7a Maf Pecam1 Slc7a5 Map2k3 Pias1 Snx9 Med29 Pik3ip1 Sod1 Mex3c Pip4k2a Spg7 Mgat2 Pmel1 Spry1 Mpeg1 Pphln1 Stat3 Mrpl43/Sema4g/Peo1 Ppm1h Stx1a Mrps33 Ppp1r9b Tap1 Mxd4 Prdm2 Tatdn1/Rnf139 Nacc1 Prkar2b Tceal3/Tceal6/Tceal5 Nae1 Pspc1 Tfg Nbeal2 Pten Tmcc1 Nfyb Ptgr2 Tnfrsf9 Nr4a2 Pts Topors/2010003O02Rik Nr4a3 Qdpr Tox Ociad1 Rab14 Traf3 Odc1 Rab40b Traf5 Padi2 Rab7l1 Trib1 Pdcd1 Rad9b Tspyl2 Plekha2 Ramp1 Tuba4a Plk3 Rapgef6 Vta1 Pop4 Rasa3 Ypel5 Ppih/Gm17748 Rcbtb1 Zc3h12a Ppp3cb Rcbtb2 Zc3h12c Ppp3cc Recql/Pyroxd1 Zfand5 Pqlc3 Rfx3 Zfp36l1 Prdx1/LOC100862012 Rnf115 Zmym2 Prkcd Rnf41 Znrf1 Prl8a8 Rpf1 Prrc2c Rpl18a Psmd8 Rps15a Ptger4 Rps6ka1 Ptpre Rps6kc1 Rab19 Rsph4a Rab8b Rwdd4a Rbbp7 Rxrb Rbm18/Mrrf Sbds Rbm22 Sdad1 Rgs1 Sdha Rgs16 Sec13 Rpl22l1/Gm15421 Sec22b Rpl27 Sema4f Rpl27-ps2 Sfxn2 Rpl36a/Rpl36al She S100a10 Skap1 S100a4 Slamf6 S100a6 Slc26a10/B4galnt1 Samd3 Slc43a2 Sars2 Slfn5 Setd8 Snx12 Sft2d1 Spcs2 Slc16a6/LOC100503496 Spna2 Slc25a3/LOC100046151 Stard5 Slc38a2 Stau1 Slc3a2 Stx6 Smarcc1 Suv420h1 Smarce1 Tardbp/Masp2 Smek1 Tas2r114 Snrpd2 Tbc1d22b Snx10 Tcf12 Son Tcf3 Spata13 Tcf7 Ssty2 Tcirg1 Stat3 Tcra-V8 Stat6 Tcrb-J Surf1/Surf2 Tex9 Taf15 Timm8a1 Tcf4 Tlr1 Tcrg-V2 Tm6sf1/Hdgfrp3 Tex10/Invs Tnik Tex2 Trak2 Tigit Trav12n-1 Tmed2 Trpm7 Tmem50b Tuba3a/Tuba3b Tmem63a Tubal3 Tmem88/Lsmd1 Tubgcp4/Trp53bp1 Tmpo Utp14a Tnfaip3 Vat1 Tnfrsf9 Vbp1 Trappc6b Vmn2r96 Trf/Srprb Vps26b Ttc38 Vps8 Txn1 Wapal Ubash3a Wdfy1 Ube2l3 Wtap Ubl3 Xist Ubp1/Fbxl2 Xlr5a/Xlr5b/Xlr5c Uhrf2 Xrcc3 Usp48 Xrcc5 Vim/LOC100862060 Xrn1 Wdr45l Yeats4 Wipi2 Yipf6 Xlr4b/Xlr4a/Xlr4c Zbtb22 Zbtb44 Zcchc7 Zc3h12d Zfml Zc3h13 Zfp157 Zfp36 Zfp330 Zmiz2 Zfp826 Zmynd11 Zgpat Supplemental Table 1. Transcription profiles of published T cell programs compared to tumor-specific TIL. Gene sets were compiled from previously published lists of differentially expressed genes associated with CD8+ T cell programs that are most relevant to TASPR T cells.

Supplemental Table 2. Overlap between hypofunctional CD8+ T cell programs that share increased gene expression with TASPR TIL 75 (a) Anln Cdca3 E2f1 Kif11 Ndrg1 Rrm1 Apitd1 Cdca5 Ecm1 Kif15 Nmral1 Scamp1 Aurka Cdca7 Espl1 Kif18b Nrgn Sgol1 Bcat1 Cenpa Fancd2 Kif22 Pbk Sh3bp2 Birc5 Cenph Fen1 Kif2c Plk1 Spag5 Bub1b Cenpm Galk1/Itgb4 Kntc1 Ppa1 Spc25 Capg Cenpn Gtse1 Lig1 Ppic Tcf19 Ccdc99 Chaf1b H2afx Lmnb1 Psat1/LOC100047252 Tk1 Cdc20 Chtf18 Hist1h2ak Mcm10 Ptpla Tpi1 Cdc45 Clspn Hist1h2bh Mcm2 Rad51 Tyms-ps Cdc6 D17H6S56E-5 Hist1h2bm Mcm5 Rad54b/Fsbp Tyms/Tyms-ps Cdca2 Dctpp1 Hmgn3 Ncapd2 Rad54l Uhrf1 Ncaph Rcc1 Xcl1 110 (b) 1110067D22Rik Cd160 Entpd1 Isg20 Nrp1 S100a11 1810035L17Rik/Nrp Cd200 Etf1 Itgav Nucb1 S100a4 5730469M10Rik Cd244/LOC677008 (2B4) Fgl2 Itih5 Pawr Serpinb6a Acot7 Cd9 Gapdh Klra3/LOC100862437/LOC100862410/Klra9Pdcd1 (PD-1) Serpinb9 Alcam Chek1 Gas2 Klra9/Klra3/Klra10 Penk Sh3bgrl Anxa2 Chl1 Gdpd5 Kpna2 Perp Slc29a1 Atf1/Gm1862 Cit Glrx Lat2 Pglyrp1 Smc2 BC055004 Clic4 Gm12070/Gapdh Lclat1 Plin2 Sypl Bhlhe40 Cox17 Gm5068 Lgals1 Plk4 Tacc3 Bub1 Cpsf2 Gm5177 Lgals3 Plscr1 Tank C330027C09Rik Cpt2 Gm5523/Gapdh/Gm12070 Lilrb4 Pqlc3 Tfdp1 Casp3 Cxcl10 Gpd2 Lman2 Prdm1 (Blimp-1) Tmem109 Casp4 Cyfip1 Gpr56 Ly6a Ptger2 Tnfrsf1b Ccdc50 Dock7 Gpr65 Mdfic Ptpn13 Trim47 Ccl3 E2f8 Gzmb Mki67 Rcn1 Txn1 Ccnb2 Ect2 Ifih1 Mx1 Rhoq/Pigf Ube2t Ccrl2 Eea1 Irf8 Nfil3 Romo1 Vamp8 Ell2 Isg15 Npcd/Cbx6/Nptxr Rpa2 Vmp1 Nr4a2 Rsad2 Wbp5 12 (c ) 30 (d) 11 (e) 3 (f) 0 (g) Crem Atf3 Parvb 2810417H13Rik Endod1 X Cyth3 Bst2 Parvg Ccnb1/Gm5593 Nrn1 Ephx1 Cblb Pes1 Cdk1 Ptprs Gpm6b Cct6a Phlda1 Fignl1 Hspa4l Cltb Ptprj Lag3 Ikzf2 Dusp16 Rab27a Mad2l1/LOC100045924 Marcks Dusp4 Sema7a Nusap1/Oip5 Nab2 Hdgf Slc7a5 Prc1 Prmt2 Hip1 Snx9 Rrm2 Soat1 Hspd1 Spry1 Tnfrsf9 (4-1BB) Synj2 Ier3ip1/Hdhd2 Stat3 Top2a Ypel2 Lix1l Tatdn1/Rnf139 Ndufc2/LOC675851/LOC635087 Tfg Nfat5 Trib1 Otud7b Zc3h12c 3 (h) 4 (i) 1 (j) 1 (k) 0 (l) 0 (m) Itm2a Ctla4 Tox Rgs16 X X Mcm6 Gem Pkm Irf4 Ndfip1 0 (n) 0 (O) X X Supplemental Table 2. Overlap between hypofunctional CD8+ T cell programs that share increased gene expression with TASPR TIL. Overlap of genes up-regulated by hypofunctional CD8+ T cells that comprised the Leading Edge [30] of gene sets enriched among genes up-regulated by TASPR TIL were compared for overlap by the Venn diagram in Figure 3B. An “X” indicates that no genes were in this category.

Supplemental Table 3. The molecular profile of TIL consists of 1164 differentially expressed genes between tumor-specific CD8+ T cells from the tumor and spleen Cluster Probe ID Gene Symbol Fold-change Gene type(s)

TILTOT / SpTOT Cluster 01 10407072 Elovl7 -5.60 Acyltransferase Cluster 01 10420131 Rabggta -2.60 Acyltransferase Cluster 01 10351197 Sell -13.09 Adpolipoprotein, receptor, metalloprotease, serine protease, complement component, cell adhesion molecule Cluster 01 10356880 St8sia4 -2.57 Alpha-N-acetylneuraminate alpha-2,8-sialyltransferase activity Cluster 01 10473367 Slc43a1 -2.75 Amino acid transporter Cluster 01 10471154 Ass1/Gm5424 -3.21 Argininosuccinate synthase Cluster 01 10363541 Ass1/Gm5424 -3.18 Argininosuccinate synthase Cluster 01 10549102 Kcnj8 -3.24 ATP binding, ATP-activated inward rectifier potassium channel activity Cluster 01 10479463 Slc17a9 -2.98 Cation transporter Cluster 01 10443764 Slc37a1 -2.28 Cation transporter Cluster 01 10522335 Atp10d -6.32 Cation transporter, hydrolase Cluster 01 10468898 Lax1 -3.66 Cation transporter, hydrolase Cluster 01 10491486 Atp11b -2.21 Cation transporter, hydrolase Cluster 01 10406111 Slc12a7 -3.24 Cation: chloride symporter activity Cluster 01 10359816 Pogk -2.15 Centromere DNA-binding protein Cluster 01 10389207 Ccl5 (RANTES) -3.29 Chemokine Cluster 01 10565218 Il16 -2.45 Cytokine, interleukin superfamily Cluster 01 10378549 Rtn4rl1 -4.79 Cytokine, receptor, extracellular matrix protein Cluster 01 10499655 Il6ra (CD126) -2.68 Cytokine, type I cytokine receptor, defense/immunity protein Cluster 01 10477649 Acss2 -2.97 Dehydrogenase ligase Cluster 01 10488482 Acss1 -2.28 Dehydrogenase ligase Cluster 01 10387316 Chd3 -3.80 DNA helicase, helicase Cluster 01 10503190 Chd7 -2.98 DNA helicase, helicase Cluster 01 10377537 Chd3 -2.77 DNA helicase, helicase Cluster 01 10494428 Txnip -2.89 Enzyme inhibitor activity Cluster 01 10472235 Dapl1 -5.90 Enzyme modulator Cluster 01 10402730 Ppp1r13b -2.68 Enzyme modulator Cluster 01 10469867 Pnpla7 -2.28 Esterase Cluster 01 10411235 Iqgap2 -3.14 G-protein coupled modulator Cluster 01 10364650 Hmha1 -2.82 G-protein coupled modulator Cluster 01 10501586 S1pr1 -38.24 G-protein coupled receptor Cluster 01 10591494 S1pr5 -10.63 G-protein coupled receptor Cluster 01 10422493 Gpr18 -6.35 G-protein coupled receptor Cluster 01 10371217 S1pr4 -3.83 G-protein coupled receptor Cluster 01 10459288 Adrb2 -3.57 G-protein coupled receptor Cluster 01 10422496 Gpr183 -3.21 G-protein coupled receptor Cluster 01 10411226 F2rl1 -2.80 G-protein coupled receptor Cluster 01 10590365 Vipr1 -2.64 G-protein coupled receptor, antibacterial response protein Cluster 01 10590381 Vipr1 -2.30 G-protein coupled receptor, antibacterial response protein Cluster 01 10606058 Cxcr3 -16.15 G-protein coupled receptor, immunoglobulin receptor superfamily, protein phosphatase, immunoglobulin superfamily cell adhesion molecule Cluster 01 10357472 Cxcr4 -13.47 G-protein coupled receptor, immunoglobulin receptor superfamily, protein phosphatase, immunoglobulin superfamily cell adhesion molecule Cluster 01 10590620 Ccr9 -2.13 G-protein coupled receptor, immunoglobulin receptor superfamily, protein phosphatase, immunoglobulin superfamily cell adhesion molecule Cluster 01 10441601 Tagap -6.81 G-protein modulator Cluster 01 10382228 Axin2 -3.78 G-protein modulator Cluster 01 10455259 Arhgap26 -3.57 G-protein modulator Cluster 01 10388310 Rap1gap2 -2.50 G-protein modulator Cluster 01 10565994 Art2b -7.94 Glycosyltransferase Cluster 01 10556598 Xylt1 -2.28 Glycosyltransferase Cluster 01 10429160 St3gal1 -2.27 Glycosyltransferase Cluster 01 10555303 Pgm2l1 -2.77 Glycosyltransferase, mutase Cluster 01 10604751 Fgf13 -4.16 Growth factor Cluster 01 10407281 Esm1 -2.11 Growth factor Cluster 01 10460968 Rasgrp2 -13.86 Guanyl-nucleotide exchange factor Cluster 01 10472860 Rapgef4 -2.47 Guanyl-nucleotide exchange factor Cluster 01 10569733 Arhgef18 -5.21 Gyanyl-nucleotide exchange factor Cluster 01 10413419 Arhgef3 -4.40 Gyanyl-nucleotide exchange factor Cluster 01 10386951 Hs3st3b1 -2.25 Heparan sulfate-glucosamine 3-sulfotransferase 1 Cluster 01 10544150 Jhdm1d (Kdm7a) -3.52 Histone demethylase activity (H3-K27 specific), histone demethylase activity (H3-K9 specific) Cluster 01 10432619 Pou6f1 -2.28 Homeobox transcription factor Cluster 01 10424213 Zhx2 -2.89 Homeobox transcription factor, nucleic acid binding Cluster 01 10443535 Tbc1d22b -2.27 Hydrolase, G-protein modulator Cluster 01 10394054 Cd7 -6.66 Immunoglobulin receptor superfamily Cluster 01 10362097 H60b -4.10 Immunoglobulin receptor superfamily, major histocompatibility complex antigen Cluster 01 10393408 Tmc6 -2.04 Ion channel Cluster 01 10373542 Dgka -5.10 Kinase Cluster 01 10518686 Pik3cd -2.21 Kinase Cluster 01 10383010 Socs3 -2.25 Kinase inhibitor Cluster 01 10362275 Samd3 -5.60 Kinase modulator Cluster 01 10549282 Itpr2 -2.52 Ligand-gated ion channel Cluster 01 10511739 Cpne3 -2.53 Membrane traffic protein Cluster 01 10446777 Ehd3 -2.87 Membrane traffic protein, G-protein modulator, calcium-binding protein Cluster 01 10402142 Ccdc88c -2.79 Membrane traffic protein, kinase modulator Cluster 01 10545608 Sema4f -2.11 Membrane-bound signaling molecule Cluster 01 10542120 Clec2i -3.90 Membrane-bound signaling molecule, receptor, defense/immunity protein Cluster 01 10380135 Mir142 -6.19 microRNA Cluster 01 10350247 Kif21b -3.73 Microtubule binding motor protein Cluster 01 10566258 Hbb-b1/Hbb-b2/Beta-s -66.23 Misc. Cluster 01 10566254 Hbb-b1/Hbb-b2/Beta-s -54.36 Misc. Cluster 01 10375051 Hba-a1/Hba-a2 -27.88 Misc. Cluster 01 10375058 Hba-a1/Hba-a2 -27.74 Misc. Cluster 01 10497337 Car1 -24.15 Misc. Cluster 01 10563099 Snord35b -17.65 Misc. Cluster 01 10404132 Cmah -17.12 Misc. Cluster 01 10404152 Fam65b -14.49 Misc. Cluster 01 10389190 Gm11435 -9.40 Misc. Cluster 01 10533182 Dtx1/Gm19706 -7.80 Misc. Cluster 01 10468527 5830416P10Rik -7.34 Misc. Cluster 01 10388488 Fam101b -7.20 Misc. Cluster 01 10573054 Gypa -7.13 Misc. Cluster 01 10570516 Kbtbd11 -6.79 Misc. Cluster 01 10490923 Car2 -6.14 Misc. Cluster 01 10375019 Nsg2 -6.13 Misc. Cluster 01 10603814 Slc9a7 -6.02 Misc. Cluster 01 10427454 Card6 -5.83 Misc. Cluster 01 10378833 Ssh2 -5.65 Misc. Cluster 01 10378855 Ssh2 -5.52 Misc. Cluster 01 10570513 Kbtbd11 -5.48 Misc. Cluster 01 10569719 A430078G23Rik -5.46 Misc. Cluster 01 10392910 C630004H02Rik -5.40 Misc. Cluster 01 10377265 Pik3r5 -5.28 Misc. Cluster 01 10414731 10414731 -5.21 Misc. Cluster 01 10543785 AB041803 -5.02 Misc. Cluster 01 10367532 Tespa1 -4.92 Misc. Cluster 01 10358717 1700025G04Rik -4.83 Misc. Cluster 01 10455784 Gramd3 -4.76 Misc. Cluster 01 10501007 Chi3l7 -4.40 Misc. Cluster 01 10591739 Acp5 -4.32 Misc. Cluster 01 10531126 Igj -4.25 Misc. Cluster 01 10394593 Fam49a -4.15 Misc. Cluster 01 10382449 Rab37 -4.11 Misc. Cluster 01 10393544 Cyth1 -3.91 Misc. Cluster 01 10545184 10545184 -3.81 Misc. Cluster 01 10463164 Frat1 -3.73 Misc. Cluster 01 10587854 Slc9a9 -3.62 Misc. Cluster 01 10356932 D1Ertd622e -3.62 Misc. Cluster 01 10498998 D930015E06Rik -3.53 Misc. Cluster 01 10400137 Dnajb9 -3.31 Misc. Cluster 01 10557481 Ypel3 -3.26 Misc. Cluster 01 10584762 Bcl9l -3.19 Misc. Cluster 01 10416371 Lpar6/Rb1 -3.16 Misc. Cluster 01 10481574 Fam78a -3.16 Misc. Cluster 01 10401428 C130039O16Rik (Elmsan1) -3.14 Misc. Cluster 01 10467650 Frat2 -3.01 Misc. Cluster 01 10533007 Ccdc64 -3.00 Misc. Cluster 01 10565990 Art2a-ps -3.00 Misc. Cluster 01 10546010 Arhgap25 -2.95 Misc. Cluster 01 10520388 Rbm33 -2.94 Misc. Cluster 01 10545065 Gprin3 -2.92 Misc. Cluster 01 10397476 2310044G17Rik -2.90 Misc. Cluster 01 10538123 Gimap9 -2.89 Misc. Cluster 01 10425799 Rnu12 -2.86 Misc. Cluster 01 10530612 Fryl -2.85 Misc. Cluster 01 10534570 Orai2 -2.84 Misc. Cluster 01 10568586 Fam53b -2.84 Misc. Cluster 01 10500100 Tnfaip8l2 -2.81 Misc. Cluster 01 10379630 Slfn2 -2.78 Misc. Cluster 01 10550365 Prkd2 -2.76 Misc. Cluster 01 10425410 Grap2 -2.73 Misc. Cluster 01 10497237 Pag1 -2.72 Misc. Cluster 01 10409767 Golm1 -2.71 Misc. Cluster 01 10430649 Cbx7 -2.70 Misc. Cluster 01 10359303 4930562F07Rik -2.70 Misc. Cluster 01 10447629 Tagap1/Tagap/Fgfr1op -2.66 Misc. Cluster 01 10549162 St8sia1 -2.65 Misc. Cluster 01 10406905 Ccdc125 -2.61 Misc. Cluster 01 10436426 Orai2 -2.59 Misc. Cluster 01 10571344 D8Ertd82e (SGK223) -2.59 Misc. Cluster 01 10350977 4930523C07Rik/4930562F07Rik -2.55 Misc. Cluster 01 10530592 Fryl -2.54 Misc. Cluster 01 10532150 Fam69a -2.51 Misc. Cluster 01 10439058 Lrrc33 (Nrros) -2.50 Misc. Cluster 01 10566438 Fam160a2 -2.49 Misc. Cluster 01 10440099 St3gal6/Dcbld2 -2.46 Misc. Cluster 01 10443027 A930001N09Rik -2.45 Misc. Cluster 01 10469695 Apbb1ip -2.45 Misc. Cluster 01 10458098 Pkd2l2/Fam13b -2.44 Misc. Cluster 01 10380514 Fam117a -2.43 Misc. Cluster 01 10468525 5830416P10Rik (Mirt1) -2.43 Misc. Cluster 01 10520043 Lhfpl3 -2.42 Misc. Cluster 01 10530563 Fryl -2.42 Misc. Cluster 01 10434302 Klhl24 -2.41 Misc. Cluster 01 10358978 Ier5 -2.41 Misc. Cluster 01 10390574 Fbxl20 -2.37 Misc. Cluster 01 10382935 Tnrc6c -2.36 Misc. Cluster 01 10460787 Atg2a -2.36 Misc. Cluster 01 10528036 10528036 -2.35 Misc. Cluster 01 10583669 AB124611 -2.35 Misc. Cluster 01 10380059 Rnu3b1/Rnu3b2/Rnu3b4/Rnu3b3 -2.34 Misc. Cluster 01 10380061 Rnu3b1/Rnu3b2/Rnu3b4/Rnu3b3 -2.34 Misc. Cluster 01 10380063 Rnu3b1/Rnu3b2/Rnu3b4/Rnu3b3 -2.34 Misc. Cluster 01 10380065 Rnu3b1/Rnu3b2/Rnu3b4/Rnu3b3 -2.34 Misc. Cluster 01 10528723 Mll3 (Kmt2c) -2.34 Misc. Cluster 01 10426042 Gramd4 -2.34 Misc. Cluster 01 10456719 10456719 -2.33 Misc. Cluster 01 10591263 Fbxl12/Ubl5 -2.32 Misc. Cluster 01 10571302 Tmem66 (Saraf) -2.31 Misc. Cluster 01 10358191 Camsap2 -2.31 Misc. Cluster 01 10492826 Fbxw7/Dear1 -2.29 Misc. Cluster 01 10524229 A630023P12Rik -2.29 Misc. Cluster 01 10358713 1700025G04Rik -2.28 Misc. Cluster 01 10441115 Brwd1 -2.28 Misc. Cluster 01 10587012 Ccpg1 -2.27 Misc. Cluster 01 10560202 Gltscr1 -2.26 Misc. Cluster 01 10457022 Mbp -2.25 Misc. Cluster 01 10390186 Abi3/Phospho1 -2.24 Misc. Cluster 01 10375083 Stk10 -2.24 Misc. Cluster 01 10577230 Erich1 -2.23 Misc. Cluster 01 10493626 1700094D03Rik -2.22 Misc. Cluster 01 10380477 Ppp1r9b -2.20 Misc. Cluster 01 10567171 Snord14a/Rps13 -2.19 Misc. Cluster 01 10487595 10487595 -2.19 Misc. Cluster 01 10528090 Rundc3b -2.18 Misc. Cluster 01 10432473 Nckap5l -2.18 Misc. Cluster 01 10456721 10456721 -2.18 Misc.

Cluster 01 10585905 Parp6 -2.17 Misc. Cluster 01 10449854 Akap8l -2.13 Misc. Cluster 01 10426535 Fam113b/Amigo2 -2.13 Misc. Cluster 01 10568780 Mapk1ip1 -2.10 Misc. Cluster 01 10503484 Fam82b -2.09 Misc. Cluster 01 10407370 4833420G17Rik -2.02 Misc. Cluster 01 10439583 Sidt1 -13.82 Misc. Cluster 01 10503259 Trp53inp1 (S1P) -7.63 Misc. Cluster 01 10419966 Zfhx2/Zfhx2as -2.58 Misc. Cluster 01 10552276 Ube2h -2.47 Misc. Cluster 01 10509379 Usp48 -2.46 Misc. Cluster 01 10512480 Sit1 -2.43 Misc. Cluster 01 10543686 Ube2h -2.40 Misc. Cluster 01 10383982 Znrf3 -2.25 Misc. Cluster 01 10412038 Zswim6 -2.23 Misc. Cluster 01 10503551 Usp45 -2.17 Misc. Cluster 01 10378649 Slc43a2 -2.15 Misc. Cluster 01 10506569 Usp24 -2.13 Misc. Cluster 01 10383632 Sfi1/LOC100861749 -2.08 Misc. Cluster 01 10494160 Tmod4 -2.17 Non-motor actin binding protein Cluster 01 10575120 Sntb2 -2.10 Non-motor actin binding protein Cluster 01 10357604 Ikbke -3.05 Non-receptor serine/theronine protein kinase Cluster 01 10420413 Lats2 -2.34 Non-receptor serine/theronine protein kinase, annexin, calmodulin Cluster 01 10500813 Hipk1 -2.61 Non-receptor serine/theronine protein kinase, non-receptor tyrosine protein kinase Cluster 01 10372745 Dyrk2 -2.57 Non-receptor serine/theronine protein kinase, non-receptor tyrosine protein kinase Cluster 01 10449527 Stk38 -3.89 Non-receptor serine/theronine protein kinase, transfer/carrier protein, annexin, calmodulin Cluster 01 10362073 Sgk1 -3.51 Non-receptor serine/theronine protein kinase, transfer/carrier protein, annexin, calmodulin Cluster 01 10479852 Camk1d -2.20 Non-receptor serine/threonine protein kinase Cluster 01 10362596 Fyn -2.68 Non-receptor tyrosine kinase Cluster 01 10381082 Rara -2.40 Nuclear hormone receptor Cluster 01 10417734 Nr1d2 -4.49 Nuclear hormone receptor, receptor, nucleic acid binding Cluster 01 10385776 Tcf7 -20.02 Nucleic acid binding Cluster 01 10496091 Lef1 -7.64 Nucleic acid binding Cluster 01 10505911 Dmrta1 -2.91 Nucleic acid binding Cluster 01 10422321 Dzip1 -2.66 Nucleic acid binding Cluster 01 10443063 Phf1 -2.60 Nucleic acid binding Cluster 01 10401454 Entpd5 -2.14 Nucleotide phosphatase, lyase, nucleic acid binding Cluster 01 10506786 Zcchc11 -2.50 Nucleotidyltransferase Cluster 01 10350697 Nmnat2 -2.25 Nucleotidyltransferase Cluster 01 10364460 Gzmm -6.69 Peptide hormone, receptor, serine protease, protease inhibitor, annexin, calmodulin Cluster 01 10373740 Pik3ip1 -3.82 Peptide hormone, receptor, serine protease, protease inhibitor, annexin, calmodulin Cluster 01 10362811 Sesn1 -2.81 Peroxidase Cluster 01 10555510 Pde2a -15.99 Phosphodiesterase Cluster 01 10497399 Pde7a -2.80 Phosphodiesterase Cluster 01 10360173 Slamf7 -3.21 Protein kinase, membrane-bound signaling molecule, immunoglobulin receptor superfamily, cell adhesion molecule Cluster 01 10360158 Ly9 (CD229) -2.82 Protein kinase, membrane-bound signaling molecule, immunoglobulin receptor superfamily, cell adhesion molecule Cluster 01 10357191 Ptpn4 -2.44 Protein phosphatase Cluster 01 10563338 Ppp1r15a -2.60 protein phosphatase binding, protein phosphatase regulator activity Cluster 01 10484941 Madd -2.53 Rab guanyl-nucleotide exchange factor activity Cluster 01 10362896 Cd24a -27.60 Receptor Cluster 01 10365344 Tcp11l2 -8.00 Receptor Cluster 01 10557895 Itgax (CD11c) -5.12 Receptor Cluster 01 10541246 Il17ra (CD217) -3.74 Receptor Cluster 01 10473125 Itga4 (CD49d) -2.65 Receptor Cluster 01 10432957 Itgb7 (Ly69) -4.43 Receptor, extracellular matrix glycoprotein, cell adhesion molecule Cluster 01 10530145 Tlr1 -5.65 Receptor, extracellular matrix protein Cluster 01 10374560 Zrsr1 -2.62 Ribonucleoprotein Cluster 01 10453272 Zfp36l2 -3.15 RNA binding protein Cluster 01 10561453 Zfp36 -2.53 RNA binding protein Cluster 01 10401238 Zfp36l1 -2.40 RNA binding protein Cluster 01 10412211 Gzma -8.46 Serine protease Cluster 01 10598467 Pim2 -2.54 Serine/threonine protein kinase receptor, protein kinase Cluster 01 10517301 Ldlrap1 -2.49 Signaling molecule Cluster 01 10577190 Rasa3 -7.61 Signaling molecule, G-protein modulator Cluster 01 10415725 Spata13 -2.49 Signaling molecule, guanyl-nucleotide exchange factor Cluster 01 10392177 Icam2 -2.88 Signaling molecule, immunoglobulin superfamily cell adhesion molecule Cluster 01 10380381 Tob1 -3.22 SMAD binding, transcription corepressor activity Cluster 01 10390535 Arl5c -7.19 Small GTPase Cluster 01 10356475 Arl4c -4.50 Small GTPase Cluster 01 10567043 Rras2 -2.12 Small GTPase Cluster 01 10411126 Jmy -3.12 Transcription coactivator activity Cluster 01 10365428 Btbd11 -2.11 Transcription cofactor, growth factor, serine protease, non-motor actin binding protein Cluster 01 10544891 Nod1 -5.20 Transcription cofactor, nucleic acid binding Cluster 01 10474006 Phf21a -2.81 Transcription cofactor, ubiquitin-protein ligase Cluster 01 10594404 Smad3 -4.40 Transcription factor Cluster 01 10354111 Aff3 -4.19 Transcription factor Cluster 01 10583207 Maml2 -3.10 Transcription factor Cluster 01 10598101 Maml2 -2.73 Transcription factor Cluster 01 10466865 Rfx3 -2.52 Transcription factor Cluster 01 10489961 Nfatc2 -2.36 Transcription factor Cluster 01 10574962 Nfatc3 -2.26 Transcription factor Cluster 01 10606989 Tsc22d3 -2.24 Transcription factor Cluster 01 10362861 Scml4 -3.52 Transcription factor, chromatin/chromatin-binding protein Cluster 01 10467420 Pdlim1 -2.82 Transcription factor, non-motor actin binding protein Cluster 01 10520950 Pdlim1 -2.44 Transcription factor, non-motor actin binding protein Cluster 01 10522051 Klf3 (Bklf) -8.67 Transcription factor, nucleic acid binding Cluster 01 10572800 Klf2 (Lklf) -4.64 Transcription factor, nucleic acid binding Cluster 01 10546661 Foxp1 -3.49 Transcription factor, nucleic acid binding Cluster 01 10530142 Klf3 (Bklf) -2.59 Transcription factor, nucleic acid binding Cluster 01 10557488 Tbx6 -2.51 Transcription factor, nucleic acid binding Cluster 01 10397346 Fos -2.41 Transcription factor, nucleic acid binding Cluster 01 10589994 Eomes -2.17 Transcription factor, nucleic acid binding Cluster 01 10441003 Runx1 -2.09 Transcription factor, nucleic acid binding Cluster 01 10438445 Klhl6 -5.33 Transcription factor, serine protease, non-motor actin binding protein Cluster 01 10594001 Arid3b -2.71 Transcription regulator Cluster 01 10396205 Arid4a -2.18 Transcription regulator Cluster 01 10481103 Lcn4 -2.25 Transfer/carrier protein, isomerase Cluster 01 10524345 Tpst2 -2.87 Transferase Cluster 01 10547513 Cecr5 -2.18 Transferase Cluster 01 10463997 Pdcd4 -2.79 Translation elongation factor Cluster 01 10407327 Emb -16.65 Transmembrane receptor regulatory/adaptor protein Cluster 01 10605357 Gab3 -4.64 Transmembrane receptor regulatory/adaptor protein Cluster 01 10412376 Emb -3.92 Transmembrane receptor regulatory/adaptor protein Cluster 01 10558410 Ptpre -2.24 Transmembrane receptor reguloatory/adaptor protein, receptor, protein phosphatase Cluster 01 10564631 Slco3a1 -6.08 Transporter Cluster 01 10391649 Slc4a1 -4.53 Transporter Cluster 01 10439239 Dirc2 -3.11 Transporter Cluster 01 10382532 Slc16a5 -2.41 Transporter Cluster 01 10569485 Tnfrsf26 -8.06 Tumor necrosis factor receptor Cluster 01 10427628 Il7r (CD127) -5.74 Type I cytokine receptor Cluster 01 10436830 Ifnar2 -2.43 Type I cytokine receptor, type II cytokine receptor Cluster 01 10351691 Slamf6 -17.97 Tyrosine protein kinase receptor, membrane-bound signaling molecule, immunoglobulin receptor superfamily, tyrosine protein kinase receptor, protein kinase, immunoglobulin receptor superfamily, cell adhesion molecule Cluster 01 10404874 Mylip -3.40 Ubiquitin protein ligase activity, ubiquitin-protein transferase activity Cluster 01 10374485 Peli1 -2.53 Ubiquitin protein ligase activity, ubiquitin-protein transferase activity Cluster 01 10454202 Rnf138 -3.11 Ubiquitin-protein ligase Cluster 01 10501879 Usp53 -2.36 Ubiquitin-protein ligase Cluster 01 10511755 Wwp1 -2.15 Ubiquitin-protein ligase Cluster 01 10580452 Siah1a -2.23 Ubiquitin-protein transferase activity, zinc ion binding

Cluster 01 10350046 Kdm5b -2.40 Zinc finger transcription factor Cluster 01 10479159 Zfp831 -3.29 Zinc finger transcription factor, nucleic acid binding Cluster 01 10402554 Bcl11b -2.85 Zinc finger transcription factor, nucleic acid binding Cluster 01 10361338 Ipcef1 -2.72 Cluster 02 & 03 10560304 Calm1/Calm2/Calm3 2.09 Calmodulin Cluster 02 & 03 10461452 Fen1 2.02 Damaged DNA-binding protein, endodeoxyribonuclease, exodeoxyribonuclease, nuclease, hydrolase Cluster 02 & 03 10389395 Brip1 3.88 DNA helicase, helicase Cluster 02 & 03 10510687 Acot7 (Bach) 2.80 Esterase Cluster 02 & 03 10452648 Emilin2 3.08 Extracellular matrix glycoprotein Cluster 02 & 03 10540298 Chl1 2.55 G-protein coupled receptor, immunoglobulin receptor superfamily, protein phosphatase, immunoglobulin superfamily cell adhesion molecule Cluster 02 & 03 10485963 Arhgap11a 3.60 G-protein modulator Cluster 02 & 03 10584710 H2afx 2.42 Histone Cluster 02 & 03 10520521 Cenpa 2.79 Histone Cluster 02 & 03 10600017 Hmgb3 2.03 HMG-box transcription factor, signaling molcule, chromatin/chromatin-binding protein Cluster 02 & 03 10352954 Hmgb3 2.28 HMG-box transcription factor, signaling molcule, chromatin/chromatin-binding protein Cluster 02 & 03 10497689 Gnb4 2.09 Hydrolase, heterotrimeric G-protein Cluster 02 & 03 10441436 Snx9 8.06 Membrane trafficking regulatory protein Cluster 02 & 03 10585331 Exph5 2.00 Misc. Cluster 02 & 03 10439881 5330426P16Rik 2.08 Misc. Cluster 02 & 03 10469505 Commd3 2.14 Misc. Cluster 02 & 03 10503617 Mms22l 2.18 Misc. Cluster 02 & 03 10420730 Fdft1/Ctsb 2.20 Misc. Cluster 02 & 03 10416155 Kctd9 2.22 Misc. Cluster 02 & 03 10515090 Cdkn2c 2.27 Misc. Cluster 02 & 03 10412909 Fdft1 2.28 Misc. Cluster 02 & 03 10588482 Poc1a 2.29 Misc. Cluster 02 & 03 10380289 Mmd 2.35 Misc. Cluster 02 & 03 10605431 Rab39b 2.39 Misc. Cluster 02 & 03 10345074 Cetn4 2.55 Misc. Cluster 02 & 03 10465861 Incenp 2.56 Misc. Cluster 02 & 03 10404380 Dusp22 2.66 Misc. Cluster 02 & 03 10575733 Cenpn 2.93 Misc. Cluster 02 & 03 10403413 Idi1/Gm13502 2.94 Misc. Cluster 02 & 03 10548086 Rad51ap1 2.96 Misc. Cluster 02 & 03 10560695 Ceacam16 3.02 Misc. Cluster 02 & 03 10482762 Idi1/Gm13502 3.04 Misc. Cluster 02 & 03 10482528 Neb 3.45 Misc. Cluster 02 & 03 10414315 Cdkn3 3.46 Misc. Cluster 02 & 03 10474825 D2Ertd750e 3.48 Misc. Cluster 02 & 03 10505299 Bspry 3.76 Misc. Cluster 02 & 03 10608686 10608686 4.99 Misc. Cluster 02 & 03 10360985 Cenpf 5.21 Misc. Cluster 02 & 03 10538356 Chn2/9130019P16Rik 5.68 Misc. Cluster 02 & 03 10442219 Zfp52 2.08 Misc. Cluster 02 & 03 10534301 Syna 3.36 Misc. Cluster 02 & 03 10511363 Penk 9.61 Neuropeptide Cluster 02 & 03 10480432 Mastl 6.11 Non-receptor serine/theronine protein kinase, transfer/carrier protein, calmodulin Cluster 02 & 03 10544106 Zc3hav1 2.11 Nucleic acid binding Cluster 02 & 03 10563323 Nucb1 2.35 Nucleic acid binding, annexin, calmodulin Cluster 02 & 03 10456383 Impa2 3.85 Phosphatase Cluster 02 & 03 10513158 Ptpn3 4.00 Protein phosphatase Cluster 02 & 03 10563685 Ptpn5 4.31 Protein phosphatase Cluster 02 & 03 10557156 Plk1 3.88 Protein serine/threonine kinase activity Cluster 02 & 03 10503098 Lyn/Gm11787 4.56 Protein tyrosine kinase activity Cluster 02 & 03 10547906 Lag3 (CD223) 4.96 Transmembrane signaling receptor activity, MHC class II protein binding Cluster 02 & 04 10396421 Hif1a 2.11 Basic helix-loop-helix transcription factor, nucleic acid binding Cluster 02 & 04 10531724 Plac8 2.66 Chromatin binding Cluster 02 & 04 10477052 Slc52a3 2.08 G-protein coupled receptor Cluster 02 & 04 10421517 Cysltr2 2.49 G-protein coupled receptor Cluster 02 & 04 10414271 Ptger2 2.85 G-protein coupled receptor Cluster 02 & 04 10597279 Ccrl2 4.77 G-protein coupled receptor, immunoglobulin receptor superfamily, protein phosphatase, immunoglobulin superfamily cell adhesion molecule Cluster 02 & 04 10411332 Hmgcr 2.03 Hydroxymethylglutaryl-CoA reductase (NADPH) Cluster 02 & 04 10441195 Dscam 3.55 Immunoglobulin receptor superfamily, protein phosphatase, immunoglobulin receptor superfamily cell adhesion molecule Cluster 02 & 04 10548532 Klra5/Klra9/Klra19 3.26 Membrane-bound signaling molecule, receptor, defense/immunity protein Cluster 02 & 04 10548450 Klra5 4.17 Membrane-bound signaling molecule, receptor, defense/immunity protein Cluster 02 & 04 10406982 Adamts6 2.97 Metalloprotease, extracellular matrix glycoprotein, serine protease inhibitor Cluster 02 & 04 10398039 Serpina3f/Serpina3g 2.07 Misc. Cluster 02 & 04 10523923 Ccdc18 2.09 Misc. Cluster 02 & 04 10461553 A430093F15Rik 2.28 Misc. Cluster 02 & 04 10446229 Tnfsf9 2.64 Misc. Cluster 02 & 04 10447341 Rhoq/Pigf 2.98 Misc. Cluster 02 & 04 10502791 Ifi44 3.32 Misc. Cluster 02 & 04 10558090 Tacc2 2.00 Misc. Cluster 02 & 04 10548573 Styk1 2.84 Non-receptor tyrosine protein kinase Cluster 02 & 04 10513145 Ptpn3 2.16 Protein phosphatase Cluster 02 & 04 10513164 Ptpn3 2.35 Protein phosphatase Cluster 02 & 04 10513166 Ptpn3 2.74 Protein phosphatase Cluster 02 & 04 10513162 Ptpn3 2.74 Protein phosphatase Cluster 02 & 04 10346790 Ctla4 (CD152) 2.48 Receptor Cluster 02 & 04 10381809 Itgb3 (CD61) 2.40 Receptor, extracellular matrix glycoprotein, cell adhesion molecule Cluster 02 & 04 10511290 Tnfrsf18 2.26 Tumor necrosis factor receptor Cluster 02 & 04 10469278 Il2ra (CD25) 5.32 Type I cytokine receptor Cluster 02 & 05 10495405 Slc25a24 3.50 Amino acid transporter, mitochondrial carrier protien, transfer/carrier protein, ribosomal protein, calmodulin Cluster 02 & 05 10606436 Hmgn5 2.22 Chromatin/chromatin-binding protein Cluster 02 & 05 10419296 Wdhd1 2.12 DNA binding protein Cluster 02 & 05 10578690 Neil3 3.97 DNA glycosylase, damaged DNA-binding protein, endodeoxyribonuclease, nuclease, lyase, esterase, glycosidase Cluster 02 & 05 10546163 Mcm2 2.58 DNA helicase, hydrolase Cluster 02 & 05 10550102 Lig1 3.30 DNA ligase Cluster 02 & 05 10568050 Aldoa/Aldoart1 2.40 Fructose-bisphosphate aldolase Cluster 02 & 05 10587107 Myo5a 2.76 G-protein coupled modulator, actin binding motor protein, cell junction protein Cluster 02 & 05 10397645 Gpr65 2.10 G-protein coupled receptor Cluster 02 & 05 10362294 Arhgap18 2.06 G-protein modulator Cluster 02 & 05 10482059 Ggta1 2.84 Glycosyltransferase Cluster 02 & 05 10462973 Hells (LSH) 3.74 Helicase Cluster 02 & 05 10511416 Tox 4.29 HMG box transcription factor, signaling molecule, chromatin/chromatin-binding protein Cluster 02 & 05 10491780 Hspa4l 2.59 Hsp70 family chaperone Cluster 02 & 05 10472212 Pkp4 2.68 Intermediate filament binding protein Cluster 02 & 05 10459481 Lman1 3.17 Membrane traffic protein Cluster 02 & 05 10486396 Ehd4 4.27 Membrane traffic protein, G-protein modulator, calcium-binding protein Cluster 02 & 05 10559261 Cd81 5.98 Membrane-bound signaling molecule, receptor, cell adhesion molecule Cluster 02 & 05 10594251 Kif23 2.76 Microtubule binding motor protein Cluster 02 & 05 10353733 Prim2 2.22 Misc. Cluster 02 & 05 10580771 Ciapin1 2.30 Misc. Cluster 02 & 05 10400189 10400189 2.31 Misc. Cluster 02 & 05 10361760 Timm8a1 2.36 Misc. Cluster 02 & 05 10606689 Timm8a1 2.37 Misc. Cluster 02 & 05 10474437 Ccdc34 2.45 Misc. Cluster 02 & 05 10556640 6330503K22Rik (Ccp110) 2.55 Misc. Cluster 02 & 05 10548585 Csda 2.60 Misc. Cluster 02 & 05 10544644 Dfna5 2.88 Misc. Cluster 02 & 05 10479811 Mcm10 2.97 Misc. Cluster 02 & 05 10365933 Eea1 3.07 Misc. Cluster 02 & 05 10409990 6720489N17Rik 3.20 Misc. Cluster 02 & 05 10503315 Rad54b/Fsbp 3.28 Misc. Cluster 02 & 05 10452709 Ndc80 3.35 Misc. Cluster 02 & 05 10554325 5730590G19Rik 3.36 Misc. Cluster 02 & 05 10455967 2610318N02Rik 3.44 Misc. Cluster 02 & 05 10499035 Mnd1/Gm3833 3.46 Misc. Cluster 02 & 05 10352756 Lpgat1 3.80 Misc. Cluster 02 & 05 10483401 Spc25 4.05 Misc. Cluster 02 & 05 10524169 Pole 4.23 Misc. Cluster 02 & 05 10497520 Ect2 5.18 Misc. Cluster 02 & 05 10408600 Serpinb6a 6.27 Misc. Cluster 02 & 05 10424221 Wdr67 (Tbc1d31) 3.15 Misc. Cluster 02 & 05 10466410 Psat1/LOC100047252 4.62 Misc. Cluster 02 & 05 10412562 Flnb 2.44 Non-motor actin binding protein Cluster 02 & 05 10421877 Diap3 4.90 Non-motor actin binding protein Cluster 02 & 05 10473919 Ckap5 2.21 Non-motor microtubule binding protein Cluster 02 & 05 10557459 Mapk3 2.57 Non-receptor serine/theronine protein kinase Cluster 02 & 05 10452980 Eif2ak2 2.70 Non-receptor serine/theronine protein kinase Cluster 02 & 05 10524790 Cit 2.96 Non-receptor serine/theronine protein kinase Cluster 02 & 05 10588243 Ryk 3.62 NOT protein tyrosine kinase activity Cluster 02 & 05 10582809 Tk1 2.50 Nucleotide kinase Cluster 02 & 05 10538253 Mpp6 3.02 Nucleotide kinase, cell junction protein Cluster 02 & 05 10371888 Tmpo 2.05 Peptide hormone Cluster 02 & 05 10578703 Spcs3 2.51 Protease Cluster 02 & 05 10458195 Cdc25c 4.13 Protein phosphatase Cluster 02 & 05 10439895 Alcam (CD166) 2.68 Receptor, immunoglobulin superfamily cell adhesion molecule Cluster 02 & 05 10428672 Dscc1 4.00 Replication origin binding protein Cluster 02 & 05 10438378 Cdc45 4.26 Replication origin binding protein Cluster 02 & 05 10601854 Wbp5 2.41 Transcription factor Cluster 02 & 05 10408477 E2f3 2.16 Transcription factor, nucleic acid binding Cluster 02 & 05 10570373 Tfdp1 2.35 Transcription factor, nucleic acid binding Cluster 02 & 05 10355246 Acadl 2.67 Transferase dehydrogenase oxidase Cluster 02 & 05 10592058 Tuba1b 2.06 Tubulin Cluster 02 & 05 10581800 Rfwd3 2.04 Ubiquitin-protein ligase Cluster 02 & 05 10568461 Rfwd3 2.08 Ubiquitin-protein ligase Cluster 02 & 05 10428534 Trps1 3.70 Zinc finger transcription factor, nuclease Cluster 02 & 06 10468992 Frmd4a 3.25 Actin family cytoskeletal protein Cluster 02 & 06 10379535 Ccl8 10.85 Chemokine Cluster 02 & 06 10515744 Cdc20 2.79 Enzyme modulator Cluster 02 & 06 10436169 Ift57 2.34 Enzyme modulator, cytoskeletal protein Cluster 02 & 06 10445293 Pla2g7 4.98 Esterase Cluster 02 & 06 10449000 Msln 3.12 Extracellular matrix glycoprotein Cluster 02 & 06 10597413 Crtap 2.84 Extracellular matrix protein Cluster 02 & 06 10460108 Gnpnat1 2.40 Glucosamine 6-phosphate N-acetyltransferase Cluster 02 & 06 10547807 Eno2 2.64 Lyase Cluster 02 & 06 10548030 Cd9 4.32 Membrane-bound signaling molecule, receptor, cell adhesion molecule Cluster 02 & 06 10474381 Kif18a 2.71 Microtubule binding motor protein Cluster 02 & 06 10548422 Klri2 5.84 Microtubule binding motor protein Cluster 02 & 06 10515943 Ctps 2.36 Misc. Cluster 02 & 06 10600797 Apoo/Apoo-ps 2.40 Misc. Cluster 02 & 06 10381445 Tmem106a 2.51 Misc. Cluster 02 & 06 10429387 Ptplb (Hacd2) 2.51 Misc. Cluster 02 & 06 10365716 Ikbip/LOC100862563 2.52 Misc. Cluster 02 & 06 10453715 Rab18 2.54 Misc. Cluster 02 & 06 10383479 Hmga1/Hmga1-rs1 2.58 Misc. Cluster 02 & 06 10582241 Zcchc14 2.61 Misc. Cluster 02 & 06 10394735 Pdia6 2.62 Misc. Cluster 02 & 06 10499366 Pmf1 2.65 Misc. Cluster 02 & 06 10523529 Cops4 2.67 Misc. Cluster 02 & 06 10582020 Gcsh 2.75 Misc. Cluster 02 & 06 10412036 Apoo/Apoo-ps 2.75 Misc. Cluster 02 & 06 10443009 Ergic1 3.02 Misc. Cluster 02 & 06 10538269 Dfna5 3.02 Misc. Cluster 02 & 06 10455873 Slc12a2 3.03 Misc. Cluster 02 & 06 10602068 Mid2 3.27 Misc. Cluster 02 & 06 10506680 Tmem48 (Ndc1) 3.38 Misc. Cluster 02 & 06 10381588 Grn 4.37 Misc. Cluster 02 & 06 10498584 Rarres1 4.39 Misc. Cluster 02 & 06 10494445 Lix1l 4.43 Misc. Cluster 02 & 06 10430358 C1qtnf6 4.48 Misc. Cluster 02 & 06 10487011 Gatm 6.36 Misc. Cluster 02 & 06 10379727 Gm11428 7.76 Misc. Cluster 02 & 06 10403821 10403821 18.40 Misc. Cluster 02 & 06 10407940 Trgv2 26.65 Misc. Cluster 02 & 06 10605437 Pls3 3.73 Non-motor actin binding protein Cluster 02 & 06 10523468 Bmp2k 2.52 Non-receptor serine/theronine protein kinase Cluster 02 & 06 10430255 Foxred2 4.83 Oxygenase Cluster 02 & 06 10521709 Lap3 3.02 Peptidase Cluster 02 & 06 10526520 Plod3/Znhit1 2.56 Procollagen glucosyltransferase activity, procollagen-lysine 5- dioxygenase activity Cluster 02 & 06 10605711 Pdk3 2.39 Protein kinase Cluster 02 & 06 10592515 Ubash3b 2.83 Protein tyrosine phosphatase activity Cluster 02 & 06 10363498 Ppa1 2.77 Pyrophosphatase Cluster 02 & 06 10544596 Tmem176b 5.69 Receptor Cluster 02 & 06 10587554 Tpbg 4.84 Receptor, extracellular matrix protein Cluster 02 & 06 10541034 Zfand4 2.72 Ribosomal protein Cluster 02 & 06 10423654 Osr2 2.79 RNA polymerase II regulatory region sequence-specific DNA bindin, sequence-specific DNA binding, transcriptional activator activity, RNA polymerase II transcription regulatory region sequence-specific binding Cluster 02 & 06 10431424 Plxnb2 2.91 Semaphorin receptor activity Cluster 02 & 06 10420280 Gzmg 9.91 Serine protease Cluster 02 & 06 10420268 Gzmd/Gzme 31.46 Serine protease Cluster 02 & 06 10550650 Ercc1/Cd3eap 2.55 TFIID-class transcription factor binding Cluster 02 & 06 10361007 Smyd2 2.86 Transcription factor Cluster 02 & 06 10373313 Nab2 2.51 Transcription factor binding Cluster 02 & 06 10406934 Etv1 3.79 Transcription factor, signaling molecule, nucleic acid binding Cluster 02 & 06 10547100 Plxnd1 3.63 Tyrosine protein kinase receptor, signaling molecule, protein kinase Cluster 02 & 07 10464128 Casp7 2.04 Cysteine protease, protease inhibitor Cluster 02 & 07 10353420 Mcm3 2.92 DNA helicase, hydrolase Cluster 02 & 07 10390707 Top2a 5.43 DNA topoisomerase, isomerase, enzyme modulator Cluster 02 & 07 10542079 Foxm1/4933413G19Rik 4.56 DNA-binding protein Cluster 02 & 07 10501699 Agl 2.66 Glycosyltransferase, glycosidase Cluster 02 & 07 10587023 Rab27a 2.15 GTP binding, GTPase activity Cluster 02 & 07 10562637 Ccnb1/Gm5593 4.44 Kinase Cluster 02 & 07 10515836 Ccnb1/Gm5593 4.62 Kinase Cluster 02 & 07 10411739 Ccnb1/Gm5593 4.67 Kinase Cluster 02 & 07 10543067 Asns 5.26 Ligase Cluster 02 & 07 10474984 Nusap1/Oip5 4.85 Microtubule binding Cluster 02 & 07 10350297 Kif14 3.11 Microtubule binding motor protein Cluster 02 & 07 10462632 Kif20b 3.15 Microtubule binding motor protein Cluster 02 & 07 10568150 Kif22 3.67 Microtubule binding motor protein Cluster 02 & 07 10391811 Kif18b 3.81 Microtubule binding motor protein Cluster 02 & 07 10515431 Kif2c 5.20 Microtubule binding motor protein Cluster 02 & 07 10496204 Cenpe 5.94 Microtubule binding motor protein Cluster 02 & 07 10590494 Kif15 8.33 Microtubule binding motor protein Cluster 02 & 07 10561706 2200002D01Rik (H2RSP) /Yif1b 2.02 Misc. Cluster 02 & 07 10389134 Slfn9 2.03 Misc. Cluster 02 & 07 10355115 Prelid1 2.14 Misc. Cluster 02 & 07 10402650 Cinp/LOC640972 2.18 Misc. Cluster 02 & 07 10457729 Dsc1/Tpi1 2.21 Misc. Cluster 02 & 07 10489078 Dsn1 2.21 Misc. Cluster 02 & 07 10430825 Cenpm 2.30 Misc. Cluster 02 & 07 10511617 Fam92a 2.44 Misc. Cluster 02 & 07 10484425 2700094K13Rik 2.59 Misc. Cluster 02 & 07 10426098 Creld2 2.60 Misc. Cluster 02 & 07 10426016 Gtse1 2.63 Misc. Cluster 02 & 07 10593483 Fdx1 2.68 Misc. Cluster 02 & 07 10367282 Cnpy2 2.68 Misc. Cluster 02 & 07 10548699 10548699 2.96 Misc. Cluster 02 & 07 10355327 Bard1 2.98 Misc. Cluster 02 & 07 10538832 Mad2l1/LOC100045924 3.27 Misc. Cluster 02 & 07 10406968 Cenpk/Ppwd1 3.81 Misc. Cluster 02 & 07 10446027 Chaf1a/Ubxn6 3.83 Misc. Cluster 02 & 07 10379127 Spag5 4.25 Misc. Cluster 02 & 07 10541729 Cdca3 4.40 Misc. Cluster 02 & 07 10400589 Mis18bp1 4.49 Misc. Cluster 02 & 07 10507112 Stil 4.64 Misc. Cluster 02 & 07 10591556 Spc24 4.69 Misc. Cluster 02 & 07 10577508 Ckap2 5.17 Misc. Cluster 02 & 07 10487340 Ncaph 5.41 Misc. Cluster 02 & 07 10501229 Gstm1 5.52 Misc. Cluster 02 & 07 10474875 Casc5 5.57 Misc. Cluster 02 & 07 10521731 Ncapg 5.93 Misc. Cluster 02 & 07 10399087 Ncapg2 6.11 Misc. Cluster 02 & 07 10474769 Bub1b 6.58 Misc. Cluster 02 & 07 10350838 2810417H13Rik 6.70 Misc. Cluster 02 & 07 10385248 Hmmr 6.76 Misc. Cluster 02 & 07 10411728 Cenph 6.77 Misc. Cluster 02 & 07 10487480 Bub1 10.75 Misc. Cluster 02 & 07 10353032 Vcpip1 2.20 Misc. Cluster 02 & 07 10528915 Tyms/Tyms-ps 2.97 Misc. Cluster 02 & 07 10459755 Ska1 3.12 Misc. Cluster 02 & 07 10451805 Sgol1 3.38 Misc. Cluster 02 & 07 10521090 Tacc3 4.24 Misc. Cluster 02 & 07 10346365 Sgol2 5.25 Misc. Cluster 02 & 07 10554445 Prc1 3.57 Non-motor actin binding protein Cluster 02 & 07 10350392 Aspm 4.89 Non-motor actin binding protein Cluster 02 & 07 10477187 Tpx2 6.58 Non-motor microtubule binding protein Cluster 02 & 07 10561854 Tbcb 2.62 Non-motor microtubule binding protein, chaperone Cluster 02 & 07 10490104 Aurka 2.55 Non-receptor serine/theronine protein kinase Cluster 02 & 07 10504470 Melk 3.61 Non-receptor serine/theronine protein kinase Cluster 02 & 07 10377405 Aurkb 5.33 Non-receptor serine/theronine protein kinase Cluster 02 & 07 10581813 Mlkl 4.33 Non-receptor serine/theronine protein kinase, non-receptor tyrosine protein kinase Cluster 02 & 07 10369815 Cdk1 4.83 Non-receptor serine/theronine protein kinase, non-receptor tyrosine protein kinase Cluster 02 & 07 10356859 Dtymk 2.08 Nucleotide kinase Cluster 02 & 07 10533090 Rfc5 2.27 Nucleotidyltransferase, DNA-directed DNA polymerase Cluster 02 & 07 10571399 Zdhhc2 4.51 Palmitoyltransferase activity Cluster 02 & 07 10607475 Prdx4 4.59 Peroxidase Cluster 02 & 07 10516956 Smpdl3b 2.01 Phosphodiesterase Cluster 02 & 07 10453214 Nutf2/Nutf2-ps1 2.66 Protein transporter activity Cluster 02 & 07 10457463 Nutf2/Nutf2-ps1 2.71 Protein transporter activity Cluster 02 & 07 10468531 Nutf2/Nutf2-ps1 2.71 Protein transporter activity Cluster 02 & 07 10428690 Mrpl13 2.14 Ribosomal protein Cluster 02 & 07 10346651 Bmpr2 2.39 Serine/theronine protein kinase, TGF-beta receptor, protein kinase Cluster 02 & 07 10576883 Shcbp1 6.49 SH2 domain binding Cluster 02 & 07 10493812 S100a4 2.17 Signaling molecule, calmodulin Cluster 02 & 07 10550509 Pglyrp1 2.46 Signaling molecule, defense/immunity protein Cluster 02 & 07 10369252 Sept10 2.51 Small GTPase, cytoskeletal protein Cluster 02 & 07 10359890 Nuf2 8.21 Structural protein Cluster 02 & 07 10363901 Etv5 2.44 Transcription factor, signaling molecule, nucleic acid binding Cluster 02 & 07 10392284 Kpna2 2.88 Transfer/carrier protein Cluster 02 & 07 10453512 Kpna2 2.92 Transfer/carrier protein Cluster 02 & 07 10497503 Kpna2 3.12 Transfer/carrier protein Cluster 02 & 07 10595664 Tmed3 2.31 Transfer/carrier protein, vesicle coat protein Cluster 02 & 07 10469035 Sephs1 2.40 Transferase Cluster 02 & 07 10419323 Dlgap5 4.06 Ttransmembrane receptor regulatory/adaptor protein Cluster 02 & 07 10534660 Ap1s1 2.62 Vesicle coat protein Cluster 02 & 08 10472782 Hat1 2.21 Acetyltransferase Cluster 02 & 08 10551614 Ech1 2.39 Acetyltransferase, acyltransferase, dehydrogenase, hydratase, ligase, epimerase/racemase Cluster 02 & 08 10594911 Tex9 2.55 Actin binding motor protein Cluster 02 & 08 10363475 Prf1 3.42 Apolioprotein, receptor, metalloprotease, serine protease, metalloprotease, serine protease, complement component cell adhesion molecule Cluster 02 & 08 10497894 Cetn4 2.15 Calcium ion binding, heterotrimeric G-protein binding Cluster 02 & 08 10601390 Pgk1 3.80 Carbohydrate kinase Cluster 02 & 08 10399379 Pgk1 3.83 Carbohydrate kinase Cluster 02 & 08 10498313 Pgk1 3.83 Carbohydrate kinase Cluster 02 & 08 10399391 Gen1 3.37 Damaged DNA-binding protein, endodeoxyribonuclease Cluster 02 & 08 10562044 Zbtb32 (PLZP) 8.95 DNA binding, transcription corepressor activity, zinc ion binding Cluster 02 & 08 10357436 Mcm6 2.66 DNA helicase, hydrolase Cluster 02 & 08 10483249 Galnt3 7.43 Glycosyltransferase Cluster 02 & 08 10408083 Hist1h3i/Hist1h3a/Hist1h3h/Hist1h3g/Hi 2.23 st1h3d/Hist2h3b/Hist1h3e/Hist1h3b/Hist 1h3c/Hist1h3f/Hist2h3c2/Hist2h3c1 Histone Cluster 02 & 08 10408202 Hist1h3e/Hist2h3b/Hist1h3b/Hist1h3d/Hi 2.26 st1h3c/Hist1h3f/Hist2h3c2/ Hist2h3c1/Hist1h3h/Hist1h3a/Hist1h3i/H ist1h3g Histone Cluster 02 & 08 10403943 Hist1h2bm 2.39 Histone Cluster 02 & 08 10494405 Hist2h3b/Hist1h3e/Hist1h3b/Hist1h3d/Hi 2.50 st1h3c/Hist1h3f/Hist2h3c2/ Hist2h3c1 Histone Cluster 02 & 08 10494402 Hist2h3c1/Hist2h3b/Hist1h3e/Hist1h3b/ 2.53 Hist1h3d/Hist1h3c/Hist1h3f/ Hist2h3c2 Histone Cluster 02 & 08 10408081 Hist1h1b 3.12 Histone Cluster 02 & 08 10406663 Arsb 5.87 Hydrolase Cluster 02 & 08 10406672 Arsb 6.27 Hydrolase Cluster 02 & 08 10554863 Sytl2 2.45 Membrane trafficking regulatory protein Cluster 02 & 08 10400383 2700097O09Rik 3.15 Methyltransferase Cluster 02 & 08 10603431 Suv39h1 2.28 Methyltransferase, DNA binding protein Cluster 02 & 08 10521136 Whsc1 2.86 Methyltransferase, DNA binding protein Cluster 02 & 08 10462796 Kif11 5.81 Microtubule binding motor protein Cluster 02 & 08 10601011 Kif4/Pdzd11 6.15 Microtubule binding motor protein Cluster 02 & 08 10603567 Dynlt3 3.38 Microtubule family cytoskeletal protein Cluster 02 & 08 10424676 Ly6e (Sca-2) 2.08 Misc. Cluster 02 & 08 10543319 Fam3c 2.12 Misc. Cluster 02 & 08 10405427 Prelid1/Rab24 2.14 Misc. Cluster 02 & 08 10377605 1810027O10Rik 2.16 Misc. Cluster 02 & 08 10582190 Gins2/Gse1 2.29 Misc. Cluster 02 & 08 10462136 Cycs 2.33 Misc. Cluster 02 & 08 10528170 Cycs 2.42 Misc. Cluster 02 & 08 10601473 Apool 2.47 Misc. Cluster 02 & 08 10532944 Mlec 2.52 Misc. Cluster 02 & 08 10544660 Osbpl3 2.60 Misc. Cluster 02 & 08 10532954 Mlec 2.81 Misc. Cluster 02 & 08 10568217 Dctpp1 2.84 Misc. Cluster 02 & 08 10355050 Raph1 2.92 Misc. Cluster 02 & 08 10573451 Syce2 2.94 Misc. Cluster 02 & 08 10495945 4930422G04Rik 3.04 Misc. Cluster 02 & 08 10508986 Stmn1 3.11 Misc. Cluster 02 & 08 10378053 Fam64a 3.21 Misc. Cluster 02 & 08 10426669 Troap 3.22 Misc. Cluster 02 & 08 10513818 Stmn1 3.26 Misc. Cluster 02 & 08 10590909 Endod1 3.36 Misc. Cluster 02 & 08 10429564 Ly6a (TAP, Sca-1) 3.45 Misc. Cluster 02 & 08 10409190 Cenpp 3.81 Misc. Cluster 02 & 08 10508151 Clspn 3.94 Misc. Cluster 02 & 08 10568714 Mki67 (Ki67) 4.18 Misc. Cluster 02 & 08 10420426 F630043A04Rik 4.87 Misc. Cluster 02 & 08 10410560 Trip13 5.04 Misc. Cluster 02 & 08 10411359 Plp2/Prickle3 5.10 Misc. Cluster 02 & 08 10525591 Kntc1 5.13 Misc. Cluster 02 & 08 10473022 Plp2 5.39 Misc. Cluster 02 & 08 10485674 2700007P21Rik 5.77 Misc. Cluster 02 & 08 10499639 Cks1b/Gm6340 5.90 Misc. Cluster 02 & 08 10516246 Cdca8 6.11 Misc. Cluster 02 & 08 10475517 AA467197/Mir147 6.92 Misc. Cluster 02 & 08 10389606 Prr11 7.39 Misc. Cluster 02 & 08 10462866 Cep55 7.55 Misc. Cluster 02 & 08 10534456 Hip1 2.58 Non-motor actin binding protein Cluster 02 & 08 10427166 Espl1 2.87 Nucleic acid binding Cluster 02 & 08 10547943 Ncapd2 2.89 Nucleic acid binding Cluster 02 & 08 10373515 Suox 3.21 Oxidase Cluster 02 & 08 10475610 Dut 2.81 Phosphatase Cluster 02 & 08 10367076 Prim1 4.46 Primase Cluster 02 & 08 10382998 Birc5 5.00 Protease inhibitor Cluster 02 & 08 10394978 Rrm2 4.69 Reductase Cluster 02 & 08 10455595 Eno1/Gm5506 2.71 RNA binding, phosphopyruvate hydratase activity Cluster 02 & 08 10473240 Eno1/Gm5506 2.75 RNA binding, phosphopyruvate hydratase activity Cluster 02 & 08 10584435 Vwa5a 2.51 Serine protease inhibitor Cluster 02 & 08 10519983 Fgl2 6.67 Signaling molecule Cluster 02 & 08 10433088 Cbx5 3.23 Transcription factor, chromatin/chromatin-binding protein Cluster 02 & 08 10366277 E2f7 2.12 Transcription factor, nucleic acid binding Cluster 02 & 08 10587792 Plscr1 4.82 Transfer/carrier protein Cluster 02 & 08 10517336 Clic4 3.35 Transferase, signaling molecule, reductase, translation elongation factor, epimerase/racemase, cytoskeletal protein Cluster 02 & 08 10354389 Slc39a10 2.81

Transporter Cluster 02 & 08 10491091 Tnfsf10 2.96 Tumor necrosis factor receptor Cluster 02 & 08 10446074 Uhrf1 3.87 Ubiquitin-protein ligase Cluster 02 & 08 10355967 Ap1s3 2.39 Vesicle coat protein Cluster 02 & 09 10428857 Mtss1 2.04 Actin family cytoskeletal protein Cluster 02 & 09 10534303 Lat2 7.88 Amino acid transporter Cluster 02 & 09 10552697 Napsa 3.59 Aspartic protease Cluster 02 & 09 10592330 Nrgn 13.60 calmodulin binding Cluster 02 & 09 10553501 Slc17a6 11.02 Cation transporter Cluster 02 & 09 10573261 Asf1b 3.27 Chromatin/chromatin-binding protein Cluster 02 & 09 10385325 Pttg1 2.14 Cysteine-type endopeptidase inhibitor activity Cluster 02 & 09 10527801 Brca2 2.34 Damaged DNA-binding protein Cluster 02 & 09 10352048 Exo1 2.79 Damaged DNA-binding protein, endodeoxyribonuclease, exodeoxyribonuclease, nuclease, hydrolase Cluster 02 & 09 10463064 Gapdh 2.09 Dehydrogenase Cluster 02 & 09 10368612 Gapdh 2.15 Dehydrogenase Cluster 02 & 09 10474239 Gapdh 2.16 Dehydrogenase Cluster 02 & 09 10425903 Gapdh 2.30 Dehydrogenase Cluster 02 & 09 10553301 Ldha 2.67 Dehydrogenase Cluster 02 & 09 10472162 Gpd2 6.49 Dehydrogenase Cluster 02 & 09 10452815 Xdh 4.71 Dehydrogenase, oxidase Cluster 02 & 09 10574427 Impdh2 2.54 Dehydrogenase, reductase Cluster 02 & 09 10485294 Hsd17b12 3.70 Dehydrogenase, reductase Cluster 02 & 09 10572906 Mcm5 3.01 DNA helicase, hydrolase Cluster 02 & 09 10474902 Rad51 7.39 DNA strand-pairing protein, hydrolase Cluster 02 & 09 10379363 Atad5 2.97 DNA-binding protein Cluster 02 & 09 10435581 Polq 2.26 DNA-directed DNA polymerase activity Cluster 02 & 09 10605674 Pola1 2.66 Exodeoxyribonuclease, nuclease Cluster 02 & 09 10523717 Spp1 87.69 Extracellular matrix binding Cluster 02 & 09 10423293 Myo10 10.78 G-protein coupled modulator, actin binding motor protein, cell junction protein Cluster 02 & 09 10407985 Gpr141 2.63 G-protein coupled receptor Cluster 02 & 09 10546853 Srgap3 9.23 G-protein modulator Cluster 02 & 09 10546855 Srgap3 10.24 G-protein modulator Cluster 02 & 09 10408225 Hist1h4c 2.32 Histone Cluster 02 & 09 10547830 Tpi1 7.60 Isomerase Cluster 02 & 09 10509441 Ece1 2.35 Metalloprotease Cluster 02 & 09 10454709 Kif20a/Cdc23 3.45 Microtubule binding motor protein Cluster 02 & 09 10444505 Rdbp (Nelfe) 2.07 Misc. Cluster 02 & 09 10593490 Gm6981/Gapdh 2.31 Misc. Cluster 02 & 09 10465912 Fen1 2.32 Misc. Cluster 02 & 09 10451547 10451547 2.36 Misc. Cluster 02 & 09 10388880 Tmem97 2.40 Misc. Cluster 02 & 09 10482432 10482432 2.42 Misc. Cluster 02 & 09 10554817 10554817 2.43 Misc. Cluster 02 & 09 10414431 10414431 2.49 Misc. Cluster 02 & 09 10547916 Ptms 2.50 Misc. Cluster 02 & 09 10463632 Tmem180 2.59 Misc. Cluster 02 & 09 10364102 Chchd10 2.59 Misc. Cluster 02 & 09 10393823 P4hb 2.60 Misc. Cluster 02 & 09 10578916 Sc4mol 2.60 Misc. Cluster 02 & 09 10486255 Oip5/Nusap1 2.65 Misc. Cluster 02 & 09 10528480 Rpl17 2.72 Misc. Cluster 02 & 09 10548565 Magohb 2.82 Misc. Cluster 02 & 09 10440393 Samsn1 3.10 Misc. Cluster 02 & 09 10369885 Cisd1 3.26 Misc. Cluster 02 & 09 10597461 Cmtm7 3.27 Misc. Cluster 02 & 09 10535065 Adap1 3.41 Misc. Cluster 02 & 09 10407946 Stard3nl 3.51 Misc. Cluster 02 & 09 10519324 Cdk6 3.83 Misc. Cluster 02 & 09 10515257 Rad54l 3.83 Misc. Cluster 02 & 09 10420877 Esco2 3.93 Misc. Cluster 02 & 09 10490838 Fabp5 3.99 Misc. Cluster 02 & 09 10355329 Bard1 4.03 Misc. Cluster 02 & 09 10371591 4930547N16Rik/Pmch 4.07 Misc. Cluster 02 & 09 10585699 Fabp5 4.21 Misc. Cluster 02 & 09 10568785 Bnip3 4.39 Misc. Cluster 02 & 09 10545958 Anxa4 4.55 Misc. Cluster 02 & 09 10568873 Adam8 4.59 Misc. Cluster 02 & 09 10592201 Chek1 5.21 Misc. Cluster 02 & 09 10471844 Nek6 5.41 Misc. Cluster 02 & 09 10407955 Epdr1 6.14 Misc. Cluster 02 & 09 10586744 Anxa2 6.59 Misc. Cluster 02 & 09 10429140 Ndrg1 7.43 Misc. Cluster 02 & 09 10461587 Ms4a4a 8.83 Misc. Cluster 02 & 09 10414269 Bnip3 10.01 Misc. Cluster 02 & 09 10595298 Filip1 13.26 Misc. Cluster 02 & 09 10439845 Gm5486 17.88 Misc. Cluster 02 & 09 10582549 Mela 64.18 Misc. Cluster 02 & 09 10582545 Mela/Kcnma1 169.29 Misc. Cluster 02 & 09 10594426 Zwilch/Lctl 3.29 Misc. Cluster 02 & 09 10452508 Twsg1 4.68 Misc. Cluster 02 & 09 10601705 Cenpi 2.98 mRNA splicing factor Cluster 02 & 09 10518561 Apitd1 3.01 Neuropeptide Cluster 02 & 09 10539135 Capg 13.60 Non-motor actin binding protein, calcium-binding protein Cluster 02 & 09 10454786 Ctnna1 6.07 Non-motor actin binding protein, cell adhesion molecule Cluster 02 & 09 10587508 Ttk 6.18 Non-receptor serine/theronine protein kinase Cluster 02 & 09 10515295 Mast2 2.06 Non-receptor serine/theronine protein kinase, transfer/carrier protein, calmodulin Cluster 02 & 09 10393431 Tk1 3.68 Nucleotide kinase Cluster 02 & 09 10463070 Entpd1 7.00 Nucleotide phosphatase, lyase, nucleic acid binding Cluster 02 & 09 10408531 Gmds 2.45 Oxidoreductase, dehydratase, epimerase/racemase Cluster 02 & 09 10424349 Sqle 2.41 Oxygenase Cluster 02 & 09 10350516 Ptgs2 18.45 Oxygenase Cluster 02 & 09 10510260 Nppb 2.69 Peptide hormone Cluster 02 & 09 10592001 St14 7.32 Peptide hormone, receptor, serine protease, protease inhibitor, annexin, calmodulin Cluster 02 & 09 10367945 Phactr2 4.22 Phosphatase modulator Cluster 02 & 09 10374236 Upp1 5.33 Phosphorylase Cluster 02 & 09 10416037 Pbk 5.87 Protein kinase Cluster 02 & 09 10483381 Stk39 4.14 Protein kinase activity Cluster 02 & 09 10501629 Cdc14a 3.75 Protein phosphatase Cluster 02 & 09 10585932 Pkm 2.52 Pyruvate kinase activity Cluster 02 & 09 10351644 Cd244 (2B4)/LOC677008 3.56 Receptor Cluster 02 & 09 10375443 Havcr2 (TIM-3, CD366) 19.74 Receptor Cluster 02 & 09 10473281 Itgav (CD51) 5.73 Receptor, contributes to opsonin binding, contributes to protein kinase C binding Cluster 02 & 09 10583732 Ldlr 2.42 Receptor, extracellular matrix glycoprotein Cluster 02 & 09 10555695 Rrm1 3.28 Reductase Cluster 02 & 09 10586604 Rps27l 2.93 Ribosomal protein Cluster 02 & 09 10372877 Xpot 2.61 RNA binding protein Cluster 02 & 09 10420308 Gzmb 2.30 Serine protease Cluster 02 & 09 10420295 Gzmf 161.81 Serine protease Cluster 02 & 09 10404439 Serpinb9b 21.68 Serine protease inhibitor Cluster 02 & 09 10414360 Lgals3 4.46 Signaling molecule, cell adhesion molecule Cluster 02 & 09 10472501 Lass6 (Cers6) 4.85 Sphingosine N-acyltransferase Cluster 02 & 09 10371959 Elk3/Cdk17 2.48 Transcription factor, DNA-binding protein Cluster 02 & 09 10569057 Rnh1 2.16 Transcription factor, nucleic acid binding Cluster 02 & 09 10582981 Tfdp1 2.96 Transcription factor, nucleic acid binding Cluster 02 & 09 10521913 Rbpj 6.04 transcription factor, nucleic acid binding Cluster 02 & 09 10576034 Irf8 13.65 Transcription factor, nucleic acid binding Cluster 02 & 09 10438626 Etv5 5.75 Transcription factor, signaling molecule, nucleic acid binding Cluster 02 & 09 10391461 Brca1 3.15 Transcription regulator, ubiquitin-protein ligase Cluster 02 & 09 10383502 Slc16a3 4.61 Transporter Cluster 02 & 09 10510580 Tnfrsf9 (4-1BB) 25.87 Tumor necrosis factor receptor Cluster 02 & 09 10345752 Il1r2 (CD121b) 16.75 Type I cytokine receptor Cluster 02 & 10 10556426 Parva 6.27 Actin and actin related protein Cluster 02 & 10 10416800 Lmo7 3.60 Actin family cytoskeletal protein Cluster 02 & 10 10489204 Tgm2 3.43 Acyltransferase Cluster 02 & 10 10456522 Tcf4 3.19 Basic helix-loop-helix transcription factor, nucleic acid binding Cluster 02 & 10 10498018 Pcdh18 3.43 Cadherin Cluster 02 & 10 10533050 Hspb8 2.77 Chaperone Cluster 02 & 10 10348632 Twist2 2.49 Chromatin binding, transcription factor activity, RNA polymerase II transcription factor binding, sequence-specific DNA binding Cluster 02 & 10 10595371 Hmgn3 2.51 Chromatin/chromatin-binding protein Cluster 02 & 10 10516735 Tinagl1 2.66 Cysteine protease Cluster 02 & 10 10395320 Twist1 4.40 E-box binding, protein heterodimerization activity, transcription factor activity, sequence-specific DNA binding Cluster 02 & 10 10531794 Wdfy3 3.66 Enzyme modulator Cluster 02 & 10 10571142 Gpr124 2.98 G-protein coupled receptor, antibacterial response protein Cluster 02 & 10 10588037 Rbp1 3.19 G-protein modulator Cluster 02 & 10 10363173 Gja1 7.28 Gap junction Cluster 02 & 10 10475643 Fgf7 2.94 Growth factor Cluster 02 & 10 10476395 Bmp2 4.05 Growth factor Cluster 02 & 10 10427402 Ghr 5.03 Growth hormone receptor Cluster 02 & 10 10358733 Rgl1 7.61 guanyl-nucleotide exchange factor Cluster 02 & 10 10465820 Gng3 4.69 Heterotrimeric G-protein Cluster 02 & 10 10458663 Dpysl3 2.54 Hydrolase Cluster 02 & 10 10481592 Dnm1 3.33 Hydrolase, small GTPase, microtubule family cytoskeletal protein Cluster 02 & 10 10594110 Neo1 3.02 Immunoglobulin receptor superfamily, protein phosphatase, immunoglobulin superfamily cell adhesion molecule Cluster 02 & 10 10501608 Vcam1 (CD106) 5.02 Immunoglobulin receptor superfamily, protein phosphatase, immunoglobulin superfamily cell adhesion molecule Cluster 02 & 10 10523727 Pkd2 3.35 Ion channel, membrane-bound signaling molecule, G-protein modulator Cluster 02 & 10 10458906 Ppic 3.81 Isomerase Cluster 02 & 10 10538547 Fkbp9 2.65 isomerase, chaperone, calcium-binding protein Cluster 02 & 10 10560190 Ehd2 4.87 Membrane traffic protein, G-protein modulator, calcium-binding protein Cluster 02 & 10 10423548 Sdc2 4.58 Membrane-bound signaling molecule, extracellular matrix glycoprotein, defense/immunity protein, cytoskeletal protein, cell adhesion molecule Cluster 02 & 10 10574027 Mt1 6.33 Metal ion binding Cluster 02 & 10 10415052 Mmp14 3.21 Metalloendopeptidase Cluster 02 & 10 10476648 Dstn 2.29 Misc. Cluster 02 & 10 10500204 Ecm1 2.35 Misc. Cluster 02 & 10 10421697 9030625A04Rik 2.44 Misc. Cluster 02 & 10 10467191 Ankrd1 2.52 Misc. Cluster 02 & 10 10356968 Pam 2.54 Misc. Cluster 02 & 10 10403727 Gli3 2.60 Misc. Cluster 02 & 10 10485948 Grem1 2.60 Misc. Cluster 02 & 10 10485607 Qser1 2.84 Misc. Cluster 02 & 10 10433264 Glis2 2.87 Misc. Cluster 02 & 10 10567995 Nupr1 3.00 Misc. Cluster 02 & 10 10381122 Fkbp10/Nt5c3l 3.03 Misc. Cluster 02 & 10 10427744 Rai14 3.42 Misc. Cluster 02 & 10 10527965 Cldn12 3.45 Misc. Cluster 02 & 10 10527940 Cdk14 4.07 Misc. Cluster 02 & 10 10532741 Tmem119 4.35 Misc. Cluster 02 & 10 10431935 Amigo2/Fam113b 4.73 Misc. Cluster 02 & 10 10427125 Igfbp6 5.55 Misc. Cluster 02 & 10 10394534 Osr1 6.33 Misc. Cluster 02 & 10 10447951 Thbs2 6.86 Misc. Cluster 02 & 10 10417759 Ube2e2/LOC100861885 2.88 Misc. Cluster 02 & 10 10438603 Igf2bp2 2.42 mRNA splicing factor, ribonucleoprotein, enzyme modulator Cluster 02 & 10 10472097 Fmnl2 2.53 Non-motor actin binding protein Cluster 02 & 10 10495659 Cnn3 2.65 Non-motor actin binding protein Cluster 02 & 10 10345241 Dst 2.84 Non-motor actin binding protein Cluster 02 & 10 10527158 Fscn1 3.50 Non-motor actin binding protein Cluster 02 & 10 10537184 Cald1 5.72 Non-motor actin binding protein Cluster 02 & 10 10352194 Cdc42bpa 2.31 Non-receptor serine/threonine protein kinase Cluster 02 & 10 10528015 Steap1 3.66 NOT cupric reductase activity, NOT ferric-chelate reductase (NADPH) activity Cluster 02 & 10 10564527 Nr2f2 6.27 Nuclear hormone receptor, receptor, nucleic acid binding Cluster 02 & 10 10477061 Srxn1 2.39 Oxidoreductase activity, acting on a sulfur group of donors Cluster 02 & 10 10356084 Irs1 2.69 phosphatidylinositol 3-kinase binding, protein kinase binding Cluster 02 & 10 10424370 Trib1 2.29 Protein kinase Cluster 02 & 10 10536505 Met 2.89 Protein kinase activity, hepatocyte growth factor-activated receptor activity Cluster 02 & 10 10427095 Tenc1 2.53 Protein phosphatase Cluster 02 & 10 10538150 Tmem176a 5.50 Receptor Cluster 02 & 10 10439651 Cd200 6.34 Receptor Cluster 02 & 10 10373223 Lrp1 (CD91) 3.21 Receptor, extracellular matrix glycoprotein Cluster 02 & 10 10417212 Itgbl1 2.76 Receptor, extracellular matrix glycoprotein, cell adhesion molecule Cluster 02 & 10 10490384 Lama5 2.24 Receptor, extracellular matrix linker protein Cluster 02 & 10 10395163 Lamb1 3.91 Receptor, extracellular matrix linker protein Cluster 02 & 10 10375175 Slit3 3.30 Receptor, extracellular matrix protein Cluster 02 & 10 10463836 Gsto1 2.60 Serine protease Cluster 02 & 10 10355984 Serpine2 4.18 Serine protease inhibitor Cluster 02 & 10 10388430 Serpinf1 5.04 Serine protease inhibitor Cluster 02 & 10 10446928 Ltbp1 3.95 Signaling molecule Cluster 02 & 10 10422728 Dab2 8.72 Signaling molecule Cluster 02 & 10 10484227 Sestd1 3.03 Signaling molecule, guanyl-nucleotide exchange factor Cluster 02 & 10 10564220 Gkap1 2.24 Structural protein Cluster 02 & 10 10416175 Nefl 2.40 Structural protein, intermediate filament Cluster 02 & 10 10421100 Nefm 3.66 Structural protein, intermediate filament Cluster 02 & 10 10416522 Tsc22d1 3.59 Transcription factor Cluster 02 & 10 10432540 Lima1 2.59 Transcription factor, nuclease, actin family cytoskeletal protein Cluster 02 & 10 10576049 Foxf1 4.49 Transcription factor, nucleic acid binding Cluster 02 & 10 10372796 Hmga2 5.49 Transcriptional activator activity, DNA-binding protein Cluster 02 & 10 10548905 Eps8 5.04 Transmembrane receptor regulatory/adaptor protein Cluster 02 & 10 10536494 Cav2 5.27 Transmembrane receptor regulatory/adaptor protein, structural protein, membrane traffic protein, G-protein modulator Cluster 02 & 10 10387791 Slc16a13 2.95 Transporter Cluster 02 & 10 10346843 Nrp2 2.71 Transporter, apolipoprotein, membrane-bound signaling molecule, receptor, metalloprotease, serine protease, oxidase, metalloprotease, serine protease, extracellular matrix protein, enzyme modulator, cell adhesion molecule Cluster 02 & 10 10490989 Cp 3.01 Transporter, apolipoprotein, membrane-bound signaling molecule, receptor, metalloprotease, serine protease, oxidase, metalloprotease, serine protease, extracellular matrix protein, enzyme modulator, cell adhesion molecule Cluster 02 & 10 10536220 Col1a2 3.38 Transporter, surfactant, receptor, extracellular matrix structural protein, antibacterial response protein Cluster 02 & 10 10440091 Col8a1 3.24 Transporter, surfactant, receptor, extracellular matrix structural protein, antibacterial response protein, cell adhesion molecule Cluster 02 & 11 10351206 Selp 5.66 Adpolipoprotein, receptor, metalloprotease, serine protease, complement component, cell adhesion molecule Cluster 02 & 11 10536818 Calu 2.91 Calmodulin Cluster 02 & 11 10605766 Maged1 8.12 Cell adhesion molecule Cluster 02 & 11 10360315 Fcrl6 10.72 Cell adhesion molecule Cluster 02 & 11 10379511 Ccl2 21.26 Chemokine Cluster 02 & 11 10528008 Steap2 8.35 Cupric reductase activity, ferric-chelate reductase (NADPH) activity Cluster 02 & 11 10521667 Bst1 2.39 Cyclase, glycosidase Cluster 02 & 11 10410124 Ctsl 16.42 Cysteine protease Cluster 02 & 11 10364375 Cstb 2.26 Cysteine protease inhibitor Cluster 02 & 11 10545672 Mthfd2 2.26 Dehydrogenase, ligase, hydrolase Cluster 02 & 11 10460585 Fosl1/Ccdc85b 3.74 DNA-binding protein Cluster 02 & 11 10553598 Cyfip1 3.07 G-protein modulator Cluster 02 & 11 10350725 Rgs8 8.05 G-protein modulator Cluster 02 & 11 10458340 Hbegf 6.27 Growth factor Cluster 02 & 11 10502655 Cyr61 8.77 Growth factor Cluster 02 & 11 10523175 Ereg 11.01 Growth factor Cluster 02 & 11 10417095 Farp1 4.58 Guanyl-nucleotide exchange factor Cluster 02 & 11 10359624 Prrx1 11.05 Homeobox transcription factor, DNA binding protein Cluster 02 & 11 10565315 Fah 3.01 Isomerase, hydrolase Cluster 02 & 11 10569646 Ccnd1 9.76 Kinase activator Cluster 02 & 11 10473356 Ube2l6 2.85 Ligase Cluster 02 & 11 10377938 Eno3 6.08 Lyase Cluster 02 & 11 10467258 Myof 7.47 Membrane traffic protein Cluster 02 & 11 10423520 Sema5a 5.16 Membrane-bound signaling molecule Cluster 02 & 11 10519717 Sema3a 10.77 Membrane-bound signaling molecule Cluster 02 & 11 10498576 Lxn 7.57 Metalloendopeptidase inhibitor Cluster 02 & 11 10598976 Timp1 20.58 Metalloprotease inhibitor Cluster 02 & 11 10585214 Cryab/Hspb2 2.10 Misc. Cluster 02 & 11 10525887 Bri3bp 2.13 Misc. Cluster 02 & 11 10386947 10386947 2.33 Misc. Cluster 02 & 11 10539263 Loxl3/Htra2 2.42 Misc. Cluster 02 & 11 10389238 Dusp14 2.47 Misc. Cluster 02 & 11 10359929 Ddr2 2.51 Misc. Cluster 02 & 11 10361887 Perp 2.58 Misc. Cluster 02 & 11 10542791 Ppfibp1 2.89 Misc. Cluster 02 & 11 10428103 Stk3 3.06 Misc. Cluster 02 & 11 10368508 Cenpw 3.23 Misc. Cluster 02 & 11 10346747 Cyp20a1 3.37 Misc. Cluster 02 & 11 10411519 Mtap1b 3.42 Misc. Cluster 02 & 11 10501222 Gstm2 3.73 Misc. Cluster 02 & 11 10417124 B930095G15Rik 4.47 Misc. Cluster 02 & 11 10547740 C1s/Gm5077 4.79 Misc. Cluster 02 & 11 10439009 Apod 4.97 Misc. Cluster 02 & 11 10561104 Axl 5.48 Misc. Cluster 02 & 11 10360398 Ifi202b 5.57 Misc. Cluster 02 & 11 10498273 Tm4sf1 6.00 Misc. Cluster 02 & 11 10497817 Anxa5 6.44 Misc. Cluster 02 & 11 10346164 Sdpr 6.95 Misc. Cluster 02 & 11 10577641 1810011O10Rik 8.86 Misc. Cluster 02 & 11 10388254 Aspa 8.88 Misc. Cluster 02 & 11 10405063 Ogn 9.17 Misc. Cluster 02 & 11 10405047 Aspn 9.56 Misc. Cluster 02 & 11 10523451 Anxa3 11.17 Misc. Cluster 02 & 11 10608724 10608724 16.00 Misc. Cluster 02 & 11 10360070 Fcer1g 18.33 Misc. Cluster 02 & 11 10408689 Nrn1 20.96 Misc. Cluster 02 & 11 10535559 Baiap2l1 (IRTKS) 24.42 Misc. Cluster 02 & 11 10502224 Sgms2 3.37 Misc. Cluster 02 & 11 10348354 Ugt1a9/Ugt1a1/Ugt1a5/Ugt1a10/Ugt1a2/ 5.52 Misc. Ugt1a6a/Ugt1a7c Cluster 02 & 11 10453825 Snrpd1 2.02 mRNA splicing factor, ribonucleoprotein Cluster 02 & 11 10523511 Prkg2 8.92

Non-receptor serine/theronine protein kinase Cluster 02 & 11 10390117 Itga3 (CD49c) 2.68 Receptor Cluster 02 & 11 10521759 Slit2 19.17 Receptor, extracellular matrix protein Cluster 02 & 11 10537146 Akr1b8 5.58 Reductase Cluster 02 & 11 10433480 Rpl39l 16.63 Ribosomal protein Cluster 02 & 11 10420274 Gzmd 95.97 serine protease Cluster 02 & 11 10565794 Serpinh1 2.97 Serine protease inhibitor Cluster 02 & 11 10484463 Serping1 5.36 Serine protease inhibitor Cluster 02 & 11 10403834 Sfrp4 2.27 Signaling molecule, G-protein coupled receptor Cluster 02 & 11 10605797 Arhgef9 4.41 Signaling molecule, gyanyl-nucleotide exchange factor Cluster 02 & 11 10495054 Rhoc 3.11 small GTPase Cluster 02 & 11 10548879 Mgp 9.25 Structural protein, signaling molecule, extracellular matrix structural protein, annexin, calmodulin Cluster 02 & 11 10466745 Tjp2 2.43 Tight junction Cluster 02 & 11 10442327 Cldn6 3.20 Tight junction Cluster 02 & 11 10400006 Ahr 3.29 Transcription factor Cluster 02 & 11 10391332 Ptrf 3.60 Transcription factor Cluster 02 & 11 10353010 Mybl1 3.14 Transcription factor, DNA binding protein Cluster 02 & 11 10514054 Nfib 3.79 Transcription factor, nucleic acid binding Cluster 02 & 11 10458555 Spry4 3.86 Transcription factor, signaling molecule Cluster 02 & 11 10562905 Atf5/Nup62/Nup62-il4i1/Il4i1 2.10 Transcription regulator Cluster 02 & 11 10536499 Cav1 20.84 Transmembrane receptor regulatory/adaptor protein, structural protein, membrane traffic protein, G-protein modulator Cluster 02 & 11 10487021 Slc30a4 7.81 Transporter Cluster 02 & 11 10534862 Pcolce 22.29 Transporter, apolipoprotein, membrane-bound signaling molecule, receptor, metalloprotease, serine protease, oxidase, metalloprotease, serine protease, extracellular matrix protein, enzyme modulator, cell adhesion molecule Cluster 02 & 11 10456400 Tubb6 5.81 Tubulin Cluster 02 & 11 10585395 Siva1 2.78 Virus receptor activity Cluster 02 & 11 10398874 Siva1 2.82 Virus receptor activity Cluster 02 & 12 10519555 Abcb1b 2.51 ATP-binding cassette (ABC) transporter Cluster 02 & 12 10544002 Creb3l2 3.35 CREB transcription factor, nucleic acid binding Cluster 02 & 12 10385912 Csf2 5.30 Cytokine Cluster 02 & 12 10501164 Csf1 9.94 Cytokine Cluster 02 & 12 10590242 Ccr8 4.14 G-protein coupled receptor, immunoglobulin receptor superfamily, protein phosphatase, immunoglobulin superfamily cell adhesion molecule Cluster 02 & 12 10350733 Rgs16 14.23 G-protein modulator Cluster 02 & 12 10424543 Wisp1 3.06 Growth factor Cluster 02 & 12 10404053 Hist1h2bc/Hist1h2bg/Hist1h2be 7.38 Histone Cluster 02 & 12 10355312 Ikzf2 5.79 KRAB box transcription factor Cluster 02 & 12 10548430 Gm156 39.76 Membrane-bound signaling molecule, receptor, defense/immunity protein Cluster 02 & 12 10459905 Setbp1 4.45 Methyltransferase, DNA binding protein Cluster 02 & 12 10542205 Klre1 7.99 Microtubule binding motor protein Cluster 02 & 12 10481491 Ier5l 2.25 Misc. Cluster 02 & 12 10462398 Pdcd1lg2 (PD-L2) 2.28 Misc. Cluster 02 & 12 10539617 Alms1 2.31 Misc. Cluster 02 & 12 10366043 Dusp6 2.42 Misc. Cluster 02 & 12 10544250 E330009J07Rik 2.44 Misc. Cluster 02 & 12 10444890 Ier3 2.55 Misc. Cluster 02 & 12 10361995 Fam54a 2.79 Misc. Cluster 02 & 12 10369290 Ddit4 3.24 Misc. Cluster 02 & 12 10536794 Hilpda 3.25 Misc. Cluster 02 & 12 10349081 Phlpp1 3.29 Misc. Cluster 02 & 12 10513190 D630039A03Rik 3.46 Misc. Cluster 02 & 12 10548735 Dusp16 3.83 Misc. Cluster 02 & 12 10605792 Spin4 4.39 Misc. Cluster 02 & 12 10356866 Pdcd1 (PD1) 13.60 Misc. Cluster 02 & 12 10442224 Zfp948 2.07 Misc. Cluster 02 & 12 10544732 Skap2 2.34 Misc. Cluster 02 & 12 10587829 Plod2 8.37 Oxygenase Cluster 02 & 12 10571321 Ppp1r3b 3.64 Phosphatase modulator Cluster 02 & 12 10463168 Pgam1 2.46 Phosphoglycerate mutase activity Cluster 02 & 12 10561302 Pgam1 2.46 Phosphoglycerate mutase activity Cluster 02 & 12 10497321 Pgam1 2.65 Phosphoglycerate mutase activity Cluster 02 & 12 10513154 Ptpn3 3.08 Protein phosphatase Cluster 02 & 12 10407126 Plk2 5.04 Protein serine/threonine kinase activity Cluster 02 & 12 10435704 Cd80 4.95 Receptor Cluster 02 & 12 10404422 Serpinb6b 2.73 Serine protease inhibitor Cluster 02 & 12 10404429 Serpinb9 3.69 Serine protease inhibitor Cluster 02 & 12 10503334 Gem 4.87 Small GTPase Cluster 02 & 12 10536472 Mdfic 2.15 Transcription factor Cluster 02 & 12 10437687 Litaf 7.45 Transcription factor Cluster 02 & 12 10391301 Stat3 2.24 Transcription factor, nucleic acid binding Cluster 02 & 12 10422227 Spry2 4.87 Transcription factor, signaling molecule Cluster 02 & 12 10356082 Plscr1 4.12 Transfer/carrier protein Cluster 02 & 12 10511282 Tnfrsf4 10.45 Tumor necrosis factor receptor Cluster 02 & 12 10545135 Il12rb2 4.48 Type I cytokine receptor, defense/immunity protein Cluster 02 & 12 10462454 Uhrf2 2.50 Ubiquitin-protein ligase Cluster 02 & 13 10447190 Plekhh2 2.45 Actin binding Cluster 02 & 13 10594661 Tpm1 2.36 Actin binding motor protein Cluster 02 & 13 10408870 Tbc1d7 2.59 Activation of GTPase activity, negative regulation of TOR signaling Cluster 02 & 13 10379518 Ccl7 5.67 Chemokine Cluster 02 & 13 10415844 Ctsb/Fdft1 2.40 Cysteine protease Cluster 02 & 13 10488415 Cst3 2.70 Cysteine protease inhibitor Cluster 02 & 13 10437639 Emp2 6.66 Cytoskeletal protein Cluster 02 & 13 10497001 Cryz 3.11 Dehydrogenase, reductase Cluster 02 & 13 10476362 Mcm8 2.16 DNA helicase, hydrolase Cluster 02 & 13 10534974 Mcm7 2.71 DNA helicase, hydrolase Cluster 02 & 13 10417273 Esd 2.56 Esterase Cluster 02 & 13 10493259 Ssr2 2.41 G-protein coupled receptor Cluster 02 & 13 10539861 Rpn1 2.46 Glycosyltransferase Cluster 02 & 13 10555041 Alg8 2.63 Glycosyltransferase Cluster 02 & 13 10466521 Gcnt1 8.92 Glycosyltransferase Cluster 02 & 13 10386058 Sparc 8.39 Growth factor, extracellular matrix glycoprotein, cell adhesion molecule Cluster 02 & 13 10481518 Ptges 6.98 Isomerase Cluster 02 & 13 10432243 Fkbp11 2.65 Isomerase, chaperone, calcium-binding protein Cluster 02 & 13 10367436 Cd63 79.88 Membrane-bound signaling molecule, receptor Cluster 02 & 13 10574023 Mt2 7.84 Metal ion binding Cluster 02 & 13 10467979 Scd1 21.69 Metal ion bindingl, palmitoyl-CoA 9-desaturase activity, stearoyl- CoA 9-desaturase activity Cluster 02 & 13 10393559 Timp2 5.22 Metalloprotease inhibitor Cluster 02 & 13 10372410 Glipr1/Krr1 2.00 Misc. Cluster 02 & 13 10554281 Fanci/Polg 2.19 Misc. Cluster 02 & 13 10419223 Fermt2 2.21 Misc. Cluster 02 & 13 10460603 Efemp2 2.25 Misc. Cluster 02 & 13 10538617 Lancl2 2.26 Misc. Cluster 02 & 13 10528143 Ppp1r14b 2.29 Misc. Cluster 02 & 13 10497548 Fndc3b 2.64 Misc. Cluster 02 & 13 10465895 Fads2 2.79 Misc. Cluster 02 & 13 10585338 Kdelc2 2.92 Misc. Cluster 02 & 13 10472916 Cdca7 2.96 Misc. Cluster 02 & 13 10398240 Eml1 3.03 Misc. Cluster 02 & 13 10480381 Arhgap21 3.20 Misc. Cluster 02 & 13 10421029 Cdca2 3.62 Misc. Cluster 02 & 13 10485622 Qser1 4.00 Misc. Cluster 02 & 13 10603346 Plp2/Prickle3 5.01 Misc. Cluster 02 & 13 10436106 C330027C09Rik 5.16 Misc. Cluster 02 & 13 10569707 Myadm 5.50 Misc. Cluster 02 & 13 10450374 D17H6S56E-5 (G7e) 5.83 Misc. Cluster 02 & 13 10466606 Anxa1 6.74 Misc. Cluster 02 & 13 10553299 Ifitm2 7.15 Misc. Cluster 02 & 13 10591781 Anln 7.76 Misc. Cluster 02 & 13 10383756 Ifitm2 7.94 Misc. Cluster 02 & 13 10569014 Ifitm2 10.27 Misc. Cluster 02 & 13 10558769 Ifitm1 12.93 Misc. Cluster 02 & 13 10569017 Ifitm3 14.75 Misc. Cluster 02 & 13 10384373 Fignl1 4.64 Non-motor microtubule binding protein Cluster 02 & 13 10504672 Tdrd7 2.14 Nuclease Cluster 02 & 13 10418171 Zcchc24 3.45 Nuclease Cluster 02 & 13 10476989 Gins1 4.82 Nucleic acid binding Cluster 02 & 13 10563883 Depdc1a 7.02 Nucleic acid binding Cluster 02 & 13 10497122 Depdc1a 7.39 Nucleic acid binding Cluster 02 & 13 10524621 Oasl2 2.84 Nucleotidyltransferase, nucleic acid binding, defense/immunity protein Cluster 02 & 13 10471721 Ptgs1 8.34 Oxygenase Cluster 02 & 13 10484318 Nckap1 3.54 Protein complex binding, contributes to Rac GTPase binding Cluster 02 & 13 10458382 Cd14 5.34 Receptor Cluster 02 & 13 10474671 Spred1 6.78 Receptor Cluster 02 & 13 10352905 Cd34 13.31 Receptor Cluster 02 & 13 10510546 Eno1/Gm5506 2.65 RNA binding, phosphopyruvate hydratase activity Cluster 02 & 13 10355403 Fn1 26.38 Signaling molecule Cluster 02 & 13 10551638 Lgals7 5.72 Signaling molecule, cell adhesion molecule Cluster 02 & 13 10391339 Psmc3ip 2.53 Signaling molecule, DNA-binding protein, enzyme modulator Cluster 02 & 13 10525983 Ran 2.30 Small GTPase Cluster 02 & 13 10523281 Sept11 2.74 Small GTPase, cytoskeletal protein Cluster 02 & 13 10469322 Vim/LOC100862060 2.20 Structural molecule activity Cluster 02 & 13 10564343 Tjp1 5.52 Tight junction Cluster 02 & 13 10385716 0610009B22Rik 2.56 Transcription regulator Cluster 02 & 13 10377790 Slc16a11 2.45 Transporter Cluster 02 & 14 10572932 Naa20 2.07 Acetyltransferase Cluster 02 & 14 10476775 Naa20 2.13 Acetyltransferase Cluster 02 & 14 10350136 Csrp1 2.25 Actin family cytoskeletal protein Cluster 02 & 14 10447317 Epas1 3.93 Basic helix-loop-helix transcription factor, nucleic acid binding Cluster 02 & 14 10592266 Slc37a2 3.78 Cation transporter Cluster 02 & 14 10531407 Cxcl9 4.24 Chemokine Cluster 02 & 14 10571696 Casp3 3.47 Cysteine protease, protease inhibitor Cluster 02 & 14 10393047 Galk1/Itgb4 2.66 Galactokinase activity Cluster 02 & 14 10531737 Hpse 9.61 Glycosidase Cluster 02 & 14 10403743 Inhba 2.66 Growth factor Cluster 02 & 14 10344713 Ahcy 2.14 Hydrolase Cluster 02 & 14 10488816 Ahcy 2.18 Hydrolase Cluster 02 & 14 10439762 Ahcy 2.26 Hydrolase Cluster 02 & 14 10521832 Pi4k2b 2.42 Kinase Cluster 02 & 14 10353899 Sema4c 2.19 Membrane-bound signaling molecule Cluster 02 & 14 10369431 Adamts14 2.73 Metalloprotease, extracellular matrix glycoprotein, serine protease inhibitor Cluster 02 & 14 10608652 10608652 2.08 Misc. Cluster 02 & 14 10458794 Ccdc112 2.12 Misc. Cluster 02 & 14 10425966 Atxn10 2.17 Misc. Cluster 02 & 14 10593913 1700017B05Rik 2.26 Misc. Cluster 02 & 14 10461439 Fads1 2.29 Misc. Cluster 02 & 14 10585610 Ptpn9/Gm10658 2.34 Misc. Cluster 02 & 14 10406733 Zbed3 2.35 Misc. Cluster 02 & 14 10488589 Fam110a 2.53 Misc. Cluster 02 & 14 10450519 Tcf19 2.67 Misc. Cluster 02 & 14 10479379 Slco4a1 2.68 Misc. Cluster 02 & 14 10467907 Erlin1 2.74 Misc. Cluster 02 & 14 10495935 4930422G04Rik (Zgrf1) 2.76 Misc. Cluster 02 & 14 10527920 Cyp51 2.78 Misc. Cluster 02 & 14 10506822 Orc1 2.78 Misc. Cluster 02 & 14 10441864 Mllt4 2.92 Misc. Cluster 02 & 14 10540738 Fancd2 3.00 Misc. Cluster 02 & 14 10361110 Dtl 3.05 Misc. Cluster 02 & 14 10459071 2010002N04Rik (Smim3) 3.14 Misc. Cluster 02 & 14 10550906 Plaur (CD87) 3.58 Misc. Cluster 02 & 14 10355567 Tmbim1 3.76 Misc. Cluster 02 & 14 10426689 Spats2 3.94 Misc. Cluster 02 & 14 10532669 2900026A02Rik 4.12 Misc. Cluster 02 & 14 10502816 Gipc2/D630002J18Rik 4.39 Misc. Cluster 02 & 14 10406581 Dhfr/Msh3 4.57 Misc. Cluster 02 & 14 10460738 Cdca5 4.99 Misc. Cluster 02 & 14 10530615 Ociad2 5.67 Misc. Cluster 02 & 14 10571312 Dusp4 6.55 Misc. Cluster 02 & 14 10411459 Tmem171 7.33 Misc. Cluster 02 & 14 10357579 Mapkapk2 2.42 Non-receptor serine/theronine protein kinase, non-motor microtubule binding protein Cluster 02 & 14 10535979 Rfc3 3.52 Nucleotidyltransferase, DNA-directed DNA polymerase Cluster 02 & 14 10419198 Ero1l 3.02 Oxidoreductase Cluster 02 & 14 10463355 Scd2 6.47 Palmitoyl-CoA 9-desaturase activity, stearoyl-CoA 9-desaturase activity Cluster 02 & 14 10452047 Ptprs 2.98 Protein phosphatase Cluster 02 & 14 10491805 Plk4 2.66 Protein serine/threonine kinase activity Cluster 02 & 14 10374464 Spred2 2.83 Receptor Cluster 02 & 14 10439527 Tigit 3.68 Receptor, defense/immnity protein Cluster 02 & 14 10403584 Nid1 2.50 Receptor, extracellular matrix glycoprotein Cluster 02 & 14 10358816 Lamc1 2.95 Receptor, extracellular matrix linker protein Cluster 02 & 14 10420302 Gzmc 54.76 serine protease Cluster 02 & 14 10469151 Itih5 9.86 Serine protease inhibitor Cluster 02 & 14 10379262 Nf1 2.45 Signaling molecule, G-protein modulator Cluster 02 & 14 10409660 Gkap1 2.74 Structural protein Cluster 02 & 14 10455813 Lmnb1 2.27 Structural protein, intermediate filament Cluster 02 & 14 10549222 Bcat1 6.18 Transaminase Cluster 02 & 14 10408321 Gmnn 2.46 Transcription corepressor activity Cluster 02 & 14 10576639 Nrp1 2.13 Transporter, apolipoprotein, membrane-bound signaling molecule, receptor, metalloprotease, serine protease, oxidase, metalloprotease, serine protease, extracellular matrix protein, enzyme modulator, cell adhesion molecule Cluster 02 & 14 10556216 Ipo7 2.09 Transporter, small GTPase Cluster 02 & 14 10519488 Tubb4b 2.09 Tubulin Supplemental Table 3. The molecular profile of TIL consists of 1164 differentially expressed genes between all tumor-specific CD8+ T cells from the tumor (TILTOT) and spleen (SPTOT) from Figure 2A. Differentially-expressed genes were narrowed as described in Figure 4 to determine the molecular profile of TASPR TIL. The gene list is ordered by K-means cluster (Figure 5A), gene type, and fold-change. Genes without a definitive gene type were labeled “Misc.” for miscellaneous.