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• W2550291749 abstract "•Transcriptome analysis performed on tumor-resident CD4+ Th1, Th17, and Treg cells•Tumor-infiltrating Treg cells are defined by the expression of signature genes•Treg-specific signature genes correlate with patients’ survival in both CRC and NSCLC Tumor-infiltrating regulatory T lymphocytes (Treg) can suppress effector T cells specific for tumor antigens. Deeper molecular definitions of tumor-infiltrating-lymphocytes could thus offer therapeutic opportunities. Transcriptomes of T helper 1 (Th1), Th17, and Treg cells infiltrating colorectal or non-small-cell lung cancers were compared to transcriptomes of the same subsets from normal tissues and validated at the single-cell level. We found that tumor-infiltrating Treg cells were highly suppressive, upregulated several immune-checkpoints, and expressed on the cell surfaces specific signature molecules such as interleukin-1 receptor 2 (IL1R2), programmed death (PD)-1 Ligand1, PD-1 Ligand2, and CCR8 chemokine, which were not previously described on Treg cells. Remarkably, high expression in whole-tumor samples of Treg cell signature genes, such as LAYN, MAGEH1, or CCR8, correlated with poor prognosis. Our findings provide insights into the molecular identity and functions of human tumor-infiltrating Treg cells and define potential targets for tumor immunotherapy. Tumor-infiltrating regulatory T lymphocytes (Treg) can suppress effector T cells specific for tumor antigens. Deeper molecular definitions of tumor-infiltrating-lymphocytes could thus offer therapeutic opportunities. Transcriptomes of T helper 1 (Th1), Th17, and Treg cells infiltrating colorectal or non-small-cell lung cancers were compared to transcriptomes of the same subsets from normal tissues and validated at the single-cell level. We found that tumor-infiltrating Treg cells were highly suppressive, upregulated several immune-checkpoints, and expressed on the cell surfaces specific signature molecules such as interleukin-1 receptor 2 (IL1R2), programmed death (PD)-1 Ligand1, PD-1 Ligand2, and CCR8 chemokine, which were not previously described on Treg cells. Remarkably, high expression in whole-tumor samples of Treg cell signature genes, such as LAYN, MAGEH1, or CCR8, correlated with poor prognosis. Our findings provide insights into the molecular identity and functions of human tumor-infiltrating Treg cells and define potential targets for tumor immunotherapy. The combination of genetic mutations and epigenetic modifications that are peculiar to all tumors generate antigens that T and B lymphocytes can use to specifically recognize tumor cells (Jamal-Hanjani et al., 2013Jamal-Hanjani M. Thanopoulou E. Peggs K.S. Quezada S.A. Swanton C. Tumour heterogeneity and immune-modulation.Curr. Opin. Pharmacol. 2013; 13: 497-503Crossref PubMed Scopus (28) Google Scholar). It is increasingly clear that T lymphocytes recognizing tumor-derived peptides presented by major histocompatibility complex (MHC) molecules play a central role in immunotherapy and in conventional chemo-radiotherapy of cancer (Galluzzi et al., 2015Galluzzi L. Buqué A. Kepp O. Zitvogel L. Kroemer G. Immunological Effects of Conventional Chemotherapy and Targeted Anticancer Agents.Cancer Cell. 2015; 28: 690-714Abstract Full Text Full Text PDF PubMed Scopus (970) Google Scholar). In fact, anti-tumor T cell responses arise in cancer patients but are disabled upon tumor progression by suppressive mechanisms triggered by the interplay between malignant cells and the tumor microenvironment (Munn and Bronte, 2016Munn D.H. Bronte V. Immune suppressive mechanisms in the tumor microenvironment.Curr. Opin. Immunol. 2016; 39: 1-6Crossref PubMed Scopus (321) Google Scholar). The tumor-dependent immunosuppressive mechanisms depend on the integrated action of infiltrating leukocytes and lymphocytes that upregulate a range of modulatory molecules, collectively called immune checkpoints, whose function is only partially characterized (Pardoll, 2012Pardoll D.M. The blockade of immune checkpoints in cancer immunotherapy.Nat. Rev. Cancer. 2012; 12: 252-264Crossref PubMed Scopus (8958) Google Scholar). Therefore, the search for agonists of co-stimulatory complexes or antagonists of inhibitory molecules to potentiate antigen-specific T cell responses is a primary goal of current anti-tumor research (Sharma and Allison, 2015Sharma P. Allison J.P. Immune checkpoint targeting in cancer therapy: toward combination strategies with curative potential.Cell. 2015; 161: 205-214Abstract Full Text Full Text PDF PubMed Scopus (1520) Google Scholar, Zitvogel et al., 2013Zitvogel L. Galluzzi L. Smyth M.J. Kroemer G. Mechanism of action of conventional and targeted anticancer therapies: reinstating immunosurveillance.Immunity. 2013; 39: 74-88Abstract Full Text Full Text PDF PubMed Scopus (647) Google Scholar). Indeed, clinical trials have unequivocally shown that the blockade of immune checkpoints unleashes the spontaneous anti-tumor immune responses in such a powerful way that it has created a paradigm shift in cancer therapy (Śledzińska et al., 2015Śledzińska A. Menger L. Bergerhoff K. Peggs K.S. Quezada S.A. Negative immune checkpoints on T lymphocytes and their relevance to cancer immunotherapy.Mol. Oncol. 2015; 9: 1936-1965Crossref PubMed Scopus (58) Google Scholar, Topalian et al., 2015Topalian S.L. Drake C.G. Pardoll D.M. Immune checkpoint blockade: a common denominator approach to cancer therapy.Cancer Cell. 2015; 27: 450-461Abstract Full Text Full Text PDF PubMed Scopus (2646) Google Scholar). Among the immune checkpoints targeted by blocking strategies, CTLA-4 has been one of the first to be translated into therapeutic applications. Anti-CTLA-4 monoclonal antibodies (mAb) show remarkable success in metastatic melanoma, and more recently in non-small-cell lung cancer, prostate cancer, renal cell carcinoma, urothelial carcinoma, and ovarian cancer (Carthon et al., 2010Carthon B.C. Wolchok J.D. Yuan J. Kamat A. Ng Tang D.S. Sun J. Ku G. Troncoso P. Logothetis C.J. Allison J.P. Sharma P. Preoperative CTLA-4 blockade: tolerability and immune monitoring in the setting of a presurgical clinical trial.Clin. Cancer Res. 2010; 16: 2861-2871Crossref PubMed Scopus (357) Google Scholar, Hodi et al., 2010Hodi F.S. O’Day S.J. McDermott D.F. Weber R.W. Sosman J.A. Haanen J.B. Gonzalez R. Robert C. Schadendorf D. Hassel J.C. et al.Improved survival with ipilimumab in patients with metastatic melanoma.N. Engl. J. Med. 2010; 363: 711-723Crossref PubMed Scopus (11264) Google Scholar, van den Eertwegh et al., 2012van den Eertwegh A.J. Versluis J. van den Berg H.P. Santegoets S.J. van Moorselaar R.J. van der Sluis T.M. Gall H.E. Harding T.C. Jooss K. Lowy I. et al.Combined immunotherapy with granulocyte-macrophage colony-stimulating factor-transduced allogeneic prostate cancer cells and ipilimumab in patients with metastatic castration-resistant prostate cancer: a phase 1 dose-escalation trial.Lancet Oncol. 2012; 13: 509-517Abstract Full Text Full Text PDF PubMed Scopus (302) Google Scholar, Yang et al., 2007Yang J.C. Hughes M. Kammula U. Royal R. Sherry R.M. Topalian S.L. Suri K.B. Levy C. Allen T. Mavroukakis S. et al.Ipilimumab (anti-CTLA4 antibody) causes regression of metastatic renal cell cancer associated with enteritis and hypophysitis.J. Immunother. 2007; 30: 825-830Crossref PubMed Scopus (590) Google Scholar). However, the fraction of patients that do not respond remains high, prompting a deeper investigation of the mechanisms underpinning the modulation of immune responses by tumors. Recent experimental evidence shows that anti-CTLA-4 mAb efficacy depends on FcγR-mediated depletion of CD4+ regulatory T cells (Treg cells) within the tumor microenvironment (Peggs et al., 2009Peggs K.S. Quezada S.A. Chambers C.A. Korman A.J. Allison J.P. Blockade of CTLA-4 on both effector and regulatory T cell compartments contributes to the antitumor activity of anti-CTLA-4 antibodies.J. Exp. Med. 2009; 206: 1717-1725Crossref PubMed Scopus (699) Google Scholar, Selby et al., 2013Selby M.J. Engelhardt J.J. Quigley M. Henning K.A. Chen T. Srinivasan M. Korman A.J. Anti-CTLA-4 antibodies of IgG2a isotype enhance antitumor activity through reduction of intratumoral regulatory T cells.Cancer Immunol. Res. 2013; 1: 32-42Crossref PubMed Scopus (604) Google Scholar, Simpson et al., 2013Simpson T.R. Li F. Montalvo-Ortiz W. Sepulveda M.A. Bergerhoff K. Arce F. Roddie C. Henry J.Y. Yagita H. Wolchok J.D. et al.Fc-dependent depletion of tumor-infiltrating regulatory T cells co-defines the efficacy of anti-CTLA-4 therapy against melanoma.J. Exp. Med. 2013; 210: 1695-1710Crossref PubMed Scopus (998) Google Scholar, Twyman-Saint Victor et al., 2015Twyman-Saint Victor C. Rech A.J. Maity A. Rengan R. Pauken K.E. Stelekati E. Benci J.L. Xu B. Dada H. Odorizzi P.M. et al.Radiation and dual checkpoint blockade activate non-redundant immune mechanisms in cancer.Nature. 2015; 520: 373-377Crossref PubMed Scopus (1637) Google Scholar). Treg cells, which are physiologically engaged in the maintenance of immunological self-tolerance and immune homeostasis (Josefowicz et al., 2012Josefowicz S.Z. Lu L.F. Rudensky A.Y. Regulatory T cells: mechanisms of differentiation and function.Annu. Rev. Immunol. 2012; 30: 531-564Crossref PubMed Scopus (1970) Google Scholar, Sakaguchi et al., 2008Sakaguchi S. Yamaguchi T. Nomura T. Ono M. Regulatory T cells and immune tolerance.Cell. 2008; 133: 775-787Abstract Full Text Full Text PDF PubMed Scopus (3696) Google Scholar), are potent suppressors of effector cells and are found at high frequencies in various types of cancers (Fridman et al., 2012Fridman W.H. Pagès F. Sautès-Fridman C. Galon J. The immune contexture in human tumours: impact on clinical outcome.Nat. Rev. Cancer. 2012; 12: 298-306Crossref PubMed Scopus (3141) Google Scholar, Nishikawa and Sakaguchi, 2010Nishikawa H. Sakaguchi S. Regulatory T cells in tumor immunity.Int. J. Cancer. 2010; 127: 759-767PubMed Google Scholar). Treg cells adapt their transcriptional program to the various cytokines to which they are exposed in the inflammatory milieu (Campbell and Koch, 2011Campbell D.J. Koch M.A. Phenotypical and functional specialization of FOXP3+ regulatory T cells.Nat. Rev. Immunol. 2011; 11: 119-130Crossref PubMed Scopus (608) Google Scholar). This versatility is controlled by transcription factors generally associated with the differentiation of other effector CD4+ T cell subsets, resulting in various Treg cell populations with unique features and immunomodulatory functions (Duhen et al., 2012Duhen T. Duhen R. Lanzavecchia A. Sallusto F. Campbell D.J. Functionally distinct subsets of human FOXP3+ Treg cells that phenotypically mirror effector Th cells.Blood. 2012; 119: 4430-4440Crossref PubMed Scopus (283) Google Scholar, Geginat et al., 2014Geginat J. Paroni M. Maglie S. Alfen J.S. Kastirr I. Gruarin P. De Simone M. Pagani M. Abrignani S. Plasticity of human CD4 T cell subsets.Front. Immunol. 2014; 5: 630Crossref PubMed Scopus (183) Google Scholar). Moreover, Treg cells infiltrating non-lymphoid tissues are reported to exhibit unique phenotypes and transcriptional signatures, because they can display functions beyond their well-established suppressive roles, such as metabolic modulation in adipose tissue (Cipolletta et al., 2012Cipolletta D. Feuerer M. Li A. Kamei N. Lee J. Shoelson S.E. Benoist C. Mathis D. PPAR-γ is a major driver of the accumulation and phenotype of adipose tissue Treg cells.Nature. 2012; 486: 549-553Crossref PubMed Scopus (813) Google Scholar) or regulation of tissue repair in skeletal muscle (Burzyn et al., 2013Burzyn D. Kuswanto W. Kolodin D. Shadrach J.L. Cerletti M. Jang Y. Sefik E. Tan T.G. Wagers A.J. Benoist C. Mathis D. A special population of regulatory T cells potentiates muscle repair.Cell. 2013; 155: 1282-1295Abstract Full Text Full Text PDF PubMed Scopus (746) Google Scholar) and in lung tissue (Arpaia et al., 2015Arpaia N. Green J.A. Moltedo B. Arvey A. Hemmers S. Yuan S. Treuting P.M. Rudensky A.Y. A Distinct Function of Regulatory T Cells in Tissue Protection.Cell. 2015; 162: 1078-1089Abstract Full Text Full Text PDF PubMed Scopus (575) Google Scholar). Treg cell depletion has been reported to increase anti-tumor specific immune responses and to reduce tumor burden (Marabelle et al., 2013Marabelle A. Kohrt H. Sagiv-Barfi I. Ajami B. Axtell R.C. Zhou G. Rajapaksa R. Green M.R. Torchia J. Brody J. et al.Depleting tumor-specific Tregs at a single site eradicates disseminated tumors.J. Clin. Invest. 2013; 123: 2447-2463Crossref PubMed Scopus (295) Google Scholar, Teng et al., 2010Teng M.W. Ngiow S.F. von Scheidt B. McLaughlin N. Sparwasser T. Smyth M.J. Conditional regulatory T-cell depletion releases adaptive immunity preventing carcinogenesis and suppressing established tumor growth.Cancer Res. 2010; 70: 7800-7809Crossref PubMed Scopus (152) Google Scholar, Walter et al., 2012Walter S. Weinschenk T. Stenzl A. Zdrojowy R. Pluzanska A. Szczylik C. Staehler M. Brugger W. Dietrich P.Y. Mendrzyk R. et al.Multipeptide immune response to cancer vaccine IMA901 after single-dose cyclophosphamide associates with longer patient survival.Nat. Med. 2012; 18: 1254-1261Crossref PubMed Scopus (650) Google Scholar). Although promising clinical results have been achieved with Treg cell depleting strategies, some relevant issues are to be addressed, for a safer, more effective, and wider clinical application of these therapies. First, severe autoimmunity can occur following systemic Treg cells depletion (Nishikawa and Sakaguchi, 2010Nishikawa H. Sakaguchi S. Regulatory T cells in tumor immunity.Int. J. Cancer. 2010; 127: 759-767PubMed Google Scholar), which could be avoided if selective depletion of tumor infiltrating Treg cells were feasible. A second issue concerns the specificity of targeting. Indeed, Treg cells share with effector lymphocytes most of the molecules targeted for therapy, which can possibly deplete also the tumor-specific effector cells. Therefore, the molecular characterization of Treg cells at different tumor sites should help to better define therapeutic targets through a better description of their signature molecules and of the network that regulates Treg cell functions in the tumor microenvironment. Non-small-cell lung cancer (NSCLC) and colorectal cancer (CRC) are the two most frequent cancers in both genders (Torre et al., 2015Torre L.A. Bray F. Siegel R.L. Ferlay J. Lortet-Tieulent J. Jemal A. Global cancer statistics, 2012.CA Cancer J. Clin. 2015; 65: 87-108Crossref PubMed Scopus (23667) Google Scholar). NSCLC has the worst prognosis due to its high mortality rate even in early stages. Although CRC survival rate is highly dependent on the tumor stage at diagnosis, about 50% of patients will progress to metastatic cancer (Gonzalez-Pons and Cruz-Correa, 2015Gonzalez-Pons M. Cruz-Correa M. Colorectal Cancer Biomarkers: Where Are We Now?.BioMed Res. Int. 2015; 2015: 149014Crossref PubMed Scopus (127) Google Scholar). Both tumors have been targeted with therapies based on monoclonal antibodies to checkpoint inhibitors, but the outcomes have been different. While remarkable clinical success has been obtained in NSCLC, evidence of durable response in CRC is scarce with the exception of mismatch repair-deficient CRC lesions (Jacobs et al., 2015Jacobs J. Smits E. Lardon F. Pauwels P. Deschoolmeester V. Immune Checkpoint Modulation in Colorectal Cancer: What’s New and What to Expect.J. Immunol. Res. 2015; 2015: 158038Crossref PubMed Scopus (45) Google Scholar, Kroemer et al., 2015Kroemer G. Galluzzi L. Zitvogel L. Fridman W.H. Colorectal cancer: the first neoplasia found to be under immunosurveillance and the last one to respond to immunotherapy?.OncoImmunology. 2015; 4: e1058597Crossref PubMed Scopus (56) Google Scholar, Le et al., 2015Le D.T. Uram J.N. Wang H. Bartlett B.R. Kemberling H. Eyring A.D. Skora A.D. Luber B.S. Azad N.S. Laheru D. et al.PD-1 Blockade in Tumors with Mismatch-Repair Deficiency.N. Engl. J. Med. 2015; 372: 2509-2520Crossref PubMed Scopus (6169) Google Scholar). Here we provide a comprehensive transcriptome analysis of human CD4+ Treg cells and effector cells (Th1 and Th17) infiltrating NSCLC or CRC and their matched normal tissues. We defined molecular signatures of tumor-infiltrating Treg cells in these two cancer types and confirmed the relevance of these signatures by single-cell analyses. These data could help a better understanding of Treg functional role at tumor sites and pave the way to the identification of therapeutic targets for more specific and safer modulation of Treg cells in cancer therapy. To assess the gene expression landscape of tumor infiltrating CD4+ T cells, we isolated different CD4+ lymphocytes subsets from two different tumors, NSCLC and CRC, from the adjacent normal tissues, and from peripheral blood samples. From all these tissues, we purified by flow cytometry (Figure 1A and S1A and S1B) CD4+ Treg (36 samples from 18 individuals), Th1 (30 samples from 21 individuals), and Th17 (22 samples from 14 individuals) cells (Table 1 and Table S1). To assess Treg cell function, we tested their suppressor activity and showed that Treg cells infiltrating either type of tumor tissues have a remarkably stronger suppressive activity in vitro compared to Treg cells isolated from the adjacent normal tissue and peripheral blood of the same patients (Figure 1B).Table 1Purification and RNA-Sequencing of Human Primary Lymphocyte SubsetsTissueSubsetSorting PhenotypeNumber of SamplesMapped Reads (M)NSCLCCD4+ TregCD4+ CD127− CD25+8587CD4+ Th1CD4+ CXCR3+ CCR6−8409CD4+ Th17CD4+ CCR6+ CXCR3−6206CRCCD4+ TregCD4+ CD127− CD25+7488CD4+ Th1CD4+ CXCR3+ CCR6−5266CD4+ Th17CD4+ CCR6+ CXCR3−5308Lung (normal tissue)CD4+ TregCD4+ CD127− CD25+1 (pool of 6)73CD4+ Th1CD4+ CXCR3+ CCR6−1 (pool of 6)76Colon (normal tissue)CD4+ TregCD4+ CD127− CD25+7404CD4+ Th1CD4+ CXCR3+ CCR6−6352CD4+ Th17CD4+ CCR6+ CXCR3−6284PB (healthy donor)CD4+ TregCD4+ CD127− CD25+8259CD4+ Th1CD4+ CXCR3+ CCR6−570CD4+ Th17CD4+ CCR6+ CXCR3−577For each cell subsets profiled by RNA-sequencing tissue of origin, surface marker combinations used for sorting, number of profiled samples, as well as number of mapped sequencing reads are indicated. M, million; CRC, colorectal cancer; NSCLC, non-small cell lung cancer; PB, peripheral blood.See also Table S1. Open table in a new tab For each cell subsets profiled by RNA-sequencing tissue of origin, surface marker combinations used for sorting, number of profiled samples, as well as number of mapped sequencing reads are indicated. M, million; CRC, colorectal cancer; NSCLC, non-small cell lung cancer; PB, peripheral blood. See also Table S1. The polyadenylated RNA fraction extracted from the sorted CD4+ Treg, Th1, and Th17 cells was then analyzed by paired-end RNA sequencing obtaining about 4 billion mapped “reads” (Table 1). First, we interrogated RNA-sequencing data of CD4+ T cells infiltrating both CRC and NSCLC and their matched normal tissues, to quantitate mRNA expression of known immune checkpoints and their ligands. Second, we analyzed RNA-seq data of CRC and NSCLC, as well as of normal colon and lung samples. We found that several immune checkpoints and their ligands transcripts were strikingly upregulated in tumor infiltrating Treg cells compared to both normal tissue and peripheral blood-derived Treg cells, as well as to T and B lymphocyte subsets purified from peripheral blood mononuclear cells (PBMCs) (Figures 1C and S1C and Table S5). Our findings highlight the specific expression patterns of immune checkpoints and their ligands in tumor infiltrating Treg and effector cells and suggest that their functional relevance should be investigated directly at tumor sites. We then asked whether tumor infiltrating Treg cells could be defined by specific gene-expression patterns. First, in order to capture the overall similarity between the tumor infiltrating lymphocytes, we performed a principal components analysis (PCA) on the whole transcriptomes. Tumor-infiltrating Treg cells purified from CRC and NSCLC tissues clustered together and were clearly separated from Th1 and Th17 cells purified from CRC and NSCLC tissues (Figures S2A and S2B). PCA showed a distinct grouping of Treg cells purified from different sites; in fact, separation along the first principal component (PC1) clearly divided peripheral blood Treg cells from tissue infiltrating Treg cells (Figure 2A), whereas normal-tissue and tumor-tissue infiltrating Treg cells are mostly divided by the second component (PC2). These findings indicate that tumor-infiltrating Treg cells have specific expression patterns compared not only to other CD4+ T cell subsets but also compared to Treg cells isolated from normal tissues. In order to identify genes that are preferentially expressed in tumor-infiltrating lymphocytes, we performed self-organizing maps (SOM) analyses that provide a powerful way to define coordinated gene-expression patterns that are visualized in spatial proximity in a 2D mosaic grid heatmap (Wirth et al., 2012Wirth H. von Bergen M. Binder H. Mining SOM expression portraits: feature selection and integrating concepts of molecular function.BioData Min. 2012; 5: 18Crossref PubMed Scopus (45) Google Scholar). In this way, we analyzed 7,763 genes that were differentially expressed between the different CD4+ T cell subsets purified from PBMCs and tumor tissues (DESeq2 package; FDR < 0.05). Among the different CD4+ T cell subsets (Th1, Th17, and Treg) assessed with SOM, only the tumor-infiltrating Treg cells displayed peculiar gene-expression patterns that were similar between NSCLC and CRC samples (Figures 2B and S2C), thus allowing the identification (FDR < 0.1) of transcripts upregulated in both CRC and NSCLC infiltrating Treg cells (Figure 2C and Table S2). Gene-ontology (GO) analyses of those genes upregulated in tumor infiltrating Treg cells showed significant enrichment for terms related to lymphocytes activation (Figure 2C and Table S3). To identify signature transcripts of tumor-infiltrating Treg cells, we included in the expression pattern analyses the transcriptome datasets we previously obtained from different T and B lymphocyte subsets purified from PBMCs (Ranzani et al., 2015Ranzani V. Rossetti G. Panzeri I. Arrigoni A. Bonnal R.J. Curti S. Gruarin P. Provasi E. Sugliano E. Marconi M. et al.The long intergenic noncoding RNA landscape of human lymphocytes highlights the regulation of T cell differentiation by linc-MAF-4.Nat. Immunol. 2015; 16: 318-325Crossref PubMed Scopus (246) Google Scholar). In so doing, we obtained a signature of 309 transcripts whose expression is higher in tumor infiltrating Treg cells (Wilcoxon Mann Whitney test p < 2.2 × 10–16) (Figures 2D and S2D and Table S4) compared to the other lymphocyte subsets purified from non-tumoral tissues and from PBMCs of healthy or neoplastic patients. Altogether, the data show that Treg cells display the most pronounced differences in transcripts expression among CD4+ T cell subsets infiltrating normal and tumor tissues. We defined a subset of signature genes that describe the specific gene-expression profile of tumor infiltrating Treg cells. We then look at the single cell level for the differential expression profile of signature genes of tumor infiltrating Treg cells. We isolated CD4+ T cells from 5 CRC and 5 NSCLC tumor samples, as well as from 5 PBMCs of healthy individuals (Table S1), purified Treg cells, and using an automated microfluidic system (C1 Fluidigm) captured single cells (a total of 858 Treg cells: 320 from CRC and 286 from NSCLC; 252 from PBMCs of healthy individuals). We then assessed by high throughput RT-qPCR (Biomark HD, Fluidigm) the expression of 79 genes selected among the highly expressed (> 10 FKPM) tumor Treg cell signature genes (Figures 3A, S3A and S3B). Notably, we found that the vast majority (75 over 79; 95%) of the tumor-infiltrating Treg cell signatures were co-expressed with bona fide Treg cell markers (i.e., FOXP3+ and IL2RA) (Figure 3B). The percentage of co-expression between these Treg cell markers and the 79 genes selected among the tumor-infiltrating-Treg-cell signature genes ranged between 81% of TIGIT and 0.59% of CGA (Figure 3B). The expression of Treg signature genes in the RNA-seq of the whole Treg cell population correlated with the percentage of single cells expressing the different genes (Figure 3C). In order to reduce the “drop-out” effect of the single cell data (i.e., events in which a transcript is detected in one cell but not in another one because the transcript is “missed” during the reverse-transcription step) (Kharchenko et al., 2014Kharchenko P.V. Silberstein L. Scadden D.T. Bayesian approach to single-cell differential expression analysis.Nat. Methods. 2014; 11: 740-742Crossref PubMed Scopus (695) Google Scholar), we defined a threshold (median value t = 8.4%) based on the expression distribution for each transcript and discarded genes below this threshold (see the Supplemental Experimental Procedures). The forty-five signature transcripts of tumor infiltrating Treg cells detected above this threshold were in most cases significantly overexpressed in Treg cells from both tumors (39 over 45, 87%; Wilcoxon Mann Whitney test p < 0.05) or in one tumor type (43 over 45, 96%; Figure 3D). Homogeneity of the purified tissue infiltrating Treg cells can be affected by the carry-over of cells from other lymphocyte subsets. To quantitate this possible contamination, the single cell RT-qPCR analyses of Treg cells was performed including markers specific for other lymphocytes subsets (i.e., Th1, Th2, Th17, Tfh, CD8 T cells, B cells) (Figure S3C). Our data showed that only a very low fraction of the purified single cells displayed markers of lymphocytes subsets different from Treg cells (Figure S3C). The overlap between the signature genes in the CRC and NSCLC infiltrating Treg cells (Figure 2D) prompted us to assess whether this signature were also enriched in Treg cells infiltrating other tumors. RNA was thus extracted from Treg cells infiltrating breast cancer, gastric cancer, brain metastasis of NSCLC, and liver metastasis of CRC. We found by RT-qPCR that tumor infiltrating Treg signatures genes were mostly upregulated also in these tumors (Figure 3E). Overall these data show that the tumor-infiltrating Treg cell signature genes are co-expressed at single cell level with FOXP3 and IL2RA and that several primary and metastatic human tumors express the tumor-infiltrating Treg cell signature. We then assessed at the single cell level by flow cytometry the protein expression of ten representative signature genes present in CRC and NSCLC infiltrating Treg cells, adjacent normal tissues, and patients PBMCs. Of the ten proteins, two were proteins (OX40 and TIGIT) whose relevance for Treg cells biology has been demonstrated (Joller et al., 2014Joller N. Lozano E. Burkett P.R. Patel B. Xiao S. Zhu C. Xia J. Tan T.G. Sefik E. Yajnik V. et al.Treg cells expressing the coinhibitory molecule TIGIT selectively inhibit proinflammatory Th1 and Th17 cell responses.Immunity. 2014; 40: 569-581Abstract Full Text Full Text PDF PubMed Scopus (515) Google Scholar, Voo et al., 2013Voo K.S. Bover L. Harline M.L. Vien L.T. Facchinetti V. Arima K. Kwak L.W. Liu Y.J. Antibodies targeting human OX40 expand effector T cells and block inducible and natural regulatory T cell function.J. Immunol. 2013; 191: 3641-3650Crossref PubMed Scopus (73) Google Scholar), seven are proteins (BATF, CCR8, CD30, IL-1R2, IL-21R, PDL-1, and PDL-2) whose expression has never been described in tumor-infiltrating Treg cells, and one protein, 4-1BB, is a co-stimulatory receptor expressed on several hematopoietic cells, whose expression on Treg cells has been shown to mark antigen-activated cells (Schoenbrunn et al., 2012Schoenbrunn A. Frentsch M. Kohler S. Keye J. Dooms H. Moewes B. Dong J. Loddenkemper C. Sieper J. Wu P. et al.A converse 4-1BB and CD40 ligand expression pattern delineates activated regulatory T cells (Treg) and conventional T cells enabling direct isolation of alloantigen-reactive natural Foxp3+ Treg.J. Immunol. 2012; 189: 5985-5994Crossref PubMed Scopus (88) Google Scholar). Our findings showed that all these proteins were upregulated (Figure 4A), to different extent, in tumor infiltrating Treg cells compared to the Treg cells resident in normal tissues. Given the increasing interest in the PD1 - PDLs axis as targets for tumor immunotherapy, we assessed the effect of antibodies against PDL-1 and PDL-2 on the suppressive function of tumor-infiltrating Treg cells toward effector CD4+ T cell proliferation in vitro. We found that preincubation of tumor infiltrating Treg cells with monoclonal antibodies against PDL-1 or PDL-2 reduced their suppressive activity as demonstrated by the increased proliferation of effector CD4+ T cells (Figure 4B). Altogether, our data show there is a molecular signature of tumor infiltrating Treg cells, which can be detected both at the mRNA and at the protein levels. In an attempt to correlate our findings with clinical outcome, we asked whether the expression of the tumor-Treg signature transcripts correlated with disease prognosis in CRC and NSCLC patients. We therefore interrogated for expression of Treg signature genes transcriptomic datasets obtained from resected tumor tissues of a cohort of 177 CRC patients (GSE17536; Smith et al., 2010Smith J.J. Deane N.G. Wu F. Merchant N.B. Zhang B. Jiang A. Lu P. Johnson J.C. Schmidt C. Bailey C.E. et al.Experimentally derived metastasis gene expression profile predicts recurrence and death in patients with colon cancer.Gastroenterology. 2010; 138: 958-968Abstract Full Text Full Text PDF PubMed Scopus (490) Google Scholar) and of a cohort of 263 NSCLC patients (GSE41271; Sato et al., 2013Sato" @default.
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• W2550291749 date "2016-11-01" @default.
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• W2550291749 title "Transcriptional Landscape of Human Tissue Lymphocytes Unveils Uniqueness of Tumor-Infiltrating T Regulatory Cells" @default.
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• W2550291749 doi "https://doi.org/10.1016/j.immuni.2016.10.021" @default.
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