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.

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.

Non-small-cell lung cancer (NSCLC) and colorectal cancer (CRC) are the two most frequent cancers in both genders (). 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 (). 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 ().

Colorectal cancer: the first neoplasia found to be under immunosurveillance and the last one to respond to immunotherapy?.

Treg cell depletion has been reported to increase anti-tumor specific immune responses and to reduce tumor burden (). 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 (), 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.

Treg cells, which are physiologically engaged in the maintenance of immunological self-tolerance and immune homeostasis (), are potent suppressors of effector cells and are found at high frequencies in various types of cancers (). Treg cells adapt their transcriptional program to the various cytokines to which they are exposed in the inflammatory milieu (). This versatility is controlled by transcription factors generally associated with the differentiation of other effector CD4T cell subsets, resulting in various Treg cell populations with unique features and immunomodulatory functions (). 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 () or regulation of tissue repair in skeletal muscle () and in lung tissue ().

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 (). 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 CD4regulatory T cells (Treg cells) within the tumor microenvironment ().

Among the immune checkpoints targeted by blocking strategies, CTLA-4 has been one of the first to be translated into therapeutic applications.

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 (). 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 (). 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 (). 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 (). 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 (). 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 ().

In conclusion, high expression in the whole-tumor samples of three genes (LAYN, MAGEH1, and CCR8) that are specifically and highly expressed in tumor infiltrating Treg cells correlates with a poor prognosis in both NSCLC and CRC patients.

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;) and of a cohort of 263 NSCLC patients (GSE41271;) and correlated high and low gene expression with the 5-year survival data. Among those genes whose expression is highly enriched in tumor-infiltrating Treg cells, we selected LAYN, MAGEH1, and CCR8 that are the three genes more selectively expressed ( Figure S5 A). To normalize for differences in T cell densities within the resected tumor tissues, we used the ratio between expression of the selected signature genes and CD3G. We found that high expression of the three signature genes is in all cases correlated with a significantly reduced survival ( Figure 5 A). We also observed that expressions of the three signature genes increased with tumor staging of CRC patients ( Figure 5 B).

(B) Expression distributions of CCR8, MAGEH1, and LAYN according to tumor staging at the time of surgery in the cohort of CRC patients.

(A) Kaplan-Meier survival curve comparing the high and low expression of the tumor Treg signature transcripts (CCR8, MAGEH1, LAYN) normalized to the CD3G for the CRC (n = 177) and NSCLC (n = 263) studies. Univariate analysis confirmed a significant difference in overall survival curve comparing patients with high and low expression. Statistical significance was determined by the log-rank test. (CRC: p = 0.05 for CCR8, p = 1.48 × 10 −3 for MAGEH1, p = 2.1 × 10 −4 for LAYN; NSCLC: p = 0.0125 for CCR8, p = 0.035 for MAGEH1, p = 0.0131 for LAYN.) Each table depicts the Kaplan-Meier estimates at the specified time points.

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.

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 (), 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 (). Our findings showed that all these proteins were upregulated ( Figure 4 A), 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 CD4T 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 CD4T cells ( Figure 4 B).

(B) Flow cytometry plots representative of four independent experiments showing suppressive activity of CRC infiltrating Treg cells on proliferation (shown as CFSE dilution) of CD4 + effector T cells. First panel shows the inhibitory effect of Treg cells on the effector T cell proliferation in the presence of an isotype control antibody. The other panels show the inhibitory effect of Treg cells that have been preincubated with anti PD-L1 or PD-L2 antibodies. Percentage of proliferating cells are indicated. The calculated division index is 0.26 in the presence of the control antibody; 0.57 in the presence of anti-PDL-1 and 0.39 in the presence of anti-PDL-2. Data are representative of four independent experiments.

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.

The overlap between the signature genes in the CRC and NSCLC infiltrating Treg cells ( Figure 2 D) 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 3 E).

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., FOXP3and IL2RA) ( Figure 3 B). 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 3 B). 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 3 C). 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) (), 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 3 D). 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 S3 C). Our data showed that only a very low fraction of the purified single cells displayed markers of lymphocytes subsets different from Treg cells ( Figure S3 C).

We then look at the single cell level for the differential expression profile of signature genes of tumor infiltrating Treg cells. We isolated CD4T 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 3 A, S3 A and S3B).

(D) Expression distribution (violin plots) in Treg cells infiltrating CRC, NSCLC, or PB. Plots representing the ontology classes of receptors, signaling and enzymatic activity, cytokine activity, and transcription factors are shown (Wilcoxon Mann Whitney test p < 0.05). Color gradient indicates the percentage of cells expressing each gene in Treg cells isolated from the three tissues.

(A) Schematic representation of the experimental workflow. Experiments were performed on Treg cells infiltrating CRC, NSCLC, or isolated from peripheral blood of healthy donors (PB); five samples were collected for each tissue.

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.

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 (). 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 2 D and S2 D and Table S4 ) compared to the other lymphocyte subsets purified from non-tumoral tissues and from PBMCs of healthy or neoplastic patients.

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 (). In this way, we analyzed 7,763 genes that were differentially expressed between the different CD4T cell subsets purified from PBMCs and tumor tissues (DESeq2 package; FDR < 0.05). Among the different CD4T 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 2 B and S2 C), thus allowing the identification (FDR < 0.1) of transcripts upregulated in both CRC and NSCLC infiltrating Treg cells ( Figure 2 C 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 2 C and Table S3 ).

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 S2 A 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 2 A), 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 CD4T cell subsets but also compared to Treg cells isolated from normal tissues.

(D) Z-score normalized expression values of genes that are preferentially expressed in tumor-infiltrating Treg cells (Wilcoxon Mann Whitney test p < 2.2 × 10–16) over the listed cell subsets are represented as boxed plots. Colon tissues are indicated as C, lung tissues as L, and peripheral blood as B.

(C) Group-centered analysis for the identification of upregulated spot (FDR < 0.1) in Treg cells infiltrating both NSCLC and CRC is described as 2D heatmap. Heatmap representing Z-score normalized expression values of genes selected from the upregulated spot is shown on the right side of the figure. Top enriched GO term (DAVID) for genes assigned to upregulated spot is reported with the corresponding significance p value. Colon tissues are indicated as C, lung tissues as L, and peripheral blood as B.

The polyadenylated RNA fraction extracted from the sorted CD4Treg, 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 CD4T 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 1 C and S1 C 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.

To assess the gene expression landscape of tumor infiltrating CD4T cells, we isolated different CD4lymphocytes 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 1 A and S1 A and S1B) CD4Treg (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 1 B).

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.

(C) Z-score normalized RNA-seq expression values of immune checkpoints genes are represented as a heatmap. Cell populations are reported as a color code in the upper part of the graph, while gene names have been assigned to heatmap rows. Hierarchical clustering results are shown as a dendrogram drawn on the left side of the matrix. Colon tissues are indicated as C, lung tissues as L, and peripheral blood as B.

(B) Representative flow cytometry plots showing suppressive activity of Treg cells isolated from tumor (NSCLC or CRC), normal tissue and blood of the same patient. 4 × 10 5 carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeled CD4 + naive T cells from healthy donors were cocultured with an equal number of Treg cells for 4 days with a CD3-specific mAb and CD1c + CD11c + dendritic cells. Percentage of proliferating cells is indicated. Data are representative of three independent experiments.

Discussion

Panduro et al., 2016 Panduro M.

Benoist C.

Mathis D. Tissue Tregs. Diversity of tumor-infiltrating Treg cells should be fully elucidated to understand their functional relevance and prognostic significance in different types of cancer and to possibly improve the therapeutic efficacy of Treg cell modulation through the selective depletion of tumor infiltrating Treg cells. The transcriptome analysis we performed on CRC- and NSCLC-infiltrating T cells showed that tumor-infiltrating Treg cells are different from both circulating and normal tissue-infiltrating Tregs, suggesting that the tumor microenvironment influences specific gene expression in Treg cells. Our findings further support the view that Treg cells from different tissues are instructed by environmental factors to display different gene-expression profiles (). Indeed the list of signature genes includes a number of molecules that are consistently upregulated in tumor-infiltrating Treg cells isolated from different tumor types, and these signature genes would have not been identified if we had not profiled specifically tumor infiltrating Treg cells.

The number of genes highly expressed in tumor infiltrating cells, as defined by differential expression and SOM analyses, was significantly higher in Treg than in Th17 and Th1 cells, suggesting that Treg cells are more susceptible than other T cell subsets to external cues they are exposed to in tumor tissues. We found that tumor-infiltrating-Treg signature genes are not only largely shared between CRC- and NSCLC-infiltrating cells but are also conserved in breast and gastric cancers, as well as in CRC and NSCLC metastatic tumors (in liver and brain, respectively) suggesting that expression of these genes is a common feature of tumor infiltrating Treg cells that might correlate with Treg cell-specific function within the tumor microenvironment.

Although our knowledge on the function of immune checkpoints on lymphocytes is still incomplete, agonist or antagonist monoclonal antibodies targeting checkpoints are in clinical development. We have found that some of these checkpoints (such as GITR, OX40, TIGIT, LAG-3, and TIM-3) and some of their ligands (such as OX40LG, Galectin-9, CD70) are upregulated also in tumor-infiltrating Treg cells, and this fact should be taken into account in interpreting clinical results with checkpoint inhibitors. Indeed, it is likely that assessment of the expression of checkpoints and of their ligands on the various subsets of tumor infiltrating lymphocytes will help to elucidate conflicting results and provide the rationale for combination therapies. Therefore, expression pattern of checkpoints should be evaluated both in tumor-infiltrating lymphocytes and in tumor cells.

Boussiotis et al., 2014 Boussiotis V.A.

Chatterjee P.

Li L. Biochemical signaling of PD-1 on T cells and its functional implications. Lesterhuis et al., 2011 Lesterhuis W.J.

Steer H.

Lake R.A. PD-L2 is predominantly expressed by Th2 cells. Messal et al., 2011 Messal N.

Serriari N.E.

Pastor S.

Nunès J.A.

Olive D. PD-L2 is expressed on activated human T cells and regulates their function. + T cells differentiation ( Murphy et al., 2013 Murphy T.L.

Tussiwand R.

Murphy K.M. Specificity through cooperation: BATF-IRF interactions control immune-regulatory networks. + T cells is induced by IL-21 ( Xin et al., 2015 Xin G.

Schauder D.M.

Lainez B.

Weinstein J.S.

Dai Z.

Chen Y.

Esplugues E.

Wen R.

Wang D.

Parish I.A.

et al. A Critical Role of IL-21-Induced BATF in Sustaining CD8-T-Cell-Mediated Chronic Viral Control. Single-cell analysis on selected tumor Treg signature genes confirmed the whole transcriptomic data and provided information on the expression frequency of these genes. Tumor-infiltrating Treg cells express with high frequency genes that are associated with increased suppressor activity, such as the well characterized OX40, CTLA4, and GITR. Moreover, there were a number of interesting and less expected genes the specific expression of which was validated also at the protein level. For example, IL-1R2 upregulation could be another mechanism that tumor resident Treg cells employ to dampen anti-tumor immune responses through the neutralization of IL-1β function on effector cells. PD-L1 and PD-L2 expression has been recently reported on activated T cells or APCs () but, to the best of our knowledge, neither PD-L2 nor PD-L1 expression has ever been reported in Treg cells, and our finding that they are overexpressed in tumor infiltrating Treg cells adds an additional level of complexity to the PD1 - PD-Ls immunomodulatory axis within the tumor microenvironment. BATF is a transcription factor that has been mainly associated to Th17 development and CD8T cells differentiation (). Our findings revealed that BATF transcript is upregulated in tumor-infiltrating Treg cells more than in tumor infiltrating Th17 cells ( Figure S4 ). Expression of BATF in CD8T cells is induced by IL-21 (), and we found that IL21R is highly expressed in tumor-infiltrating Treg cells ( Figure 4 ).

Schoenbrunn et al., 2012 Schoenbrunn 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. We showed that tumor-infiltrating Treg cells express high amounts of 4-1BB (CD137) a marker of TcR-mediated activation () and have shown they display very high suppressor function on effector T cell proliferation. It could be that expression of the signature genes correlated with the enhanced suppressive ability and so contributed to the establishment of a strong immunosuppressive environment at tumor sites.

Borowsky and Hynes, 1998 Borowsky M.L.

Hynes R.O. Layilin, a novel talin-binding transmembrane protein homologous with C-type lectins, is localized in membrane ruffles. Weon and Potts, 2015 Weon J.L.

Potts P.R. The MAGE protein family and cancer. Islam et al., 2013 Islam S.A.

Ling M.F.

Leung J.

Shreffler W.G.

Luster A.D. Identification of human CCR8 as a CCL18 receptor. Chen et al., 2011 Chen J.

Yao Y.

Gong C.

Yu F.

Su S.

Chen J.

Liu B.

Deng H.

Wang F.

Lin L.

et al. CCL18 from tumor-associated macrophages promotes breast cancer metastasis via PITPNM3. Schutyser et al., 2005 Schutyser E.

Richmond A.

Van Damme J. Involvement of CC chemokine ligand 18 (CCL18) in normal and pathological processes. A corollary to our findings would have that increased number of Treg cells in the tumor environment should associate with a worst clinical outcome. In fact, when LAYN, MAGEH1, and CCR8 (which represent three of the most enriched genes in tumor-infiltrating Treg cells) are highly detected in whole-tumor samples there is a significant worsening of the 5-year survival of both CRC and NSCLC patients. Although, the functional roles in Treg cells of LAYN, a transmembrane protein with homology to c-type lectin (), and of MAGEH1, a member of the melanoma antigen gene family (), are unknown, the high expression of the chemokine receptor CCR8 is instead intriguing. Indeed, CCL18, the ligand of CCR8 (), is highly expressed in different tumors including NSCLC (). The high specificity of CCR8 expression on tumor-infiltrating Treg cells suggests it could be an interesting therapeutic target to inhibit Treg cells trafficking to tumor sites, without disturbing recruitment of other effector T cells that do not express CCR8.