γδ T cells in human colon adenocarcinomas comprise mainly Vδ1, Vδ2, and Vδ3 cells with distinct phenotype and function.

Γδ T cell infiltration into tumours usually correlates with improved patient outcome, but both tumour-promoting and tumoricidal effects of γδ T cells have been documented. Human γδ T cells can be divided into functionally distinct subsets based on T cell receptor (TCR) Vδ usage. Still, the contribution of these different subsets to tumour immunity remains elusive. Here, we provide a detailed γδ T cell profiling in colon tumours, using mass and flow cytometry, mRNA quantification, and TCR sequencing. δ chain usage in both the macroscopically unaffected colon mucosa and tumours varied considerably between patients, with substantial fractions of Vδ1, Vδ2, and non-Vδ1 Vδ2 cells. Sequencing of the Vδ complementarity-determining region 3 showed that almost all non-Vδ1 Vδ2 cells used Vδ3 and that tumour-infiltrating γδ clonotypes were unique for every patient. Non-Vδ1Vδ2 cells from colon tumours expressed several activation markers but few NK cell receptors and exhaustion markers. In addition, mRNA analyses showed that non-Vδ1 Vδ2 cells expressed several genes for proteins with tumour-promoting functions, such as neutrophil-recruiting chemokines, Galectin 3, and transforming growth factor-beta induced. In summary, our results show a large variation in γδ T cell subsets between individual tumours, and that Vδ3 cells make up a substantial proportion of γδ T cells in colon tumours. We suggest that individual γδ T cell composition in colon tumours may contribute to the balance between favourable and adverse immune responses, and thereby also patient outcome.

Intratumoral regulatory T cells from colon cancer patients comprise several activated effector populations.

BACKGROUND: Intratumoral regulatory T cells (Treg) in colon cancer are a heterogeneous cell population, with potential impact on patient outcome. Generally, a high Treg infiltration has been correlated to a worse patient outcome, but it is still unclear how the composition of different Treg subsets affects patient relapse and survival. In this study, we used mass and flow cytometry to characterize Treg in colon tumors and corresponding unaffected tissue, followed by a correlation to clinical parameters and patient outcome. RESULTS: Using mass cytometry, we defined 13 clusters of intestinal Treg, three of which were enriched in the tumors. The two most enriched clusters were defined by their expression of the proliferation marker Ki67 and CD56, respectively. The Treg accumulating in the tumors expressed inducible T-cell co-stimulator (ICOS), OX-40, and CD39, indicating that they were effector Treg (eTreg). Intratumoral CD39+ Treg also had a higher expression of Foxp3, suggesting a higher suppressive activity, and we subsequently used CD39 as a marker for eTreg. Our further studies showed that colon tumors can be divided into two tumor groups, based on the proportion of CD39+ putative eTreg in the tumors. This property was independent of both tumor microsatellite status and tumor stage, which are important factors in predicting cancer disease progression. In a prospective study of forty-four colon cancer patients, we also showed that patients with a high CD39 expression on tumor-infiltrating Treg have a tendency towards a less favorable patient outcome in terms of cumulative cancer-specific survival. CONCLUSIONS: This study uncovers novel subsets of tumor-infiltrating Treg in colon cancer, and suggests that CD39 may be a potential therapeutic target in patients with microsatellite stable colon tumors, which are usually refractory to checkpoint blockade therapy.

Exhaustion in tumor-infiltrating Mucosal-Associated Invariant T (MAIT) cells from colon cancer patients.

Mucosal-associated invariant T (MAIT) cells are unconventional T cells recognizing microbial metabolites, presented by the invariant MR1 protein. Upon activation, MAIT cells rapidly secrete cytokines and exert cytotoxic functions, and may thus be highly relevant also in tumor immunity. MAIT cells accumulate in colon tumors, but in contrast to other cytotoxic T cell subsets, their presence in tumors has been associated with worse patient outcome. Here we investigated if exhaustion may contribute to reduced anti-tumor immunity by MAIT cells. Freshly isolated lymphocytes from colon tumors, unaffected tissue and blood from the same patients were analyzed by flow cytometry to detect MAIT cells with effector functions that are relevant for tumor immunity, and their expression of inhibitory receptors and other exhaustion markers. Our studies show that MAIT cells with a PD-1highTim-3+CD39+ terminally exhausted phenotype and an increased proliferation accumulate in colon tumors. The exhausted MAIT cells have reduced polyfunctionality with regard to production of important anti-tumor effector molecules, and blocking antibodies to PD-1 partly improved activation of tumor-infiltrating MAIT cells in vitro. We conclude that the tumor microenvironment leads to exhaustion not only of conventional T cells, but also MAIT cells, and that checkpoint blockade therapy may be useful also to reinvigorate tumor-infiltrating MAIT cells.

Regulatory T cells specifically suppress conventional CD8αß T cells in intestinal tumors of APCMin/+ mice.

The presence of activated T cells in colorectal cancer tissues is a strong predictor of patient survival. Our previous studies have shown that regulatory T cells (Treg) are able to reduce T cell transendothelial migration in vitro and accumulation of effector T cells in intestinal tumors in vivo in the murine APCMin/+ model for microsatellite stable intestinal tumors. In this study, we investigated the effect of Treg depletion on the density and effector functions of different TCRαß+ and TCRγδ+ T cell populations in intestinal tumors. We used the APCMin/+\DEREG mouse model, which harbor a diphtheria toxin receptor under the control of the FOXP3 promoter, to deplete Treg in tumor bearing mice. We found that the density of conventional TCRαß+CD8αß+ T cells was significantly increased in Treg-depleted tumors in comparison with Treg-proficient tumors. Furthermore, TCRαß+CD8αß+ T cells showed increased proliferation and activation as well as increased Granzyme B and IFN-γ production in Treg-depleted tumors. In sharp contrast, the densities and effector functions of TCRαß+CD8αα+ T cells and TCRγδ+ T cells remained unchanged by Treg depletion. We also documented a distinct population of IL-17A+TNF+ TCRγδ+CD8- T cells in tumors, which were not affected by Treg depletion. We conclude that Treg depletion affects only conventional TCRαß+CD8αß+ T cells in intestinal tumors, while unconventional T cells and T cells in unaffected tissue are not altered. Immunotherapies aimed at depleting Treg from tumors may thus be a viable option for reinvigoration of conventional cytotoxic T cells with a Th1 cytokine profile.

Immune checkpoint blockade improves the activation and function of circulating mucosal-associated invariant T (MAIT) cells in patients with non-small cell lung cancer.

Mucosal-associated invariant T (MAIT) cells constitute one of the most numerous unconventional T cell subsets, and are characterized by rapid release of Th1- and Th17-associated cytokines and increased cytotoxic functions following activation. MAIT cells accumulate in tumor tissue but show an exhausted phenotype. Here, we investigated if immune checkpoint blockade (ICB) with antibodies to PD-1 or PD-L1 affects the function of circulating MAIT cells from non-small cell lung cancer patients. ICB increased the proliferation and co-expression of the activation markers HLA-DR and CD38 on MAIT cells in most patients after the first treatment cycle, irrespective of treatment outcome. Furthermore, production of cytokines, especially TNF and IL-2, also increased after treatment, as did MAIT cell polyfunctionality. These results indicate that MAIT cells respond to ICB, and that MAIT cell reinvigoration may contribute to tumor regression in patients undergoing immune checkpoint therapy.

Single-cell BCR and transcriptome analysis after influenza infection reveals spatiotemporal dynamics of antigen-specific B cells.

B cell responses are critical for antiviral immunity. However, a comprehensive picture of antigen-specific B cell differentiation, clonal proliferation, and dynamics in different organs after infection is lacking. Here, by combining single-cell RNA and B cell receptor (BCR) sequencing of antigen-specific cells in lymph nodes, spleen, and lungs after influenza infection in mice, we identify several germinal center (GC) B cell subpopulations and organ-specific differences that persist over the course of the response. We discover transcriptional differences between memory cells in lungs and lymphoid organs and organ-restricted clonal expansion. Remarkably, we find significant clonal overlap between GC-derived memory and plasma cells. By combining BCR-mutational analyses with monoclonal antibody (mAb) expression and affinity measurements, we find that memory B cells are highly diverse and can be selected from both low- and high-affinity precursors. By linking antigen recognition with transcriptional programming, clonal proliferation, and differentiation, these finding provide important advances in our understanding of antiviral immunity.

Isolation and Characterization of MAIT Cells from Tumor Tissues.

Mucosal-associated invariant T (MAIT) cell infiltration has been demonstrated in colorectal and hepatocellular carcinoma, and their ability to produce Th1- and Th17-associated cytokines, as well as their cytotoxic function, suggests that MAIT cells may have important functions in both reducing and promoting protective tumor immunity. Here, we describe enzymatic methods to isolate intraepithelial and lamina propria lymphocyte single cell suspensions from colon tissue and tumors containing viable MAIT cells, which can be used for further purification, flow cytometry analysis, or culture.