| Background:Tumor microenvironment promotes immune suppression through recruiting and expanding suppressive immune cells such as regulatory T cells (Tregs) to facilitate cancer progression. CD4+Tregs are considered as the major immunosuppressive T cells in many cancers including human colorectal cancer (CRC). However, this conclusion is not definitive as most of those studies did not extensively examine the whole suppressive T cell landscape. In fact, the role of CD4+ Tregs in CRC is controversial in terms of their prognostic value. Therefore, it is drastically needed to understand the exact role of those regulatory T cells in human CRC. Since regulatory T cells are heterogeneous population, it is tempting to speculate that there may be another immunosuppressive T cell population that plays a pivotal role in facilitating CRC progression and immune evasion.Our previous studies have demonstrated that innate IL-17-producing γδT cells (y8T17) could recruit and expand polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) in the tumor that inhibit effector T cell function and establish an immunosuppressive microenvironment thus promoting CRC progression, suggesting that y8T cell subpopulation exhibited specific significance in immune regulation of human colorectal cancer. Further research showed that approximately 50% of tumor-infiltrating γδT cells expressed high levels of CD39 molecule. CD39~+ γδT cells were significantly increased in the tumor tissues compared to the paired normal tissues. Only a few model studies have unraveled that CD39~+ γδT cells could in vitro inhibit CD3+T cell proliferation, implicating CD39 is a specific marker of γδTregs. Thus, based on our previous findings, we believe that human CRC-infiltrating CD39~+ γδT cells are likely to be a novel immunosuppressive subset.Our preliminary functional assay shows that tumor-infiltrating CD39~+y8T cells conventional CD4+Tregs and paired normal tissue sourced CD39~+y8T cells. This suggests tumor-infiltrating CD39~+ γδT cells may be the predominant immunosuppressive T cells and tumor microenvironment can promote the immunosuppressive activity of CD39~+ γδT cells. However, the role, underlying mechanism of induction and immunosuppressive activity of CD39~+y8T cells in human CRC immune microenvironment still remains unknown. The aim of this study is to investigate the role and mechanism of CD39~+ γδT cells in human CRC immune microenvironment and their clinical significance.Methods:Firstly, we used primary cell separation technology and multicolor flow cytometry analysis to study the phenotype and cytokines of CD39~+y8T cells in human fresh tumor and paired normal tissues. Then we used fluorescence-activated cell sorting, ELISA, HPLC and in vitro co-cuture experiments to study the immunosuppressive activity of CD39~+γδT cells and the adenosine-mediated mechanism on allogeneic peripheral blood CD3+ T, CD4+ T and CD8+ T cells. To investigate the mechanism of CD39~+ γδTreg induction in tumor microenvironment, we still used fluorescence-activated cell sorting, gene silencing, RT-PCR, ELISA, HPLC, and in vitro co-cuture experiments to study the impact of tumor supernatant, recombinant human TGF-β1, colorectal SW480 cells on CD39~+ γδT cells induction. Finally, we analyzed the correlation between tumor-infiltrating CD39~+ γδTregs and malignant clinicopathological features of CRC patients.Results:1), In human CRC tissues, we found that approximately 50% of γδT cells expressed high level of CD39. CD39~+y8T cells were significantly increased in the tumor tissues compared to the paired normal tissues. Tumor-infiltrating CD39~+ γδT cells expressed remarkably higher levels of CTLA-4, PD-1, FoxP3, CD25 and CD161, and their absolute number in CD3+ T cells was significantly more than CD4+ CD25+ CD127low conventional CD4+ Treg cells in the tumor tissues. CD39~+ γδT cells exhibited potent immunosuppressive function on T cells. Surprisingly, CD39~+ γδT cells exerted more potent immunosuppressive activity than conventional CD4+ Tregs, suggesting that CD39~+ γδT cells, but not CD4+Tregs are the predominant immunosuppressive T cells in human CRC.2), Tumor-infiltrating CD39~+ γδT cells have potent immunosuppressive activity compared to other regulatory T cells in human CRC. Thus, CD39~+ γδT regs are the predominant regulatory T cells in human CRC not only in quantity but also in quality.3), CD39~+ γδT regs function directly via the adenosine-mediated pathway but independent of TGF-P or IL-10. They can also secrete cytokines including IL-17A and GM-CSF which chemoattract polymorphonuclear myeloid-derived suppressive cells (PMN-MDSCs) thus establishing an immunosuppressive network.4), We further demonstrate that tumor-derived TGF-β1 induces CD39~+ γδT cells from paired normal colon tissues to produce more adenosine and become potent immunosuppressive T cells.5), Moreover, CD39~+ γδTreg infiltration is positively correlated with TNM stage and other unfavorable clinicopathological features of CRC patients.Conclusion:Our study has unraveled tumor-infiltrating CD39~+ γδTregs as the predominant regulatory T cells and a key mediator in human CRC. These findings suggest that CD39~+ γδTregs may be a prognostic factor of human CRC and eradication of these cells may have a potential for effective human CRC treatment.Novelty1, Our study has demonstrated that novel tumor-infiltrating CD39~+ γδTregs are the predominant immunosuppressive T cells rather than conventional CD4+ Tregs in human CRC;2, Our study has unraveled a novel "TGF-β1 of tumor cells-CD39~+y8Tregs polarization-adenosine-immunosuppression-tumor progression" tumor regulatory mechanism in CRC microenvironment;3, Our study shows that CD39~+y8Tregs infiltration may be a poor prognostic factor of human CRC. |
