| In recent years,immunotherapy targeting immune checkpoints has opened up a new era in cancer treatment and achieved sustained immune responses in many cancers,including in melanoma,kidney cancer,Hodgkin’s disease,and lung cancer and a limited fraction of colorectal cancer(CRC)cases.Immune checkpoint inhibitors eliminate tumor cells by restoring immune activity and associated immune function of exhausted T cells.T cell exhaustion was first described more than a decade ago as a state of T cell dysfunction and subsequent physical deletion of antigen-specific T cells during chronic viral infection of LCMV in mice.CD8+T cell exhaustion is the most extensive and indepth study.T cell exhaustion is associated with progressive and hierarchical differentiation,characterized by the sustained upregulation and co-expression of several inhibitory receptors,and limited proliferative capacity.Emerging evidence has demonstrated that T cell exhaustion is a continuous developmental process.Exhausted T cells differentiate from progenitor or precursor exhausted T cells(Texprog),via transitional or intermediate exhausted T cells(Textran),into terminally exhausted T cells(Texterm).Texprog cells are reinvigorated by immune checkpoint blockade(ICB),regaining their effector function and ability to control tumor progression.ICB also increases the proliferation and improves the function of Texan cells.However,Texterm cells are difficult to reactivate with ICB.Therefore,T cell exhaustion,especially the inactivation state of terminal exhausted T cells that is difficult to prevent or reverse,has become a bottleneck in the entire field of tumor immunotherapy.γδ T cells are a unique group of lymphocytes whose T cell receptors consist of a y chain and δ chain,setting them aside from conventional αβ T cells.By contrast to βα T cells,γδ T cells lack major histocompatibility complex restriction.Instead,γδ T cells bear a plethora of NK cell receptors(NKRs),such as NKG2A,NKG2D,DNAM-1,NKp30,and NKp44 on their surface.These unique features enable γδ T cells to rapidly respond to infected or transformed cells,thus contributing to the first line of defense that precedes antigen-specific αβ T cell responses.Low numbers of γδ T cells are found in peripheral blood and lymph nodes,as these cells predominantly reside in the mucosal and epithelial barriers.Indeed,γδ T cells represent～20%of the total lymphocyte population in the colonic mucosal epithelium.γδT cells play an important role in maintaining intestinal mucosal homeostasis,immune regulation and exerting cytotoxic effects,and is a key point to study the immune microenvironment of colorectal cancer.However,the vast majority of patients with CRC(85%)are insensitive to immune checkpoint blocking therapy represented by CTLA-4 and PD-1/PD-L1 antibodies,indicating that targeting CTLA-4 or PD-1/PD-L1 alone can’t fully inhibit tumor progression,suggesting that colorectal cancer tumors can also evade immune cell responses by increasing the expression of other immune checkpoint molecules.It has been found that in the AOM/DSS-induced colorectal cancer mouse model,TCRδ-/-mice are more likely to form tumors than TCRα-/-mice,which indicates that γδ T cells play a role suppression of the formation and progression of colorectal adenocarcinoma in mice,and the role is more important than CD8+T cells.Whether γδ T cell exhaustion exists in the tumor microenvironment(TME)of CRC and the characteristics and the regulation mechanism of these putative exhausted cells are still unclear.Based on the above,we carried out this study and obtained the following results:Results:Part 1:γδ T cells play a key role in the formation and progression of CRCTo explore whether γδT cells play an important role in immune surveillance and immune clearance in the occurrence and development of CRC,on the one hand,we analyzed the RNAseq data of CRC in The Cancer Genome Atlas(TCGA)to explore the correlation between γδ T cell abundance and survival of CRC patients.There is a positive correlation between the γδT cells infiltration and the survival of CRC patients,especially in MSS-CRC patients.That is,the higher of γδ T cells infiltration,the longer of the survival of CRC patients.On the other hand,we established azoxymethane(AOM)/dextran sodium sulfate(DSS)mouse model of colitis-associated cancer,which can simulate the naturally occurring process of CRC.We found that the tumor numbers and tumor weights of AOM/DSS-treated TCRδ-/-mice were significantly higher than those of AOM/DSStreated WT mice.In addition,the survival of AOM/DSS-treated TCRδ-/-mice was significantly shorter than that of AOM/DSS-treated WT mice.Collectively,these results suggest that γδT cells play an important role in inhibiting the development and progression of CRC.Part 2:Tumor infiltrating γδT cells experience functional exhaustionIn order to determine the immune exhaustion state of colorectal cancer microenvironment and whether γδ T cells experience functional exhaustion,we used the multicolor flow cytometry(FACS)to detect the phenotype and function characteristics of tumor infiltrating γδ T,CD8+T and NK cells from AOM/DSS induced mice,and then verified in melanoma and hepatocellular carcinoma(HCC)mice.The results of this study are as follows:1.Tumor infiltrating γδ T cells exhibit exhaustion phenotype and experience functional exhaustionThe expression levels of several inhibitory receptors,including PD-1,TIM-3,CTLA-4,LAG-3,TIGIT,and VISTA on γδ T cells were all significantly higher in the tumor than in the spleen and the MLN,or within the colon IEL population.In addition,the ability of γδ T cells to produce IFN-γ or TNF-α was significantly lower in the tumor than in the spleen or the MLN.IFN-y production by tumor-derived γδ T cells was also lower than that of colon IEL-derived γδT cells,implying that the antitumor function of tumor infiltrating γδ T cells was markedly impaired2.Tumor infiltrating γδ T cells experience more serious functional exhaustion in the TME of CRC than CD8+T cells and NK cellsExcept for CTLA-4,we also observed higher expression levels of PD-1,TIM-3,LAG-3,TIGIT,and VISTA on CD8+T cells in the tumor than on CD8+T cells derived from the spleen,the MLN,or colon IELs.Except for LAG-3 and VISTA,the expression levels of PD-1,TIM-3,CTLA-4,and TIGIT on tumor-derived NK cells were all significantly higher than on NK cells from the spleen,the MLN,or than on colon IELs.In addition,the effector function of tumor infiltrating CD8+T and NK cells was also impaired to varying degrees,as the expression of IFN-y and/or TNF-α and/or Perforin was significantly down-regulated.We found that tumor infiltrating γδ T cells,CD8+T cells,and NK cells all exhibited exhausted phenotypes,which were characterized by the high expression of several inhibitory receptors.Notably,γδ T cells expressed much higher levels of PD-1,TIM-3 and produced lower levels of IFN-y and TNF-α than the CD8+T cells or NK cells.Taken together,these results suggest that tumor infiltrating γδ T cells experience more serious functional exhaustion in the TME of CRC than CD8+T cells and NK cells.3.Tumor infiltrating γδ T cells experience less serious functional exhaustion than CD8+T cells in the TME of melanoma and hepatocellular carcinomaTumor infiltrating γδ T,CD8+T,and NK cells were all exhibited exhaustion phenotype in the TME of melanoma and HCC,but the tumor infiltrating γδ T cells was showed less severe exhaustion than CD8+ T cells in the TME of melanoma and HCC.In conclusion,in the TME of CRC,the tumor infiltrating γδ T,CD8+ T and NK cells are all exhausted,but their phenotypes and functional states are different.The tumor infiltrating γδ T cells experience more serious functional exhaustion in the TME of CRC than CD8+T cells and NK cells.In addition,tumor infiltrating γδ T,CD8+T and NK cells exhibited different exhaustion degree and phenotypes in different TME,including colorectal cancer,melanoma,and hepatocellular carcinoma.In addition,the exhaustion of γδ T cells also exists in the TME of melanoma and hepatocellular carcinoma,but the exhaustion degree of γδ T cells is lower than that of CRC TME.Part 3:scRNA-seq analysis of exhausted tumor infiltrating γδ T cellsTo better characterize the phenotypes of exhausted TI-γδ T cells,γδ T cells were FACS-sorted from the AOM/DSS induced tumor and the colonic IEL population.Theγδ T cells were then subjected to scRNA-seq using the 10x Genomics platform.The differential gene expression profiles of IE-γδ T and TI-γδ T were analyzed by unsupervised clustering(UMAP).UMAP dimension reduction analysis was performed for TI-γδ T,and the characteristic expression gene profile of TI-γδ T subgroup was analyzed.Monocle2 was used to conduct pseudo-time series analysis of mouse TI-γδ T cells to explore the dynamic differentiation process of each subgroup of TI-γδ T cells in the microenvironment of colorectal cancer,and visualize their differentiation trajectory.Flow cytometry analysis of the expression of inhibitory receptors PD-1 and TIM-3 could determine the severity of γδ T cell depletion.The results of this study are as follows:1.The UMAP analysis of tumor infiltrating γδ T cellsOn generating a UMAP diagram,we discovered that there was a marked difference in the special distributions of TI-γδT cells and IE-γδT cells,with only a small area of overlap,which indicates that there was a big difference between the TI-and IE-γδ T cells.2.The gene expression profiles analysis of tumor infiltrating γδ T cellsThere was a different gene expression profiles between TI-γδ T cells and IE-γδ T cells.Specifically,TI-γδ T cells expressed higher levels of inhibitory receptor genes including Pdcd1,Ctla4,Havcr2,Btla,and Klrc1,and higher levels expressed higher levels of TF-encoding genes,including Zbtb32,Eomes,Hoxa5,Cebpd,Tox2,Sox4,Maf,Vdr,Sox13,Batf3,Rbpj,Batf,Prdm1,Bhlhe40,Atf3,Tgif1,Klf4,Nfil3,Nr4a3,and Foxol than IE-γδ T cells,and lower levels of the activatory receptor gene Cd69 and the co-stimulatory genes Cd27 and Tnfrsf18 than IE-γδ T cells.TI-γδ T cells expressed lower levels of genes associated with cytotoxicity(including Gzmb,Nkg7,Gzma,Prf1,Gzmk,and Gzmf)and pro-inflammatory cytokine production(including Ifng and Tnf)than IE-γδ T cells.The number of TI-γδ T cells co-expressing two,three and four inhibitory receptor genes was much higher than that of IE-γδ T cells.The results showed that TI-γδT-cells contained a higher amount of γδ T cells with co-expression of multiple IR genes compared with IE-γδ T cells(38.97%vs.7.14%coexpressing 2 IRs;25.48%vs.0.83%co-expressing 3 IRs;6.05%vs.0.01%co-expressing the 4 IRs),implying TI-γδ T cells had obvious exhaustion characteristics than IE-γδ T cells.3.The heterogeneity analysis of tumor infiltrating γδ T cellsBy UMAP dimensionality analysis,TI-γδ T and IE-γδ T cells were divided into 8 clusters.We performed a preliminary annotation of the cell types in each cluster,based on their gene expression signatures.We found that tumor infiltrating γδ T cells are heterogeneous cells.According to their gene expression profiles,we speculate that three of them are exhausted,identified cluster 6,cluster 5 and cluster 4.4.The dynamic immune states analysis of tumor infiltrating γδ T cellsUsing the na?ve-like cell related genes(such as Cd28,Lef1,Ccr7,Sell,S1pr1 and Klf2)as the pseudo-time starting point to reconstruct the differentiation trajectory of TI-γδ T cells,we found that naive-like γδ T cells and Trm cells were at the beginning of the trajectory,and three exhausted γδ T subsets were at the end of the trajectory.Next,we investigated the transcriptional changes associated with transitional states and observed that the γδ T cell clusters could be categorized into 4 phases.Cluster 6 cells were predominantly phase 2 cells,characterized by lower expression Havcr2,Pdcdl,Ctla4 and Klrc1,which was consistent with the characteristics of early exhaustion cells,suggesting that they were Texprog cells.Cluster 5 cells were predominantly phase 3 cells,characterized by upregulated expression Havcr2,Pdcd1,Ctla4 and Klrcl,which was consistent with the characteristics of transitional or intermediate exhausted cells,suggesting that they were Textran cells.Cluster 4 cells were predominantly phase 4 cells,characterized by highest expression Havcr2,Pdcdl,Ctla4 and Klrc1,which was consistent with the characteristics of terminally exhausted cells,suggesting that they were Texterm cells.5.PD-1+TIM-3+γδ T cells were terminally exhausted cellsIn order to verify whether the expression levels of PD-1 and TIM-3 can define the exhaustion stage of tumor infiltrating γδ T cells,we established AOM/DSS induced colorectal cancer model and analyzed the exhaustion stage of γδ T cells by flow cytometry.The results showed that compared with PD-1+TIM-3-γδ T and PD-1-TIM-3γδ T,PD-1+TIM-3+γδ T cells expressed the highest level of CTLA4 and the lowest level of activating receptor NKG2D and proliferation markers(Ki67),which confirmed that PD-1+TIM-3+γδ T cells were the most severe exhausted group,which was identified as the terminal exhausted γδT cells.6.The characteristics of exhausted tumor infiltrating γδ T cells of clinical specimens of colorectal cancer patientsIn order to determine whether tumor infiltrating γδ T cells in patients of CRC have a similar phenomenon seen in mouse,we specifically analyzed the scRNA-seq data(GSE161277)from tumor tissues of CRC patients.The results showed that there were also different exhaustion phenotypes among tumor infiltrating γδ T,CD8+T,and NK cells.Tumor infiltrating γδ T cells highly expressed PDCD1 and CTLA4,CD8+T cells highly expressed PDCD1,CTLA4 and HAVCR2,and NK cells highly expressed TIGIT,LAG3 and HAVCR2.Collectively,these results demonstrate that tumor infiltrating γδ T cells are heterogeneous cells.In the TME of CRC,there were three kinds of exhausted γδ T cells,which were characterized by early exhaustion,middle exhaustion and terminal exhaustion,respectively.The phenotype of the terminal exhausted γδ T cells was PD1+TIM-3+,accounting for the highest proportion of the total exhausted γδ T cells.Exploring the characteristics of γδ T cells exhaustion in the TME of CRC deeply will help us to understand the phenomenon and mechanism of γδ T cell exhaustion,so as to develop more effective immunotherapy methods for target γδ T cell exhaustion,improving the overall prognosis of CRC patients,and providing more solid theoretical basis and evidence support.Part 4:Immune checkpoint blockade can effectively restore the antitumor activity of tumor infiltrating γδ T cellsTo determine whether function reversal of γδ T cells can be achieved by blocking PD-1/PD-L1 and TIM-3 in vivo,we established a mouse colorectal cancer model(MC38 subcutaneous tumor)and investigated the therapeutic effect of blocking PD1/PD-L1 and TIM-3 alone or in combination in vivo.Then the effects of blocking PD1/PD-L1 and TIM-3 on the phenotypes and function of tumor infiltrating γδ T,CD8+T and NK cells were studied.The results of this study are as follows:1.Blockade of PD-1/PD-L1 and TIM-3 inhibit tumor growthThe anti-PD-1,anti-PD-L1,and anti-TIM-3 antibodies were used to treat CRC mice with single and combined block therapy.The results showed that the tumor growth was inhibited regardless of whether PD-1,PD-L1 or TIM-3 were blocked alone.The combined blocking therapy(PD-1 combined with TIM-3,PD-L1 combined with TIM3)was more effective in inhibiting tumor growth.2.Blockade of PD-1/PD-L1 and TIM-3 prevent exhaustion of tumor infiltratingγδ T cellsBlocking therapy was performed in CRC mice and the immune status of tumor infiltrating γδ T cells was detected.Compared with the untreated group,the exhaustion status of tumor infiltrating γδ T cells in the treatment group(PD-1,PD-L1 and TIM-3 alone)was significantly reduced,and the expression level of NKG2D and Ki67 were significantly increased.The expression of inhibitory receptors PD-1 and TIM-3 decreased significantly.PD-1 and TIM-3 combination therapy or PD-L1 and TIM-3 combination therapy prevent exhaustion of tumor infiltrating γδ T cells more effectively.Similar to tumor infiltrating γδ T cells,blockade of PD-1/PD-L1 and TIM-3 also prevent exhaustion of tumor infiltrating CD8+T and NK cells.3.Blockade of PD-1/PD-L1 and TIM-3 can effectively restore the anti-tumor activity of tumor infiltrating γδ T cellsCompared with the untreated group,the antitumor function of tumor infiltratingγδ T cells in the treatment group(PD-1,PD-L1 and TIM-3 alone)was significantly increased,and the production level of IFN-y and TNF-α were significantly increased.PD-1 and TIM-3 combination therapy or PD-L1 and TIM-3 combination therapy restore the anti-tumor activity of tumor infiltrating γδ T cells more effectively.Similar to tumor infiltrating γδ T cells,blockade of PD-1/PD-L1 and TIM-3 also restore the anti-tumor activity of tumor infiltrating CD8+T and NK cells.In summary,blocking PD-1/PD-L1 and TIM-3 in CRC mice can significantly prevent or reverse the exhaustion of γδ T cells,thus enabling γδ T cells can better exert their antitumor function.These results provide a theoretical basis for the development and application of γδ T cell immunotherapy.Part 5:Exploring the upstream regulatory genes of tumor infiltrating γδ T cell exhaustion In order to explore the transcriptional regulation mechanism of exhausted γδ T cells,we first mined the scRNA-seq data of AOM/DSS induced colorectal cancer mice,and TCGA database of CRC patients and the scRNA-seq data of CRC patient.1.Analyzing the transcription factor expression of tumor infiltrating γδ T cells in CRC miceBy scRNA-seq analysis,we found three exhausted γδ T subsets were characterized by different transcription factor genes expression profiles.Of note,the expression of some TF-encoding genes(Maf Bhlhe40,Nfil3,Rbpj,Batf3,Nr4a3,and Batf)was upregulated along the pseudotime of γδ T cell exhaustion,especially Maf(encoding cMaf)2.Transcription factor gene expression positively correlates with inhibitory receptor gene expression in CRC patients within TCGATo determine whether the phenomenon found in scRNA-seq data of mouse can be extended to CRC patients,we analyzed the genetic association of CRC patients(n=620)in TCGA database.Analyzing the correlations between gene expression of CRC in The Cancer Genome Atlas(TCGA),we found that the expression of some transcription factor-encoding genes(MAF,BATF3,BATF and NR4A)was positively correlated with the inhibitory receptors-encoding genes(PDCD1,HAVCR2 and CTLA4),especially MAF(encoding c-MAF).3.Analyzing the transcription factor expression of tumor infiltrating γδ T cells in patients with CRCIn order to determine whether tumor infiltrating γδ T cells in patients of CRC have a similar phenomenon seen in mouse,we specifically analyzed the scRNA-seq data(GSE161277)from tumor tissues of CRC patients.We found that tumor tissue infiltrating γδ T cells in CRC patient had high expression of transcription factor genes MAF,NR4A3 and SOX4,among which MAF was the strongest expression.4.c-Maf expression was correlated with the severity of tumor infiltrating γδ T cell exhaustionTo verify whether transcription factor c-Maf is associated with γδ T cell exhaustion,we used FACS to evaluate c-Maf expression and correlated it with the severity of tumor infiltrating γδ T cell exhaustion.We found that c-Maf expression was correlated with the severity of tumor infiltrating γδ T cell exhaustion not only in TME of CRC,but also in TME of melanoma and HCC.Further,we observed similar correlation in CD8+T and NK cells.5.c-Maf facilitated the expression of PD-1 and TIM-3 and induced the functional exhaustion of γδ T cellsTo determine whether the transcription factor c-Maf can directly regulate the exhaustion of γδ T cells,we firstly used the JASPAR database(used to predict TF binding sites)and the UCSC database(used to identify gene promoter regions)to show that there were c-Maf binding sites～2 kb upstream of the transcription start site(TSS)on the mouse and human inhibitory receptor(IR)genes promoters.ChIP assay showed that the anti-c-Maf antibody efficiently precipitated the promoter fragments of both Pdcdl and Havcr2,whereas the IgG control did not,suggesting that c-Maf directly bound to the Pdcdl and Havcr2 promoters.Furthermore,the luciferase reporter assay showed that c-Maf activated Pdcd1 and Havcr2 gene transcription.These results suggested that by binding to the promoters of Pdcdl and Havcr2 directly,c-Maf facilitated the expression of these genes and induced the functional exhaustion of γδ T cells.Collectively,c-Maf may be a transcription factor related to γδ T cell exhaustion.It can promote γδ T cell exhaustion by regulating the expression of PD-1 and TIM-3.cMaf promotes γδ T cell exhaustion not only in CRC microenvironments,but also in B16 and HCC microenvironments.In addition,c-Maf not only promotes γδ T cell exhaustion,but also plays an important regulation role in CD8+T and NK cell exhaustion.This suggests that c-Maf may be a universal transcription factor in tumor immunotherapy that can be used as a target to reverse γδ T,CD8+T,and NK cell exhaustion.Combining the biological information,animal models and clinical samples to study the exhaustion status,exhaustion phenotype and exhaustion mechanism of γδ T cells systematically and comprehensively in the TME of CRC.We found that tumor infiltrating γδ T cells experience more serious functional exhaustion in the TME of CRC than CD8+T cells and NK cells.Moreover,there is heterogeneity of γδ T cells in the TME of CRC,including three exhaustion γδ T cell subsets in different stages,and PD1+TIM-3+terminal exhausted γδ T cell accounted for the highest proportion.We also found that c-Maf promotes γδ T cell exhaustion by up-regulating the expression of PD1 and TIM-3,and was involved in the regulation of CD8+T and NK cell exhaustion.The revealed features of exhausted tumor infiltrating γδ T cells may provide help for understanding the mechanisms and the association of γδ T cell exhaustion with tumor development and pathogenesis. |
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