Id2 Regulates Antitumor Immunity In Exhausted CD8~+T Cells

In recent years,tumor immunotherapy has achieved great progress,but there are obvious individual differences in its efficacy,and the overall effective rate is still not high.How to improve the efficacy of tumor immunotherapy,and stratify or expand the beneficiary population has become the focus of current tumor immunotherapy research.The main reason for the failure of tumor immunotherapy is that tumor cells evade the surveillance and killing of the immune system by various mechanisms,that is,tumor immune escape.CD8⁺T cell exhaustion is a key factor in tumor immune escape.Therefore,elucidating the key regulatory molecules and mechanisms of CD8⁺T cell exhaustion is of great theoretical and practical significance for inhibiting tumor immune escape and improving the efficacy of tumor immunotherapy.As a transcriptional regulator,Id2 plays a crucial role in regulating the development and differentiation of immune cells such as T cells.However,it remains to be elucidated that whether Id2 can regulate the antitumor immunity of exhausted CD8⁺T cells.Therefore,we delved into the scientific issue of"the role and molecular mechanism of Id2 in regulating the antitumor immunity of exhausted CD8⁺T cells".We performed bioinformatics analysis and constructed various gene-modified mouse and cell models.By using cell sorting,T cell adoptive transfer,co-immunoprecipitation,chromatin immunoprecipitation and other experimental techniques,we investigated:1)the expression profiles of Id2 in tumor-infiltrating immune subpopulations,the correlation between Id2 expression level and the number of immune cells,the correlation between Id2 expression level and inhibitory receptor expression,and its clinical significance on the prognosis of tumor patients;2)the effect of Id2 on antitumor immunity of CD8⁺T cells,the effect of Id2 on biological functions and immune characteristics of CD8⁺T cells,and the effect of Id2 on generation and conversion of exhausted subpopulations of CD8⁺T cells;3)the key Id2 interactomes in regulating the generation of progenitor exhausted CD8⁺T cells,and the transcriptional and epigenetic regulatory mechanisms.The accomplishment of this study is expected to provide new ideas and ways for inhibiting tumor immune evasion and improving the efficacy of tumor immunotherapy.The study falls into three parts.Part Ⅰ:Expression profiles of Id2 in the tumor microenvironment of tumor patients and its clinical significance.Using the TISCH database and TCGA database,we used the CIBERSORT algorithm for bioinformatics analysis and found that Id2 is highly expressed in CD8⁺T cells and exhausted CD8⁺T cells,and the number of infiltrating CD8⁺T cells in tumor patients is in significant correlation with the expression level of Id2.In addition,the expression level of Id2 is in significant correlation with the expression levels of PD-1,CTLA-4,TIGIT and other inhibitory receptors,and can be used as an important indicator for the prognosis of tumor patients.These results indicate that the Id2 expression level is distinct among tumor-infiltrating immune cell subsets,especially high in tumor-infiltrating CD8⁺T cells and tumor-infiltrating exhausted CD8⁺T cells,and can significantly indicate the prognosis of tumor patients.Part Ⅱ:Regulation of Id2 in antitumor immunity of CD8⁺T cells,and generation and conversion of exhausted CD8⁺T cell subpopulations.By constructing various T-cell conditional Id2-modified mouse models,we used flow cytometry sorting,adoptive transfer of immune cells and DEN-induced liver cancer models,and found that knockout of Id2significantly inhibited antitumor immunity of CD8⁺T cells,manifested as a significant decrease in the expression of effector cytokines,especially IFN-γ.Knockout of Id2accelerated the tumor growth and shortened the survival time of the tumor-bearing mice.These results suggest that Id2 may inhibit tumor development by enhancing the immune effector function of CD8⁺T cells.By constructing a melanoma model,we found that knockout of Id2 in T cells significantly inhibited the expression of major effector cytokines such as IFN-γand granzyme B in adoptively transferred OT-I CD8⁺T cells that specifically recognize OVA antigen.In the tumor microenvironment,deletion of Id2 significantly decreased the proportion of adoptively transferred TNF-α⁺IFN-γ⁺OT-I CD8⁺T cells.Furthermore,we found that the Id2 can maintain the stemness of adoptive CD8⁺T cells.Id2deletion in T cells led to the decreased expression of Tcf1,a key molecule for maintaining cell stemness,and also decreased expression of Slamf6,a marker of progenitor exhaustion.And the proportion of low-expression subsets of inhibitory receptor Tim-3 was significantly decreased.The results showed that the Id2 could maintain the immune properties and functional status of adoptively transferred T cells in the tumor microenvironment.By constructing mice with distinct expression of immune homologous marker CD45.1 and CD45.2,using OVA-expressing B16-F10 melanoma model,and combining flow cytometry sorting,in vitro activation and reactivation and other experimental techniques,we found that Id2 deletion led to the reduced conversion of OT-I CD8⁺T cells to the terminally exhausted subpopulation（Slamf6^-Tim-3⁺）,while the generation of the progenitor exhausted subpopulation（Slamf6⁺Tim-3^-）was also inhibited.Since Id2^TKO mice lack the progenitor exhausted subpopulation that can respond to PD-1 immune checkpoint blockade,Id2ablation inhibited the antitumor effect of anti-PD-1 therapy.These results showed that Id2can regulate the generation of progenitor exhausted CD8⁺T cells and their conversion to terminally exhausted cells.Part Ⅲ:The molecular mechanism of Id2-mediated transcriptional and epigenetic modification during CD8⁺T cell exhaustion.Using Co-IP,dual-luciferase reporter assay,Ch IP and other experimental techniques,we further investigated the key interactomes and transcriptional regulatory mechanisms of Id2 on the generation of progenitor exhausted CD8⁺T cells.We found that Id2 could interact with the transcription factor Tcf3 to inhibit the formation of the Tcf3-Tal1 transcription complex.The Tcf3-Tal1 transcription complex can bind to the E-box regions of the Slamf6 promoter and thus inhibit the expression of Slamf6.Deletion of the HLH domain of Id2 can increase the binding of Tcf3 to the four E-box regions of the Slamf6 promoter and inhibit the transcription of Slamf6.These results showed that Id2 could interact with Tcf3 to regulate the expression of Slamf6.We further used mass spectrometry,dual-luciferase reporter assay,Co-IP,chromatin fractionation,Ch IP and other experiments,and found that the transcription factor Tcf3 can recruit histone lysine demethylase LSD1.Deletion of the HLH domain of Id2 significantly increased the recruitment of LSD1 by Tcf3 and attenuated dimethylation of H3K4（H3K4me2）in the E-box regions of Slamf6 promoter,thereby inhibiting Slamf6 transcription and regulating the generation of exhausted CD8⁺T cell subpopulation.These results showed that Id2 enhanced the H3K4me2 modification in the E-box regions of the Slamf6 promoter by inhibiting the assembly of the Tcf3-LSD1 complex,thereby regulating the epigenetic process of CD8⁺T cell exhaustion.In conclusion,in this study,we conducted in-depth research on the scientific issue of"the role and molecular mechanism of Id2 in regulating antitumor immunity of exhausted CD8⁺T cells".We investigated the role and clinical significance of Id2 in antitumor immunity of CD8⁺T cells,discovered the key Id2 interactomes in regulating the generation and conversion of exhausted CD8⁺T cell subpopulations,elucidated the Id2-mediated transcriptional and epigenetic mechanism in CD8⁺T cell exhaustion via Tcf3-LSD1complex.It is of great theoretical and practical significance for providing novel targets for tumor immunotherapy and improving the efficacy of immunotherapy.