Study Of The Effect Of Silencing Histone Deacetylase 11 On Immune Function Of NKG2D-CAR-T

With the approval of FDA for two CD19-CAR-T products from Novartis and Kite Pharma,CAR-T cell immunotherapy has rapidly become the key point of research in the field of anti-cancer.However,there are still many problems restricting the development of this therapy,such as the high price of products,poor treatment effect on solid tumors,and the difficulty of proliferation and survival in vivo.Therefore,we design an enhanced CAR-T to solve the above problems.Histone deacetylase 11（HDAC11）can negatively regulate the immune function of T lymphocytes by negatively regulating the acetylation levels of T-bet and Eomes’s promoters.Based on the killing effect of NKG2D-CAR-T on prostate cancer cells,this study explored the effect of knockdown of HDAC11 on the immune function of NKG2D-CAR-T cells by RNAi.Firstly,four shRNAs against HDAC11 gene and one negative control（NC）sequence were synthesized according to the mechanism of RNA interference and the principle of shRNA design.Sequencing results showed that the above sequences were accurately inserted into the vector pLL3.7-U6-EF1α-EGFP.The lentiviruses were packaged successfully and the positive rate of infected Jurkat cells was close to 100%.The interference sequence shRNA-D was identified to have the best interference effect at the level of mRNA and protein,and the interference efficiency was nearly 60%.Then we successfully inserted the optimal interference sequence shRNA-D into pLL3.7vector with NKG2D-CAR targeting sequence,the recombinant plasmid was identified by sequencing analysis and named as pLL3.7-shRNA-D-NKG2D-CAR.The lenti-viruses were packaged and the titer of the concentrated viruses was over 2?10⁸ TU/ml.The infection efficiency of T cells was up to 80%and the expression of HDAC11protein in NKG2D-CAR-T was down-regulated by nearly 80%.Cytotoxicity data showed that silencing of HDAC11 could enhance the killing ability of NKG2D-CAR-T cells on prostate cancer cells.Furthermore,the expression level of CD69,IFN-?and GzmB was significantly up-regulated,and higher proporation of CD107a-positive T cells were detected in shRNA-D-NKG2D-CAR-T cells,suggesting that the knockdown of HDAC11 could enhance the cytotoxic activity,activation level and degranulation ability of NKG2D-CAR-T cells.Therefore,silencing of HDAC11 could significantly enhance the immune function of NKG2D-CAR-T.In addition,we also explored the effects of silencing HDAC11 on proliferation,cell exhaustion and differentiation of memory T cells of NKG2D-CAR-T cells.The results showed that the proliferation of NKG2D-CAR-T cells was significantly enhanced and the exhausting markers of PD-1 and TIM-3 was significantly decreased after HDAC11 protein was down-regulated,suggesting that NKG2D-CAR-T cells had stronger survival ability and longer survival time in vivo after silencing of HDAC11.Futhermore,silencing of HDAC11 could increase the proportion of central memory T(T_CM)cells,indicating that the down-regulation of HDAC11 protein can prolong the time of maintaining immune memory.To explore its mechanism,we found that the expression level of transcription factors T-bet and Eomes in NKG2D-CAR-T cells increased after silencing of HDAC11,suggesting that HDAC11 affected the function of NKG2D-CAR-T cells by regulating the expression of T-bet and Eomes.In conclusion,the study has designed and prepared an enhanced CAR-T cell by silencing of HDAC11,and the novel CAR-T cells had stronger ability of cytotoxicity and cell proliferation,longer exhausting period and higher proporation of T_CM,which provides a new idea to prepare function-enhancing CAR-T cells.