Study On Induced Dedifferentiation Of Terminal Effector T Cells By Overexpression Of Specific Transcription Factors

Objective:To study the feasibility of reprogramming effector T cells(T_EFF)directly into memory T cells by overexpressing multiple T cells early differentiation-specific transcription factors and inducing the dedifferentiation of terminally differentiated T_EFF cells.Background:The poor proliferative potential and viability of genetically engineered T cells in vivo markedly limits the long-term effect of adoptive cell therapy on tumors.The therapeutic efficacy and proliferative potential of T cells have reported to be dependent on the differentiation status of T cells.The T cells at the early stage of progressive differentiation have a longer lifespan,stronger proliferative potential,and the ability to reconstruct intact T cell subsets.Thus,they are more suitable for adoptive immunotherapy.However,a large number of terminal T_EFF cells were obtained after in vitro stimulation and expansion.Currently,it is difficult to obtain a sufficient number of early differentiated T cells by inhibiting the progressive differentiation of early T cells or by“terminal T cells-induced pluripotent stem cells(i PSCs)-early T cells”two-step reprogramming.Therefore,it has important research significance and potential application value to induce T_EFF cells reverse differentiation by overexpression of early T cells specific transcription factors,which can provide a new strategy for obtaining adequate less-differentiated T cells.Method:First,the in vitro transcription kit(T7 promoter RNA polymerase)was used to synthesize capped and tailed mRNA which can efficiently translate in T cells,and the enhanced green fluorescent protein(EGFP)mRNA were used to verify the RNA expression and optimize the electroporation conditions of T cell.Then,we attempted to simultaneously introduce eight early differentiation-specific transcription factors mRNA(LEF1,KLF7,ID3,EOMES,BCL6,TCF7,FOXP1,and FOXO1)into the T_EFF cells which were activated by in vitro stimulation with?-CD3/CD28and high concentration of IL-2.Next,we evaluated the effect of inducing T_EFF cell dedifferentiation through RNA sequencing,detection of T cell differentiation-characteristic phenotypic markers,and cell function experiments(such as ELISA of cytokines,cell cycle and apoptosis).Result:The results of electroporation optimization experiments showed that the best mRNA electrotransfection conditions were 1?g mRNA 200V,2ms,and 1 pulse.Under these conditions,the transfection efficiency of T cells is more than 90%.Transcriptomics analysis revealed significant differences in gene expression patterns between the control group and the co-transfection group.The functions of differentially expressed genes mainly focus on the cell cycle,cell proliferation and effector functions.Many genes related to early differentiated T cells(such as BCL2,PIM1,E2F1,and E2F2)were significantly upregulated,while those related to the effector function of T_EFF cells were significantly downregulated(such as GZMB,PRF1,GNLY,and PDCD1).More importantly,the transcription factors overexpressed T_EFF cells showed a gene expression pattern that was highly similar to that of early differentiated T cells(na(?)ve T cells and stem cell-like memory T cells).In addition,after the specific transcription factors mRNA were simultaneously introduced into the mature T_EFF cells,the expression pattern of T cells differentiation markers exhibited a tendency to change to the pattern observed during early differentiation as evidenced by the upregulation of CCR7 expression ratio and the downregulation of CD45RO expression ratio.We also found that the T_EFF cells exhibited an enhanced anti-apoptotic capability and a decreased secretion of T cells effector function related cytokines(IFN-?and TNF-?)after overexpression of characteristic transcription factors.Conclusion:Based on these results,we preliminarily believe that the T_EFF cells have a tencency to transform to the early stage of differentiation after overexpression of early T cell differentiation-specific transcription factors.