Overexpression of early T cell differentiation-specific transcription factors transforms the terminally differentiated effector T cells into less differentiated state.

ABSTRACT

The in vivo proliferation and viability of transfused engineered T cells markedly limits the long-term effect of adoptive cell therapy on tumors. The therapeutic efficacy and proliferative potential of T cells are reported to be dependent on the differentiation status of T cells. The T cells at the early stage of progressive differentiation have a long lifespan, strong proliferative potential, and the ability to reconstruct intact T cell subsets. Thus, they are more suitable for adoptive immunotherapy. Previously, it was difficult to obtain a sufficient number of early differentiated T cells by inhibiting the progressive differentiation of T cells or by two-step programming. A more effective strategy is to directly reprogram and dedifferentiate the easily available terminal effector T (TEFF) cells, which are generated in large numbers, into early T cells. This study attempted to overexpress eight (candidate) early differentiation-specific transcription factors (TFs) (LEF1, KLF7, ID3, EOMES, BCL6, TCF7, FOXP1, and FOXO1) in the TEFF cells, which were activated by in vitro stimulation, to promote dedifferentiation into early T cells. In the mature TEFF cells simultaneously overexpressing these specific TFs, the expression pattern of T cell differentiation markers (CCR7 and CD45RO) exhibited a tendency to change to the pattern observed during early differentiation. The transcriptome analysis revealed that the function of differentially expressed genes was mainly concentrated in the cell cycle, growth and development, and effector function. Moreover, many genes related to early differentiated T cells (such as BCL2 and PIM1) were significantly upregulated, while those related to the effector function of TEFF cells were significantly downregulated (such as GZMB, PRF1, and GNLY). Additionally, the TEFF cells overexpressing characteristic TFs exhibited enhanced anti-apoptotic capabilities and decreased secretion of cytokines (IFN-γ and TNF-α). Based on these results, we believe that the TEFF cells were reprogrammed into a less differentiated state after overexpression of the eight specific TFs.