MLL Histone Methyltransferase Restrains Effector Differentiation and PD-1 Expression in Human CD8⁺ T Cells While Promoting Their Proliferation

Adoptive cellular therapy (ACT) for cancer requires amplification and persistence of tumor-specific T cells. To completely eliminate malignant tumor cells, infused tumor-reactive T cells must retain the capacity to expand over weeks and months in order to produce sufficient effector cells. However, T-cell potency can be blunted by excessive differentiation and upregulation of PD-1, which mediates exhaustion and impairs their proliferative capacity. Histone methylation is thought to be central in directing transcriptional programs important for effector proliferation, survival and differentiation; however, the epigenetic regulator(s) of this process are not well characterized. We report that the histone methyltransferases Mixed Lineage Leukemia 1 (MLL1) and MLL4, which catalyze trimethylation of histone H3 at lysine 4 (H3K4me3), play important roles in restraining effector differentiation and promoting proliferation of activated human CD8⁺ T cells. Upon T cell receptor (TCR) activation, human CD8⁺ T cells upregulated MLL1 and MLL4, however, down-regulated the global level of H3K4me3. To assess the specific effect of MLL1 in CD8⁺ T cell differentiation, we produced lentivirus encoding short hairpin RNA (shRNA) specific to MLL1 or MLL4. Knockdown of either protein increased the frequency of IFN-γ-producing cells by 50% to 100%, with silencing of MLL1 having the more potent effect. Pharmacological inhibition with MI-2-2, which simultaneously inhibits the menin-MLL1 and menin-MLL4 interactions, increased the frequency of IFN-γ ⁺ CD8⁺ T cells three-fold and reduced cell proliferation from 94% to 64%. Using a second MLL1 inhibitor MM-401, which affects MLL1 specifically, we confirmed that inhibiting MLL1 resulted in a two-to-four-fold increase in the expression of numerous effector molecule transcripts, including IFNG, TNFA, PRF1, FASL and GZMB. Our results suggest that while both MLL1 and MLL4 are important for proliferation of activated CD8⁺ T cells, MLL1 potently restrains effector differentiation. T-BET and EOMES are two transcription factors critical for mediating effector differentiation. We found that inhibition of MLL1 in cultured, TCR-activated CD8⁺ T cells using either MI-2-2 or MM-401 led to a significant increase in expression of EOMES, but had no significant effect on T-BET expression. Flow cytometric analysis showed that silencing MLL1 also increased the expression of EOMES protein in activated CD8⁺ T cells. Interestingly, MLL1 knockdown impaired subsequent persistence of ex vivo expanding CD8⁺ T cells, which was associated with a substantial increase of CD45RO⁺CCR7^- short-lived effector CD8⁺ T cells. Remarkably, knockdown of MLL1 in proliferating CD8⁺ T cells led to their upregulation of PD-1. Taken together, these data suggest that MLL1 may play an important role in restraining precocious effector differentiation and exhuastion in CD8⁺ T cells. Future studies will investigating the impact of both MLL1 and MLL4 in regulating the CAR CD8⁺ T cell response in vivo and in vitro. Results from these experiments will allow us to identify epigenetic mechanisms that regulate the generation and persistence of antitumor effector and memory CD8⁺ T cells.