The Phosphorylation State of Ezh2 Determines Its Capacity to Maintain CD8+ Memory T Cells for Antitumor Immunity

Abstract

Memory T cells are under active investigation in the context of inhibitory checkpoint blockade therapy and vaccination. In adoptive cell therapies (ACT), minimally differentiated CD8+ T cells, including naïve and central memory T cells, exhibit superior persistence and enhanced antitumor activity compared with highly differentiated effector T cells. Thus, developing strategies to enrich minimally differentiated subsets in cellular products could significantly enhance efficacy and durability of ACT. T cell signal strength, along with costimulatory signals and cytokines, are known to substantially shape the amplitude and quality of the T cell response. Downstream of these signals, epigenetic regulation is thought to critically mold transcriptional programs of memory precursors, however the specific epigenetic regulator(s) that establish and maintain these states are not well characterized. We report that Ezh2 is a key molecular gatekeeper for generation of memory CD8+ T cells, acting to both restrain terminal differentiation and maintain recall response capability. Using experimental ACT models, we found that transfer of melanoma-associated antigen gp100-specific CD8+ T cell receptor transgenic Pmel-1 cells repressed the growth of pre-established B16 melanoma. In contrast, loss of Ezh2 in Pmel-1 T cells failed to repress the tumor growth. Ezh2 deficiency caused a preferential decrease of the central memory precursor (TCMP) pool independent of cell apoptosis early during expansion phase (3 days post-stimulation), while markedly increasing the fraction of terminal effector differentiation. Seven days after priming, Ezh2-deficient CD8+ T cells exhibited increased apoptosis, decreased generation of mature memory T cells and impaired recall response capability upon rechallenge with tumor-associated antigen. Since both the first division of activated CD8+ T cells and their subsequent differentiation early after antigen priming may influence the ratio of TCMP and effector cells, our findings suggest that Ezh2 is important for preserving the TCMP pool and restraining precocious terminal differentiation. Genome-wide gene profiling analysis and functional assessment revealed that Ezh2 achieved these effects by orchestrating the expression of transcription factors critical for effector and memory differentiation. Ezh2 activated Id3 while silencing Id2, Prdm1 and Eomes, promoting the expansion of memory precursor cells and their differentiation into functional memory cells. However, these data could not explain our previous observations (Blood 2012, Blood 2013, Blood 2017) that during the immune response, normal CD8+ T cells expressed high levels of Ezh2 upon antigen activation, yet, they still underwent a “programmed” differentiation into terminal effector cells. We hypothesized that Ezh2 may undergo a chemical modification capable of altering its function in regulating memory in T cells. Our chromatin immunoprecipitation analysis showed that Ezh2 was dissociated from the promoter regions of these key transcription factors (e.g., Id3, Id2, Prdm1 and Eomes), which occurred 3 days after activation and persisted throughout 7 days of in vitro stimulation. This decreased presence of Ezh2 was paralleled by a reduction of H3K27me3 at the Prdm1 and Eomes loci. In these proliferating T cells, active Akt mediated phosphorylation of Ezh2. Inhibiting Akt activity significantly increased the amount of Ezh2 and H3K27me3 at the promoter regions of Id3, Id2, Eomes and Prdm1 loci. Furthermore, we found that retroviral engineering CD8+ T cells with Akt-phosphorylation-resistant Ezh2 significantly enhanced their generation of long-lasting memory T cells and antitumor activity in vivo. In conclusion, our findings are conceptually and translationally innovative, because they identify the importance of Ezh2 in regulating memory formation and function, and discover a transcription factor network targeted by Ezh2 crucial for regulating the balanced generation of effector and memory T cells. Furthermore, these findings illuminate the Akt-dependent signaling mechanisms responsible for controlling Ezh2-mediated epigenetic control of step-wise differentiation of effector and memory T cells. We propose that Akt-mediated phosphorylation of Ezh2 is a critical target to potentiate anti-tumor immunotherapeutic strategies. DisclosuresNo relevant conflicts of interest to declare.