Transcriptional and epigenetic regulation of CD8+ T cell differentiation and function

CD8+ cytotoxic T lymphocytes (CTL) are directly responsible for the elimination of intracelullarly infected cells and tumorigenic cells. The biological mechanism and importance of contact-mediated CTL cytotoxicity has been well documented. Fully differentiated effector CTL can directly kill target cells by releasing the contents of cytotoxic granules, cytoplasmic compartments loaded with the pore-forming protein perforin (Prf) and a class of proteases known as granzymes. However, the precise factors that regulate the differentiation of naïve, antigen-inexperienced CD8+ T cells into either cytotoxic effector CTL or memory cells, as well as the mechanisms that control Prf expression during this differentiation process, are incompletely understood. Many factors have been implicated in CTL differentiation. Of these, the cytokine interleukin (IL)-2 plays a prominent role because it is present during clonal expansion, is required for the protective secondary expansion of memory CTL, and has been shown to affect Prf expression. We have taken advantage of an in vitro system to systematically study the effects of different IL-2 receptor (IL-2R) signal strengths during clonal expansion. Our results delineate two phases of CTL differentiation: an early phase initiated by TCR signals that enables re-induction of cytokine and cytotoxic genes upon TCR restimulation, and a subsequent phase in which high or low IL-2R signals regulate reciprocal activation or silencing of IL-7Rα and Prf, expressed by memory-precursor cells and effector cells respectively. T-bet is induced early in response to TCR stimulation, whereas 1L-2R signals upregulate Eomesodermin (Eomes) expression at a later phase. Eomes and Stat5 directly bind the Prf1 locus, promoting epigenetic changes and recruitment of RNA polymerase II (Pol-II). Thus, IL-2R signals and sequential expression of Tbox transcription factors control divergent transcriptional responses that may resemble the programs of effector and memory CTL differentiation in vivo. Furthermore, we find that restimulation-dependent induction of Prf is highly dependent on NFAT and is mediated by increased transcriptional elongation of RNA Pol-II. Additionally, CTL deficient in the transcription factor Runx3 fail to induce Eomes and Prf upregulation in the late phase of clonal expansion. Taken together, these results provide a more detailed view of the transcriptional networks that regulate differentiation of TCR-stimulated CD8+ T cells and cytotoxic gene activation.