STAT3, STAT4, NFATc1, and CTCF Regulate PD-1 through Multiple Novel Regulatory Regions in Murine T Cells

Abstract

Programmed cell death-1 (PD-1) is a crucial negative regulator of CD8 T cell development and function, yet the mechanisms that control its expression are not fully understood. Through a non-biased DNase I hypersensitivity assay, four novel regulatory regions within the Pdcd1 locus were identified. Two of these elements flank the locus, bind the transcriptional insulator protein CTCF, and interacted with each other, creating a potential regulatory compartmentalization of the locus. In response to T cell activation signaling, NFATc1 bound to two of the novel regions that function as independent regulatory elements. STAT binding sites were identified in these elements as well. In splenic CD8 T cells, TCR-induced PD-1 expression was augmented by interleukin 6 and 12, inducers of STAT3 and STAT4 activity, respectively. IL-6 or IL-12 on its own did not induce PD-1. Importantly, STAT3/4 and distinct chromatin modifications were associated with the novel regulatory regions following cytokine stimulation. The NFATc1/STAT regulatory regions were found to interact with the promoter region of the Pdcd1 gene providing a mechanism for their action. Together these data add multiple novel distal regulatory regions and pathways to the control of PD-1 expression and provide a molecular mechanism by which proinflammatory cytokines, such as IL-6 or IL-12 can augment PD-1 expression.

IL10 Release upon PD-1 Blockade Sustains Immunosuppression in Ovarian Cancer

“…2E–F). These results are consistent with the recent findings that implicate activated STAT3 as an important transcription factor in PD-1 expression (28). …”

“…The IL-10-mediated increase in PD-1 expression on DCs was dependent on STAT-3 activity, as use of STAT-3 inhibitor or siRNA mediated knockdown of STAT3 both resulted in inability of IL-10 to increase PD-1 expression on DCs. These results support the previous findings implicating STAT-3 in mediating PD-1 expression on CD3/CD28 stimulated T cells upon IL-6 treatment (28). …”

Genetic and Epigenetic Regulation of PD-1 Expression

“…These elements encode STAT binding sites and were found to interact directly with the Pdcd1 promoter following ex vivo cell activation and cytokine treatment (62). Additional elements at −26.7 and +17.5 kb bind the mammalian transcriptional insulator CCCTC-binding factor (CTCF) and form constitutively interacting chromatin loops (Figure 1) (62).…”

“…These elements encode STAT binding sites and were found to interact directly with the Pdcd1 promoter following ex vivo cell activation and cytokine treatment (62). Additional elements at −26.7 and +17.5 kb bind the mammalian transcriptional insulator CCCTC-binding factor (CTCF) and form constitutively interacting chromatin loops (Figure 1) (62). Because transcriptional insulators prevent the actions of distant enhancers from controlling a downstream gene (67), these CTCF sites likely define the extreme ends of the Pdcd1 regulatory locus.…”

“…Thus, NFATc1 is necessary for initial activation-induced expression of PD-1 in CD4 and CD8 T cells. PMA/Io mediated NFATc1 binding and transcriptional activity was also found at the −3.7 and +17.1 sites, suggesting that multiple NFATc1 elements are required for driving Pdcd1 expression or that there is genetic redundancy in the mechanism by which Pdcd1 is induced (62). Intriguingly, NFATc1 expression is repressed at extended time points after induction and this is likely part of the mechanism for restricting PD-1 expression during acute antigen exposure (53).…”