PD-1 Signals Inhibit Cell Cycle Progression by Mediating Upregulation of Both KIP and INK Family of Cdk Inhibitors

Abstract 585

The PD-1 pathway plays a critical role in the inhibition of T cell activation and the maintenance of T cell tolerance. PD-1 is expressed on activated T cells and limits T cell clonal expansion and effector function upon engagement with its ligands PD-L1 and PD-L2. PD-1 signals are vital for inhibition of autoimmunity whereas PD-1 ligation by PD-L1 and PD-L2 expressed on malignant cells has a detrimental effect on tumor-specific immunity. Furthermore, PD-1 signals result in T cell exhaustion in several chronic viral infections. The mechanism via which PD-1 signals mediate inhibition of T cell expansion is currently poorly understood. Here, we sought to determine the effects of PD-1 signals on mechanistic regulation of cell cycle progression mediated via TCR/CD3 and CD28 in primary human CD4⁺ T cells using anti-CD3/CD28 with or without agonist anti-PD-1 mAb conjugated to magnetic beads. Cell cycle analysis by ethynyl-deoxyuridine incorporation revealed that PD-1 induced blockade of cell cycle progression at the early G1 phase. To determine the molecular mechanisms underlying the blocked cell cycle progression we examined the expression and activation of cyclins and cdks and the regulation of cdk inhibitors that counterbalance the enzymatic activation of cyclin/cdk holoenzyme complexes. Our studies revealed that PD-1 mediated signals inhibited upregulation of Skp2, the SCF ubiquitin ligase that leads p27^kip1 cdk inhibitor to ubiquitin-dependent degradation, and resulted in accumulation of p27^kip1. Expression of cyclin E that is induced at the G1/S phase transition, and cyclin A that is synthesized during the S phase of the cell cycle, was dramatically reduced in the presence of PD-1 signaling. Strikingly, although expression of cdk4 and cdk2 was comparable between cells cultured in the presence or in the absence of PD-1, cdk2 enzymatic activation was significantly reduced in the presence of PD-1 signaling. Smad3 is a novel critical cdk substrate. Maximum cdk-mediated Smad3 phosphorylation occurs at the G1/S phase junction and requires activation of cdk2. Phosphorylation by cdk antagonizes TGF-β-induced transcriptional activity and antiproliferative function of Smad3 whereas impaired phosphorylation on the cdk-specific sites renders Smad3 more effective in executing its antiproliferative function. Based on those findings, we examined the effects of PD-1 signaling on Smad3 phosphorylation on cdk-specific and TGF-β-specific sites using site-specific phospho-Smad3 antibodies. Compared to anti-CD3/CD28 alone, culture in the presence of PD-1 induced impaired cdk2 activity, reduced levels of Smad3 phosphorylation on the cdk-specific sites and increased Smad3 phophorylation on the TGF-b-specific site. To determine whether the differential phosphorylation of Smad3 might differentially regulate Smad3 transcriptional activity in CD4+ T cells cultured in the presence versus the absence of PD-1, we examined expression of the INK family cdk4/6 inhibitor p15, a known downstream transcriptional target of Smad3. Expression of p15 was upregulated in CD4+ T cells cultured in the presence of PD-1 but not in cells cultured in the presence of CD3/CD28-coated beads alone. These results indicate that PD-1 signals inhibit cell cycle progression by mediating upregulation of both KIP and INK family of cdk inhibitors and Smad3 is a critical component of this mechanism, regulating blockade at the early G1 phase.