PSGL-1 attenuates early TCR signaling to suppress CD8+ T cell progenitor differentiation and elicit terminal CD8+ T cell exhaustion

Highlights

• •PSGL-1 restrains TCR signaling and glycolytic potential of CD8⁺ T cells
• •PSGL-1 deficiency limits CD8⁺ T cell exhaustion and supports precursor populations
• •Co-ligation of PSGL-1 and the TCR promotes T cell exhaustion in CD8⁺ T cells
• •PSGL-1 therapeutic blockade promotes T cell responses and melanoma tumor control

Summary

PSGL-1 (P-selectin glycoprotein-1) is a T cell-intrinsic checkpoint regulator of exhaustion with an unknown mechanism of action. Here, we show that PSGL-1 acts upstream of PD-1 and requires co-ligation with the T cell receptor (TCR) to attenuate activation of mouse and human CD8⁺ T cells and drive terminal T cell exhaustion. PSGL-1 directly restrains TCR signaling via Zap70 and maintains expression of the Zap70 inhibitor Sts-1. PSGL-1 deficiency empowers CD8⁺ T cells to respond to low-affinity TCR ligands and inhibit growth of PD-1-blockade-resistant melanoma by enabling tumor-infiltrating T cells to sustain an elevated metabolic gene signature supportive of increased glycolysis and glucose uptake to promote effector function. This outcome is coupled to an increased abundance of CD8⁺ T cell stem cell-like progenitors that maintain effector functions. Additionally, pharmacologic blockade of PSGL-1 curtails T cell exhaustion, indicating that PSGL-1 represents an immunotherapeutic target for PD-1-blockade-resistant tumors.

Introduction

CD8⁺ effector T cells (T_EFF) are vital in protecting against and clearing virally infected cells and tumor cells. However, in settings of chronic viral infection and cancer, multiple factors including sustained antigen exposure promote T cell exhaustion. Accompanied by progressive decline in effector functions and proliferative capacity, this state is linked to increased co-expression of multiple inhibitory receptors including PD-1, CTLA-4, LAG3, and TIM-3.¹ The discovery of immune checkpoint blockade (ICB) monoclonal antibody therapy to promote antitumor immunity by inhibiting CTLA-4 and/or PD-1 on T cells is one of most significant cancer therapeutic breakthroughs of the last decade, with efficacy shown across many cancers.² Despite dramatic success in some patients, a significant population fails to respond to ICB. It is therefore essential to identify novel mechanisms regulating the differentiation of exhausted T cells (T_EX) that could expand ICB’s applicability to more patients. In particular, it will be critical to develop ICB strategies that support the development and maintenance of stem cell-like (T_SC) or progenitor T_EX (T_PEX) that retain the capacity for proliferation and T_EFF function.³

PSGL-1 (P-selectin glycoprotein ligand-1) is expressed on most hematopoietic cells, including T cells.^4,5 We identified that genetic deletion of PSGL-1 prevented development of T_EX and supported viral clearance in the chronic lymphocytic choriomeningitis virus (LCMV) clone 13 (Cl13) model⁶ due to increased T_EFF function that was accompanied by decreased inhibitory receptor expression. PSGL-1 deficiency also supported growth control of a PD-1-blockade-resistant melanoma tumor.⁶ Furthermore, PSGL-1 deficiency with acute LCMV Armstrong infection promoted greater T_EFF and memory progenitor T cell formation,⁷ underscoring the fundamental role PSGL-1 plays as a regulator of T cell responses. In this study, we investigated cellular and molecular mechanisms by which PSGL-1 signaling intrinsically regulates T_EX differentiation including the impact of PSGL-1 on T cell receptor (TCR) signaling, glycolysis, and the promotion of T_SC and T_PEX. We demonstrate herein that PSGL-1 ligation concomitant with TCR ligation drives T_EX differentiation in human and mouse CD8⁺ T cells. Critically, we show that blockade of PSGL-1 recapitulates PSGL-1 deficiency in antitumor and antiviral responses, and its distinct mechanism of T cell inhibition underscores the translational potential of targeting PSGL-1 by ICB.

Results

PSGL-1 restrains TCR signaling magnitude and duration

Since PSGL-1 deficiency enhanced T_EFF responses, we addressed whether PSGL-1 modulates CD8⁺ T cell responses to TCR engagement. TCR stimulation of naive wild-type (WT) and PSGL-1^−/− OT-I CD8⁺ T cells showed that PSGL-1 deficiency enabled higher expression of the T cell activation markers CD25, CD69, CD44, and PD-1 (Figure 1A)^8,9,10,11 and greater functionality (Figure S1A). To directly evaluate TCR signaling, we assessed phosphorylation of Zap70, Erk1/2, and Akt by western blot (Figure 1B) and found greater activation of each of these signaling molecules in PSGL-1^−/− T cells (Figure S1B), with the greatest effect on pAkt levels in PSGL-1^−/− compared with WT T cells (Figure 1C), demonstrating that PSGL-1 expression inherently limits T cell activation from the time of initial TCR engagement…