PD-1/SHP-2 negatively regulate Tc1/Th1 phenotypic responses and activation of T cells in the tumor microenvironment

Abstract

Rejection of tumor cells by a robust cellular immune response relies on production of type 1 cytokines (such as IFN-γ) and cytolytic activity of T cells. Programmed Death 1 (PD-1), a co-inhibitory receptor proposed to represent T cell dysfunction, is highly expressed on tumor infiltrating lymphocytes (TIL) [1], and may reflect T cell exhaustion marked by decreased proliferation, production of type 1 cytokines and poor cytolytic activity [2]. T-bet, a T-box transcription factor which can be activated by phosphorylated signal transducers and activators of transcription 1 (p-STAT1), plays an important role in Tc1/Th1 skewing. Although anti-PD-1 antibodies enhance IFN-γ secretion after TCR stimulation [3], the mechanistic link between PD-1 and Tc1/Th1 skewing remains unclear. In prospectively collected cancer tissues, TIL manifested dampened Tc1/Th1 skewing and activation compared to PBL (Figure ​(Figure11 and ​and2).2). In addition, PD-1 triggering using PD-L1 coated beads further suppressed TCR-stimulated upregulation of p-STAT1, T-bet and p-S6 as well as Th1 cytokines, while PD-1 blockade reversed suppressive effects of PD-1: PD-L1 ligation (Figure ​(Figure3).3). We also found that Src homology-2 domain-containing phosphatase (SHP-2) is higher in TIL than in PBL, tightly correlates with PD-1 expression (Figure ​(Figure4),4), and negatively regulates STAT1 and T-bet activation (Figure ​(Figure5).5). Thus, the PD-1/SHP-2/p-STAT1/T-bet axis provides an important mechanism for PD-1 suppression of type 1 immunity at tumor sites. PD-1 blocking Abs, which are clinically effective in several solid cancers, should improve T cell-based cancer immunotherapy by restoring robust type 1 immunity and T cell activation to reverse immunosuppression in the tumor microenvironment. SHP-2 inhibitory strategies may also be useful to improve type 1-biased TIL. Figure 1 CD8+ TIL have dampened Tc1 phenotypic responses and activation compared to PBL. p-STAT1, T-bet, p-STAT4 and p-S6 levels in CD8+ PBL and TIL from HNC patients were analyzed by intracellular flow cytometry. Representative figures (A) and summary data (B) ... Figure 2 CD4+ TIL show abortive Th1 differentiation and low activation compared with PBL. p-STAT1, T-bet, p-STAT4 and p-S6 levels in CD4+ PBL and TIL from HNC patients were analyzed by intracellular flow cytometry. Representative figures (A) and summary data (B) ... Figure 3 PD-1 ligation with bead-coated PD-L1 suppressed TCR-stimulated up-regulation of p-STAT1, T-bet and production of Th1 cytokines, while anti-PD-1 blockade could reverse the suppressive effects of PD-1. Total TIL were stimulated with anti-CD3/-CD28/hIgG1 ... Figure 4 SHP-2 activation by fusaruside suppresses p-STAT1/T-bet and production of Th1 cytokines upon TCR stimulation. Total TIL were stimulated with anti-CD3/-CD28/hIgG1 beads (bead: cell = 10:1) or anti-CD3/-CD28/PD-L1 beads plus 100 ug/mL anti-PD-1 blockade ... Figure 5 SHP-2 activation by fusaruside suppresses p-STAT1/T-bet and production of Th1 cytokines upon TCR stimulation. Total TIL were stimulated with anti-CD3/-CD28/hIgG1 beads (bead: cell = 10:1) or anti-CD3/-CD28/PD-L1 beads plus 100 ug/mL anti-PD-1 blockade ...