PD-1 Inhibits TCR Proximal Signaling By Sequestering SHP-2 Phosphatase and Facilitating Csk-Mediated Inhibitory Phosphorylation of Lck
Authors: Bardhan Kankana , Patsoukis Nikolaos , Sari Duygu , Anagnostou Theodore , Chatterjee Pranam , Freeman Gordon J. , Li Lequn , Boussiotis Vassiliki A. 
Published: October 2019

Abstract
Programmed death (PD)-1 is a negative regulator of T cell responses, which controls peripheral tolerance but also suppresses anti-viral and anti-tumor immunity. The biochemical mechanisms via which PD-1 inhibits T cell activation remain poorly understood. The cytoplamsic tail of PD-1 contains two structural motifs, an immunoreceptor tyrosine-based inhibitory motif (ITIM) and an immunoreceptor tyrosine-based switch motif (ITSM). It has been reported that SHP-2 tyrosine phosphatase constitutively interacts with the ITSM and has a critical role in PD-1-mediated inhibitory function but the mechanism remains unclear. Although phosphatases are generally considered negative regulators of activation, somatic mutations of SHP-2 leading to gain of function have been identified in MDS and AML, where they induce activation of Ras-MAPK cascade. In contrast, fibroblasts and T cells from SHP-2 deficient mice display impaired activation of MAPK in response to growth factor-mediated activation and TCR/CD3-mediated activation, respectively. These studies suggest that SHP-2 has a role in activating some signaling pathways. In the present study we sought to determine the mechanism via which PD-1: SHP-2 interaction leads to inhibition of T cell activation. SHP-2 has two src homology domains, (N-SH2 and C-SH2) and one PTP domain. To identify the region of SHP-2 that interacts with PD-1, we generated five different GST-fusion proteins in which GST was fused with either SHP-2 full length (FL), SHP-2-N-SH2, SHP-2-C-SH2, SHP-2-ΔN-SH2 (lacking the N-terminus SH2 domain) or SHP-2-PTP. Pull-down assays using lysates from naive or activated primary human T cells revealed that PD-1 interacted with full length GST-SHP-2 fusion protein only after TCR/CD3-mediated activation and simultaneous PD-1 ligation. Pull-down assays with each GST-fusion protein showed that this interaction was mediated selectively via the SH2 domains of SHP-2, indicating that PD-1 requires prior tyrosine phosphorylation for interaction with SHP-2. The TCR itself lacks kinase activity but constitutively interacts with Fyn, the most TCR-proximal tyrosine kinase, which induces TCR phosphorylation upon oligomerization of the TCR/CD3 chains. To determine whether Fyn can also induce PD-1 phosphorylation during simultaneous ligation with TCR/CD3, we used COS cells to express PD-1 together with empty vector, Fyn, or a kinase inactive mutant of Fyn, followed by pull-down with GST-SHP-2 fusion protein. No interaction between PD-1 and GST-SHP-2 fusion protein was detected in lysates from COS cells expressing empty vector or kinase inactive Fyn mutant. In contrast, in the presence of active Fyn, PD-1 underwent phosphorylation and association with SHP-2. A key event required for the activation of the TCR/CD3 downstream signaling cascade is the downregulation of Csk-mediated inactivating C-terminal phosphorylation of Lck on Tyr-505. In epithelial cells, upon growth factor stimulation, SHP-2 is recruited to the plasma membrane and abrogates Csk recruitment and Csk-mediated inactivating C-terminal phosphorylation of Src kinase. We examined whether SHP-2 might be recruited to the plasma membrane during TCR/CD3-mediated stimulation and whether this event might be affected by PD-1: SHP-2 interaction. Using nitrogen cavitation to fractionate cytoplasmic and membranous compartments, we determined that stimulation of primary human T cells by TCR/CD3 resulted in robust membrane translocation of SHP-2. This event correlated with downregulation of the inactivating phosphorylation of Lck on Tyr-505. In contrast, stimulation of T cells with simultaneous ligation of PD-1 resulted in PD-1: SHP-2 association and sequestration, and prevented the translocation of SHP-2 to the membranous fraction and the downregulation of the inactivating Lck phosphorylation on Tyr-505. Moreover, phosphorylation of Lck substrates including PLC-γ1 was impaired. Consistent with the key role of PLC-γ1 in the activation of Ras/MAPK via RasGRP1, activation of Ras and MAPK was also defective. Our results unravel a previously unidentified mechanism via which PD-1 inhibits T cell activation by sequestering SHP-2 and preventing its translocation to the plasma membrane where SHP-2 plays a key role in activating the TCR signaling cascade by reversing Csk-mediated inactivating phosphorylation of Lck.

Disclosures
No relevant conflicts of interest to declare.