SHP-1 and SHP-2 Associate with Immunoreceptor Tyrosine-Based Switch Motif of Programmed Death 1 upon Primary Human T Cell Stimulation, but Only Receptor Ligation Prevents T Cell Activation

TLDR

The cytoplasmic tail of PD‑1 contains two structural motifs, an ITIM and an immunoreceptor tyrosine‑based switch motif (ITSM). The study aimed to identify cis‑ and trans‑acting factors that mediate PD‑1 signaling in primary human CD4 T cells by constructing a chimeric molecule comprising the murine CD28 extracellular domain fused to the human PD‑1 cytoplasmic tail. The authors used this CD28–PD‑1 chimeric construct to probe PD‑1 signaling in primary human CD4 T cells. The construct suppressed T cell expansion and cytokine production; mutation of the ITIM had little effect, whereas mutation of the ITSM abolished suppression, and biochemical analysis revealed that recruitment of SHP‑1 and SHP‑2 to the ITSM—rather than SHP‑1/2 association alone—was required for inhibition, indicating that PD‑1 must be ligated to block T cell activation.

Abstract

Abstract To study the cis- and trans-acting factors that mediate programmed death 1 (PD-1) signaling in primary human CD4 T cells, we constructed a chimeric molecule consisting of the murine CD28 extracellular domain and human PD-1 cytoplasmic tail. When introduced into CD4 T cells, this construct mimics the activity of endogenous PD-1 in terms of its ability to suppress T cell expansion and cytokine production. The cytoplasmic tail of PD-1 contains two structural motifs, an ITIM and an immunoreceptor tyrosine-based switch motif (ITSM). Mutation of the ITIM had little effect on PD-1 signaling or functional activity. In contrast, mutation of the ITSM abrogated the ability of PD-1 to block cytokine synthesis and to limit T cell expansion. Further biochemical analyses revealed that the ability of PD-1 to block T cell activation correlated with recruitment of Src homology region 2 domain-containing phosphatase-1 (SHP-1) and SHP-2, and not the adaptor Src homology 2 domain-containing molecule 1A, to the ITSM domain. In TCR-stimulated T cells, SHP-2 associated with PD-1, even in the absence of PD-1 engagement. Despite this interaction, the ability of PD-1 to block T cell activation required receptor ligation, suggesting that colocalization of PD-1 with CD3 and/or CD28 may be necessary for inhibition of T cell activation.