[Delta]42PD1 is a novel negative immune regulator of human B cell

Abstract

Inhibitory receptors such as PD1, CTLA4, and Tim3, play essential roles in regulating T cell activity, including T cell proliferation, cytokines secretion and cytotoxic activity. During virus chronic infection or in the tumor environment, the upregulated inhibitory receptors are associated with the functional exhaustion of T cells, which contributes to virus and tumor survival. Based on this conception, the strategies to treat virus-infected patients and tumor patients by blocking the inhibitory receptors pathway have been widely adopted. Some blocking antibodies have been approved for clinical use and shown encouraging results. Compare to T cells, the inhibitory receptors on B cells are less studied. Recently, a novel PD1 alternatively splice isoform named Δ42PD1 was discovered. Previous studies indicate that Δ42PD1 is expressed on human B cell, and its expression is upregulated on human B cell of HIV infected patients. However, its function was not well understood.

In this study, we find that B cell receptor (BCR) stimulation upregulates Δ42PD1 on the human primary B cell. Expression of Δ42PD1 suppresses B cell proliferation and cell cycle progression. Further investigation reveals that Δ42PD1 recruits both SHP1 and SHP2 in 293T cells, but only SHP1 in B cell, through its intracellular ITSM motif. AKT1-FOXO1 pathway is a critical pathway involved in B cell cycle regulation. Using 293T cells, we find that SHP1 directly interacts with AKT1, inhibits AKT1 activity, and its substrate FOXO1 phosphorylation. Overexpression of Δ42PD1 in B cells inhibits AKT1 activation and downstream FOXO1 signaling, while blockade of Δ42PD1 with a Δ42PD1 specific monoclonal antibody (CH34) can promote B cell proliferation, reduce Δ42PD1 associated SHP1 and reverse Δ42PD1 inhibitory effects on AKT1-FOXO1 signaling. Inhibition of SHP1 with the commercial drug also promote B cell proliferation and promote AKT1-FOXO1 signaling.

SHP1 functions through dephosphorylating phospho-tyrosine residues of target proteins. The regulation of AKT1 by SHP1 has not been reported. To study the underlying mechanism of how SHP1 regulated AKT1 activity, I generate several tyrosine mutations of AKT1. I find that SHP1 dephosphorylate an unidentified phospho-tyrosine site of AKT1. Phosphorylation of this tyrosine site is SRC kinase-dependent. Moreover, AKT1 Tyr474 residue is critical for its activation and phosphorylation of this residue positively correlates with AKT1 activation (Ser473 phosphorylation). Tyr474 residue likely contributes to the stabilization of AKT1 active form by intramolecular interaction with the Leu213 site, which will antagonize the effects of related phosphatase.

Finally, we find that Δ42PD1 was upregulated on B cell of HBV chronically infected patients and its expression negatively correlates with B cell replication in HBV patients. In conclusion, our study demonstrates that Δ42PD1 is a novel inhibitory receptor of human B cell. It inhibits B cell proliferation by recruiting SHP1 and suppression of the AKT1-FOXO1 pathway. Our study also indicates the clinical significance Δ42PD1 on chronic HBV infected patients and the potential use of CH34 as a Δ42PD1 blockade antibody for immunotherapy. (473 words)