Abstract Immune checkpoints (ICPs) exert inhibitory or stimulatory effects on immune defense, surveillance, regulation, and self-tolerance through their ligand-receptor interactions. ICPs exist in both membrane-bound and soluble forms in vivo. Imbalances between inhibitory and stimulatory membrane-bound ICPs (mICPs) in malignant cells and immune cells in the peripheral blood and tissues have been well documented. Blockade of inhibitory mICPs such as CTLA-4, PD-1, and PD-L1 has become a revolutionary treatment for advanced stages of malignancies such as melanoma. However, the origin, regulation, and biological significance of both mICPs and soluble ICPs (sICPs) in the central nervous system (CNS) have not yet been systematically studied. We used a multiplex immunoassay to simultaneously quantify the concentrations of 16 sICPs in cerebrospinal fluid (CSF) samples from 33 HIV-negative individuals (HNIs) and 105 people living with HIV (PLWH), including 80 cases with HIV-associated neurocognitive disorder (HAND) and 25 cases without HAND. We found that 10 sICPs (sHVEM, sCD27, sGITR, sICOS, sLAG-3, sPD-1, sTIM-3, sBTLA, sCD28, & sCD40) were consistently detected in all CSF samples, and most of them were highly elevated in PLWH. Strikingly, CSF sHVEM was significantly increased in HAND when compared to PLWH without HAND. These 10 sICPs were also detectable in the CSF samples from rhesus macaques (RMs). Immunohistochemistry (IHC) analysis of brain tissues (frontal cortex and hippocampus) from RMs with and without simian immunodeficiency virus (SIV) infection showed that mHVEM was expressed at a basal level on neurons, but not on astrocytes or microglia, and mHVEM expression was highly increased in SIV-infected RMs (SIV/RMs). Furthermore, we have recently reported that PD-L1 expression in U87MG cells (a human astrocyte cell line) can be robustly induced by a mixture of IFN-γ/IL-1β/TNF-α, and induced PD-L1 controls production of MCP-1 (the most important chemokine that regulates migration of monocytes/macrophages) via PD-L1 intrinsic signaling. Thus, we hypothesize that both sICPs and mICPs are dysregulated in the CNS of PLWH, which are associated with HAND neuropathogenesis. We have two Specific Aims to test this central hypothesis: (1) To determine sICP profiles and biological functions in the CNS of PLWH and SIV/RMs, and (2) To characterize the cellular niches and spatial associations of ICP receptor/ligand-positive neural cells and infiltrating immune cells in the brain of PLWH and SIV/RMs. Our results will provide insights into the roles of ICPs in the CNS neuroimmune dysregulation and neuroinflammation of PLHIV with and without HAND. The CSF levels and profiles of sICPs such as sHVEM can potentially be used as a biomarker for HAND severity and progression.

PROJECT NARRATIVE Our research aims to study the levels, profiles, and clinical relevance of immune checkpoints (ICPs), key regulators of immune homeostasis and defense, in the brain of HIV-infected people with or without neurocognitive disorder (HAND). Our study will not only lead to the discovery of potential HAND biomarkers but also uncover the roles of ICPs in the pathogenesis of HAND.