Single-cell transcriptional analysis of CD45+ cells in the rhesus brain during acute SIV infection.

People living with HIV are particularly vulnerable to developing a spectrum of neurocognitive abnormalities referred to as HIV-associated neurocognitive disorders. These disorders have been linked to neurological impairment driven by inflammation within areas of the brain controlling cognition. Current data suggest that HIV establishment within the central nervous system (CNS) occurs within the first weeks following infection and drives subsequent neuroinflammatory processes. Various studies have implicated HIV-target cells (i.e., monocytes, macrophages, and CD4 T cells) as key facilitators of HIV CNS establishment via a “trojan horse” mechanism, in which infected cells cross CNS barrier tissues and contribute to viral seeding. Cellular entry is mediated by integrins, specifically a4 integrins, which are expressed at the blood brain barrier and blood cerebrospinal fluid barrier and allow circulating immune cells to enter the CNS. To investigate the contribution of CNS infiltrating cells to early neurological disease, we utilized an acute rhesus macaque SIVmac251 infection model, where animals were treated with an anti-a4 integrin monoclonal antibody to inhibit immune cell trafficking to the CNS. To gain insights into neuro inflammatory transcriptional programs induced following SIV infection and how this might be modulated by a4 integrin blockade, we utilized single cell (sc) RNA sequencing of FACS sorted CD45+ cells isolated from brain and spleen. Overall design: Rhesus macaques were infected intravenously with (10^4 TCID50) SIVmac251 and euthanized at three weeks post-infection. Infected animals were divided into two cohorts and treated with either: (1) 25mg/kg rhesus IgG1 anti-a4 integrin blocking monoclonal antibody or (2) 25mg/kg rhesus IgG1 control antibody (specific for Desipramine). The antibody course was initiated 1 week prior to infection and approximately every 10 days subsequently over the course of the study. Furthermore, a cohort of age matched, untreated, uninfected animals were included as additional controls. At three weeks post-infection, animals were euthanized, and mononuclear cells were collected from the spleen and the right brain hemisphere. Once mononuclear cells were isolated, they were cryopreserved in DMSO and 10% FBS. Cells were subsequently thawed and CD45+ cells enriched using Miltenyi Biotec CD45 Microbeads for non-human primates. Live CD45+ cells were further enriched by fluorescent activated cell sorting for subsequent single cell RNA-seq analyses.