T cell-mediated SIV dissemination into the CNS: a single-cell transcriptomic analysis

HIV infection disrupts immune function in both the periphery and central nervous system by depleting CD4+ T cells and inducing uncontrolled inflammation. CNS infection is initiated when infected blood leukocytes, thought to be monocytes and/or CD4+ T cells, infiltrate the brain and facilitate viral entry. However, the composition and phenotypic characteristics of these "Trojan horse" cells during acute infection remain poorly understood. To address this gap, we performed single-cell RNA sequencing (scRNA-seq) on the brain and blood of rhesus macaques 12 days post-inoculation (p.i.) with simian immunodeficiency virus (SIV). Our analysis of brain immune cells revealed a distinct cell cluster co-expressing both myeloid and lymphoid genes, raising the possibility that brain myeloid cells may engulf CD4+ T cells entering the brain from the blood, and that the lymphocytes represent the Trojan horse. To examine this finding, we conducted scRNA-seq on a macrophage–T cell (Mac-T) coculture system. Interestingly, we identified a substantial population of Mac-T cells under different culture conditions. Furthermore, our analysis suggested that specific chemokines could serve as markers to distinguish Mac-T cells from technical doublet artifacts in scRNA-seq data. Beyond this intriguing cell population, our study identified additional lymphocyte subsets and uncovered phenotypic T cell shifts during acute infection. Notably, we observed a sharp decline in proportion of CD4+ memory T cells alongside an expansion of proliferating CD4+ cytotoxic T cells in both the blood and brain. These cytotoxic CD4+ T cells exhibited high expressions of viral entry receptors (e.g., CD4 and CCR5) and adhesion molecules (e.g., LFA-1 and VLA-4), and had the highest number and proportion of infected cells in the brain, suggesting their role as "Trojan horse" cells that facilitate viral dissemination into the CNS. Additionally, their elevated expression of cytotoxic molecules indicates potential pathogenic effects on the brain. Comparative transcriptomic analysis further revealed that brain lymphocytes were more mature than their blood counterparts, with naïve T cells being nearly absent in the brain. To further examine these findings and assess their temporal dynamics, we integrated publicly available scRNA-seq data from brain lymphocytes at 21 days p.i. This analysis corroborated our key observations and highlighted progressive changes in lymphocyte phenotypes and transcriptomic profiles over time. Overall, this study provides a comprehensive transcriptomic characterization of brain lymphocytes and blood CD4+ T cells during acute SIV infection, identifying a candidate lymphocyte phenotype that can contribute to CNS infection Overall design: For studying brain lymphocyte responses in acute SIV infection, 92T was non-infected control samples and other three (93T, 94T, 95T) were in acute-infected group. The rhesus macaques in the acute-infected group were infected with Simian Immunodeficiency Virus (SIV) for 12 days. For studying monocyte-derived macrophages (MDMs) and jurkats cocultivation, we had four different groups: NoApop, CD95, Campto, NoCoCul groups. The MDMs and jurkats were cocultured in NoApop, CD95, and Campto groups. For NoCoCul groups, the jurkats and MDMs were mixed before library preparation to assess the artefactual dual capture. In CD95 and Campto groups, the jurkats were induced apoptosis by CD95 or camptothecin prior to cocultivation.