a4 Blockade Reduces CD8 TEM Cells in the Brain and Elevates CNS Viral Loads

Background. The blood-brain barrier (BBB) regulates immune cell entry into the CNS, and various strategies have been developed to target this barrier in efforts to alleviate neuroinflammation in neurodegenerative diseases. a4ß1 is crucial for T cell influx into the CNS. In the context of HIV, CD4 T cells drive viral infiltration, while CD8 T cells are essential for viral clearance. This dual role complicates strategies aimed at targeting T cell entry during HIV infection, yet it remains an area that has not been thoroughly explored. Methods. We investigated the impact of disrupting a4-mediated T cell entry into the CNS in SIVmac251-infected rhesus macaques treated with an a4-blocking antibody (n=4) versus an isotype control (n=4). Our comprehensive approach included single-cell analysis of CD45+ cells in the brain and spleen, spatial transcriptomics of the hippocampus, viral load measurements, and flow cytometry on brain tissue. Results. Single-cell analysis revealed a marked reduction in the brain CD8 TEM cluster, particularly in cells expressing Granulysin, CCL5, and STAT4, while CD4 T cell clusters remained unchanged, and two monocyte clusters were enriched. This shift was accompanied by an increase in brain SIV RNA+ CD45+ CD4+ T cells in the a4-treated group (vRNA+ cells = 60) compared to controls (n=11). Spatial transcriptomics of SIV vRNA+ versus SIV vRNA- regions in the hippocampus of a4-treated macaques showed enrichment of CD4 TCM and monocyte gene signatures. Correspondingly, quantitative PCR revealed higher vRNA levels in the gray matter of the PFC, STS, and Hp. These findings suggest that a4 blockade selectively disrupts CD8 TEM populations while sparing CD4 T cells, leading to elevated viral loads and heightened neuroinflammation. The increase in SIV RNA+ CD4+ T cells and viral loads in the gray matter furthermore underscores the compartmentalized effect of a4-mediated immune modulation on viral dynamics across distinct brain regions Conclusion. These results underscore the critical need to evaluate immune-targeting therapies, with implications for strategies aimed at controlling T cell influx in the context of viral infections in the brain. Overall design: Sections from the prefrontal cortex and hippocampus of six rhesus macaques were collected 3 weeks post-infection with SIVmac251. Animals were treated with either a monoclonal antibody targeting a4 integrin (n=3) or an isotype control (n=3). Morphological markers, including CD45, CD3, and NeuN, were used to define regions of interest. RNAscope was employed to further delineate SIV-positive and SIV-negative regions for transcriptomic analysis.