α4 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. α4β1 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 α4-mediated T cell entry into the CNS in SIVmac251-infected rhesus macaques treated with an α4-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 α4-treated group (vRNA+ cells = 60) compared to controls (n=11). Spatial transcriptomics of SIV vRNA+ versus SIV vRNA- regions in the hippocampus of α4-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 α4 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 α4-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. 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 α4 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.

研究背景。血脑屏障（blood-brain barrier, BBB）可调控免疫细胞进入中枢神经系统（central nervous system, CNS），学界已开发多种靶向该屏障的策略，以缓解神经退行性疾病中的神经炎症。α4β1整合素对于T细胞向中枢神经系统的浸润至关重要。在人类免疫缺陷病毒（human immunodeficiency virus, HIV）感染情境中，CD4阳性T细胞介导病毒侵入，而CD8阳性T细胞则是病毒清除的关键。这种双重作用使得靶向HIV感染期间T细胞中枢神经系统浸润的治疗策略变得复杂，而该领域仍未得到充分探索。
研究方法。本研究针对感染猴免疫缺陷病毒251株（SIVmac251）的恒河猴，分别给予α4整合素阻断型抗体（n=4）与同型对照抗体（n=4），以此探究阻断α4介导的T细胞中枢神经系统浸润的影响。本研究采用的综合分析手段包括：脑与脾脏中CD45阳性细胞的单细胞分析、海马体空间转录组学检测、病毒载量测定，以及脑组织流式细胞术。
研究结果。单细胞分析显示，脑内CD8阳性效应记忆T细胞（CD8 effector memory T cells, CD8 TEM）簇显著减少，尤其是表达颗粒溶素（Granulysin）、趋化因子配体5（CCL5）以及信号转导与转录激活因子4（STAT4）的细胞亚群；而CD4阳性T细胞簇未发生明显变化，同时两个单核细胞簇出现富集。与对照组（vRNA阳性细胞数为11）相比，α4阻断组脑内SIV RNA阳性的CD45阳性CD4阳性T细胞数量显著升高（该组vRNA阳性细胞数为60）。对α4阻断组恒河猴海马体中SIV vRNA阳性与阴性区域的空间转录组学分析显示，CD4阳性中央记忆T细胞（CD4 central memory T cells, CD4 TCM）与单核细胞的基因特征出现富集。相应地，定量PCR检测显示，恒河猴前额叶皮层（prefrontal cortex, PFC）、颞上回（superior temporal sulcus, STS）以及海马体（hippocampus, Hp）的灰质中病毒RNA水平更高。上述结果表明，α4阻断可选择性破坏CD8阳性效应记忆T细胞群，而保留CD4阳性T细胞，进而导致病毒载量升高与神经炎症加剧。SIV RNA阳性CD4阳性T细胞的增加以及灰质中病毒载量的升高，进一步凸显了α4介导的免疫调控对不同脑区病毒动态的分区效应。
研究结论。本研究结果凸显了评估免疫靶向治疗的必要性，该结论对旨在调控中枢神经系统T细胞浸润以控制脑部病毒感染的治疗策略具有重要参考价值。
我们收集了6只感染SIVmac251三周后的恒河猴的前额叶皮层与海马体组织样本。受试动物分别接受靶向α4整合素的单克隆抗体（n=3）或同型对照抗体（n=3）处理。研究采用CD45、CD3与NeuN等形态学标记物划定感兴趣区域，并使用RNAscope原位杂交技术进一步区分SIV阳性与阴性区域，以开展转录组学分析。