CD68+ Follicular Macrophages Harbor HIV Reservoirs in Human Lymph Node Tissues During Suppressive ART

Uncertainty persists regarding the contribution of tissue macrophages to HIV reservoirs, largely due to insufficient characterization of these reservoirs within their native tissue microenvironments. This study aimed to characterize and quantify macrophage reservoirs in human lymph node (LN) tissues in terms of their phenotype, location, and their potential for sustained productive infection during suppressive antiretroviral therapy. We examined the topology, nature, and size of macrophage reservoirs in lymph nodes (LNs) from 45 PLWH subtype C on suppressive ART and 14 matched controls using in situ imaging and multiplexed immunofluorescence microscopy. Germinal center CD68+ macrophages harbored HIV gag-pol DNA, HIV gag-pol RNA and Gagp24 protein. Digital droplet PCR confirmed the presence of proviral reservoirs in myeloid cells within LNs. High-resolution imaging techniques revealed that infected macrophages within GCs displayed distinct morphological characteristics, featuring larger and irregular shapes. In contrast, phagocytic macrophages exhibited intracellular staining for CD4+ T cells, had regular shapes, and were predominantly found outside the GCs. Our findings provide detailed quantitative, spatial, and phenotypic characterization of macrophage reservoirs in LNs, offering a clear estimation of the extent to which macrophages contribute to persistent HIV reservoirs in these tissues. These findings establish a basis for developing targeted strategies aimed at the elimination of these reservoirs in LN tissues. Author summaryHIV hides in reservoirs within immune cells across the body in the blood and various tissues, making it a complex challenge to cure. Therefore, it is essential to identify and understand all sources of HIV reservoirs aid the development of an HIV cure. Macrophages are increasingly recognized as key contributors of viral reservoir persistence. However, the role of macrophages as latent HIV reservoirs remains unclear due to limited studies in human tissues. In this study we investigated macrophage reservoirs in human lymph nodes to answer key questions: where do they hide, how can we identify them, what is their contribution to the lymph node reservoir burden, and are they truly able to support productive viral replication? We found that macrophages residing within lymph node germinal centers, identified by CD68 expression, contained HIV DNA, RNA, and Gagp24 protein. Moreover, using high resolution microscopy, we were able to distinguish between productively infected macrophages from those that engulfed T cells. By unveiling these unique features of macrophage reservoirs, our research paves the way for the design of targeted therapy aimed at eliminating these reservoirs, towards an HIV cure.