Moodley, M.; Chasara, C.; Khaba, T.; Mahlobo, B.; Reddy, N.; Nxele, S.; Msipa, S.; Reddy, K.; Ngubane, T.; Jajbhay, I.; Pansegrouw, J.; Ndung'u, T.; Ndhlovu, Z. M.

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.