Spatial Constraints Limit Natural Killer Cell Access to Germinal Center HIV Reservoirs in Lymph Nodes

BACKGROUND: Antiretroviral therapy effectively suppresses HIV-1 replication in blood but fails to eradicate viral reservoirs in lymphoid tissues, particularly in germinal centres (GCs) of lymph nodes (LNs). While CD8+ T cells are largely excluded from GCs, the role of natural killer (NK) cells, potential alternative effectors, remains poorly defined in human LNs during HIV infection. We investigated NK cell phenotype, function, and localization in paired LN and peripheral blood samples from South African women living with HIV (WLWH) and HIV-negative controls. METHODS: Using immunofluorescence microscopy, flow cytometry, and spatial analysis, we characterized NK cell subsets, assessed CXCR5 expression, and mapped their distribution relative to HIV-infected GC regions. RESULTS: NK cells displayed distinct compartmentalized phenotypes, with CD56+CD16+ cells enriched in blood and CD56+CD16- cells predominating in LNs, irrespective of HIV status. Within LNs, NK cells were largely excluded from GCs and localized to the extrafollicular and mantle zones. CXCR5+ NK cells, with follicular homing potential, were rare across all donors and not enriched in WLWH. NK cell cytolytic activity was predominantly restricted to CXCR5- subsets outside follicles during viraemia, despite elevated granzyme B expression. Spatial modelling confirmed NK cell segregation from HIV-infected GC regions, limiting their antiviral potential. CONCLUSION: LN-resident NK cells are functionally and spatially constrained during HIV infection, highlighting a disconnect between NK cell effector capacity and the localization of the HIV reservoir. Enhancing NK cell trafficking or function within lymphoid tissues may therefore represent a promising avenue for HIV remission or cure strategies.