The last gift cohort: advancing HIV reservoir research through single-cell and spatial approaches

Purpose of review Despite effective antiretroviral therapy (ART), persistent HIV reservoirs remain the primary obstacle to achieving an HIV cure. Understanding persistence requires defining not only reservoir size but also the identity and state of infected cells and their anatomical microenvironments – features that blood-based bulk measurements cannot resolve. We synthesize published findings from the Last Gift end-of-life cohort, in which longitudinal sampling and tissue donation via rapid autopsy have advanced our understanding of HIV persistence across anatomical compartments. We then highlight how integrated single-cell and spatial profiling can link proviral features to host-cell states and local tissue neighborhoods, helping to define the cellular and microenvironmental niches that sustain persistence. Recent findings The Last Gift studies have mapped HIV DNA and envelope diversity across up to 33 anatomical compartments, inferred lymphoid and gastrointestinal tissues to be dominant sources of viral dispersal, shown that >85% of expanded proviral clones are shared across tissues, and recovered inducible, replication-competent virus from parietal-cortex microglia. Single-cell profiling has resolved heterogeneous microglial transcriptional and chromatin states containing rare HIV RNA or accessible proviral DNA and has identified a transcriptomic definition of cells expressing HIV RNA despite viral suppression on ART. Additionally, mass cytometry of viral sensors and restriction factors has defined tissue- and cell subset-specific antiviral programs, including restriction-factor profiles in lymph-node follicular helper T-cells predicted to favor HIV persistence. In parallel, imaging-based spatial transcriptomics with custom HIV probes, immunoPET/CT-guided tissue sampling, and duplex RNAscope/DNAscope have made direct localization of viral nucleic acids within annotated tissue neighborhoods technically feasible. No single assay, however, establishes proviral intactness, transcriptional activity, cellular identity, spatial position, and neighborhoods at once. Summary Reservoir measurements should be reported by what each assay can establish rather than as interchangeable estimates of reservoir size. Cohorts such as the Last Gift, which yield matched blood and multiorgan tissue from the same donor with longitudinal clinical context, are well suited to applying direct proviral characterization, single-cell phenotyping and spatially resolved viral detection in parallel on the same specimens. This combination defines the resolution – intact provirus, in an identified cell, in a defined tissue niche – that cure trials should increasingly be expected to measure.