Background
Identifying biomarkers of latently infected cells is essential for eliminating the HIV reservoir. Since HIV proviruses exhibit activity even during latency, single-cell (sc) RNA-seq can detect gene expression patterns in these cells. Our prior in vitro study (PMID 38156317) revealed that transcriptional profiles of latent HIV+ cells vary depending on the environment, though certain genes were consistently expressed across conditions. We now aim to validate these biomarkers in CD4+ T cells from people with HIV (PWH) and investigate their tissue specificity.
Methods
We collected lymph nodes, spleen, and liver via rapid research autopsy (<6h postmortem) from four PWH enrolled in the Last Gift cohort, all on ART with undetectable HIV RNA at death. CD4+ T cells were isolated by negative selection from dissociated tissues and subjected to scRNA-seq (10X Genomics). Reads were mapped to a combined human and autologous HIV genomic reference using CellRanger, and data were analyzed with Seurat v5 in Bioconductor R. Genes with a Bonferroni-adjusted p<0.05 were considered differentially expressed between HIV+ and HIV- cells. Gene set enrichment analysis was performed using the Database for Annotation, Visualization and Integrated Discovery v2024q2.
Results
Participants included three men and one woman, with a median CD4+ T cell count of 317 before death. Across tissues, a total of 78 CD4+ T cells with low-level HIV RNA were detected. Differential gene expression analysis between HIV+ and HIV- CD4+ T cells revealed 84 genes in blood, 217 in lymph nodes, 141 in spleen, and 129 in liver. Two of the markers identified in vitro, LGALS1 and S100A11, were differentially expressed in blood and all tissues (Figure). Proteins encoded by these genes, along with proteins encoded by CD2 and ITGAL, function in cell-cell adhesion. Tissue dependencies were also observed (Figure). For example, the previously reported marker CD2 and another transcript, ITGAL, were validated in all tissues but liver, and cytotoxic genes KLRD1, GZMA and HCST were validated in all tissues but lymph nodes.
Conclusions
This study validated biomarkers of HIV persistence in PWH using scRNA-seq, highlighting the influence of tissue microenvironments on latently infected CD4+ T cells. While several in vitro markers were confirmed, tissue-dependent variations underscore the complexity of HIV latency. These findings indicate that tissue-specific biomarkers must be considered when designing strategies to target and eliminate the HIV reservoir.
Table or Figure
