Roadmap for spatial transcriptomics of HIV in tissues.

Purpose of review: Mechanisms of HIV persistence in tissues are distinct from that in the blood. Spatial transcriptomic profiling examines HIV-infected cells, surrounding neighborhoods, and tissue microenvironment in unprecedented resolution. Spatial profiling captures cytokine gradients, distances between HIV-infected cells and immune effectors (and their function versus exhaustion), and cell-cell interactions. We present an overview of spatial transcriptomic platforms and a workflow of quality controls, sanity check, and bioinformatic analysis.

Recent findings: The selection of spatial profiling methods should base on the research question, resolution, breadth of coverage, the expression level of RNA of interest, tissue quality, and tissue size. Advanced spatial transcriptomic profiling can capture RNA molecules at high resolution (<1 μm) and thus enable near-single cell profiling at genome-wide (~20 000 genes) breadth. Specifically, poly-A-based mRNA capture can identify previously unknown targets, while targeted RNA capture increases sensitivity in low-quality tissues. In targeted capture, however, the increase in target numbers frequently decreases sensitivity. Coupling ATAC-seq, protein capture, and T cell receptor sequencing to spatial platforms is ongoing.

Summary: Spatial transcriptomic profiling uncovers mechanisms of HIV persistence in tissues and informs therapeutic strategies. Investigators should ensure the rigor of analysis, validate findings, and avoid reporting signatures with unknown biological significance.