Background
In HIV/SIV infection, CD8 T and natural killer (NK) cells are critical antiviral effectors, yet the exact mechanisms for their inability to mediate durable viral control remains unclear and a key challenge for curative strategies. We used single-cell transcriptomics to dissect the heterogeneity of CD8 T and NK cells within lymph nodes (LNs) of SIV-infected, ART-treated rhesus macaques (RMs). Defining effector transcriptional programs in LNs, a key anatomic reservoir, is needed to better understand their contribution to HIV control and for implementing strategies promoting the ability of those cells to control HIV after ART interruption (ATI).
Methods
We characterized transcriptional signatures of CD8 T and NK cell subsets in LN samples from ART-suppressed, SIV-infected RMs dual treated with IL-15+IL-21, in which post-treatment controllers (PTC) and non-controllers (NC) emerged after ATI. Single-cell RNA sequencing was performed on CD3 T (CD3⁺NKG2A⁻) and NKG2A⁺ cells FACS-sorted from LNs collected at the final day of ART from PTC (n=3) and NC (n=3) RMs.
Results
Beyond conventional CD8 T cell clusters, we identified NK-like CD8 T cells expressing NK-associated markers, which we classified as early, intermediate, or mature subsets based on differential expression of LEF1, TIGIT, GZMB, GZMK, CCL4L1, and CCL5. In parallel, early, transitional, and terminally differentiated NK clusters were identified based on the differential expression of NKG7, cytotoxic mediators (PRF1, GZMA, GZMB, GZMK, GZMM, GZMH), and the transcription factor ID2. Interestingly, transitional NK cells were significantly enriched in PTC RMs. While frequencies of other clusters were comparable, transcriptional programs diverged: in many cell subsets, PTC displayed elevated interferon-stimulated genes (ISGs), whereas NC showed higher TGF-β signaling and lipid metabolism pathways. Additional analysis revealed JAK-STAT signaling enrichment in mature NK-like CD8 T cells and apoptotic signaling in transitional and terminally differentiated NK cells specifically in NC RMs.
Conclusions
This study uncovers transcriptionally distinct NK-like CD8 T cell and NK cell subsets within lymphoid tissues, providing a high-resolution map of effector heterogeneity in ART-treated, SIV-infected RMs. Our findings suggest that distinct transcriptional programs, rather than subset frequencies, are associated with post-ATI outcomes. This work refines our understanding of immune mechanisms relevant to HIV persistence and control.