Extensive evidence has indicated that peripheral blood HIV-specific CD8+ T cell cytolytic activity and effector functions are associated with the control of HIV replication in HIV elite controllers (EC). However, the majority of HIV replication in EC likely occurs in lymphoid tissue, where CD8+ T cell immune surveillance mechanisms are undefined. Here we performed single-cell RNA sequencing (scRNAseq) analyses to determine the transcriptional signature of HIV-specific CD8+ T cells that control viral replication in lymphoid tissue of EC.
We isolated human lymph node (LN) mononuclear cells from inguinal LN of HIV-infected EC and cervical LN of chronic progressors (CP) from the SCOPE cohort at UCSF and the Center for Investigation of Infectious Diseases (INER-CIENI) in Mexico City, respectively. We index sorted single HIV-specific CD8+ T cells, as identified by MHC-class I tetramers, and subjected these cells for scRNAseq using the SMARTseq-v2 platform. Functional assays were analyzed on a BD LSR II flow cytometer. The results were analyzed using RStudio, FlowJo, and GraphPad Prism.
Using an unsupervised scRNAseq analysis approach, we observed distinct clustering between EC cells and CP cells driven by 2264 differentially expressed genes. Compared to CP, EC cells expressed lower levels of cytolytic genes, and heightened expression of several secreted molecules with potential anti-HIV activity, including as CCL5, TNF, and IL32. In order to determine a gene signature that could distinguish EC cells from CP cells, we next used a supervised classification approach, yielding a list of 200 genes that were enriched for immune-related and protein translation-related genes. Within this gene signature, EC cells showed a downregulation of inhibitory receptor genes and an upregulation of specific cytokines and ribosome subunits, implying that these cells are highly functional. We functionally confirmed this signature with ex vivo peptide stimulation polyfunctional cytokine and protein translation assays, finding heightened polyfunctional and protein translation capacity in lymph node CD8+ T cells from HIV EC.
Our findings suggest that protective HIV-specific CD8+ T cells in lymphoid tissue of EC are defined by unique non-cytolytic functional features with a high capacity to translate mRNA into protein upon antigen encounter, and call into question known correlates of protection mediated by peripheral blood CD8+ T cells.