scRNA-Seq reveals that previous studies in blood have largely failed to capture the major component of HIV-specific CD8+ T cell responses in lymphoid tissues

Current paradigms of CD8⁺ T cell–mediated protection in HIV infection center almost exclusively on studies of peripheral blood, which is thought to provide a window into immune activity at the predominant sites of viral replication in lymphoid tissues (LTs). Through extensive comparison of blood, thoracic duct lymph (TDL), and LTs in different species, University of Pennsylvania researchers show that many LT memory CD8⁺ T cells bear phenotypic, transcriptional, and epigenetic signatures of resident memory T cells (T_RMs). Unlike their circulating counterparts in blood or TDL, most of the total and follicular HIV-specific CD8⁺ T cells in LTs also resemble T_RMs. Moreover, high frequencies of HIV-specific CD8⁺ T_RMs with skewed clonotypic profiles relative to matched blood samples are present in LTs of individuals who spontaneously control HIV replication in the absence of antiretroviral therapy (elite controllers). Single-cell RNA sequencing analysis confirmed that HIV-specific T_RMs are enriched for effector-related immune genes and signatures compared with HIV-specific non-T_RMs in elite controllers. Together, these data indicate that previous studies in blood have largely failed to capture the major component of HIV-specific CD8⁺ T cell responses resident within LTs.

Distribution of HIV-specific clonotypes between LN and blood

(A) scRNA-seq was conducted on CD69⁺ and CD69⁻ index-sorted HLA-B*2705–restricted and/or HLA-B*5701–restricted Gag-specific CD8⁺ T cells from LNs of two elite controllers. The heat map (left) illustrates the single-cell gene expression variability of differentially expressed genes (P < 0.01), and violin plots (right) illustrate immune-related genes differentially expressed between CD69⁺ and CD69⁻ single HIV-specific CD8⁺ T cells. (B) CDR3 amino acid sequence and percent frequency are shown for each HLA-B*2705–restricted and HLA-B*5701–restricted clonotype. The frequency of CD69 expression for each clonotype was obtained from single-cell index data (right). (C) CD69 expression on LN clonotypes, where higher frequencies of specific clonotypes are present in LNs versus blood (LN > blood; n = 7) or vice versa (LN < blood; n = 7). Bars show means ± SD. (D) Correlation between CD69 expression on LN clonotypes and the distribution of clonotypes between LNs and blood (frequency of a given clonotype in LNs minus the corresponding frequency in blood). Data points were only included, where at least one clonotype was present in both compartments. N/A, not applicable. *P < 0.05, **P < 0.01. Conventional flow cytometry was used for all stains in this figure.