Background
Transcriptionally-active cells (TAC) that produce HIV RNA persist during ART contribute to chronic inflammation and co-morbidities, and can lead to viral rebound upon ART interruption. However, our understanding of these cells – particularly within tissues – is limited. We implemented HIV-seq, a technique we established to efficiently identify HIV+ cells by scRNAseq, to characterize the transcriptional profiles and antigen specificities of TAC from tissues.
Methods
Our case study focused on participant LG34 enrolled in the Last Gift rapid research autopsy program. LG34 initiated ART during chronic HIV in 2016 and remained on ART with inconsistent intake until his death in 2022. Between 2021 and 2022, he experienced multiple low-level HIV blips (<75cp/ml), received four COVID-19 vaccinations, and had a breakthrough SARS-CoV-2 infection. We hypothesized that blipping during active SARS-CoV-2-specific immune responses (from vaccination and breakthrough infection) could seed an HIV reservoir within SARS-CoV-2-specific CD4+ T cells. To investigate this, we processed LG34’s spleen and lymph nodes post-mortem, and performed multiomic HIV-seq to characterize HIV RNA+ cells through scRNAseq and VDJ-seq to evaluate SARS-CoV-2 specificities.
Results
We identified 49 and 37 TAC from 5162 and 5849 memory CD4+ T cells from spleen and lymph nodes, respectively. TAC were mainly found in Tregs with proliferative and survival transcriptional signatures, and terminally-differentiated Temra cells. Compared to uninfected Tregs, infected Tregs expressed higher levels of MHC-II and Foxp3. By contrast, compared to uninfected Temras, infected Temras expressed higher MHC-I and cytolytic effectors (granzymes B/H, perforin). VDJ-seq revealed greater clonal expansion in TAC than in uninfected cells, with some clones shared across tissues. Of all TCR matches identified by VDJMatch among HIV RNA+ cells, 83% were predicted to recognize SARS-CoV-2 antigens, and these cells resided primarily among Tregs.
Conclusions
HIV-seq enabled detection of high numbers of TAC from tissues of LG34 under ART. These TAC were predominantly Tregs and Temras. Infected Tregs upregulated Foxp3 and MHC-II which may increase immunosuppressive function and facilitate recognition by CD4+ T cells. Infected Temras expressed cytolytic molecules, and their increased MHC-I expression may facilitate recognition by CD8+ T cells. TAC exhibit shared clonotypes across tissues, and include Tregs with specificities for SARS-CoV-2 antigens.
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