Background
The HIV reservoir is enriched for clonal CD4+ T-cells harboring intact proviruses after years of antiretroviral therapy (ART). In this study, we characterize the immunophenotypes of ‘authentic reservoir-harboring clones’ (ARCs) isolated and propagated from people living with HIV (PLWH) and aim to understand the contribution of these profiles to persistence.
Methods
Individual ARCs from PLWH on ART (n=3) were isolated by limiting dilution of memory CD4+ T-cells and propagated with irradiated feeders and anti-CD3/CD28 stimulation in media containing ARVs and bNAbs. Wells were screened for the presence of ARCs by Gag-p24 ELISA and infected cell purity was validated by IPDA and single-cell TCR-sequencing. Transcriptional, surface protein profiles and HIV-expression of ARCs were characterized by CITEseq and confirmed by flow cytometry. Latency reversal activity and granzyme release from ARCs were assessed using PMA/Ionomycin.
Results
ARCs exhibited robust expression of CD8-like cytotoxic programs, contrasting with autologous uninfected clones (Figure 1A; 3 donors; ARCs n=3; Uninfected clones n=10). ARCs also displayed higher expression of interferon response genes. >90% of cells within each ARC were confirmed to be GzmA+, TNFa+ Th1s (IFNg+). While most of the cells from ARCs had low to no expression of HIV transcripts, a subpopulation of cells from donor 1 ARC displayed high expression of HIV (Figure 1B). This HIV-hi population had markedly lower expression of cytotoxic and interferon response genes. LRA-treatment of ARCs resulted in limited latency reversal (Donor 2 ARC: 0.05% +/- 0.02, Donor 3 ARC: 2.12n%+/- 2.25 Gag+ cells after treatment). LRA-treated cells that released GzmA from 2 donor ARCs showed 2.2-3 fold lower Gag-expression compared to cells that did not release granzyme.
Conclusions
Each ARC was enriched for cytotoxic and interferon response programs and determined to be inflammatory-like Th1s expressing high levels of TNFa and granzyme. A small subset of cells expressing HIV transcripts within 1 ARC differed from the majority of cells by having significantly lower expression of cytotoxic and interferon response genes. LRA-treated cells from 2 other ARCs displayed increased latency reversal in cells that did not undergo degranulation, suggesting differential latency reversal capacity based on cell-intrinsic cytotoxic properties. Our results highlight the value of using ARCs to evaluate the contribution of the immunophenotypes of reservoir-harboring cells to HIV persistence.