Background
The HIV reservoir persists within clonally expanded CD4+ T cells carrying intact proviruses. We describe the isolation and long-term in vitro expansion of these ‘authentic reservoir clones’ (ARCs) and insights into functional characteristics that may underlie their persistence.
Methods
Authentic reservoir clones (ARCs) were derived from CD4+ memory T cells (n=4) using multiple rounds of limiting dilutions with irradiated feeder cells, anti-CD3, anti-CD28 stimulations, ARVs, and bNAbs. p24 ELISA was used to screen for wells containing clones. Proviruses were characterized through integration site analysis, intact proviral DNA assay (IPDA), and near full-length sequencing with clonality also verified by TCR sequencing. Flow cytometry based functional assays were performed, assessing proliferation (n=2), latency reversal (n=2), and susceptibility to CTL-mediated killing (n=3). Transcriptomic and immunophenotypic profiles were assessed by CITE-Seq, while antigen-specificities were queried by ELISPOT.
Results
Each of the four ARCs contained a single intact provirus integrated into either ZNF561, ZNF486, ZNF721, or ATPB4. The ZNF721 ARC was poorly proliferative, exhibiting minimal reactivation (0.2±0.1% Gag+) in response to anti-CD3/anti-CD28 stimulation. The ZNF486 ARC was deeply latent, with Gag expression of 0.04±0.3% upon reactivation with anti-CD3/CD28 (0% at baseline). The ZNF721 ARC, however, showed elevated basal Gag expression (1.16±1.18%) and modest reactivation with bryostatin-1 (3.27±1.22%). Distinct transcriptional and phenotypic profiles were revealed by CITE-Seq analysis, but shared features emerged: cytotoxicity and the expression of interferon-stimulated genes. CTL killing assays using peptide-pulsed targets demonstrated differential susceptibilities across the ARCs: the ATPB4 ARC resisted killing, ZNF486 was highly susceptible, and ZNF561 and ZNF721 showed moderate susceptibilities. Notably, the ZNF721 ARC had a proliferative advantage over autologous uninfected CD4+ T cells (Figure), was determined to be EBV specific, and killed EBV-transformed B cells.
Conclusions
Each ARC featured distinct mechanisms of persistence. The ATPB4 ARC was resistant to CTL-mediated killing, poorly proliferative, but displayed the highest HIV expression. In contrast, ZNF486 was deeply latent, while ZNF721 was highly proliferative. ZNF561 was intermediate for each of these properties. ARCs serve as valuable models for understanding HIV persistence mechanisms and may guide target interventions.