Background:
HIV-1 reservoir cells persist lifelong despite ART but may be vulnerable to host immune responses that could be exploited for HIV-1 cure strategies. Here, we used a novel single-cell, next-generation sequencing approach for the direct ex vivo phenotypic profiling of individual HIV-1 infected memory CD4 (mCD4) T cells from peripheral blood (PB) and lymph nodes (LN) of people living with HIV-1.
Methods:
mCD4 T cells derived from PB and LN of 8 participants were analyzed by a novel single-cell proteogenomic profiling assay. Cells were labeled with oligomer-tagged antibodies directed against n=53 surface markers and encapsulated into single cells. Multiplex PCR was conducted to amplify small fragments of genomic HIV-1 DNA (n=18) and the viral-host chromosomal junctions of large clones, coupled with amplification of antibody-bound oligomer tags. DNA and protein libraries were sequenced and biocomputationally deconvoluted.
Results:
We analyzed n=530,143 individual mCD4 T cells from PB of 5 participants with around 10 years of suppressed viremia: n=2,859 represented HIV-infected cells; n=193 cells harbored genome-intact HIV-1; n=125 included clonally-expanded reservoir cells. Cells encoding for intact HIV or being part of large clones displayed more mature, effector memory phenotypic features and frequently expressed ensemble signatures of surface markers conferring increased resistance to immune-mediated killing (such as PVR and HVEM), paired with elevated expression of immune checkpoint markers (such as PD1 and KLRG1) likely to limit proviral gene transcription. A total of n=396,628 mCD4 T cells from LN were assayed from 3 participants who received ART for around 10-15 years: n=3,888 were HIV-1 infected cells, and n=111 cells harbored genome-intact HIV-1. Cells encoding for intact proviruses were equally distributed between T follicular helper cell (TFH) and resident memory (TRM) CD4 cells and expressed higher levels of CD44, the IL-21 receptor (CD360), and the IL-7 receptor (CD127), all of which are associated with cell survival.
Conclusions:
Cells encoding intact proviruses display distinct phenotypic features in PB and LN and upregulate markers associated with proviral transcriptional repression, resistance to immune-mediated killing, and cell survival. We propose that these phenotypic changes result from immune selection processes, implying that only small subsets of infected cells with optimal adaptation to their anatomical immune microenvironment can survive during long-term ART.