Background
Recent studies suggest that HIV-1 reservoir cells are subject to immune selection pressure, but the host immune responses that are able to engage and target viral reservoir cells are unknown. To infer mechanisms underlying immune selection activity against viral reservoir cells, we conducted a parallel, longitudinal phenotypic evaluation of HIV-1 reservoir cells and antiviral immune cells.
Methods
Longitudinal peripheral blood samples collected at ~1 year on ART (T1), at ~10 years on ART (T2), and at ~20 years on ART (T3) of 5 long-term ART-treated individuals were studied. Phenotypic and proviral sequencing (PheP-Seq) was used for the analysis of 73 markers on HIV-1 reservoir cells and CITE-seq was conducted for profiling of 177 markers on host immune cells.
Results
Using PheP-seq, we analyzed a total of 1,075,240 individual memory CD4 (mCD4) T cells from all three timepoints combined (2433 represented HIV-infected cells; 265 cells harbored genome-intact HIV-1 and 356 included clonally-expanded reservoir cells). Cells harboring clonal intact proviruses integrated into heterochromatin regions increased from 2.5% at T1 to 25% at T3. At the earliest timepoint, reservoir cells harboring intact proviruses showed few phenotypic alterations relative to HIV-uninfected cells. After 20 years of ART, the following markers were significantly (FDR-adjusted p<0.01) upregulated on mCD4 T cells containing intact HIV-1: CD127, HLA-G, 2B4, CD161, HLA-E, KLRG1, CD28, and CD48; all of these markers can be associated with reservoir persistence and resistance to immune-mediated killing. Correspondingly, CITE-Seq demonstrated statistically significant (FDR-adjusted p<0.01) downregulation of the activating markers Siglec-7, CD161, NKG2D, NKp46, DNAM1, NKp30 on NK cells on T3 relative to T1, while the inhibitory receptors KIR2DL2/3 were upregulated. Phenotypic changes in CD8 T cells over two decades of ART were more limited and included significant upregulation of activation (CD38, CD151) and immune inhibition (CD39, CD93) markers.
Conclusions
These data suggest a longitudinal evolution of viral reservoir cells with phenotypic features of resistance to immune-mediated killing, paired with a progressive increase of NK cells and, to a lesser extent, CD8 T cells with signs of immune inhibition and dysfunction. Phenotypic changes in reservoir cells and effector cells may jointly contribute to an immune microenvironment conducive to viral persistence.
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