Immunologic and Virologic Parameters Associated With Human Immunodeficiency Virus (HIV) DNA Reservoir Size in People With HIV Receiving Antiretroviral Therapy

Abstract

Background: A better understanding of the dynamics of human immunodeficiency virus (HIV) reservoirs in CD4+ T cells of people with HIV (PWH) receiving antiretroviral therapy (ART) is crucial for developing therapies to eradicate the virus.

Methods: We conducted a study involving 28 aviremic PWH receiving ART with high and low levels of HIV DNA. We analyzed immunologic and virologic parameters and their association with the HIV reservoir size.

Results: The frequency of CD4+ T cells carrying HIV DNA was associated with higher pre-ART plasma viremia, lower pre-ART CD4+ T-cell counts, and lower pre-ART CD4/CD8 ratios. During ART, the High group maintained elevated levels of intact HIV proviral DNA, cell-associated HIV RNA, and inducible virion-associated HIV RNA. HIV sequence analysis showed no evidence for preferential accumulation of defective proviruses nor higher frequencies of clonal expansion in the High versus Low group. Phenotypic and functional T-cell analyses did not show enhanced immune-mediated virologic control in the Low versus High group. Of considerable interest, pre-ART innate immunity was significantly higher in the Low versus High group.

Conclusions: Our data suggest that innate immunity at the time of ART initiation may play an important role in modulating the dynamics and persistence of viral reservoirs in PWH.

Keywords: CD4+ T-cell count; HIV reservoirs; antiretroviral therapy; immune cells; plasma viremia.

Figure 1.

Comparison of virologic parameters between the High and the Low groups. A, Levels of total human immunodeficiency virus (HIV) DNA, cell-associated HIV RNA, and inducible virion-associated HIV RNA in the High versus Low group at the post–antiretroviral therapy (ART) time point. B, Levels of intact, 5′defective, and 3′defective HIV DNA in the High versus Low group at pre- and post-ART time points. C, Correlations between pre-ART CD4⁺ T-cell counts (top row) or pre-ART plasma viremia (bottom row) and post-ART total, intact, 5′ and 3′defective HIV DNA, and inducible virion-associated HIV RNA. Triangles and circles represent pre- and post-ART time points, respectively. P values were determined using the Mann–Whitney or the Wilcoxon matched-pairs signed-rank test: ***P < .001; ****P < .0001. Correlations were determined by the Spearman method. Abbreviation: TBP, TATA-binding protein.

Figure 2.

Composition and dynamics of human immunodeficiency virus (HIV) DNA reservoir in total and CD4⁺ T-cell subsets. A, Intact, 5′defective, and 3′defective HIV DNA decay rate in the High versus Low group. The leftmost graph shows median values (HIV DNA copies/10⁶ CD4⁺ T cells) for the High and Low group at 0.5, 2, 4, and 6–8 years on antiretroviral therapy (ART). The remaining graphs show comparisons of HIV DNA half-lives. B, Levels of intact, 5′defective, and 3′defective HIV DNA in naive (T_N), central memory (T_CM), transitional memory (T_TM), and effector (T_E) T cells. C, Comparison of CD4⁺ T-cell subset frequencies between the High and Low groups. D, Cell contribution to the pool of intact proviral DNA (IPD)–infected CD4⁺ T cells. E, Comparison of HIV DNA sequence diversity calculated by average Hamming distance (left) and clonality (right) in total CD4⁺ T cells between the High and Low groups. F, Comparison of clonality among the 4 CD4⁺ T-cell subsets and between the High and Low groups. P values were determined using the Mann–Whitney or the Friedman test: *P < .05; **P < .01; ****P < .0001; ns, not significant (P ≥ .05).

Figure 3.

Pre- and post–antiretroviral therapy (ART) T-cell immune parameters in the High versus the Low group. A, Frequencies of the exhaustion (T cell immunoreceptor with Ig and ITIM domains [TIGIT] and programmed cell death 1 [PD-1]) and activation (CD38/HLA-DR) markers on CD4⁺ and CD8⁺ T cells in the High and Low groups pre- and post-ART. B–D, High-dimensional flow cytometric analysis. B, Uniform manifold approximation and projection (UMAP) plots of CD3⁺ T cells from the High and Low group volunteers pre- and post-ART (left) and UMAP visualization of expression of the indicated markers (right). C, UMAP map of T-cell clusters identified by FlowSOM clustering (left) and a heatmap showing the level of expression of individual markers in each cluster (right). Four of the 15 clusters that showed significant differences between the groups are displayed. D, Comparison of frequencies of T cells expressing markers associated with indicated clusters: cluster 2 (memory CD4⁺ T cells expressing CD28), cluster 4 (transitional memory CD8⁺ T cells expressing CD38 and HLA-DR, and TIGIT, PD-1, and 2B4), cluster 8 (naive CD4⁺ T cells), and cluster 9 (effector memory and terminally differentiated CD8⁺ T cells expressing CD226 and 2B4). E, Frequencies of polyfunctional human immunodeficiency virus (HIV) Gag-specific CD4⁺ (interferon gamma [IFN-γ]⁺tumor necrosis factor alpha [TNF-α]⁺interleukin 2⁺CD40L⁺; left) and CD8⁺ (IFN-γ⁺TNF-α⁺macrophage inflammatory protein 1β⁺; right) T cells in High and Low groups pre- and post-ART. P values were determined using the Mann–Whitney and the Wilcoxon matched-pairs signed-rank tests for unpaired and paired comparisons, respectively: *P < .05; **P < .01; ***P < .001; ****P < .0001; ns, not significant (P ≥ .05).

Figure 4.

Innate immune characteristics in High and Low groups prior to initiation of antiretroviral therapy (ART). A, Frequencies of plasmacytoid dendritic cells (pDCs; left) and levels of interferon alpha 2 (IFN-α2) expression upon CpG stimulation of peripheral blood mononuclear cells (PBMCs)/mL of cell-culture supernatant (right) pre- and post-ART. B, Frequencies of CD56^dimCD16⁺ natural killer (NK) cells (left) and levels of CD57 expression in the CD56^dimCD16⁺ NK cell compartment (right) in the High and Low group pre- and post-ART. Symbols under the dotted line indicate values below the limit of detection. P values were determined using the Mann–Whitney test: *P < .05; **P < .01; ns, not significant (P ≥ .05).

Figure 5.

Correlation between immunologic and virologic variables and their relation to human immunodeficiency virus (HIV) DNA reservoir size. Correlogram visualization shows the relationship between individual variables measured in this study in the High and Low groups pre- and post–antiretroviral therapy (ART). Each row or column represents a different variable, ordered by the strength of the correlation with post-ART HIV DNA levels. The size and color of each circle correspond to the correlation coefficient between a pair of variables. The correlation coefficients were calculated using the Spearman method. Only significant correlations (P < .05) are shown.