Selective miRNA inhibition in CD8+ cytotoxic T lymphocytes enhances HIV-1 specific cytotoxic responses

Abstract

miRNAs dictate relevant virus-host interactions, offering new avenues for interventions to achieve an HIV remission. We aimed to enhance HIV-specific cytotoxic responses-a hallmark of natural HIV control- by miRNA modulation in T cells. We recruited 12 participants six elite controllers and six patients with chronic HIV infection on long-term antiretroviral therapy ("progressors"). Elite controllers exhibited stronger HIV-specific cytotoxic responses than the progressors, and their CD8+T cells showed a miRNA (hsa-miR-10a-5p) significantly downregulated. When we transfected ex vivo CD8⁺ T cells from progressors with a synthetic miR-10a-5p inhibitor, miR-10a-5p levels decreased in 4 out of 6 progressors, correlating with an increase in HIV-specific cytotoxic responses. The effects of miR-10a-5p inhibition on HIV-specific CTL responses were modest, short-lived, and occurred before day seven after modulation. IL-4 and TNF-α levels strongly correlated with HIV-specific cytotoxic capacity. Thus, inhibition of miR-10a-5p enhanced HIV-specific CD8⁺ T cell capacity in progressors. Our pilot study proves the concept that miRNA modulation is a feasible strategy to combat HIV persistence by enhancing specific cytotoxic immune responses, which will inform new approaches for achieving an antiretroviral therapy-free HIV remission.

Keywords: HIV; cellular immunity; cytotoxicity; micro-RNA; non-coding RNA.

Figure 1

Workflow for in silico prediction of miR-10a-5p.

Figure 2

(A) Cytolytic activity of CD8⁺ T cells from EC and CP evaluated by p24 antigen quantification (n=6 in each group). Depicts the mean and stand deviation of p24 (ng/ml) levels for each patient at the different time points. HIV-suppressive capacity of CD8⁺ T cells is calculated at the peak of viral replication in CD4⁺ T cells alone, as the log decrease in p24 production when CD4⁺ T cells are cocultured with CD8⁺ T cells. Experiments run in triplicate. (B) Volcano plot depicting log2 of mean fold changes in miRNA expression (X-axis) and -log10 P values (Y-axis).

Figure 3

Expression levels of miR-10a-5p measured by qRT-PCR after modulation. Data are the mean ± SEM and are expressed as fold change using 5S levels as the reference (2−ΔΔCt). Purple histograms represent the mean of expression values from 5S, blue histograms represent the mean of expression values from day 7 and green from day 10. Expression values are normalized to the value of the control condition. Error bars represent the standard error of the mean of the three relative expressions from three biological replicates and two technical duplicates of each treatment.

Figure 4

Impact of transfected miR-10a-5p inhibitor in CD8⁺ T cells from progressors at day 7 and at day 10 post-treatment on CTL response level, measured as the reduction in log10 p24 levels at baseline and at days 7 and 10 after miRNA-10a-5p inhibition. A reduction of p24 antigen ≥ 1 log₁₀ was considered a relevant increase of CD8⁺ CTL response. For each experiment, we used three biological replicates and two technical duplicates.

Figure 5

Cytokines secretion and cytotoxicity markers production before and after transfection of CD8⁺ T cells from progressors with miR-10a-5p inhibitor. Light blue lines represent individual trajectories and red lines the mean trajectories for IL-2, IL-4, IL-6, IL-10, IL-17A, IFNg, TNFa, perforin, granzyme A and B levels from three biological triplicates and two technical duplicates. Significant changes between time points were determined using a Wilcoxon sign-rank test. *p<0.05.

Figure 6

(A) Heatmap of correlations between cytolytic activity, cytokines and cytotoxicity markers. The pie charts depict the magnitude of each individual Spearman Rho correlation coefficient in a color gradient from red (Rho -1) to blue (Rho +1). Correlations with a P<0.05 are marked with a red asterisk. (B) Correlation analysis for those variables significantly associated with CTL capacity.