The immunosuppressive tuberculosis-associated microenvironment inhibits viral replication and promotes HIV-1 latency in CD4+ T cells

Abstract

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), is the most common coinfection among people living with HIV-1. This coinfection is associated with accelerated HIV-1 disease progression and reduced survival. However, the impact of the HIV-1/TB coinfection on HIV-1 replication and latency in CD4⁺ T cells remains poorly studied. Using the acellular fraction of tuberculous pleural effusion (TB-PE), we investigated whether viral replication and HIV-1 latency in CD4⁺ T cells are affected by a TB-associated microenvironment. Our results revealed that TB-PE impaired T cell receptor-dependent cell activation and decreased HIV-1 replication in CD4⁺ T cells. Moreover, this immunosuppressive TB microenvironment promoted viral latency and inhibited HIV-1 reactivation. This study indicates that the TB-induced immune response may contribute to the persistence of the viral reservoir by silencing HIV-1 expression, allowing the virus to persist undetected by the immune system, and increasing the size of the latent HIV-1 reservoir.

Keywords: Immunology; Virology.

Graphical abstract

Figure 1

HIV-1 infection is inhibited by TB-PE in primary CD4⁺ T cells Primary CD4⁺ T cells isolated from healthy donors were infected with HIV-1 in the presence of the acellular fraction of TB-PE. Cells infected with HIV-1 in the absence of TB-PE were used as a control. (A) Schematic representation of the experimental design. (B) Cell viability assessed by live/dead staining. (C) The proportion of HIV-1 infected cells was determined at day 3 post-infection by Gag-p24 immunostaining. A representative cytometry (left) and Gag-p24 quantification (right) in cells from 5 independent donors are shown. (D) Isolated CD4⁺ T cells from 7 healthy donors were infected with HIV-1 in the presence of TB-PE from 2 independent TB patients. The quantification of Gag-p24 positive cells, relative to HIV-1-infected cells in the absence of TB-PE, is shown. (E) CD4⁺ T cells were treated with TB-PE or HF-PE and infected with HIV-1. The quantification of Gag-p24 positive cells relative to the control condition is shown. (F) Viral entry was assessed by the HIV-1/BlaM-Vpr fusion assay. Enzymatic cleavage of CCF2 by BlaM-Vpr shifts the CCF2 fluorescence emission spectrum from 520 nm to 447 nm, indicating viral uptake. (G) The HIV-1 reverse transcripts R/U5 and LTR/Gag were quantified by real-time qPCR at 6 h post-infection. (H) Integrated HIV-1 DNA was measured by real-time qPCR at 24 h post-infection. Each dot represents values obtained from an independent donor. Data are represented as mean ± SD. Statistical significance was determined by paired two-tailed t test or One-way ANOVA followed by the Tukey’s HSD post- test. ∗p ≤ 0.05, ∗∗p ≤ 0.01, ∗∗∗p ≤ 0.001.

Figure 2

Transcriptional profile of CD4⁺ T cells exposed to TB-PE The transcriptional profile of primary CD4⁺ T cells treated with TB-PE was characterized by bulk RNA-seq analysis in resting and activated cells from 3 healthy donors. (A) Principal component analysis of resting and activated CD4⁺ T cells. (B) Heatmaps displaying hierarchical clustering of the top 500 variable genes between TB-PE-treated and control CD4⁺ T cells. Data from resting (left) and activated (right) CD4⁺ T cells are shown. (C) Volcano plots representing the differentially expressed genes (DEGs) analysis. (D) Gene set enrichment analysis (GSEA) comparing TB-PE-treated and control CD4⁺ T cells in resting and activated cells (DEGs: FDR<0.05 and absolute logFC>1). Dot plots illustrating the enriched GO biological processes terms related to T cell activation. The size of the dot reflects the gene ratio, indicating the proportion of differentially expressed genes associated with the pathway. The color intensity of the dot corresponds to the significance of enrichment, as determined by the adjusted p value. (E) Heatmaps displaying the normalized expression of specific genes involved in T cell activation. Primary CD4⁺ T cells were stimulated with anti-CD3/CD28 antibodies in the presence of TB-PE or HF-PE. Cells activated in the absence of PEs were used as control. (F) The expression of the activation markers CD69, CD25 and HLA-DR was measured by flow cytometry after 24 h (CD69 and CD25) or 48 h post-activation (HLA-DR). A representative cytometry (left) and the proportion of activated cells (right) are shown. (G) Proportion of CD69 (left) or CD25 (right) positive cells stimulated in the presence of TB-PE from 2 independent TB-infected donors (red) or HF-PE (gray). Values are relative to the non-PE control condition (blue). Data are represented as mean ± SD. Each dot represents values obtained from an independent donor. Statistical significance was determined by paired two-tailed t test or one-way ANOVA followed by the Tukey’s HSD post-test. ∗p ≤ 0.05, ∗∗p ≤ 0.01.

Figure 3

TB-PE decreases glycolysis and oxidative phosphorylation Primary CD4⁺ T cells were stimulated with anti-CD3/CD28 antibodies in the presence of TB-PE or HF-PE. Cells activated in the absence of PEs were used as control. (A) Oxidative phosphorylation determined by oxygen consume rate (OCR, left) and mitochondrial-dependent ATP production rate (right). (B) Level of glycolysis was determined by proton efflux rate (PER, left) and glycolysis-dependent ATP production rate (right). (C) HIF-1α expression in activated CD4⁺ T cells was determined by flow cytometry. Each dot represents values obtained from an independent donor. Statistical significance was determined by paired two-tailed t test or One-way ANOVA followed by the Tukey’s HSD post-test. ∗p ≤ 0.05, ∗∗p ≤ 0.01.

Figure 4

TB-PE promotes latency in CD4⁺ T cells (A) Gene set enrichment analysis (GSEA) comparing TB-PE-treated and control CD4⁺ T cells in activated cells (DEGs: FDR<0.05 and absolute logFC>1). The dot plots illustrate the positively and negatively enriched GO biological processes terms involved in HIV-1 transcription and reactivation. The size of the dot reflects the gene ratio, indicating the proportion of differentially expressed genes associated with the pathway. The color intensity of the dot corresponds to the significance of enrichment, as determined by the adjusted p value. (B) Heatmaps displaying the normalized expression of specific genes involved in cellular pathways necessary for HIV-1 transcription and reactivation. (C) Schematic of the dual HIV-1 latency reporter construct. (D) CEM CD4⁺ T cells were infected with the dual HIV-1 latency reporter and the proportion productively (GFP+/iRFP+) and latent (iRFP+) infected cells was determined by flow cytometry (left). The expression of CD4 (middle) and tetherin (right) on each gated population is shown. Jurkat (E) or primary CD4⁺ T cells (F) were infected with the dual HIV-1 latency reporter virus in the presence or absence of TB-PE. Representative cytometry (left) and the ratio between latent and productively infected cells (right) are shown. Each dot represents values obtained from an independent experiment (E) or independent donor (F). Statistical significance was determined by paired two-tailed t test ∗p ≤ 0.05. See also Figures S1–S3.

Figure 5

Reversal of HIV1 latency is impaired by TB-PE J-Lat 6.3 (A) and 10.6 (B) cells were treated with different LRAs for 48 h in the presence or absence of TB-PE and GFP expression was measured by flow cytometry as a correlate for viral reactivation. A representative cytometry (left) and GFP quantification relative to the control condition (right) are shown. (A) J-Lat 6.3 cells were treated with 2 ng/ml PMA, 50 nM PEP005 or 10 ng/mL TNF-α. (B) J-Lat 10.6 cells were treated with 0.5 ng/mL PMA, 0.05 ng/mL TNF-α, 5nM PEP005, 1μM JQ1, or 10μM vorinostat. Each dot represents values obtained from an independent experiment. Statistical significance was determined by paired two-tailed t test. (C) Unspliced HIV-1 RNA (US HIV RNA) was measured in CD4⁺ T cells from 3 HIV-1-infected donors on ART after the stimulation with 2 ng/ml PMA. Values obtained by real-time qPCR (left) and relative quantification of US HIV RNA copies per 10⁶ cells (right) are shown. Each dot represents values obtained from an independent donor. Data are represented as mean ± SD. Statistical significance was determined by One-way ANOVA followed by the Tukey’s HSD post-test. ∗p ≤ 0.05, ∗∗p ≤ 0.01, ∗∗∗∗p ≤ 0.0001. See also Figure S4.