HIV-Tat upregulates the expression of senescence biomarkers in CD4+ T-cells

Abstract

Introduction: Current antiretroviral therapy (ART) for HIV infection reduces plasma viral loads to undetectable levels and has increased the life expectancy of people with HIV (PWH). However, this increased lifespan is accompanied by signs of accelerated aging and a higher prevalence of age-related comorbidities. Tat (Trans-Activator of Transcription) is a key protein for viral replication and pathogenesis. Tat is encoded by 2 exons, with the full-length Tat ranging from 86 to 101 aa (Tat₁₀₁). Introducing a stop codon in position 73 generates a 1 exon, synthetic 72aa Tat (Tat₇₂). Intracellular, full-length Tat activates the NF-κB pro-inflammatory pathway and increases antiapoptotic signals and ROS generation. These effects may initiate a cellular senescence program, characterized by cell cycle arrest, altered cell metabolism, and increased senescence-associated secretory phenotype (SASP) mediator release However, the precise role of HIV-Tat in inducing a cellular senescence program in CD4⁺ T-cells is currently unknown.

Methods: Jurkat Tet_off cell lines stably transfected with Tat₇₂, Tat₁₀₁, or an empty vector were used. Flow cytometry and RT-qPCR were used to address senescence biomarkers, and 105 mediators were assessed in cell supernatants with an antibody-based membrane array. Key results obtained in Jurkat-Tat cells were addressed in primary, resting CD4⁺ T-cells by transient electroporation of HIV-Tat-FLAG plasmid DNA.

Results: In the Jurkat cell model, expression of Tat₁₀₁ increased the levels of the senescence biomarkers BCL-2, CD87, and p21, and increased the release of sCD30, PDGF-AA, and sCD31, among other factors. Tat₁₀₁ upregulated CD30 and CD31 co-expression in the Jurkat cell surface, distinguishing these cells from Tat₇₂ and Tet_off Jurkats. The percentage of p21⁺, p16⁺, and γ-H2AX⁺ cells were higher in Tat-expressing CD4⁺ T-cells, detected as a FLAG⁺ population compared to their FLAG^- (Tat negative) counterparts. Increased levels of sCD31 and sCD26 were also detected in electroporated CD4⁺ T-cell supernatants.

Discussion: Intracellular, full-length HIV-Tat expression increases several senescence biomarkers in Jurkat and CD4⁺ T-cells, and SASP/Aging mediators in cell supernatants. Intracellular HIV-Tat may initiate a cellular senescence program, contributing to the premature aging phenotype observed in PWH.

Keywords: CD4+ T-Cell; HIV; HIV-Tat; SASP; aging; cellular senescence.

Figure 1

Full-length HIV-Tat increases the expression of senescence protein markers. Jurkat Tet_off T-cells stably expressing full-length HIV-Tat (Tat₁₀₁) or first-exon Tat (Tat₇₂) were treated with 1μg/ml Doxycycline for 48h (+DOX) or left untreated (-) and then the expression of senescence protein markers determined by flow cytometry. The percentage of live cells positive for CD87 (A), γH2AX (B), and p21 (C) and the geometric mean BCL-2 (D), KI67 (E), and Senescence associated β-GAL (SA β-GAL) (F) are shown. Symbols represent individual experiments (n=3-5) and floating bars indicate minimum to maximum values with a line at the mean value. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001. Statistical analysis was performed by one-way ANOVA with Tukey’s multiple comparisons test.

Figure 2

SASP Mediators in Jurkat cell line supernatants. Jurkat Tet_off, Tat₇₂ or Tat_101, cells were grown in round bottom 96-well plates at 5×10⁵ cells/ml in 200 μl R10, and cell supernatants were collected after 72 hours. The expression levels of 105 cytokines were addressed with an antibody-based membrane array. (A) Heatmap indicates the mean intensity values of the 43 mediators with visible spots in the membrane after ELC incubation. Mediators with mean signals over 1.4 (green boxes) or below 0.7-fold change (red boxes) expression in Tat₁₀₁ over Tet_off (dotted line) are shown in (B). Changes in Tat₇₂ over Tet_off (C) and Tat₁₀₁ over Tat₇₂ (D) are also shown. (n=3). Secreted levels of CD30 (E) and CD31 (F) were evaluated in Tet_off (n=4), Tat₇₂ (n=2-3) and Tat₁₀₁ by Luminex on the same cell supernatants as (A). The percentage of cells expressing cell surface CD30 (G) or CD31 (H) was addressed by Flow Cytometry. Graphs indicate percentages of cells within the Live cell gate after 48h cell culture. CD30 and CD31 co-expression for each of the cell lines (blue contour plot) overlayed with autofluorescence controls (grey contour plot) from a representative experiment is shown in (I). Symbols represent individual experiments (n=3), and floating bars indicate minimum to maximum values with a line at the mean. Statistical analysis was performed by one-way ANOVA with Tukey’s multiple comparisons test *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.

Figure 3

Full-length HIV-Tat increases several senescence markers at the mRNA level. Jurkat Tet_off, Tat₇₂ or Tat_101, cells were grown for 24h in 24-well plates at 4×10⁵ cells/ml in 500 μl of R10. Cell pellets were collected, and mRNA levels of indicated senescent markers were determined by RT-qPCR (A-J). Symbols represent individual experiments (n=4-5) with values representing the Fold Change versus Jurkat TET_off control cell line (dotted line) following the 2^-ΔΔCT calculation. Floating Bars indicate minimum to maximum values with a line at the mean value. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001. Statistical analysis was performed by one-way ANOVA with Tukey’s multiple comparisons test on ΔCT values.

Figure 4

Full-length HIV-Tat increases transcripts involved in cellular senescence. An RNA-seq approach was used in the same Jurkat Tet_off, Tat₇₂ or Tat_101, cells used here (Rodríguez-Agustín, under revision). Bars indicate the Log₂ Fold Change (FC) in relevant DEG (Differentially Expressed Genes) in the Tat₁₀₁ vs Tet_off comparison. Green bars indicate upregulated DEG Genes (PPDE>0.95 and FC > 2), and red bars indicate downregulated DEG genes (PPDE>0.95, FC <0,5). (A) Tat₁₀₁ stable expression increases TNF and decreases CD1E mRNA transcripts, well-known Tat target genes. (B) Senescence biomarkers and SASP targets addressed in this work that are also DEG in the RNA-seq experiment are shown. A grey bar indicates a DEG gene with an up-regulation below the 2-fold threshold (1, 8). Expression values (Log₂FC) of Tat₁₀₁ DEG genes present in the SenMayo dataset (C), in the hsa04218 KEGG Dataset (D) or in the Reactome R-HSA-2559583 dataset (E) are shown.

Figure 5

Tat-expressing CD4⁺ T-cells show increased levels of p21, γH2AX, and p16 senescence biomarkers. Resting CD4⁺T-cells isolated from buffy coats were transiently transfected with DNA plasmids encoding for Tat_72-DYK, Tat_101-DYK or a pcDNA empty vector backbone. An anti-FLAG antibody was used to detect Tat-expressing cells. The percentage of p21⁺ (A), γH2AX⁺ (B), p16⁺ (C) and CD87⁺ (D) cells within viable (Live Dead Negative) FLAG⁺ or FLAG^- populations at 72hours post electroporation are shown. Fold change values in the geometric mean of BCL2 (E), and CD4 (F) in FLAG⁺ cells compared to FLAG^- cells are indicated. An antibody membrane-based array was used to address changes in released mediators in cell supernatants. The levels (Fold-change) of detectable mediators in TAT_101-DYK cell supernatants against pcDNA cell supernatants are shown in (G). Symbols represent individual experiments (n=3-4) and Floating Bars indicate minimum to maximum values with a line at the mean. A green dotted line indicates > 1.4-fold-change and a red dotted line indicates < 0.7-fold-change. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001. Statistical analysis performed by two-way ANOVA with Tukey’s multiple comparisons test.