HIV-Sheltering Platelets From Immunological Non-Responders Induce a Dysfunctional Glycolytic CD4+ T-Cell Profile

Abstract

Immunological non-responders (InRs) are HIV-infected individuals in whom the administration of combination antiretroviral therapy (cART), although successful in suppressing viral replication, cannot properly reconstitute patient circulating CD4+ T-cell number to immunocompetent levels. The causes for this immunological failure remain elusive, and no therapeutic strategy is available to restore a proper CD4+ T-cell immune response in these individuals. We have recently demonstrated that platelets harboring infectious HIV are a hallmark of InR, and we now report on a causal connection between HIV-containing platelets and T-cell dysfunctions. We show here that in vivo, platelet-T-cell conjugates are more frequent among CD4+ T cells in InRs displaying HIV-containing platelets (<350 CD4+ T cells/μl blood for >1 year) as compared with healthy donors or immunological responders (IRs; >350 CD4+ T cells/μl). This contact between platelet containing HIV and T cell in the conjugates is not infectious for CD4+ T cells, as coculture of platelets from InRs containing HIV with healthy donor CD4+ T cells fails to propagate infection to CD4+ T cells. In contrast, when macrophages are the target of platelets containing HIV from InRs, macrophages become infected. Differential transcriptomic analyses comparing InR and IR CD4+ T cells reveal an upregulation of genes involved in both aerobic and anaerobic glycolysis in CD4+ T cells from InR vs. IR individuals. Accordingly, InR platelets containing HIV induce a dysfunctional increase in glycolysis-mediated energy production in CD4+ T cells as compared with T cells cocultured with IR platelets devoid of virus. In contrast, macrophage metabolism is not affected by platelet contact. Altogether, this brief report demonstrates a direct causal link between presence of HIV in platelets and T-cell dysfunctions typical of InR, contributing to devise a platelet-targeted therapy for improving immune reconstitution in these individuals.

Keywords: CD4+ T-cell metabolism; HIV-1; glycolysis; immunological failure; platelets; virus-containing platelets.

Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2022 Zhu, Real, Zhu, Greffe, de Truchis, Rouveix, Bomsel and Capron.

Figures

Figure 1

Platelets from immunological non-responders (InRs) form more conjugates with CD4+ T cells in vivo but do not transfer infectious virus to CD4+ T cells in vitro. (A) Platelet–CD4+ T-cell conjugates are more frequent in the circulation of InRs in vivo. Flow cytometry analysis of CD4+ T cells (CD3+CD4+) conjugated with platelets (CD41/61high) circulating in the blood of combination antiretroviral therapy (cART)-treated patients virally suppressed. Left: Gating strategy to assess platelet–T-cell conjugates. Right: Percentage of platelet–CD4+ T cell (CD3+CD4+CD41/61high) conjugates among CD4+ T cells (CD3+CD4+) as detected in healthy donor (gray) and HIV-infected patient samples categorized as immunological responder (IR, orange) and InR (blue). Kruskal–Wallis, statistically relevant differences p < 0.05 (*). (B, C) Upon direct interaction, HIV-containing (HIV+) platelets propagate infection to macrophages (MΦ), not to CD4+ T cells. (B)In vitro experimental setup. Left: HIV+ platelets are directly incubated with activated CD4+ T cells from healthy donors for a 14-day viral outgrowth assay. Right: MΦ from heathy donors interacts overnight with HIV+ platelets. At day 7, MΦ culture supernatants are collected and tested for their infectious replicative virus content by addition of the same activated CD4+ T cells from healthy donors as in left for a 14-day viral outgrowth assay. (C) Viral production quantification: HIV-1 is quantified in coculture supernatants after 14 days of viral outgrowth in CD4+ T cells that were incubated directly with HIV+ platelets in the presence or absence of polybrene (left) or incubated with supernatants of MΦ infected upon interaction with HIV+ platelets in the presence or absence of abciximab (right). LOD, limit of detection for the technique. *Statistically significant.

Figure 2

Differential transcriptomic analyses of CD4+ T cells from immunological non-responder (InR) and immunological responder (IR) patients focusing on genes associated with metabolic pathways. (A) InR, IR, and healthy donor transcriptomes have distinct profiles. Uniform Manifold Approximation and Projection (UMAP) of transcriptomic data from the GSE106792 dataset based on CD4+ T cells InR, IR, and healthy donors grouping individuals according to blood CD4+ T-cell counts and assessed by the Gene Expression Omnibus (GEO) constructed at a probability of 0.99. (B) Volcano plot from the differentially expressed gene (DEG) analysis between InR and IR CD4+ T cells. In the y axis, significance is indicated by red dotted line threshold at -Log10p value >3 for both InR and IR datasets. In the x axis, significance is indicated by red dotted line thresholds at Log2 fold change >0.5 for InR and <-0.5 for IR DEGs. (C) Differential biological pathway analysis activated in InR vs. IR CD4+ T cells using g:Profiler. (D) Network of biological pathways enriched in CD4+ T cells from InR (blue nodes and connectors) or from IR (orange nodes and connectors) (E) HumanCyC metabolic map showing genes upregulated in CD4+ T cells from InR (blue font) and upregulated in CD4+ T cells from IR (orange font) implicated in glycolysis, pyruvate oxidation, and TCA cycle biochemical reactions.

Figure 3

Metabolic profiling of CD4+ T cells after interaction with immunological non-responder (InR), immunological responder (IR), or healthy donor platelets, sheltering or not HIV in platelets. (A) Oxygen consumption rate (OCR; upper) and extracellular acidification rate (ECAR; lower) obtained throughout 1 h of data acquisition from live CD4+ T cells that interacted with InR (blue), IR (orange), or healthy donor (gray) platelets. Filled or empty circles with the same color code indicate the presence (HIVpos) or absence (HIVneg) of HIV in platelets, respectively. During the time course of data acquisition, the oxidative phosphorylation (OXPHOS) inhibitor and mitochondrial respiratory chain blocker oligomycin and rotenone/antimycin were injected at time points indicated by red and purple lines, respectively. (B) Energy phenotype map of CD4+ T cells that interacted with InR (blue), IR (orange), or healthy donor (gray) platelets. Filled or empty circles with the same color code indicate the presence (HIVpos) or absence (HIVneg) of HIV in platelets, respectively. (C) ATP production rate from CD4+ T cells (left) or macrophages (right) that interacted with InR, IR, or healthy donor platelets. The contribution of either glycolysis or mitochondria respiration to ATP production is discriminated in the bar graph by orange and red, respectively. Asterisks indicate statistically relevant differences established using a threshold of p < 0.05 calculated with a Kruskal–Wallis test on glycolysis levels (orange). Mitochondrial ATP production does not differ between groups using the same statistical test and significance threshold. (D) ATP production rate from CD4+ T cells that interacted with healthy donor platelets, IR platelets without HIV (HIVneg) in platelets, IR platelets with HIV (HIVpos) in platelets, InR HIVneg platelets, and InR HIVpos platelets. The contribution of either glycolysis or mitochondria respiration to ATP production is discriminated in the bar graph by orange and red, respectively. Asterisks indicate statistically relevant differences established by threshold of p < 0.05 using ANOVA on glycolysis levels (orange). Mitochondrial ATP production does not differ between groups using the same statistical test and significance threshold.