HIV-1 is characterised by a series of viral subtypes, with clades A, B, C, and D the most globally prevalent. Viral heterogeneity has implications for differential disease transmission and replication rates, pathogenicity, and response to ART (including the development of resistance).
Tat is a regulatory protein critical for transcription of HIV proteins within infected cells. Released into the extracellular environment, Tat can induce numerous biological effects in various cell types and is thought to be a significant mediator of viral pathogenesis. It can induce cytokine production by cells of the monocyte/macrophage lineage [1,2], with clades B and C Tat exhibiting distinct effects [3,4]. As altered cytokine production by T lymphocytes characterizes early-stage HIV infection and is predictive of disease progression , we investigated if Tat from HIV-1 clades A-D can similarly affect cytokine production by T cells. In addition to conventional T cells, we have focused on the Vγ9Vδ2 subset of γδ T cells. Vγ9Vδ2 T cells are a significant source of the immunomodulatory cytokines, tumour necrosis factor (TNF)-α and interferon (IFN)-γ , and are also thought to mediate anti-HIV effects [7,8]. Subject to depletion in HIV-infected patients , Vγ9Vδ2 T cells are currently being developed as therapeutic targets for HIV infection .
Initially, we assessed the concentration-dependent effects of recombinant Tat clade B and C proteins (ProSpec Bio) on TNF-α secretion by the monocytic cell line THP-1 (American Tissue Culture Collection). Cells were seeded in 24-well plates and exposed to various concentrations of Tat followed by stimulation for 48 h with 10 ng/ml phorbol myristate acetate (PMA). TNF-α and IFN-γ levels in cell supernatants were measured by ELISA (R&D Systems). Consistent with published data [3,4], we found that monocytes exposed to Tat B, but not Tat C, significantly upregulated secretion of TNF-α (Fig. 1a).
Adopting a single Tat concentration (200 ng/ml), we subjected peripheral blood mononuclear cells (PBMC) from healthy donors to Tat clades A, B, C and D, thus assessing the most prevalent global genetic forms of HIV-1. PBMC were seeded in 24-well plates and incubated for 24 h with Tat followed by stimulation for a further 24 h with PMA (10 ng/ml) and ionomycin (1 μg/ml). Intracellular TNF-α and IFN-γ production by total CD3+ T cells and the Vγ9Vδ2 T-cell subset was examined by flow cytometry as described previously  (Fig. 1b for TNF-α). About 0.5% of unstimulated total and Vγ9Vδ2 T cells stained positive for TNF-α and/or IFN-γ and these percentages rose to 30–80% when PBMC were stimulated with PMA and ionomycin (PMA/I). These frequencies were not altered by incubation with Tat clades A, B, C or D (Fig. 1c, d for TNF-α). When PBMC were stimulated with the pyrophosphate antigen (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMB-PP; 10 nmol/l; kindly provided by Hassan Jomaa, Justus-Liebig-Universitat, Giessen, Germany), which specifically activates Vγ9Vδ2 T cells [6,11], most Vδ2 T cell receptor-positive cells, but none of the Vδ2-negative cells, expressed TNF-α and IFN-γ. Again, these frequencies were not changed by preincubation of the cells with Tat clade A-D proteins (Fig. 1b, e for TNF-α).
To confirm that TNF-α and IFN-γ expression reflects release of these cytokines into cell supernatants, we enriched T cells from PBMC using CD3 Microbeads (Miltenyi Biotec). Lines of expanded Vγ9Vδ2 T cells were generated by stimulating PBMC with HMB-PP (10 nmol/l) and culturing them for 14–21 days in medium containing 50 U/ml interleukin-2 as described previously . Vγ9Vδ2 T cells were then enriched to more than 99% purity using a MoFlo XDP Cell Sorter (Beckman Coulter). Sorted CD3+ or Vγ9Vδ2 T cells were subjected to stimulation with PMA/I or HMB-PP as above and the levels of TNF-α (Fig. 1f, g) and IFN-γ (not shown) released into the cell supernatants were measured by ELISA. In support of our findings using flow cytometry, unstimulated CD3+ and Vγ9Vδ2 T cells secreted undetectable levels of TNF-α and IFN-γ whereas stimulation with PMA/I resulted in substantial inductions of both cytokines and HMB-PP induced TNF-α and IFN-γ secretion by Vγ9Vδ2 T cells only. However, divergent from our results with THP-1 cells, exposure to Tat A, B, C or D had no effects on cytokine secretion by unstimulated or stimulated T cells (Fig. 1f, g).
These results confirm previous studies [3,4] reporting that HIV Tat proteins exhibit clade-specific induction of cytokine production by monocytes. However, we also found that clades A-D Tat do not induce, augment, or modulate TNF-α and IFN-γ production by T cells, including the Vγ9Vδ2 subset of γδ T cells which are thought to mediate immunity against HIV [7,8]. Future analysis of the expression of Tat receptors on monocytes and T cells may ultimately reveal why Tat differentially affects these cells.
J.P.S. conceived the study. J.P.S., D.G.D. and A.K. designed the study. A.K. carried out the experiments and analysed and interpreted the results. A.P. assisted with generating Vγ9Vδ2 cell lines. A.K., J.P.S., D.G.D. and M.H. wrote the article.
Conflicts of interest
There are no conflicts of interest. This study was supported by internal funding.
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