HIV infection strongly affects cellular immunity, causing the depletion of CD4+ T cells, in particular within the naïve compartment , and dysfunction of both CD8+ and CD4+ T lymphocytes [2–6]. This status of chronic immune dysregulation involves the whole T-cell compartment, including uninfected T cells , and is not completely restored during effective antiretroviral therapy (ART). There is a general consensus on the complexity of these phenomena which seem to be due not only to viral replication and CD4+ T cells loss, but also to the immunomodulatory activity of HIV products, including Tat [5,7]. Indeed, the HIV-1 Tat protein is released extracellularly , even during ART , and enters neighboring cells affecting their functionality [10–15]. In this context, it has been shown that Tat has a strong impact on CD8+ T-cell programs and activity  and, in murine models, favors the activation of CD8+ T cells and the modulation of antiviral responses , causing dysfunctions similar to those observed in HIV-infected individuals. It is also noteworthy that naturally acquired or vaccine-induced anti-Tat immunity limits T-cell dysfunction, CD4+ T-cell loss and viral load, and is associated with the reduction of proviral DNA, resulting in the delay of disease progression [17–21]. However, whether Tat has a direct or indirect effect upon the CD4+ T-cell compartment is currently unknown. To shed light on this issue, we have determined whether extracellular bioactive Tat impacts human resting or activated CD4+ T cells. Our results show that Tat promotes the activation of CD4+ T cells as well as differentiation of naïve CD4+ T cells toward memory subtypes that may result in the generation of new targets of infection.
Materials and methods
Human cells and culture conditions
Buffy coats from healthy volunteers, who provided consent, were obtained from the University Hospital of Ferrara. Peripheral blood lymphocytes (PBLs) were separated by use of Ficoll–Hypaque (Lonza, Basel, Switzerland) density gradient centrifugation followed by 90 min of adhesion on a plastic support at 37 °C to remove monocytes.
Total and naïve CD4+ T cells were sorted by MACS magnetic selection (Miltenyi Biotec, Bergisch Gladbach, Germany) according to manufacturer's instructions and cultured, as detailed in Supplemental information, http://links.lww.com/QAD/B210, in the absence or presence of the Tat protein in 24-well flat-bottomed polystyrene plates precoated overnight at 4 °C with PBS or anti-CD3 mAb (0.5 μg/ml; R&D Systems, Minneapolis, Minnesota, USA). Naïve CD4+ T cells were cultured in nonpolarizing condition as previously described  and detailed in Supplemental information, http://links.lww.com/QAD/B210.
HIV-1 Tat from human T lymphotropic virus type IIIB isolate (BH10 clone) was expressed in Escherichia coli and purified by heparin-affinity chromatography and HPLC, as described previously . The lyophilized Tat protein was then stored at −80 °C and handled as described . Endotoxin concentration was below the detection limit (0.05 EU/μg).
Surface and intracellular staining were performed as detailed in Supplemental information, http://links.lww.com/QAD/B210.
Gene expression was evaluated by quantitative as detailed in Supplemental information, http://links.lww.com/QAD/B210.
Tat enhances CD4+ T-cell activation
The HIV-1 Tat protein, which is released by infected cells, enhances the production of proinflammatory cytokines from activated PBLs and CD8+ T cells [15,23,24]. To understand whether soluble Tat, at physiological concentration within a nanomolar range, may induce cytokine production in CD4+ T cells, resting or anti-CD3/CD28-stimulated T helper lymphocytes from healthy donors were cultured for 4 h in the absence or presence of 0.1 μg/ml of Tat protein. As shown in Fig. 1a, Tat significantly increased the expression of IL2, IFNγ and TNFα mRNAs in anti-CD3/CD28-stimulated CD4+ T cells, but not in resting lymphocytes. This effect was observed at similar levels for Tat doses ranging from 0.01 to 1 μg/ml, and it was abolished after incubation with anti-Tat positive sera (Fig. S1, http://links.lww.com/QAD/B210). This result was confirmed by cytokine intracellular staining of the cells that demonstrated increased production of IL2, IFNγ and TNFα (Fig. 1b) after 18 h of treatment with Tat when compared with untreated cells. However, the expression of early (CD69) and late (CD25, CD38, HLA-DR) activation markers was not affected by the presence of Tat (Fig. S2, http://links.lww.com/QAD/B210). As these results indicate that, in human-activated CD4+ T cells, Tat enhances the production of Th1-type cytokines, which are under the control of T-box transcription factors [25,26], we characterized the expression of T-bet and Eomes in resting and activated CD4+ T cells cultured in the absence or in the presence of Tat. As shown in Fig. 1c, Tat did not induce the mRNA expression of T-box transcription factors in unstimulated CD4+ T cells, whereas it increased significantly the expression of T-bet and Eomes transcription factors in CD3/CD28-activated CD4+ T cells as compared with CD4+ T cells activated with CD3/CD28 and cultured in the absence of Tat. Thus, at a physiological concentration, soluble Tat protein enhances the production of proinflammatory cytokines in activated CD4+ T cells and influences the expression of transcription factors crucial for T-cell programing and functionality.
Tat favors the expansion and the differentiation of naïve CD4+ T cells
The HIV-related chronic immune activation plays a major role in the increased proliferation and differentiation of naïve T cells into memory cells [1,27] leading to a decline of naïve T cells. As our data clearly indicate that Tat favors the activation of CD4+ T cells and the expression of transcription factors controlling T-cell programing, we wondered whether Tat had also an effect upon proliferation and differentiation of naïve lymphocytes, thus participating in immune activation and pathogenesis of HIV infection. To address this, purified naïve CD4+ T cells were cultured, in the presence or absence of Tat, in nonpolarizing conditions (NPC) to induce their activation and differentiation toward a memory phenotype avoiding potential biases due to polarization toward some specific T helper cell subpopulations . As shown in Fig. 2a, NPC induced the proliferation of naïve CD4+ T cells starting from day 7 and reaching the peak at day 12. The addition of Tat enhanced duration and magnitude of naïve T helper cell expansion which peaked at day 15 and remained higher until day 18. To determine whether Tat affected the differentiation of naïve CD4+ T cells cultured in NPC, the phenotype of T helper lymphocytes was assessed. Overall, NPC prompted the loss of CD45RA expression (Fig. 2b), suggesting a shift toward a nonnaïve phenotype that had started by day 12, with a more pronounced downregulation by day 18. Significantly, this phenomenon was more pronounced in the presence of Tat. In fact, higher numbers of central memory (CD45RA−, CCR7+, CD27+), transitional memory (CD45RA−, CCR7−, CD27+) and effector memory (CD45RA−, CCR7−, CD27−) CD4+ T cells were generated in the presence of Tat as compared with NPC alone (Fig. 2c). It is noteworthy that effector memory CD4+ T cells were almost absent in cultures derived from naïve CD4+ T cells activated under NPC, whereas they were strongly induced in the presence of Tat (Fig. 2c). Taken together, these data suggest that Tat supports the activation of naïve CD4+ T cells promoting their transition toward more differentiated phenotypes.
The Tat protein of HIV is released by infected cells  and interacts with neighboring cells [10–15]. We showed here that soluble Tat favors the activation of CD4+ T cells inducing the release of proinflammatory cytokines and the expression of transcription factors such as T-bet and Eomes which are crucial for T-cell activation and differentiation. In addition, Tat increased the expansion and differentiation of naïve CD4+ T cells activated in NPC. These findings, together with the observations made in CD8+ T cells [15,16,28], confirm that Tat plays an important role in the hyperactivation of the T-cell compartment, a phenomenon characterizing the progression to AIDS and possibly the residual disease observed in successfully ART-treated individuals [29,30].
Naïve CD4+ T cells are resistant to productive HIV infection due to their quiescent state [31,32]. However, their number dramatically decreases during AIDS , in part due to the status of chronic immune activation which favors their differentiation into memory and effector cells [27,33]. Tat, by favoring naïve T-cell activation, promotes their recruitment into the memory compartment and, fostering the exit from a quiescent state, might also contribute to the generation of new potential targets of infection, in line with previous observations showing higher susceptibly to HIV infection by CD4+ T cells exposed to Tat [34,35]. Tat expression has been detected in tissues from patients on ART , whose success is dependent on the levels of naïve CD4+ T cells , a compartment not always fully reconstituted by ART . Therefore, our data suggest that blocking Tat effects may favor therapy efficacy, as indeed observed in ART-treated individuals vaccinated with the Tat protein that showed restored T-cell responses against heterologous antigens and a rise in CD4+ T-cell count [18,19].
In previous works conducted with cell lines, Tat was alternatively shown to promote apoptosis or to have antiapoptotic effects, for instance promoting the release of IL2 [38–40]. On primary human CD4+ T cells, Tat immobilized on solid support, but not high concentrations of soluble Tat, was shown to mediate IL2 production [41,42]. In contrast, we showed here that soluble Tat, used at physiological concentrations , induced IL2 production in primary human CD4+ T cells. Thus, our data would argue against a direct effect of Tat on T-cell death as the main mechanism of CD4+ T-cell depletion.
Tat does not promote the exit from a quiescent state of resting lymphocytes, thus probably not affecting viral reservoirs . However, in activated T helper lymphocytes, it favors the production of IL2, IFNγ and of TNFα, whose plasmatic levels are increased in HIV-infected individuals [45,46]. Significantly, loss of naïve T cells, accumulation of differentiated lymphocytes and an increased level of proinflammatory cytokines are hallmarks of the accelerated immunosenescence characterizing HIV-infected individuals [47–49]. Our data suggest that Tat may support this phenomenon through the induction of proinflammatory cytokines and differentiation of T cell receptor-stimulated naïve CD4+ T cells toward late stages of differentiation, such as effector memory T cells. Accordingly, Tat has been shown to induce production of IL6 , which is associated with immunosenescence , reduction of telomerase activity in CD4+ T cells  and senescence of bone marrow mesenchymal stem cells .
In conclusion, our data suggest that Tat may contribute to the exacerbation of several immune dysfunctions observed during AIDS progression, such as chronic immune activation and premature aging. Therefore, the induction of anti-Tat immune responses by Tat administration can be an effective strategy for restoration of the immune system.
F.N. and R.G. conceived and designed the experiments and analyzed the data. F.N., F.S., E.G., V.F. and M.C. performed the experiments. F.N., A.C., A.C., C.G., B.E. and R.G. wrote the article. This work was supported by grants from the University of Ferrara and by the Gilead Fellowship Program. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the article.
Conflicts of interest
There are no conflicts of interest.
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