Persistent immune activation plays a central role in driving HIV disease progression and may lead to erosion, depletion, and exhaustion of the CD4 T-cell pool.1 Moreover, activation and/or proliferation of CD4 T cells create targets for the virus itself to further promote replication because activated CD4 T cells are the main source of viral infection and active replication.2 One of the mechanisms capable of controlling the activation and expansion of immune cells is the ability of regulatory T cells (Treg) to actively suppress immune responses. Treg are induced (or recruited and expanded) in most of the infections to modulate host immune responses to avoid overreactive immunity.3 In fact, several authors report that a decrease in the absolute counts of Treg results in a failure to control immune activation.4–7
The homeostatic role of Treg depends on Interleukin-2 (IL-2) and the expression of the high-affinity IL-2 receptor alpha (IL-2-Rα or CD25) at the surface of Treg.8,9 IL-2 maintains Foxp3+ natural Treg, expands them at high doses, and facilitates transforming growth factor β–dependent differentiation of naive T cells to Foxp3+ Treg.10 Thus, assuming that the main source of IL-2 is activated T cells, there is a negative feedback control of immune responses via IL-2. As the number of IL-2–producing T cells (ie, activated CD4 T cells) and the IL-2 concentration increase, Treg respond with expansion and eventually reestablish steady state. In this way, the relative proportion of Treg and activated T cells is stably maintained even when the total number of CD4 T cells is dramatically altered.8 Disruption of this IL-2–mediated feedback loop at any step has proved to promote the development of autoimmune/inflammatory disease.9 Therefore, peripheral homeostasis of Treg is in equilibrium or indexed to the number of activated T cells,8 ensuring that peripheral T-cell expansion during an immune response remains under control. However, this homeostatic mechanism seems to be disturbed in HIV-infected patients, where the values of Treg are decreased despite the increased presence of activated T cells.4,7
Here we examine the ratio between Treg and IL-2–producing T cells in a cohort of HIV-infected patients to determine the potential alterations in this homeostatic mechanism. Related to this, we analyzed the expression of IL-2-Rα (CD25) in Treg cells from HIV-infected patients, focusing on the potential effect of replicating HIV. Finally, we looked into the effect of in vitro HIV infection on the IL-2 signaling pathway of Treg.
Peripheral blood samples were obtained after informed consent in accordance with the local ethical committee approval from healthy adult volunteers, naive (untreated) HIV-infected patients with a viral load (VL) higher than 10,000 copies per milliliter, and HIV-infected patients with undetectable VL (ud-VL) (<50 copies/mL). All patients with ud-VL were under highly active antiretroviral therapy. Blood was immediately processed after extraction in the Spanish HIV BioBank11 or in the Instituto de Biomedicina de Sevilla/Hospital Universitario Virgen del Rocío Institute, and peripheral blood mononuclear cells (PBMC) were purified using Ficoll-Paque according to the manufacturer's protocol.
Cellular Immunophenotyping of Treg From Donors
PBMC from donors were washed and stained with anti-hCD4-ECD, anti-hCD25-PE-Cy5 or anti-hCD25-PE-Cy7, and anti-hCD3-FITC (Beckman Coulter, Barcelona, Spain). Fixation and permeabilization for intracellular staining were done with Anti-Human Foxp3 Staining Set (eBiosciences, San Diego, CA), and cells were stained with anti-Foxp3-PE (eBiosciences). Data acquisition was performed in a Beckman Coulter FC-500 or GALLIOS cytometer.
The frequency of IL-2–producing T cells was determined in freshly purified PBMC from HIV-infected patients as previously described.12 Briefly, Treg-depleted PBMC were stained with 5-(and -6)-carboxyfluorescein diacetate succinimidyl ester, then stimulated with phytohemagglutinin, and restimulated 5 days later for 4 additional hours in the presence of Brefeldin A. CD4+ proliferating cells (CFSElow) were analyzed by flow cytometry for intracellular IL-2 expression. Data are expressed as the percentage of IL-2+ T cells in CD4-gated T cells, and this percentage was correlated with the percentage of peripheral Treg (defined as CD4+CD25highFoxP3+) measured in each patient. Expression of IL-2-Rα (CD25) in the Treg subset from HIV-infected patients was determined in gated CD3+CD4+Foxp3+ T cells.
Culture and In Vitro Infection of Treg Cells
Untouched CD4+ T cells isolated with magnetic beads (Miltenyi Biotec) from healthy donors were stained and CD4+CD25+CD127low Treg cells isolated using an ASTRIOS cell sorter (Beckman Coulter, Bergisch Gladbach, Germany) as previously described.13 After sorting, more than 95% of the sorted cells were CD4+ without contaminating CD8+ cells and more than 90% were CD4+CD25+CD127−, from which more than 85% were CD25highFoxp3+. Freshly sorted Treg were activated with Dynabeads Human Treg Expander (Invitrogen, Paisley, United Kingdom) following manufacturer's recommendations. Treg cells were infected with 0.1 multiplicity of infection of HIVNL4-3 for 3 hours; cells were then extensively washed and cultured in the presence of 500 U/mL of IL-2. Noninfected Treg (NI-Treg) cells from the same donor were processed in parallel and used as controls. Viral entry and infection were confirmed by measuring the concentration of p24gag in the culture supernatant by enzyme-linked immunosorbent assay (Innotest HIV-1 antigen mAb; Innogenetics, Gent, Belgium) and intracellular p24 protein by flow cytometry (KC57-FITC, Beckman coulter) as previously described.13 The level of phosphorylated STAT5 (pSTAT5) was determined by flow cytometry and intracellular staining using pSTAT5-PE-Cy7 (pY694) (Beckton Dickinson, Franklin Lakes, NJ). pSTAT5 and CD25 expressions were analyzed in gated CD3+CD4+Foxp3+ T cells.
Statistical analysis was performed using statistical software SPSS. Nonparametric Mann–Whitney and Wilcoxon paired tests were used. Correlation between variables was established by Pearson correlation test. P value <0.05 by 2-sided test was considered significant.
Treg Capacity to Index to the Number of IL-2–Producing CD4 T Cells Was Disturbed in HIV-Infected Patients
We analyzed in a cross-sectional study the balance between the percentage of Treg (defined as CD4+CD25hiFoxP3+) and the frequency of IL-2–producing CD4 T cells in response to stimulation in a group of 7 HIV-infected viremic patients. All 7 patients were males, with a mean ± standard error of the mean (SEM) age = 26.73 ± 1.4 and a mean ± SEM VL = 117,486 ± 40,042 HIV-RNA copies per milliliter. We have previously published12 that healthy donors showed a direct correlation between the frequency of Treg and the IL-2–producing CD4 T cells (r = 1.000, P < 0.001) (control group in Fig. 1A), which agrees with the Treg/T-activated indexation described by other authors.8 However, this correlation was totally lost in the HIV group studied (r = −0.181, P = 0.698), and we did not find a balance between the frequency of Treg and the amount of CD4 T cells that produce IL-2 in response to stimulation (Fig. 1A). These results suggest that Treg subset in HIV-infected patients would not be able to proportionally increase in parallel to an increase in the CD4 T-cell activation.
Treg of Viremic HIV Patients Showed Lower IL-2-Rα Expression Than Those of Aviremic Patients
We further investigated if the disturbed Treg/IL-2–producing CD4 T cell ratio could be because of an altered expression of the receptor for IL-2 on Treg mediated by HIV. For that, we compared the expression of IL-2-Rα (CD25) in CD3+CD4+Foxp3+ T cells in the presence or absence of VL, studying a group of 9 HIV-infected patients with ud-VL and a new group of 9 viremic patients (mean ± SEM VL = 105,325 ± 57,253 HIV-RNA copies/mL) (high VL). There were no significant differences of sex (P = 0.436) or age (mean ± SEM age: ud-VL = 44.69 ± 3.53, high VL = 34.75 ± 4.78 years; P = 0.136) between groups. Most of the Foxp3+ Treg cells in the patients of ud-VL group expressed CD25 with high intensity, as it has also been described in Foxp3+ cells from healthy donors.14 However, in patients with high VL, we observed the appearance of a Foxp3+CD25low population (Fig. 1B), which is not present in patients with controlled viremia and was not reported in uninfected subjects.14 In fact, the density of CD25 at the surface of the Foxp3+ CD4 T cells, determined as mean fluorescence intensity, was significantly decreased in patients of the high VL group (mean ± SEM = 1.90 ± 0.25) with respect to the ud-VL group (mean ± SEM = 2.69 ± 0.21) (Fig. 1C). Therefore, a reduction in the expression of IL-2-Rα in Treg was observed in patients with replicating HIV, probably compromising the IL-2–mediated expansion of Treg in response to the proliferation of activated T cells.
In Vitro HIV Infection of Treg Affects Their CD25 Expression and Their STAT5 Phosphorylation in Response to IL-2
Because Treg from viremic patients showed decreased levels of IL-2-Rα (CD25), we investigated if this decrease could be because of the presence of the virus and a direct effect of the HIV infection on Treg cells. To confirm that replicating HIV could modulate the expression of IL-2-Rα (CD25) in Treg, we purified and infected in vitro Treg cells from 6 different male healthy donors (mean ± SEM age = 33.68 ± 1.64 years). NI-Treg and HIV-infected Treg (HIV-Treg) (pNL4.3; multiplicity of infection = 0.1), as confirmed by p24gag values in supernatant (data not shown), were cultured in the presence of IL-2. Five days after infection, we observed a marked reduction of CD25 density (mean fluorescence intensity) in HIV-Treg compared with NI-Treg (Fig. 2A). IL-2 signaling is primarily mediated through the activation of JAK1 and JAK3 with subsequent phosphorylation and activation of STAT5.15 Therefore, we measured the presence of pSTAT5 to further confirm the alteration on the IL-2 signaling because of the decreased expression of IL-2-Rα (Fig. 2B). In fact, we confirmed that both the frequency (Fig. 2C) and density (Fig. 2D) of pSTAT5 were significantly lower in HIV-Treg than in NI-Treg. These in vitro results confirmed that direct HIV infection of Treg downregulates the IL-2-Rα expression interfering in the IL-2 signaling pathway.
Here we show that the balance between Treg and IL-2–producing CD4 T cells is deeply disturbed in HIV-infected patients, and the higher frequency of IL-2–producing CD4 T cells (related to cells with an activated/memory phenotype8) was not associated to the higher frequency of Treg. We speculate that the lower expression of CD25 observed in Treg cells from viremic patients could affect their capacity of expansion in response to the IL-2 produced by activated CD4 T cells. However, we cannot rule out that it could also affect their survival or suppressive function, compromising the adequate immune homeostasis in these patients.
Different causes could explain the loss of this balance between Treg and activated CD4 T cells. We have recently proved that direct HIV infection of Treg in vitro could disrupt their phenotype and function.13 In fact, here we demonstrate that the presence of high VL in patients was associated to a downregulation of the IL-2-Rα in Treg, and this effect was further confirmed in vitro where direct HIV infection of Treg produced a decrease in the expression of IL-2-Rα. The IL-2 signaling pathway plays a key role in the homeostasis of Treg,8,15 and a decreased expression of the IL-2 receptor would implicate that Treg would not be capable of sensing and respond to the circulating IL-2 and the increase of activated T cells in patients. In addition, IL-2-Rα has demonstrated being essential for the expansion, induction, and above all for the suppressive function of these cells,8,15 and thus, decreased IL-2-Rα expression could also reduce the suppressive capacity of Treg in HIV-infected patients. In fact, Miyara et al14 have reported in healthy donors that Foxp3+ cells with a lower expression of CD25 have an impaired suppressive function and are less capable of maintaining Foxp3 expression.
In addition to the downregulation of IL-2-Rα observed in viremic HIV-infected patients, we have analyzed the effect of HIV infection in purified Treg. These in vitro experiments indicated that direct HIV infection of Treg would be responsible for the observed IL-2-Rα downregulation, which was further confirmed by a reduction in pSTAT5, the transcription factor downstream of the IL-2R signaling. The loss of STAT5 has shown to prevent the Foxp3 expression and suppressive function of Treg.16 According to our results, Kryworuchko et al17 report that viral envelope of HIV induces IL-2 unresponsiveness in total CD4 T cells. However, this is the first report in HIV-infected patients that shows a disruption of the IL-2 signaling pathway in the Treg population, which is markedly dependent on this pathway for its proliferation, survival, and functionality. Further studies would be necessary to confirm that such alterations observed in the IL-2 signaling, and in the Treg phenotype (as we recently reported in vitro13), lead to a functional impairment of Treg in HIV patients.
Hyperimmune activation in HIV-infected patients is probably a multifactorial phenomenon affected by numerous virological and immunological factors. However, the inability of conventional CD4 T cells to detect IL-2 because of defects of IL-2R expression18 or IL-2 signaling16 has been probed to result in uncontrolled CD4 T-cell activation and autoimmune disease. Therefore, the HIV-mediated CD25 downregulation in Treg, which we described here, and the functional impairment described in HIV-Treg13 could be determinants in the immune hyperactivation process of viremic HIV-infected subjects. Additionally, our previous data suggest that a suppressor antiretroviral treatment could not be able to restore such Treg-associated disturbance because the balance between Treg and IL-2–producing CD4 T cells was also found to be disrupted in aviremic HIV-infected patients, who had been treated for at least 2 years.12 Future designs are encouraged to test whether this disturbance could also be contributing to the residual hyperactivation of successfully treated HIV-infected patients. Numerous authors report that decreased absolute counts of Treg in HIV-infected patients were correlated with rising markers of immune activation.5,6 Our findings contribute to the better understanding of HIV effects on Treg cells, providing the molecular basis of Treg impairment in the presence of HIV reported by these authors.
The authors thank the patients and volunteers who participated in this study for providing blood samples. The authors thank L. Díaz and the Platform of Cytometry of Instituto de Investigación Sanitaria Gregorio Marañón for technical assistance.
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