Foxp3+ regulatory T cells in antiretroviral-naive HIV patients

Abstract

We characterized regulatory T cells from antiretroviral-naive HIV patients by flow cytometry. The proportion of CD4 cells positive for CD25 and Foxp3 was increased, mainly in those with CD4 cell counts less than 200 cells/μl. The total number of Foxp3-positive cells correlated with the CD4 cell count. Further studies are needed on whether Foxp3-positive cell numbers or function explain the susceptibility to autoimmune and inflammatory diseases seen in some patients with advanced HIV.

Patients with advanced HIV infection have an increased susceptibility to a number of immune-mediated and inflammatory conditions, including high rates of adverse reactions to medications as well as immune reconstitution inflammatory syndromes ^[1]. Regulatory T cells (T_regs) are a subgroup of CD4 T cells characterized by the upregulation of the IL-2 receptor (CD25) and the expression of the transcription factor protein Foxp3 ^[2]. In contrast to activated T cells, which are also CD4+CD25+, the biological role of regulatory cells is to dampen immune responses ^[3–5]. T_regs can be infected with HIV ^[6]. Some reports suggest that their numbers are depleted by HIV infection ^[7–9], whereas others suggest that the proportion of regulatory cells may be increased ^[10,11]. Previous studies, however, may have been confounded by methodological problems, including a failure to separate regulatory cells that highly express CD25 from other CD4+CD25+ cells, the inclusion of patients without regard to antiretroviral therapy (with unknown effects on regulatory cell numbers), and a failure to distinguish regulatory numbers and percentages. In addition, the availability of monoclonal antibodies to Foxp3 now makes it possible to study expression of Foxp3 protein at the single cell level. In the present study, we enumerated T_regs in antiretroviral-naive HIV-infected patients using flow cytometry to identify Foxp3-expressing CD4+CD25+ T cells.

To determine the effects of HIV on T_reg numbers, we recruited antiretroviral-naive patients with a wide range of CD4 cell numbers at the Instituto de Medicina Tropical Alexander von Humboldt of the Hospital Nacional Cayetano Heredia, Lima, Perú. Ethics committees from the Universidad Peruana Cayetano Heredia and Baylor College of Medicine approved the protocol and consent forms before initiation of the study.

We identified T_regs by flow cytometry after intracellular staining for Foxp3. Briefly, 10 ml blood samples were collected from consenting participants. Peripheral blood mononuclear cells were surface stained using Cy-chrome-conjugated anti-CD4 and phycoerythrine-conjugated anti-CD25 monoclonal antibodies (BD Biosciences, San Jose, California, USA). After fixing the cells and permeabilizing them, we stained for intracellular Foxp3 using a fluorescein-isothiocyanate-conjugated anti-Foxp3 monoclonal antibody according to the manufacturer's instructions (eBiosciences, San Diego, California, USA). Cells were analysed using a FACScalibur flow cytometer instrument (Becton Dickinson, Franklin Lakes, New Jersey, USA). T_regs were identified as CD25-positive and Foxp3-positive cells among CD4 cells within the lymphocyte gate. Flow cytometry analysis used FlowJo software (V. 6.4 Tree Star, Ashland, Oregon, USA). The CD4 cell count was determined by four-color surface staining using fluorescein-isothiocyanate-conjugated anti-CD3, phycoerythrine-conjugated anti-CD4, peridin chlorophyll protein-conjugated anti-CD45 and activated protein C-conjugated anti-CD8 monoclonal antibodies (BD Biosciences) using a single-platform protocol. The absolute T_reg number was determined by multiplying the proportion of CD25+Foxp3+ by the total CD4 cell count.

Twenty-three antiretroviral-naive HIV patients and 16 HIV-negative healthy individuals consented to participate between December 2005 and March 2006. In the HIV-positive group, 15 were male and eight female. Ages ranged from 19 to 67 years (median 32). Six had previously completed treatment for tuberculosis. In the HIV-negative group, 10 were male and six female, ages ranged from 21 to 49 years (median 28).

The proportion of CD4 T cells among HIV-positive subjects that was also positive for both CD25 and Foxp3 ranged from 2.9 to 28.2% (mean 9.9). This compared with 4.7–8.6% (mean 6.8) in normal controls (P < 0.03). The proportion of CD4 cells that were Foxp3 positive was significantly increased in HIV-positive patients. The proportion of cells positive for Foxp3 increased as the CD4 cell count fell. It was normal in those with CD4 cell counts above 400 cells/μl and was modestly increased in those with CD4 cell counts of 200–400 cells/μl (mean 8.5%, range 4–12.3, P = 0.1, Fig. 1). Marked increases were noted in individuals with very low CD4 cell counts (mean 11.6%, range 2.9–28.2, P = 0.04, Fig. 1). Overall, there was thus an increase in the proportion of Foxp3-positive cells that inversely correlated with CD4 cell counts (Fig. 1).

Fig. 1: The proportion of CD4+CD25+ T cells that were positive for Foxp3 was increased in advanced HIV. Bars show the mean and standard deviation for HIV-negative (black bar) or HIV-positive (grey bars) subjects. The latter are stratified by CD4 cell count. T_reg, T-regulatory. *P < 0.05.

We also analysed the total number of Foxp3-positive cells. The greater dynamic range of CD4 cell numbers overshadowed the increase in the proportion of cells positive for Foxp3 in advanced disease. There was thus an overall decline in T_regs as HIV disease progressed and a close correlation between the number of CD4 cells and the total number of FoxP3-positive cells regardless of the HIV status (Fig. 2, r² = 0.88, P < 0.001).

Fig. 2: The number of T regulatory cells declined as the number of CD4 cells decreased. Scatter distribution of all patients regardless of HIV status (n = 39) and linear regression curve with 95% confidence interval (r ² = 0.88, P < 0.0001). T_reg, T-regulatory.

Overall, our data support a complex relationship between the level of T_regs and HIV. On the one hand, there is a steady decline in the total number of T_regs as HIV progresses. This is in keeping with the data on decreased T_reg function in advanced HIV. However, there is a relative sparing of T_regs in some patients with advanced disease, as reflected by the increasing percentage of cells positive for Foxp3. The latter may reflect the relative sparing of these cells, because HIV preferentially infects activated, proliferating T cells ^[6]. T_regs have lower rates of cell division and thus may be partly spared from the consequences of HIV infection and death associated with activation-induced apoptosis ^[7]. Alternatively, there may be an expansion of these cells in the setting of the chronic immune stimulation characteristic of advanced HIV infection. Curiously, there was a marked variability in the percentage of CD4 cells expressing Foxp3 in those with advanced HIV infection. Although the overall proportion of cells positive for Foxp3 increases, some subjects with low CD4 cell numbers had a normal or even decreased proportion of cells staining for Foxp3. There is also variability in the numbers of advanced patients with immune and inflammatory manifestations in advanced HIV. Further studies are needed to determining whether T_regs are important in blunting these responses.

Sponsorship: This study was partly supported by the Baylor–UT Houston Center for AIDS Research core support grant AI36211 from the National Institute of Allergy and Infectious Diseases and a Fogarty Center training grant (D43TW006569), and also supported by the Framework agreement between the Belgian Directorate General of Aid in Development and the Institute of Tropical Medicine in Antwerp.