T-regulatory (T-reg) cells are important in the maintenance of immunologic self-tolerance and in modulating the immune response to non-self-antigens. T-reg cells can be identified by the coexpression of the interleuken (IL)-2 receptor, CD25, and the IL-7α chain receptor, CD127.1-3 In addition, the T-reg cell population is comprised of distinct subsets with unique roles. These include natural or naive T-reg cells that are directly derived from the thymus and recognize self-antigens.4 Peripherally inducible or memory T-reg cells, on the other hand, are generated from nonregulatory T cells after exposure to cognate antigens in peripheral tissues.5 These subsets may play distinct roles in diseases ranging from multiple sclerosis6 to multiple myeloma.7
Human immunodeficiency virus (HIV) infection is associated with decreased numbers of CD4+CD25+ T-reg cells8 but an increase in the percentage of CD25+CD127loCD4+ T-reg cells. T-reg cell abnormalities are associated with the elevated levels of immune activation that is a characteristic of HIV infection.3,9 The effect of HIV infection on naive and memory CD25+CD127loCD4+ T-reg cells has not been reported. Determining the effect on these subsets is important because these subsets mediate unique functions through distinct mechanisms and may be related to different aspects of HIV pathogenesis. These studies were designed to determine the association of naive and memory CD25+CD127loCD4+ T-reg cells with pathogen-specific immunity and surrogate markers of disease progression including viral load, CD4+ T-cell count, and immune activation.
Blood specimens were obtained from HIV-infected (HIV+) and uninfected (HIV−) subjects who were recruited from Rush University Medical Center. Peripheral blood mononuclear cells (PBMCs) were isolated by density centrifugation using lymphocyte separation medium (Biowhittaker, Walkersville, MD). Isolated PBMCs were either used immediately for lymphocyte proliferation assays or were cryopreserved. Plasma HIV-1 RNA and CD4+ T-cell numbers were obtained at the same time and were measured in CLIA-certified commercial laboratories.
T-cell phenotypes were determined using a 4-color flow cytometry on frozen PBMCs with FACSCalibur and CellQuest software (Becton Dickinson, San Jose, CA). Mouse anti-human monoclonal antibodies to CD4, CD8, CD25, CD127, CD45RO, CD38, HLA-DR, and isotype control antibodies conjugated to fluorescein isothiocyanate, phycoerythrin, peridinin-chlorophyll-protein, PE-Cy5, or allophycocyanin (BD-Pharmingen, San Jose, CA) were used. A representative gating strategy is shown in Figure 1. Total leukocyte counts were also done at the same time, and the absolute numbers of total, naive, and memory T-reg cells were determined.
Lymphocyte proliferation assays for pokeweed mitogen (0.1 μg/mL; Sigma Chemical, St. Louis, MO), Candida albicans skin test antigen (Candida skin test antigen 10 μg/mL; Greer Laboratory, Lenoir, NC), and HIV p24 antigen/control (5 μg/mL; Protein Sciences, Meriden, CT) were done on fresh PBMCs as described previously.10 Results were expressed as a stimulation index, defined as the ratio of the median counts per minute of the wells with antigen to the median counts per minute of the wells without antigen. A stimulation index of ≥10 was considered a positive response.
Group comparisons were done using the Kruskal-Wallis test. Continuous variables were compared using the Mann-Whitney rank sum U test. Categorical variables were compared using χ2 tests of independence. Correlations between variables were measured using the Spearman rank correlation test. All P values were 2 sided at a 0.05 significance level. All analyses were done using SPSS (Chicago, IL).
We studied 74 HIV+ subjects (14 treatment naive, 60 on highly active antiretroviral therapy (HAART)) and 10 HIV− subjects. Sex, age, CD4+ T-cell counts, and plasma HIV-1 RNA are shown in Table 1. We used the presence or absence of CD45RO on CD25+CD127loCD4+ T-reg cells to define memory (CD45RO+) and naive (CD45RO−) T-reg cell subsets. We then determined the correlation between CD4+ T-cell numbers and the relative percentages and absolute numbers of CD25+CD127loCD4+ T-reg cells. The absolute number of total, naive, and memory T-reg cells directly correlated with the absolute number of CD4+ T cells (Table 2). In contrast, as CD4+ T cells declined, the percentages of total and memory T-reg cells increased, whereas the percentage of naive T-reg cells decreased (Fig. 2). CD4+ T-cell number had a significant, inverse correlation with the percentages of total and memory T-reg cells (Table 2). Plasma HIV-1 viral load correlated inversely with the absolute number of memory and naive T-reg cells and percentage of naive T-reg cells (Table 2).
We determined the relationship between CD25+CD127loCD4+ T-reg cell subsets and immune activation, as defined by the coexpression of CD38 and HLA-DR on CD4+ and CD8+ T cells (Table 2). The absolute number of total, memory, and naive T-reg cells had a significant, inverse correlation with the percentages of CD38+HLA-DR+CD4+ and CD8+ T cells. The percentages of total, memory, and naive T-reg cells did not correlate significantly with the percent-activated CD4+ or CD8+ T cells.
We evaluated the lymphoproliferative response to HIV and Candida antigens in a subset of our HIV+ subjects (n = 26) who were on HAART and had viremia >50 copies/mL (Fig. 3). We compared the absolute number and proportion of total, naive, and memory CD25+CD127loCD4+ T-reg cells between subjects with and without a proliferative response with these antigens. The percentage of total T-reg cells was significantly lower in subjects who had a response to HIV or Candida. Also, the percentage of memory T-reg cells was significantly lower in subjects with a response to Candida. Although the absolute numbers of total, memory, and naive T-reg cells were not significantly different between subjects with or without a response to these antigens, CD4+ T cells were higher in those with a positive response.
This is the first study to evaluate the effect of HIV infection on CD25+CD127loCD4+ T-reg cell subsets. We have shown that although memory and naive T-reg cell numbers decline in parallel with CD4+ T cells, the CD4+ T-cell pool becomes enriched with CD25+CD127loCD4+ T-reg cells with a memory phenotype. This indicates not only a greater loss of CD127hiCD4+ T cells compared with CD127loCD4+ T-reg cells11 but also a greater loss of CD45RO− compared with CD45RO+ T-reg cells. Alternatively, the increase in the percentage of memory T-reg cells may reflect the expansion of T-reg cells in response to chronic exposure to antigens, such as HIV or other pathogens, that have breached an immune system weakened by HIV disease.11,12 The higher percentages of total and memory T-reg cells were associated with the absence of Candida- and HIV-specific immunity. T-reg cells from HIV+ individuals have potent suppressive capacity.13
Our findings imply that declining absolute T-reg cell numbers may contribute to the high levels of immune activation seen in HIV disease. However, we did not see a correlation between percent CD25+CD127loCD4+ T-reg cells and measures of immune activation. The latter observation is consistent with other studies9,14 but inconsistent with a study that used HLA-DR expression as the activation marker.3 We used a more reliable marker of activation, the coexpression of CD38 and HLA-DR. In aggregate, these findings suggest that the decline in peripheral T-reg cells is likely a part of the overall decline in CD4+ T cells in the setting of chronic activation and not a direct cause of increasing systemic activation.
Our study is limited by its cross-sectional nature. Prospective studies that correlate changes in the T-reg cell subsets with changes in CD4+ T cells, plasma HIV-1 RNA, immune activation, pathogen-specific immunity, and HAART will be more informative. Moreover, most of our subjects were on HAART, which may influence the HIV-mediated effects on T-reg cells. Despite this, we saw significant T-reg cell abnormalities in treated HIV+ individuals.
In aggregate, our data indicate that CD25+CD127loCD4+ T-reg cells play an important role in the immunodeficiency seen in HIV. The enrichment of the circulating CD4+ T-cell pool with CD25+CD127lo cells with a memory phenotype parallels a similar trend in lymphoid tissues12 and may lead to the suppression of pathogen-specific immunity and contributes to the lack of immune control of HIV. The therapeutic manipulation of this subset may modulate pathogen-specific immune responses in HIV disease.
The authors would like to acknowledge Julia Bienias, PhD, and Yuxiao Tang, PhD, for help with the statistical analysis and Harold Kessler, MD, Beverly Sha, MD, Joan Swiatek, RN, and Kristine Richards, RN, of the Mark Weiss Clinic for Infectious Diseases for their help in recruiting subjects for this study.
1. Seddiki N, Santner-Nanan B, Martinson J, et al. Expression of interleukin (IL)-2 and IL-7 receptors discriminates between human regulatory and activated T cells
. J Exp Med
2. Liu W, Putnam AL, Xu-Yu Z, et al. CD127 expression inversely correlates with FoxP3 and suppressive function of human CD4
+ T reg cells. J Exp Med
3. Lim A, Tan D, Price P, et al. Proportions of circulating T cells
with a regulatory cell phenotype increase with HIV
-associated immune activation
and remain high on antiretroviral therapy. AIDS
4. Sakaguchi S, Ono M, Setoguchi R, et al. Foxp3+ CD25+ CD4
+ natural regulatory T cells
in dominant self-tolerance and autoimmune disease. Immunol Rev
5. Vigouroux S, Yvon E, Biagi E, et al. Antigen-induced regulatory T cells
6. Haas J, Fritzsching B, Trübswetter P, et al. Prevalence of newly generated naive regulatory T cells
(Treg) is critical for Treg suppressive function and determines Treg dysfunction in multiple sclerosis. J Immunol
7. Beyer M, Kochanek M, Giese T, et al. In vivo peripheral expansion of naive CD4
+CD25high FoxP3+ regulatory T cells
in patients with multiple myeloma. Blood
8. Tsunemi S, Iwasaki T, Imado T, et al. Relationship of CD4
+CD25+ regulatory T cells
to immune status in HIV
-infected patients. AIDS
9. Eggena MP, Barugahare B, Jones N, et al. Depletion of regulatory T cells
infection is associated with immune activation
. J Immunol
10. Lederman MM, Connick E, Landay A, et al. Immunologic responses associated with 12 weeks of combination antiretroviral therapy consisting of zidovudine, lamivudine, and ritonavir: results of AIDS Clinical Trials Group Protocol 315. J Infect Dis
11. Nilsson J, Boasso A, Velilla PA, et al. HIV
-1-driven regulatory T-cell accumulation in lymphoid tissues is associated with disease progression in HIV
12. Andersson J, Boasso A, Nilsson J, et al. The prevalence of regulatory T cells
in lymphoid tissue is correlated with viral load in HIV
-infected patients. J Immunol
13. Aandahl EM, Michaelsson J, Moretto WJ, et al. Human CD4
+ CD25+ regulatory T cells
control T-cell responses to human immunodeficiency virus and cytomegalovirus antigens. J Virol
14. Oswald-Richter K, Grill SM, Shariat N, et al. HIV
infection of naturally occurring and genetically reprogrammed human regulatory T-cells. PLoS Biol