Analysis of CD4 + CD25 + Foxp3 + Regulatory T Cells in HIV-Exposed Seronegative Persons and HIV-Infected Persons with Different Disease Progressions

Abstract

Regulatory T cells (Tregs) are a subset of T cells that play an important role in the regulation of T-cell function. In a previous study, CD25 was used as a marker of Tregs; however, FoxP3 was recently discovered to be a valuable phenotype of Tregs. In this study, we compared the frequency of Tregs in HIV-1-infected long-term nonprogressors (LTNP), AIDS patients (AP), HIV-exposed seronegative (ES) persons, and healthy controls (HC), by using CD4⁺CD25⁺FoxP3⁺ as a marker of Tregs. The results showed that the frequency of Tregs in AP was significantly higher than in the LTNP, ES, and HC, which suggests that Tregs may play a role in disease progression. Another unique finding in this study is that we found a decrease of Tregs in ES.

Introduction

H uman immunodeficiency virus type-1 (HIV-1) infection leads to a defined pattern of disease progression in most infected individuals. After primary infection, which manifests in some individuals as an acute clinical syndrome, a prolonged period of clinical latency and then clinically overt disease characterized by the emergence of opportunistic infections and tumors occurs, with an average time range of 9–10 y (1). However, HIV-1-infected individuals present with different rates of disease progression (2,3). In some individuals viral replication is controlled, resulting in a nonprogressive phenotype (4). Moreover, documentary evidence suggests that some people can achieve some degree of resistance to HIV infection, and they remain uninfected despite exposure to HIV (5,6).

Many nonprogressors maintain vigorous and broadly directed HIV-specific cytotoxic T-lymphocyte (CTL) and CD8-mediated HIV-suppressive activity (4,7 –10), which is also seen in seronegative individuals who were exposed to HIV. These findings imply a central role for cell-mediated immunity in the control of HIV infection and disease progression. Nonetheless, the molecular bases for the accomplishment of cell-mediated immunity in those people remain largely unknown.

Regulatory T cells (Tregs) were first described in 1995 by Sakaguchi et al. (11). They can suppress antigen-specific CD4 and CD8 responses, and also control inappropriate or exaggerated immune activation induced by pathogens (12,13). Considering the critical role of Tregs in the regulation of immune cells, it was not surprising to find that Tregs may play a role in the dynamics of immune activation during HIV infection (14,15). However, only limited and inconsistent findings regarding the changes in Tregs during HIV-1 infection have been demonstrated (14,16 –19). In this article, we examined the correlation between the frequency of Tregs and HIV infection or disease progression in Chinese persons by using FoxP3⁺CD25⁺CD4⁺CD3⁺ as the phenotype of Tregs (20).

Materials and Methods

Ten HIV-exposed seronegative persons (ES),10 long-term nonprogressors persons with HIV infection (LTNP),10 AIDS patients (AP), and 10 healthy controls (HC), were screened and enrolled in the study with informed consent using the following criteria: (1) all ES individuals were mates of HIV-infected people who had unprotected sex with their spouse, but remained seronegative for more than 10 y; (2) LTNP persons were infected with HIV for more than 15 y, and had CD4 cell numbers >500 cells/μL, though all of them were treatment-naïve; (3) AP were HIV-infected treatment-naive individuals with CD4 cell numbers <200 cells/μL who had not taken any immunomodulatory agents in the year before being enrolled in the study; and (4) HC were normal control persons free of HIV risk factors who were screened with a blood test and questionnaire. The clinical data are shown in Table 1. The phylogenetic analysis of C2V3 sequences revealed that all sequences were subtype B.

Table 1.

Epidemiological and Clinical Data of the Subjects Enrolled in the Study

	LTNP	AP	ES	HC
Age (y)	29–55	37–52	37–46	30–50
Transmission route	BBT	BBT	None	None
Viral load (log)	2.87 ± 1.35	4.41 ± 0.73	None	None
CD4⁺ T cells/mm³	616.6 ± 109.0	114.6 ± 62.12	776 ± 308.5	499.1 ± 145.1
CD8⁺ T cells/mm³	1253.7 ± 522.5	865.8 ± 522.6	494.6 ± 195.8	391.5 ± 156.3
CD3⁺ T cells/mm³	1969.4 ± 522.1	1209.5 ± 489.4	1379 ± 429.1	1007.3 ± 314.9

BBT, blood-borne transmission; LTNP, long-term nonprogressors; AP, AIDS patients; ES, HIV-exposed seronegative persons; HC, healthy controls.

To identify the Tregs, 100 μL of fresh peripheral blood were first stained with anti-CD4-FITC and anti-CD25-APC (BD Biosciences, San Jose, CA) for 20 min at room temperature before the blood was lysed and washed. The isotype control was added for gating CD25⁺ T cells. PMT voltages were adjusted to set the negative population at a reproducible rate. Staining of intracellular FoxP3 was performed according to the manufacturer's protocol (eBioscience, San Diego, CA) by using rat IgG_2a-PE as the isotype control. The cells were analyzed with a FACS flow cytometer (BD Biosciences). Tregs were identified as both CD25- and FoxP3-positive cells among CD3⁺CD4⁺ T lymphocytes, and absolute Treg cell numbers were determined by multiplying the proportion of CD4⁺CD25⁺FoxP3⁺ cells by the total CD4 cell count (Fig. 1).

FIG. 1.

Levels of CD4⁺CD25⁺FoxP3⁺ T cells from subjects in the study groups differed. Fresh peripheral blood was first stained with anti-CD4-FITC and anti-CD25-APC, before the blood was lysed and washed. The isotype control was added for gating CD25⁺ T cells. Staining of intracellular FoxP3 was performed using anti-FoxP3-PE and rat IgG_2a-PE as the isotype control. (A) Representative gating strategy used for the CD4⁺CD25⁺FoxP3⁺ T-cell counts. (B) Representative flow cytometry of individuals from the study groups.

Results and Discussion

Many phenotypic markers have been identified for the discrimination of Tregs, such as CTLA-4, GITR, CD62L, CD103, LAG-3, and CD25. Among them, CD25 was mostly popularly used in the early study of Tregs; however, this marker was later shown to be nonspecific for Tregs (21,22). Most recent studies have provided strong evidence that the forkhead transcription factor, FoxP3, may be the best marker to define Tregs (23). To check the difference in defining Tregs by using these two different markers, we compared the consistency between CD4⁺CD25⁺ cells and CD4⁺FoxP3⁺ cells. A significant difference was found between the frequency of CD4⁺CD25⁺ T cells (21.15 ± 6.96%) and CD4⁺FoxP3⁺ T cells (8.23 ± 3.89%; p < 0.01) using Wilcoxon's test, although a significant correlation between them was also identified (p < 0.01) using Pearson's method, which may be one reason that inconsistent results were found in previous studies in which different markers of Tregs were used.

Our data showed that Tregs with CD3⁺CD4⁺CD25⁺FoxP3⁺ phenotype constitute a small fraction of the CD4 T cells in the healthy Chinese population, with a median of 4.47%. This percentage was slightly increased to 6.45% in AP, and decreased to 3.58% in LTNP (Table 2). A significant difference was found between the AP and LTNP (p < 0.05), which is consistent with reports published previously (24), using the Kruskal-Wallis test. The results suggested that HIV-1-infected individuals with low CD4 T-cell counts have, on average, an elevated percentage of Tregs compared to those with a high CD4 level. It was interesting that the frequency of Tregs/CD4 also decreased to 3.45% in ES, although no statistical significance was found when comparing them to healthy controls (Table 2). The absolute numbers of Tregs in AIDS patients was significantly lower than in the HC, LTNP, and ES groups (p < 0.01), using the LSD method and one-way ANOVA. The finding was consistent with that of the study of Thorborn et al., in which the reduction in absolute Treg cell number was found in chronic HIV infection (25).

Table 2.

Frequency of Tregs in LTNP, AP, ES, and HC

	LTNP	AP	ES	HC
% CD4⁺CD25⁺FoxP3⁺ T cells^*	3.58 ± 1.65	6.45 ± 4.19	3.45 ± 1.03	4.47 ± 1.16
CD4⁺CD25⁺FoxP3⁺ cells/μL^**	21.64 ± 9.41	8.15 ± 4. 32	25.88 ± 10.46	21.57 ± 5.35
% CD4⁺CD25⁺ T cells	16.77 ± 5.80	22.09 ± 8.08	17.91 ± 4.45	24.88 ± 5.72
CD4⁺CD25⁺ T cells/μL	102.94 ± 38.63	28.50 ± 16.11	133.91 ± 53.24	120.28 ± 31.69
% CD4⁺FoxP3⁺ T cells	6.80 ± 1.80	11.71 ± 5.25	6.41 ± 1.98	6.65 ± 1.54
CD4⁺FoxP3⁺ T cells/μL	41.49 ± 12.04	15.01 ± 7.30	47.63 ± 18.11	32.14 ± 8.04

*p < 0.05 for AP versus LTNP; **p < 0.01 for AP versus LTNP, ES, or HC.

LTNP, long-term nonprogressors; AP, AIDS patients; ES, HIV-exposed seronegative persons; HC, healthy controls; Tregs, regulatory T cells.

To further investigate the relationship between Tregs and disease progression, we analyzed the correlation between Tregs and CD4 cells. The absolute number of Tregs was determined by multiplying the proportion of Tregs by the CD4 T-cell count. The correlation between Tregs and CD4 cells was determined with the Spearman rank correlation test by using SPSS 10.0 software. A regression line was calculated by the least-squares method, and p < 0.05 was considered significant. Positive correlations were found between the numbers of Tregs and CD4 T cells, both in HIV-1-infected people and seronegative individuals, which is consistent with data from previous studies (26). Significantly smaller numbers of Tregs were seen in AIDS patients (15.01 ± 7.30/μL), compared to the LTNP, ES, and HC groups, among which no significant differences were found (Table 2). Taken together, our data indicate that Tregs appear to decline at a slower rate than do other CD4 T-cell subpopulations, resulting in a relative expansion of this cell subset in AIDS patients.

In summary, we analyzed the correlation between Treg frequency and disease progression, and found that the frequency of Tregs in AIDS patients was increased significantly, suggesting that Tregs may play an important role in HIV disease progression. In addition, an effect of Tregs in preventing HIV infection was also observed, one of the unique findings being that the frequency of Tregs in ES individuals was lower than normal controls, although no statistical significance was found. Positive correlations were found between the numbers of Tregs and CD4 T cells, both in HIV-1-infected persons and seronegative individuals. Although further studies are needed to better illustrate the functions of FoxP3-expressing cells, our data are promising, and help to clarify the role of Tregs in preventing HIV infection.

Footnotes

Acknowledgments

This work was supported by the National Grand Fundamental Research 973 Program of China (grant no. 2006CB504206), the National Key S&T Special Projects on Major Infectious Diseases (grant no. 2008ZX10001-002), and the National Natural Science Foundation of China (grant no. 30700706).

Author Disclosure Statement

No competing financial interests exist.

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