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Originally published online as doi:10.1189/jlb.0507281 on November 2, 2007

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(Journal of Leukocyte Biology. 2008;83:254-262.)
© 2008 by Society for Leukocyte Biology

FOXP3 expressing CD127lo CD4+ T cells inversely correlate with CD38+ CD8+ T cell activation levels in primary HIV-1 infection

Lishomwa C. Ndhlovu*,1, Christopher P. Loo*, Gerald Spotts{dagger}, Douglas F. Nixon*,2 and Frederick M. Hecht{dagger},2

* Division of Experimental Medicine and
{dagger} Positive Health Program, Department of Medicine, San Francisco General Hospital, University of California San Francisco, San Francisco, California, USA

1 Correspondence: Division of Experimental Medicine, University of California San Francisco, 1001 Potrero Avenue, San Francisco, CA 94110, USA. E-mail: lishomwa.ndhlovu{at}ucsf.edu

ABSTRACT

During the course of HIV-1 infection, the status of immune activation has been determined to be a powerful indicator of disease progression. The immune system has adopted self-regulatory mechanisms to counterbalance undesirable immune responses. CD25+CD4+ T regulatory (Treg) cells that express the transcription regulator, forkhead box P3 (FOXP3), play an important role in this immunosuppression. Using a combination of Treg cell discriminatory markers (FOXP3, CD25, CD127), we predicted that an expansion of Treg cell subsets would negatively correlate with immune activation during the early stages of HIV-1 infection. We report that FOXP3+CD127lo expressing CD4+ T cells increases in primary HIV-1 infection over time. Furthermore, the FOXP3+CD127lo CD4+ T cells may, in fact, reduce the levels of T cell activation following primary infection. It is interesting that the positive correlation between FOXP3+CD127lo CD4+ and CD25+CD127lo CD4+ T cells noted in HIV-uninfected persons is not only lost but may also be reversed in early, chronic HIV-1 infection. Unlike FOXP3+CD127lo CD4+, the level of FOXP3+CD25+CD127lo CD4+ T cells did not correlate with T cell activation, suggesting that these cells were not effective in reducing T cell activation. These observations suggest that different Treg populations may have different effects on reducing immune activation in HIV-1 infection and that the FOXP3+CD127lo CD4+ T cell population may be particularly important in limiting immune activation.

Key Words: regulation • immunosuppression • viral • AIDS

INTRODUCTION

Immune activation, a normal response to a pathogen, is now recognized as a major driving force of the CD4+ T cell depletion in HIV-1 disease [1 2 3 4 5 6 7 8 ]. Recent evidence strengthens previous suggestions that T cell activation may predict CD4+ T cell depletion during HIV-1 infection better than viral load [7 , 9 10 11 12 ]. It is important that the level of T cell activation is known to be a strong predictor of more rapid disease progression. Factors that are known to influence T cell activation include immune regulators and pathogen-derived cellular components [13 14 15 16 17 ].

Several studies indicate that regulatory T (Treg) cells are essential modulators of immunity to certain pathogens, including malaria, tuberculosis, and Hepatitis B and C [18 19 20 21 22 23 ]. Recent studies have defined Treg cells as a key population that regulates the immune response in HIV-1 and SIV-1 infection [3 , 24 25 26 27 28 29 30 31 32 33 ]. Longitudinal studies of Treg cell frequency in HIV-1 infection have shown divergent results. Some studies report a reduction in Treg cells during the course of HIV-1 infection [3 , 24 , 27 , 30 , 33 ], whereas others show an expansion of the Treg cell pool during HIV-1 infection [28 , 32 33 34 ] or little or no change [26 , 28 , 31 ]. In patients on highly activate antiretroviral therapy (HAART), there appears to be an increase in forkhead box P3 (FOXP3) mRNA, indicating an increase in Treg cell numbers [25 , 33 ]. Thus, changes in Treg cell frequency or number in HIV-1 infection remain unclear. The differences between studies may be, in part, a result of differences in how Treg cells were defined, differing tissues being evaluated, and differences in the stage of HIV infection examined.

Tregs could impact HIV-1 infection in several ways, including suppression of immune activation or suppression of effector anti-HIV-1-specific T cell responses. In acute and primary HIV-1 infection, the balance between suppression of activation and suppression of anti-CD8+ T cell responses could be particularly critical. Little is known about the dynamics of FOXP3 expressing Treg cells during HIV-1 infection, and even less is known about the changes that occur during early infection. We hypothesized that a preferential expansion of the Treg cell pool during early HIV-1 infection would correlate negatively with immune activation.

We have defined Treg cells using a combination of phenotypic markers that better delineate Treg cells [35 36 37 38 ]. In a longitudinal assessment of primary HIV-infected subjects, we analyzed FOXP3 expression, a master regulator of the suppressor properties of CD4+ Treg cells, and the phenotypic markers IL-2R{alpha} chain (CD25) and IL-7R{alpha} chain (CD127), to define the Treg cell population and to determine associations with parameters of disease progression.

In this study, we report that during the evolution of primary HIV-1 infection, an expansion in Treg cell frequencies (as defined as FOXP3+ CD127lo CD4+ T cells) and numbers occurs between the early stage when viral replication is variable and the post-acute stage when viral set point has been established. During primary infection, a weak association between CD25+CD127lo expressing CD4+ T cells and FOXP3+CD127lo CD4+ T cells was evident. In addition, over time, there is a dramatic loss in CD127 expression expressing CD4+ T cells. Associations were determined for the frequency and number of FOXP3+CD127lo expressing CD4+ T cells and T cell activation, and our results reveal a strong negative correlation between the level of T cell activation and the frequency of FOXP3+CD127lo CD4+ T cells, which was most prominent during the early, chronic stage of disease. Our findings suggest that different subsets of Treg cells have differing effects on immune activation in HIV-1 infection and that interventions that increase FOXP3+CD127lo CD4+ T cells might dampen the deleterious effects of immune activation.

MATERIALS AND METHODS

Human study subjects and blood samples
PBMC samples were obtained from eight HIV-1-infected subjects from the University of California San Francisco (UCSF; San Francisco, CA, USA) OPTIONS project at two points post-sero-conversion (Table 1 ). Control HIV-1 seronegative blood samples from eight healthy donors that were age-matched were purchased from the Blood Centers of the Pacific (San Francisco, CA, USA). At the time of blood draw, HIV-1-infected subjects were assessed for complete blood counts and viral load and immunophenotyped for T cell activation marker CD38. All samples were processed with Ficoll-Paque PLUS (Amersham Biosciences, Pittsburgh, PA, USA), and PBMCs were stored frozen in 10% DMSO in FBS prior to subsequent analysis. All HIV-1-infected subjects in this study were antiretroviral therapy-naïve and were within 6 months of HIV-1 antibody seroconversion at initial assessment, based on documented HIV-1 antibody seroconversion or a history compatible with recent HIV-1 infection and less-sensitive HIV-1 antibody results <0.75 OD [39 , 40 ]. Informed consent and approval for this study were obtained in accordance with the guidelines of the University of California San Francisco Committee on Human Research.


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  		Table 1. Patient Characteristics

Immunophenotyping analysis
Frozen PBMCs were rapidly thawed and incubated with a combination of the following conjugated anti-human mAbs: (Alexa 700) CD4, [allophycocyanin-Cy7 (APC-Cy7) and allophycocyanin (APC)] CD25 (BD Biosciences, San Jose, CA, USA), phycoerythrin-Cy5.5 (PE-Cy 5.5) CD8 (Caltag/Invitrogen, Carlsbad, CA, USA), energy coupled dye (ECD) CD3, and phycoerythrin (PE) CD127 (Beckman Coulter, Fullerton, CA, USA). This was followed by intracellular staining for (Pacific Blue) FOXP3 (Biolegend, San Diego, CA, USA) using the protocol as recommended by the manufacturer. Cells were then washed in FACS buffer PBS in 2 mM EDTA and 1% BSA and then fixed in 1% paraformaldehyde before being run on an LSRII flow cytometer (BD Biosciences). Data were analyzed by using Flowjo software, Version 6.4 (Treestar Inc., Ashland, OR, USA). At blood draw, whole blood was stained with conjugated antibodies for CD38 and evaluated by FACScan to determine the activation status of the CD4+ and CD8+ T cell populations. CD38 expression on CD8+ T cells was expressed as the median fluorescence value of less than 10. Absolute cell numbers of CD4+ expressing T cell subpopulations were calculated by multiplying the number of CD4+ T cells (as a proportion of all CD4+ T cells) with absolute CD4+ T cell counts (per mL blood).

Viral load assessment
Plasma HIV-1 viral load was determined by Versant HIV-1 RNA 3.0 assay (bDNA; Bayer, Tarrytown, NY, USA) as described previously [2 ].

Treg cell suppression assay
Putative CD25+CD4+ Treg cells (>85% purity) were obtained by a positive selection noncolumn-based method using the EasySep CD25+ selection kit (StemCell Technologies Inc., Vancouver, Canada) from PBMCs. The fluorescent intracellular dye CFSE (Molecular Probes, Eugene, OR, USA) was used to track cell division. In brief, autologous responder CD4+CD25– T cells were labeled with 1 mM CFSE in PBS and mixed periodically for 10 min at room temperature. Labeling was quenched by addition of an equal volume of complete media (15% FBS in RPMI) for 2 min. The labeled cells were then washed twice, counted, and resuspended in cell culture media and used as responders upon stimulation with Staphylococcal enterotoxin B (SEB; Sigma Aldrich, St. Louis, MO, USA) in the presence or absence of potential suppressor T cell populations. Ratio of suppressors to responders was 1:1.

Statistical analysis
The statistical significance of observed differences between subject groups was determined by a Mann-Whitney rank sum test. A paired Student’s t-test was performed for comparison within subject changes between the different stages of infection, and correlations were assessed using the Spearman rank test for nonparametric data. All results were conducted using Prism Graphpad Ver4a (Graphpad Software, San Diego, CA, USA), and the statistical significance of the findings was set at a P value of less than 0.05.

RESULTS

Characterization of CD4+ Treg cell subsets in HIV-1 infection
In the pursuit of definitive markers of natural and inducible Treg cells, numerous markers have been described to delineate this subpopulation [41 42 43 ]. The transcription factor, FOXP3, and CD25 have predominately been used to define natural Treg cells [42 , 44 ]. We and others [35 36 37 ] have shown recently that natural CD25+CD4+ Treg cells, which retain low CD127 expression from healthy persons, contain the highest expression of FOXP3 and exhibit suppression of autologous T cell proliferation in vitro. We extend these findings and show that this regulatory population is present in PBMCs derived from healthy and HIV-1-infected subjects, although at different frequencies (Fig. 1A 1B 1C ). In addition to FOXP3+CD127lo T cells and CD25+CD127lo T cells, we identify the FOXP3+CD25+CD127lo fraction of CD4 T cells and included these subsets in the overall delineation of Treg cell subpopulations (Fig. 1D 1E 1F) .


Figure 1
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  		Figure 1. Gating strategy for CD4+ T cell subsets. After thawing frozen cells, a phenotypic analysis was performed on PBMCs from healthy blood donors and early-infected, HIV-1-infected subjects (see Table 1 ). (A–D) A representative flow cytometry plot from a healthy and HIV-1-infected specimen showing (A) CD4+ T cells that were evaluated for (B) CD25, (C) FOXP3, CD127, and (D) FOXP3 and CD25 dual expression. Percentages indicate frequency of CD4+ T cells within the CD3+ T cell lymphocyte gate. Fluorescence minus one samples were used to define the gates used. Arrows indicated the gated population analyzed subsequently.

Enumeration of the frequency of CD4+ Treg cell subsets during early HIV-1 infection
We then conducted a longitudinal study of primary HIV-1-infected subjects to assess the dynamics of Treg cell subpopulations by multiparametric flow cytometry. All HIV-1-infected subjects were HAART-naïve during the early and early-chronic stages following HIV-1 infection (Table 1) . We observed no statistically significant increase in the frequency of CD25+CD127lo but observed a statistically significant expansion in the frequency of the FOXP3+CD127lo CD4+ T cell subset in comparison with HIV-1 seronegative control subjects at the earliest time-point examined postinfection (Fig. 2A and 2B ). A reduced frequency of FOXP3+CD25+ CD127lo T cells was seen in HIV-1-infected subjects as compared with healthy subjects, as the expansion of FOXP3+ CD4+ T cells appears to be expanded in the CD25– fraction (Fig. 2C) . A significant loss of CD127 expressing CD4+ T cells was apparent early during the course of the infection in comparison with healthy, control subjects (Fig. 2D) . Previous reports have shown a selective loss of CD127 during chronic HIV-1 infection on CD8+ and CD4+ T cell subsets [45 46 47 48 49 50 51 52 53 ]. Zaunders et al. [28 ] have shown recently that the targeting of CD127 expressing cells has been suggested to be restricted to a CCR5+ activated CD4+ T cell subpopulation. Collectively, these results shed light on the fluctuations in the frequency of Treg cell subsets during the early stage of HIV-1 infection and highlight the early loss of CD127 expressing CD4+ T cells.


Figure 2
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  		Figure 2. Comparison of Treg cell subsets between healthy and early HIV-1-infected subjects. Treg cell subsets were compared between healthy subjects and early HIV-1-infected subjects (A–D). Relationship between FOXP3+ CD127lo expressing T cells and CD25+ CD127lo CD4+ T cells (E–G).

Expansion in the frequency and number of FOXP3+CD127lo CD4+ T cells during the evolution of primary HIV-1 infection
By the early-chronic period of HIV-1 infection, the viral set-point has been established [2 ]. No statistically significant expansion was observed in the frequency of the CD25+CD127lo expressing CD4+ T cell or FOXP3+CD25+ CD127lo CD4 T cell subset between the primary and early-chronic stages of HIV-1 infection (Fig. 2A 2B 2C) . Despite a few HIV-1-infected subjects with a complete loss of FOXP3 expressing CD4+ and CD8+ T cells at the early or early-chronic period of infection, overall, we observed a trend toward an expansion of the frequency and number of FOXP3+ CD127lo expressing CD4+ T cells in this cohort (Fig. 2B , and data not shown). In comparison with healthy, seronegative subjects, a statistically significant, higher frequency of FOXP3+ CD127lo CD4+ T cells was seen in the HIV-1-infected subjects (Fig. 2B) .

The preferential targeting of CD127 expressing CD4+ T cells by HIV-1 has been shown recently, but the kinetics of this loss has been unclear [28 ]. We report that the frequency and number of CD127 expressing CD4+ T cells, which are diminished early in infection, are sustained during the evolution of the infection (Fig. 2D) . The observed pattern of CD127+ CD4+ T cell expression in HIV-1 infection is consistent with previous reports [45 46 47 48 49 50 51 52 53 ].

In summary, the changes in the CD4+ Treg cell subsets in early HIV-1 infection could impact the levels of immune activation, viremia, and/or CD4+ T cell loss.

No association between CD25+ CD127lo and FOXP3+ CD127lo expressing CD4+ T cells during primary HIV-1 infection
Among healthy HIV-1-uninfected subjects, a positive correlation between PBMC-derived CD25+ CD127lo expressing CD4+ T cells and FOXP3+CD127lo expressing CD4+ T cells has been described [36 , 37 ]. We observed that among the HIV-1 seronegative control subjects, a tight association was observed between CD25+CD127lo CD4+ T cells and FOXP3+ CD127lo CD4+ T cells (P=0.0218; Fig. 2E ). In contrast, during the course of primary HIV-1 infection, no statistically significant correlation between FOXP3+CD127lo and CD25+ CD127lo expressing CD4 T cells was observed during the early phase or early-chronic stage of infection (Fig. 2F and 2G) . Our findings support the data from recent studies, which showed that FOXP3 expressing Treg cells are short-lived and being renewed constantly [54 ].

Retained suppression of CD25+CD4+ T cells in primary HIV-1-infected subjects
We next determined whether the CD25+ CD4+ T cells among the HIV-1-infected subjects over time would have variability in their suppressor capacity. No marked difference in suppressor potential was observed, irrespective of the frequency of CD25+CD127lo expressing CD4 T cells. Furthermore, we did not observe any statistically significant differences in the suppressor potential of CD25+CD4+ T cells between the early and early-chronic period of the infection (Fig. 3A 3B 3C 3D ), suggesting that the threshold for suppression is relatively low, and the CD4+ Treg cell population retains potent, intrinsic, functional potential in primary HIV-1 infection.


Figure 3
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  		Figure 3. Suppressor potential of CD4+ Treg cells with variable levels of FOXP3+ CD127lo and CD25+ CD127lo expression. Different frequencies of FOXP3+ CD127lo and CD25+ CD127lo expressing CD4+ T cells following HIV-1 infection in subjects OP506 and OP565. (A) Representative plot of an in vitro coculture suppression assay follows stimulation with SEB for 4–5 days of subject OP506 (early and early-chronic stage) and OP565 (early stage). Sorted CD25– CD4+ T cells were labeled with CSFE and cocultured in the presence of (B) unlabeled CD25–CD4+ T cells (B) or an equal number of (C) unlabeled CD25+CD4+ T cells. (D) Summary of percent inhibition between the early and early-chronic period postinfection (n=5; Pts: 443, 419, 506, 565, and 548).

Relationship among CD4+ T cell count, plasma viremia, and immune T cell activation status during the evolution of primary HIV-1 infection
We then assessed the relationship among CD4+ T cell count, CD38+CD8+ T cell expression as an index of T cell activation, and HIV-1 viral load (Fig. 4A 4B 4C 4D 4E 4F 4G 4H 4I ). In this cohort, we found an inverse correlation between viral load and CD4+ T cell count during the course of primary infection, where statistical significance was achieved during the early-phase postinfection (Fig. 4A and 4B) . In addition, we found an inverse relationship between CD4+ T cell count and T cell activation early in the infection, which was statistically significant during the early-chronic period (Fig. 4B) . Conversely, a positive association was observed between viral load and T cell activation during the early and early-chronic phase of infection, where a strong statistical significance was achieved during the early-phase postinfection (Fig. 4E and 4F) .


Figure 4
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  		Figure 4. Correlation among plasma viral load, CD4+ T cell count, and T cell activation. (A and B) Correlation between CD4 T cells count and viral load. T cell activation was determined by evaluating the M.F.I. of CD8 T cells expressing CD38 prior to cryopreservation of PBMC. (C–F) Correlaation between T cell activation and CD4 count and viral load. (G-I) Longitudinal analysis of CD4 count, viral load, and T cell activation in the HIV-1-infected cohort.

The frequency and number of FOXP3+ CD127lo CD4 T cells correlated negatively with the level of T cell activation in primary HIV-1 infection
We next assessed by regression analysis if an association persisted between the Treg cell subsets and CD4+ T cell count through the course of infection. We show a statistically significant association between the number of FOXP3+CD127lo expressing CD4+ T cells and CD4+ T cell count at the early-chronic stage during the infection, which was not evident early during the infection (Fig. 5A 5B 5C 5D ). This result suggests that CD4+ T cell loss parallels the loss in the FOXP3+CD127lo expressing CD4+ T cell pool.


Figure 5
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  		Figure 5. Correlation between FOXP3+CD127lo CD4+ T cell expression; CD4 T cell count and T cell activation. The accumulation of FOXP3+CD127lo CD4+ T cells was associated with CD4 T cell count (A–D) and CD38+CD8+ T cell activation (E–H) during the early-chronic phase of HIV-1 infection. (I–K) Longitudinal analysis of the level of FOXP3+CD127lo CD4 T cells and T cell activation for comparison in the HIV-1-infected cohort.

Given the strong association between T cell activation and CD4+ T cell counts, we next addressed the question of whether the expansion of the FOXP3+CD127lo expressing CD4+ T cell pool affected T cell activation. During the early-chronic period of infection, we observed a strong inverse correlation between the frequency and number of FOXP3+CD127lo expressing T cells and the level of T cell activation that was not present at the early phase of infection (Fig. 5E 5F 5G 5H) . This association appears to be independent of viral load, as we observed no statistically significant association between viral load and the frequency or number of FOXP3+CD127lo expressing CD4+ T cells during the course of infection (data not shown). This result suggests that individuals with an expanding but renewed FOXP3+CD127lo CD4+ T cell pool will sufficiently suppress the level of T cell activation more prominently during the later stages of primary infection (Fig. 5I 5J 5K) . No association was observed with the levels of FOXP3+CD25+CD127lo CD4+ T cells and T cell activation early (r=–0.08383, P=0.84) or during the early-chronic (r=–0.5238, P=0.19) stage of infection.

DISCUSSION

In this study, we hypothesized that specific Treg cell subsets would correlate with the level of T cell activation during primary HIV-1 infection. We performed a bivariate analysis of the proportion of Treg cells to determine if any relationship occurred between different Treg cell subsets and T cell activation. Our observations suggest that the FOXP3+CD127/lo CD4+ cells may, in fact, reduce the levels of T cell activation following primary infection. It is interesting that the positive correlation between FOXP3+CD127lo CD4+ and CD25+CD127lo CD4+ noted in HIV-negative persons is not only lost but may also be reversed in early-chronic HIV infection. Unlike FOXP3+CD127lo CD4+ T cells, the level of CD25+CD127lo and FOXP3+CD25+CD127lo CD4+ T cells did not correlate with T cell activation, suggesting that these cells were not effective in reducing T cell activation in HIV infection (data not shown). Recent reports about cohorts of chronically HIV-1-infected subjects and studies in the acute SIV model have shown a negative correlation between the Treg cell population and CD8+ T cell activation [3 , 55 ] or alternatively, a weak relationship [56 ]. Our studies resolve the association partially between defined Treg subsets and T cell activation level during primary and early HIV-1 infection.

Persistent immune activation has been recognized as a major participant in driving disease progression in HIV-1 infection and is predictive of disease outcome and treatment outcome [1 2 3 4 5 6 7 8 , 11 , 12 , 57 58 59 ]. T cell activation levels, as measured by the expression of CD38 expression on CD8+ T cells, appear to be established early in HIV-1 infection. During antiretroviral therapy, activation levels decline but may remain elevated compared with uninfected persons [2 ]. Factors suppressing T cell activation could potentially be an important immunotherapeutic strategy in modulating primary HIV-1 infection.

In our cohort, most subjects developed an expansion in the FOXP3+CD127lo expressing CD4+ T cell subsets early during HIV-1 infection that paralleled the increase in overall CD4+ T cell counts. However, this was not universal. Marked fluctuations in the levels of FOXP3 over time resulted in a poor correlation between FOXP3+CD127lo and CD25+CD127lo expressing CD4+ T cells following infection. FOXP3 has been shown to mediate the control of Treg cells through pathways linked to its association with the RUNX1 gene and by a complex with NFAT, which markedly repress IL-2 and confer a suppressor phenotype that is consistent with Treg cell function [60 61 62 ]. Thus, monitoring Treg cells by the inclusion of FOXP3 expression underscores the importance of this master transcription regulator, and exclusively monitoring CD25 expression may not be an accurate reflection of the frequency of CD127lo CD4+ Treg cells during primary HIV-1 infection. We performed additional studies to demonstrate an up-regulation of FOXP3 following TCR stimulation of naïve CD4+ T cells in vitro (data not shown). These cells have been shown to be suppressive [63 ]. We expected that in addition to Treg cells, activated/effector CD4+ T cells would constitute a proportion of the FOXP3 expressing CD4+ T cell population. Nevertheless, during primary HIV-1 infection, the fraction of Treg cells was sufficient to impact the level of T cell activation, suggesting that the FOXP3 expressing CD4+ T cell population retains a sufficient number and fraction of functional FOXP3+CD127lo CD4+ Treg cells. Several studies have found CD25+ and CD25– FoxP3+ Treg cell expansion at tissue sites of HIV expression during chronic infection and little change or reductions in Treg cells in the peripheral blood [26 , 33 , 56 ]. Our results show an expansion of a defined subset of Treg cells in primary/early-chronic HIV infection in PMBCs and may differ from these studies in several ways: The definition for Treg cells used in these previous studies did not represent the CD127lo T cell fraction; and longitudinal assessment of Treg cell frequencies in acute HIV-1 infection provides a better presentation of the fluctuations that occur during the course of infection. The scope of this study was limited to PBMCs, and additional work to define Treg cell subsets from other tissue sites would be important to observe the dynamics of Treg cell frequency over time and determine if they similarly associate with the level of immune activation. Several studies provide data demonstrating the production of suppressive factors by (CD25–) FOXP3+ cells in tissue sites that may account for the strong correlation we observe in the FOXP3+CD127lo T cell subset and T cell activation [55 , 63 ]. Recent findings show that CD25– CD4+ T cells can be stimulated to express FOXP3, but these cells retain poor suppressor properties [64 ]. One divergence between these results and ours may be related to the differences in which FOXP3 is up-regulated and whether an infection versus in vitro induction may lead to differing functional properties. The suppressor potential of this subset on impacting T cell activation may be occurring at tissue sites where the impact of Treg cells is presumably more prominent. Nevertheless, these results stress the importance of identifying more definitive markers of suppressor T cells among the FOXP3 expressing population, particularly following in vivo activation in the diseased state.

Fluctuation in the frequency of FOXP3 expressing CD4+ T cells during primary infection could in part be explained by the developing hypothesis that FOXP3 expressing Treg cells may be short-lived. Vukmanovic-Stejic and colleagues [54 ] suggest that Treg cells may be undergoing rapid turnover in vivo as opposed to a static population that persists through life. We propose that Treg cells are preferentially targeted by HIV-1 infection; however, during the course of primary infection, the Treg cell pool is replenished, as demonstrated by the fluctuations in the proportion of FOXP3+CD127lo expressing CD4+ T cells. An increase in FOXP3 expression over time could not only decrease the level of immune activation but also negatively influence effector CD8+ T cell functions in vivo.

A better definitive characterization of suppressor CD25+CD4+ Treg cells has been a major area of research interest to help distinguish CD25+CD4+ Treg cells from nonsuppressor-activated CD4 T cells. The divergent results in studies of the dynamics of Treg cells in HIV-1 infection may be attributable, in part, to the different criteria used to define Treg cells [3 , 24 , 27 , 28 , 30 31 32 33 ]. Furthermore, as CTLA-4 and FOXP3, which are the current favored markers of Treg cells, are present intracellularly, evaluation and isolation of purified populations have posed a challenge [42 , 44 , 65 66 67 ].

We demonstrate that during the evolution of primary HIV-1 infection, an expansion in Treg cell frequencies (as defined as FOXP3+ CD127lo CD4+ T cells) and numbers occurs between the early stage when viral replication is variable and the post-acute early-chronic stage when viral set point has been established. The initial time-points we measured were usually 12 weeks or more after we estimate that infection occurred, and thus, we were not able to determine the relationships among Tregs, activation, and viral load during the earliest stages of primary infection. Unlike FOXP3+CD127/lo CD4+, the level of CD25+CD127lo CD4+ did not correlate with T cell activation, suggesting that these cells were not effective in reducing T cell activation in HIV. Taken together, these results favor the evaluation of Treg cells using the FOXP3+CD127lo CD4+ T cell subset and help explain some of the variability that has been observed in monitoring Treg cells. The scope of patients and this study did not enable us to examine Treg cells in gut or organized lymphoid tissues; however, we were able to observe in PBMCs a strong, negative correlation between the proportion of FOXP3+CD127lo CD4+ T cells and T cell activation during the early-chronic stage of infection.

Overall, these observations suggest that different Treg populations may have different effects on reducing immune activation in HIV infection and that manipulation of the FOXP3+CD127lo CD4+ population may be particularly important in limiting the deleterious effects of immune activation in HIV-1 infection.

ACKNOWLEDGEMENTS

This work was supported by the National Institute of Health grants AI68498 and U01 AI41531, the AIDS Biology Program of UCSF AIDS Research Institute, and by the Irvington Institute and DANA Foundation H.I. Award (to L. C. N.). We gratefully acknowledge Martin Bigos and Dr. Jason Barbour for helpful discussions and advice.

FOOTNOTES

2 These authors are equal senior coauthors. Back

Received May 4, 2007; revised October 9, 2007; accepted October 9, 2007.

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