# CD4+CD25+CD127− assessment as a surrogate phenotype for FOXP3+ regulatory T cells in HIV-1 infected viremic and aviremic subjects

Julien Saison, Julie Demaret, Fabienne Venet, Christian Chidiac, Christophe

Malcus, Francoise Poitevin-Later, Jean-Claude Tardy, Tristan Ferry,
Guillaume Monneret

► To cite this version:

Julien Saison, Julie Demaret, Fabienne Venet, Christian Chidiac, Christophe Malcus, et al. CD4+CD25+CD127− assessment as a surrogate phenotype for FOXP3+ regulatory T cells in HIV-1 infected viremic and aviremic subjects. Cytometry Part B: Clinical Cytometry, 2013, 84B (1), pp.50-54. 10.1002/cyto.b.21047. inserm-04061780

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## Brief Communication

# CD4+CD25+CD127− Assessment as A Surrogate Phenotype for FOXP3+ Regulatory T Cells in HIV-1 Infected Viremic and Aviremic Subjects

Julien Saison,<sup>1,2,3†</sup> Julie Demaret,<sup>1,2†</sup> Fabienne Venet,<sup>1,2</sup> Christian Chidiac,<sup>2,3,4</sup> Christophe Marcus,<sup>1</sup> Francoise Poitevin-Later,<sup>1</sup> Jean-Claude Tardy,<sup>5</sup> Tristan Ferry,<sup>2,3,4</sup> and Guillaume Monneret<sup>1,2*</sup>

**Background:** Although likely pivotal, the role of regulatory T cells (Tregs) in HIV pathogenesis remains elusive. This can be partly explained by analytical issues regarding their phenotypic identification in clinical studies. Instead of intracellular FOXP3 staining, CD4+CD25+CD127− phenotype has been proposed as an alternative to identify Tregs in clinical samples. However, its use remains controversial in viremic patients. Therefore, the objective of the present study was to assess the correlation between frequencies of CD4+CD25+CD127− and CD4+CD25+FOXP3+ lymphocytes in viremic and matched aviremic HIV-infected patients.

**Methods:** Peripheral blood was collected from HIV-1 infected patients. Eleven viremic patients (Viral Load > 40 copies/mL) were matched (age, sex, CD4+ cell number) with 8 aviremic patients under highly active antiretroviral therapy (HAART). Fresh whole blood was immediately stained to analyze by flow cytometry the correlation between CD4+CD25+CD127− and the reference phenotype CD4+CD25+FOXP3+ lymphocytes in the same tube (four color staining CD4/CD25/CD127/FOXP3 for concomitant analysis of cell surface and intracellular markers).

**Results:** In both groups, no significant differences were observed when comparing CD4+CD25+CD127− and CD4+CD25+FOXP3+ cell frequencies. In line, a strong correlation between CD4+CD25+CD127− and CD4+CD25+FOXP3+ lymphocyte percentages was observed in the whole patient population (r: 0.948, P < 0.001) or each group separately: aviremic (r: 0.968, P < 0.001), viremic (r: 0.9, P < 0.001). Finally, we found that most CD4+FOXP3+ cells were indeed CD25+CD127−, both in viremic and aviremic groups (88.5% and 90.9%, respectively).

**Conclusions:** We observed that CD4+CD25+CD127− phenotype is a good and easy-to-perform surrogate identification strategy for FOXP3+ regulatory T cells in both viremic and aviremic HIV-1-infected subjects. Thus, it represents a useful tool for monitoring Tregs in clinical research studies based on large cohorts of patients prospectively monitored, including HIV-infected subjects. © 2012 International Clinical Cytometry Society

**Key terms:** CD4+CD25+; FOXP3; regulatory T cells; HIV; flow cytometry

How to cite this article: Saison J, Demaret J, Venet F, Chidiac C, Marcus C, Poitevin-Later F, Tardy J, Ferry T, Monneret G. CD4+CD25+CD127− assessment as a surrogate phenotype for FOXP3+ regulatory T cells in HIV-1 infected viremic and aviremic subjects. Cytometry Part B 2013; 84B: 50–54.

<footnote n="1"><sup>1</sup>Hospices Civils de Lyon, Hôpital Edouard Herriot, Laboratoire d'Immunologie, Lyon, F-69003, France</footnote>

<footnote n="2"><sup>2</sup>Université de Lyon, Université Claude Bernard Lyon 1, Lyon, F-69008, France</footnote>

<footnote n="3"><sup>3</sup>Hospices Civils de Lyon, Hôpital de la Croix Rousse, Service de Maladies Infectieuses et Tropicales, Lyon, F-69004, France</footnote>

<footnote n="4"><sup>4</sup>INSERM, U851, 21 avenue Tony Garnier, Lyon, F-69007, France</footnote>

<footnote n="5"><sup>5</sup>Hospices Civils de Lyon, Hôpital de la Croix Rousse, Laboratoire de Virologie, Lyon, F-69004, France</footnote>

<footnote>†Julien Saison and Julie Demaret contributed equally to this work.</footnote>

<footnote>*Correspondence to: Guillaume Monneret, Immunology Laboratory, Hopital E.Herriot, Hospices Civils de Lyon, 5 places d'Arsonval, 69437 Lyon Cedex 03, France.</footnote>

<footnote>E-mail: guillaume.monneret@chu-lyon.fr</footnote>

<footnote>Received 25 April 2012; Revision 31 July 2012; Accepted 4 September 2012</footnote>

<footnote>Published online 27 September 2012 in Wiley Online Library (wileyonlinelibrary.com).</footnote>

<footnote>DOI: 10.1002/cyto.b.21047</footnote>
HIV infection severely impacts immune response ho-
meostasis, leading to development of chronic immune
activation and gradual immune cell depletion. Regula-
tory T cells (Tregs) are known to modulate the host
response to viral infections (1,2), but their role in HIV
pathogenesis is still controversial. They are either sup-
posed to suppress protective anti-HIV cell mediated im-
munity (3-5) or to control immune activation (6,7),
which plays a major role in disease progression. More
recently, Moreno et al. also reported that Tregs may con-
trol viral replication during the early phase of infection
(8). Several reasons may explain these discrepancies. On
the one hand, the heterogeneity of patients' population
has to be taken into account (level of viremia, type of
progressors, treatments duration ...). On the other
hand, analytical issues may have contributed to discor-
dances: markers used for Tregs phenotyping, staining of
whole blood or purified cells, sample conservation (i.e.,
cryopreserved or not) (9,10).

Today the most widely accepted phenotype for Tregs is the coexpression of CD4, CD25 (α-chain of the IL-2 receptor), and of FOXP3 (11). FOXP3 is the key transcriptional factor for Tregs. However, its intracellular staining is hardly usable on a daily clinical routine basis in large series of samples (several steps of incubation/washing, long incubation times, use of isotype control). As a consequence, some authors took advantage of an alternatively described phenotype: the coexpression of CD4 and CD25 associated with the absence or the low expression of CD127 (α-chain of IL-7 receptor) (i.e., CD4+CD25+CD127−) (12). A good correlation between these two phenotypes was reported in healthy subjects (13) and in septic patients (14). However, a debate remains in HIV infected patients. Indeed, this correlation was found in aviremic patients but not in viremic ones (15). In contrast, other studies, based on cryopreserved or purified cells, found an acceptable correlation in viremic patients (16,17).

Considering the potential clinical interest of monitor-
ing Tregs during HIV infection (18-20), and the need to
further investigate their role in the different steps of dis-
ease progression, (especially during the phase of vire-
mia), the objective of the present study was to assess in
fresh samples the correlation between frequencies of
CD4+CD25+CD127- and CD4+CD25+FOXP3+ lym-
phocytes in viremic and matched-aviremic HIV infected
patients.

MATERIALS AND METHODS

Study Population and Blood Samples

HIV-1 infected patients from the Hospices Civils de
Lyon HIV cohort, located in Croix-Rousse Hospital
(Lyon, France) were consecutively included in the study
between February 15th and 29th, 2012. These patients
were concomitantly matched (sex, age, CD4+ cell num-
ber) with aviremic patients under highly active antiretro-
viral therapy (HAART). Analyses were performed on
available residual blood after routine analysis (i.e., CD4+

lymphocyte count) at the Cellular Immunology Labora-
tory (Hôp. E. Herriot, Lyon, France).

Flow Cytometry

Expression of cell surface and intracellular markers
was assessed on fresh whole EDTA-anticoagulated pe-
ripheral blood. Flow cytometry protocol has been previ-
ously described (14). Briefly, lymphocytes were stained
with monoclonal antibodies according to manufacturer's
recommendation: ECD-labeled anti-CD4, PE-labeled anti-
CD127, PC5-labeled anti-CD25 (Beckman Coulter, Hia-
leah, FL), FITC-labeled anti-FOXP3 (eBioscience, San
Diego, CA). Appropriate isotypic control (Rat IgG 2a-
FITC, eBioscience) was used to evaluate nonspecific
staining and set up positive threshold for FOXP3. A four
color staining (CD4/CD25/CD127/FOXP3) for concomi-
tant analysis of cell surface and intracellular markers was
performed. Cell surface staining was performed first.
Then, after fixing and permeabilizing, samples were
stained for intracellular FOXP3. Red blood cells were
lysed using Versalys reagent (Beckman-Coulter, Miami,
FL). Data were acquired on a NAVIOS flow cytometer
and analyzed using the NAVIOS software (Beckman
Coulter). Results are expressed as percentages of
CD4+CD25+CD127- or CD4+CD25+FOXP3+ cells
out of the total CD4+ lymphocytes.

Viral Load (VL) Quantification

After extraction of 0.6 mL of plasma, reverse tran-
scription, PCR amplification, and detection/quantifica-
tion reactions were performed using Abbott RealTime
HIV-1 assay and m2000 system platform (Abbott Molec-
ular, Des Plaines, IL). The detection limit was 40 copies/
mL.

Statistical Analysis

Normality of the variables was assessed using the Kol-
mogorov-Smirnov test. Exact Fisher test for categorical
variables, and Ttest for continuous variables were per-
formed. Correlations between CD4+CD25+CD127-
and CD4+CD25+FOXP3+ cell frequencies were studied
using the Pearson's correlation coefficient test. The sta-
tistical analyses were performed using SPSS software
(version 16.0, SPSS, Chicago, IL). A significance was
assumed for *P* < 0.05.

RESULTS
Population at Baseline

Nineteen HIV-1 infected patients were included in the study. None of them were coinfected with hepatitis B or hepatitis C viruses. Of them, 11 viremic patients [men $n = 8$ (73%), median age = 33 years; Inter Quartile Range (IQR): 22-61, median CD4+ cell count = 363 cells/mm3, IQR: 115-482] were enrolled. In parallel, 8 patients receiving HAART [men $n = 6$ (75%), median age = 40 years, IQR: 24-59, median CD4+ cell count = 357 cells/mm3, IQR: 84-1196] were recruited and classified in the aviremic group. There were no statistical
<figure><img src="image_2.png" /><figcaption>FIG. 1. Representative plots and histograms (Patient 2, viremic) of the two flow cytometry staining strategies for regulatory T cells (Tregs) identification (a) Tregs are identified as CD25+CD127− cells among CD4+ lymphocytes (5.1%) (b). Most of these CD4+CD25+CD127− cells also expressed FOXP3 (threshold set up with isotype control) (c). Tregs are identified as CD25+Foxp3+ cells among CD4+ lymphocytes (4.9%). (d). Most of these CD4+CD25+Foxp3+ cells did not express CD127.</figcaption></figure>

differences at baseline between the two groups either for sex (P = 0.91), age (P = 0.70), or absolute CD4+ cell counts (P = 0.33). Aviremic patients had no detectable viremia (VL < 40copies/mL), whereas median VL in the viremic group was 27304 copies/mL (IQR 3,500-138,336) (P < 0.001).

## Tregs Phenotypes

Gating strategy and illustrative examples of CD4-CD25-CD127 and CD4-CD25-FOXP3 stainings are shown in Figure 1. Respective phenotype frequencies, percentages of CD4+CD25+CD127- lymphocytes expressing FOXP3, and percentages of CD4+CD25+FOXP3+ cells not expressing CD127 are reported in Table 1, as individual values. Percentages of Tregs identified by the two phenotypes, i.e. CD4+CD25+CD127- and CD4+CD25+FOXP3+ cells were not statistically different in the viremic group (P = 0.74), in the aviremic group (P = 0.36), or in the whole population (P = 0.45). In line, we found a strong correlation between

frequencies of CD4+CD25+CD127- and CD4+CD25+FOXP3+ cells, when considering the whole study population (r: 0.948, P < 0.001) or each group separately: aviremic (r: 0.968, P < 0.001), viremic (r: 0.94 P < 0.001). Another important observation was that most CD4+FOXP3+ cells were indeed found to be CD25+CD127-. Similar results were obtained for the viremic group and the aviremic group (88.5% and 90.9%, respectively, Table 1).

## DISCUSSION

In this observational study we compared two Tregs phenotypes in an HIV-1-infected cohort. In both viremic and aviremic HIV-infected subjects, our purpose was to investigate whether CD4+CD25+CD127- phenotype identifies FOXP3+ Tregs in these particular clinical contexts.

Indeed, we observed that most CD4+CD25+CD127- cells expressed FOXP3 (Figs. 1a and 1b), either in the viremic group (median: 88.50%, IQR: 84.10-92.90), or in the aviremic group (median: 90.9%, IQR: 80.80-97.70, Table 1).
<table><caption>Table 1
Percentages of Lymphocytes in Regard with Different Treg Phenotypes According to the Virologic Status of HIV-1 Infected Patients</caption><thead><tr><th rowspan="2">Patient</th><th colspan="3">% of CD25+CD127-(among CD4+)</th><th colspan="3">% of CD25+FOXP3+ (among CD4+)</th><th colspan="3">% of FOXP3+ (among CD4+CD25+CD127-)</th><th colspan="3">% of CD127- (among CD4+CD25+FOXP3+)</th></tr><tr><th>Individual Values</th><th>Median (IQR)</th><th>P</th><th>Individual Values</th><th>Median (IQR)</th><th>P</th><th>Individual Values</th><th>Median (IQR)</th><th>P</th><th>Individual Values</th><th>Median (IQR)</th><th>P</th></tr></thead><tbody><tr><td rowspan="12">Viremic group</td><td>1</td><td>5.80</td><td>5.50</td><td rowspan="12">0.71</td><td>5.40</td><td>5.40</td><td rowspan="12">0.59</td><td>88.50</td><td>88.50</td><td rowspan="12">0.26</td><td>78.80</td><td>76.70</td><td rowspan="12">0.42</td></tr><tr><td>2</td><td>5.00</td><td>(3.50-12.30)</td><td>4.80</td><td>(3.30-12.10)</td><td>85.80</td><td>(84.10-92.90)</td><td>88.50</td><td>(59.90-88.90)</td></tr><tr><td>3</td><td>12.30</td><td></td><td>12.10</td><td></td><td>88.00</td><td></td><td>76.70</td><td></td><td>84.80</td><td></td></tr><tr><td>4</td><td>7.30</td><td></td><td>7.10</td><td></td><td>86.80</td><td></td><td>69.80</td><td></td><td>77.30</td><td></td></tr><tr><td>5</td><td>4.20</td><td></td><td>4.40</td><td></td><td>87.70</td><td></td><td>76.60</td><td></td><td>88.90</td><td></td></tr><tr><td>6</td><td>7.80</td><td></td><td>6.80</td><td></td><td>88.60</td><td></td><td>61.80</td><td></td><td>92.90</td><td></td></tr><tr><td>7</td><td>8.80</td><td></td><td>9.50</td><td></td><td>89.70</td><td></td><td>59.90</td><td></td><td>89.50</td><td></td></tr><tr><td>8</td><td>4.40</td><td></td><td>5.40</td><td></td><td>84.10</td><td></td><td>73.60</td><td></td><td>88.90</td><td></td></tr><tr><td>9</td><td>4.60</td><td></td><td>6.30</td><td></td><td>88.40</td><td></td><td>90.9</td><td></td><td>78.20</td><td></td></tr><tr><td>10</td><td>3.50</td><td></td><td>3.30</td><td></td><td>88.40</td><td></td><td>(80.80-97.70)</td><td></td><td>78.30</td><td></td></tr><tr><td>11</td><td>5.50</td><td></td><td>4.00</td><td></td><td>95.10</td><td></td><td>(70.50-90.20)</td><td></td><td>88.50</td><td></td></tr><tr><td rowspan="12">Aviremic group</td><td>12</td><td>7.50</td><td>5.70</td><td rowspan="12"></td><td>6.90</td><td>5.70</td><td rowspan="12"></td><td>91.20</td><td></td><td>74.00</td><td></td><td>70.60</td><td></td></tr><tr><td>13</td><td>5.20</td><td>(3.60-9.50)</td><td>5.90</td><td>(3.10-9.10)</td><td>90.70</td><td></td><td>85.50</td><td></td><td>70.50</td><td></td></tr><tr><td>14</td><td>6.10</td><td></td><td>5.50</td><td></td><td>90.70</td><td></td><td>85.50</td><td></td><td>90.20</td><td></td></tr><tr><td>15</td><td>9.50</td><td></td><td>9.60</td><td></td><td>80.80</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>16</td><td>3.70</td><td></td><td>3.30</td><td></td><td>91.10</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>17</td><td>5.60</td><td></td><td>4.90</td><td></td><td>87.80</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>18</td><td>5.80</td><td></td><td>6.20</td><td></td><td>95.70</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>19</td><td>3.60</td><td></td><td>3.10</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr></tbody></table>

IQR, inter quartile range; P values: viremic vs. aviremic groups (student T test).

Conversely, using a backgating strategy (Figs. 1c and 1d), we highlighted that most CD4+CD25+FOXP3+ cells were negative for CD127 expression (Table 1). Our results are consistent with two other studies (16,17), in which a close correlation (r: 0.81, P < 0.001, and r: 0.96 P < 0.0001, respectively) between the two phenotypes was observed in viremic patients. Nevertheless, these studies did not include aviremic patients. Overall, these results are important because some authors previously used the CD4+CD25+CD127- phenotype to characterize Tregs (without assessing the FOXP3+ intracellular staining). Our results confirmed that their conclusions on roles of Tregs in the pathophysiology of HIV infection are relevant for both the viremic and the aviremic subjects (21,22).

However, the present report contrasts with Del Pozo and colleagues' results (15). They observed a lack of correlation between the two phenotypes in viremic subjects. This discrepancy could be attributed to different study protocols (both analytical and regarding inclusion criteria). First, we used fresh whole blood samples rather than isolated peripheral blood mononuclear cells. Second, we included patients with lower absolute CD4+ value, and not only those presenting with CD4+ count higher than 350 cells/mm³. Third, in our study, there was no statistical difference in age between the two groups while the aviremic group was older than the viremic one in Del Pozo's study (median age 43 and 31 years respectively, P = 0.001). This may explain the higher CD4+CD25+FOXP3+ frequency observed in the aviremic group (4.89 versus 3.90, P = 0.039).

Our study has some limitations. First, we did not specifically analyzed Tregs phenotypes and frequencies in long-term nonprogressors or in case of early HIV infection. We cannot rule out that some specificity may relate to these conditions. Second, the number of patients included is relatively modest. However, the very strong correlations between CD4+CD25+ FOXP3+ and CD4+CD25+CD127- percentages obtained in such small cohort tend to support our conclusion. Third, it has to be mentioned that a small percentage of FOXP3+ cells was not included in the CD4+CD25+CD127- cell population (around 10%, no significant difference between groups, Fig. 1d). These cells belong either to CD4+CD25+CD127+ or to CD4+CD25-CD127- populations for which published data remain very scarce. These cells deserve further investigation as their potential role in HIV pathophysiology has not yet been elucidated. In that sense, since FOXP3 is to date the universal marker for Tregs cells, development of a more practical technique to stain intracellular FOXP3 remains highly desirable to monitor those cells in clinical research dealing with large numbers of patients. An important step would be to shorten the intracellular FOXP3 staining in the future.

## CONCLUSIONS

We confirm here that CD4+CD25+CD127- phenotype is a good surrogate phenotype for FOXP3+ regulatory T cells identification in both viremic and aviremic HIV-1-infected subjects. Awaiting for some robust and
rapid intracellular FOXP3 staining, we suggest that the CD4+CD25+CD127- phenotype remains a reliable and easy tool (whole blood, no wash, less than 30 min to be completed, isotype control not necessary) for Tregs measurement in clinical research on HIV-infected patients.

## ACKNOWLEDGMENTS

The authors thank Anne Portier, Caroline Guignant, and Celine Griffon-Renoux for technical assistance.

## LITERATURE CITED

1. Lund JM, Hsing L, Pham TT, Rudensky AY. Coordination of early protective immunity to viral infection by regulatory T cells. Science 2008;320:1220-1224.

2. Li S, Gowans EJ, Chougnet C, Plebanski M, Dittmer U. Natural tregs and persistent viral infections. J Virol 2008;82:21-30.

3. Kinter A, McNally J, Riggin L, Jackson R, Roby G, Fauci AS. Suppression of HIV-specific T cell activity by lymph node CD25+ regulatory T cells from HIV-infected individuals. Proc Natl Acad Sci USA 2007;104:3390-3395.

4. Kinter AL, Horak R, Sion M, Riggin L, McNally J, Lin Y, Jackson R, O'Shea A, Roby G, Kovacs C, Connors M, Migueles SA, Fauci AS. CD25_ regulatory T cells isolated from HIV-infected individuals suppress the cytolytic and nonlytic antiviral activity of HIV-specific CD8_ T cells in vitro. AIDS Res Hum Retroviruses 2007;23:438-450.

5. Shaw JM, Hunt PW, Critchfield JW, McConnell DH, Garcia JC, Pollard RB, Somsouk M, Deeks SG, Shacklett BL. Increased frequency of regulatory T cells accompanies increased immune activation in rectal mucosae of HIV-positive noncontrollers. J Virol 2011;85:11422-11434.

6. Prendergast A, O'Callaghan M, Menson E, Hamadache D, Walters S, Klein N, Goulder P. Factors influencing T cell activation and programmed death 1 expression in HIV-infected children. AIDS Res Hum Retroviruses. 2012;28:465-468.

7. Chase AJ, Yang HC, Zhang H, Blankson JN, Siliciano RF. Preservation of FoxP3R regulatory T cells in the peripheral blood of human immunodeficiency virus type 1-infected elite suppressors correlates with low CD4R T-cell activation. J Virol 2008;82:8307-8315.

8. Moreno-Fernandez ME, Rueda CM, Rusie LK, Chougnet CA. Regulatory T cells control HIV replication in activated T cells through a cAMP-dependent mechanism. Blood 2011;117:5372-5380.

9. Sattui S, de la Flor C, Sanchez C, Lewis D, Lopez G, Rizo-Patrón E, White AC Jr, Montes M. Cryopreservation modulates the detection of regulatory T cell markers. Cytometry B Clin Cytom 2012;82B:54-58.

10. Venet F, Malcus C, Ferry T, Poitevin F, Monneret G. Percentage of regulatory T cells CD4+CD25+CD127- in HIV-infected patients is not reduced after cryopreservation. J Immunol Methods 2010;357:55-58.

11. Sakaguchi S, Miyara M, Costantino CM, Hafler DA. FOXP3+ regulatory T cells in the human immune system. Nat Rev Immunol 2010;10:490-500.

12. Liu W, Putnam AL, Xu-Yu Z, Szot GL, Lee MR, Zhu S, Gottlieb PA, Kapranov P, Gingeras TR, Fazekas de St Groth B, Clayberger C, Soper DM, Ziegler SF, Bluestone JA. CD127 expression inversely correlates with FoxP3 and suppressive function of human CD4+ Treg cells. J Exp Med 2006;203:1701-1711.

13. Seddiki N, Santner-Nanan B, Martinson J, Zaunders J, Sasson S, Landay A, Solomon M, Selby W, Alexander SI, Nanan R, Kelleher A, Fazekas de St Groth B. Expression of interleukin (IL)-2 and IL-7 receptors discriminates between human regulatory and activated T cells. J Exp Med 2006;203:1693-1700.

14. Venet F, Chung CS, Kherouf H, Geeraert A, Malcus C, Poitevin F, Bohe J, Lepape A, Ayala A, Monneret G. Increased circulating regulatory T cells (CD4(+)-CD25 (+)-CD127 (-)) contribute to lymphocyte anergy in septic shock patients. Intensive Care Med 2009;35:678-686.

15. Del Pozo-Balado Mdel M, Leal M, Méndez-Lagares G, Pacheco YM. CD4+CD25+/hiCD127lo Phenotype Does Not Accurately Identify Regulatory T Cells in All Populations of HIV Infected Persons. J Infect Dis 2010;201:331-335.

16. Montes M, Sanchez C, Lewis DE, Graviss EA, Seas C, Gotuzzo E, White AC Jr. Normalization of FoxP3(+) regulatory T cells in response to effective antiretroviral therapy. J Infect Dis 2011;203:496-499.

17. Schulze Zur Wiesch J, Thomssen A, Hartjen P, Tóth I, Lehmann C, Meyer-Olson D, Colberg K, Frerk S, Babikir D, Schmiedel S, Degen O, Mauss S, Rockstroh J, Staszewski S, Khaykin P, Strasak A, Lohse AW, Fätkenheuer G, Hauber J, van Lunzen J. Comprehensive analysis of frequency and phenotype of T regulatory cells in HIV infection: CD39 expression of FoxP3+ T regulatory cells correlates with progressive disease. J Virol 2011;85:1287-1297.

18. Méndez-Lagares G, Del Mar Del Pozo-Balado M, Genebat González M, García-Perganeda Sánchez A, Leal Noval M, Pacheco López YM. Severe immune dysregulation affects CD4+CD25hiFoxP3+ regulatory T-cells in HIV-infected patients with low-level CD4 T-cell repopulation despite suppressive HAART. J Infect Dis 2012;205:1501-1509.

19. Elahi S, Dinges WL, Lejarcegui N, Laing KJ, Collier AC, Koelle DM, McElrath MJ, Horton H. Protective HIV-specific CD8+ T cells evade Treg cell suppression. Nat Med 2011;17:989-995.

20. Simonetta F, Lecuroux C, Girault I, Goujard C, Sinet M, Lambotte O, Venet A, Bourgeois C. Early and long lasting alteration of Effector CD45RA-Foxp3high Regulatory T cells homeostasis during HIV infection. J Infect Dis 2012;205:1510-1519.

21. Hunt PW, Landay AL, Sinclair E, Martinson JA, Hatano H, Emu B, Norris PJ, Busch MP, Martin JN, Brooks C, McCune JM, Deeks SG. A low T regulatory cell response may contribute to both viral control and generalized immune activation in HIV controllers. PLoS One 2011;6.

22. Tenorio AR, Spritzler J, Martinson J, Gichinga CN, Pollard RB, Lederman MM, Kalayjian RC, Landay AL. The effect of aging on T-regulatory cell frequency in HIV infection. Clin Immunol 2009;130:298-303.