Common variable immunodeficiency disorder (CVID) is a heterogeneous syndrome, characterized by deficient antibody production and recurrent bacterial infections in addition abnormalities in T cells. CD4+CD25high regulatory T cells (Treg) are essential modulators of immune responses, including down-modulation of immune response to pathogens, allergens, cancer cells and self-antigens.
In this study we set out to investigate the frequency of Treg cells in CVID patients and correlate with their immune activation status.
Materials and Methods
Sixteen patients (6 males and 10 females) with CVID who had been treated with regular intravenous immunoglobulin and 14 controls were enrolled. Quantitative analyses of peripheral blood mononuclear cells (PBMC) were performed by multiparametric flow cytometry using the following cell markers: CD38, HLA-DR, CCR5 (immune activation); CD4, CD25, FOXP3, CD127, and OX40 (Treg cells); Ki-67 and IFN-γ (intracellular cytokine).
A significantly lower proportion of CD4+CD25highFOXP3 T cells was observed in CVID patients compared with healthy controls (P<0.05). In addition to a higher proportion of CD8+ T cells from CVID patients expressing the activation markers, CD38+ and HLA-DR+ (P<0.05), we observed no significant correlation between Tregs and immune activation.
Our results demonstrate that a reduction in Treg cells could have impaired immune function in CVID patients.
Citation:Melo KM, Carvalho KI, Bruno FR, Ndhlovu LC, Ballan WM, et al. (2009) A Decreased Frequency of Regulatory T Cells in Patients with Common Variable Immunodeficiency. PLoS ONE 4(7): e6269. doi:10.1371/journal.pone.0006269
Editor: Derya Unutmaz, New York University School of Medicine, United States of America
Received: March 9, 2009; Accepted: June 2, 2009; Published: July 29, 2009
Copyright: © 2009 Melo et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding:This work was partially supported by the National Institutes of Health, grants R01-AI52731 and AI60379 (Nixon) and The Fogarty International Center, grant #D43 TW00003; KIC's Ph.D. scholarship has been provided by the Conselho Nacional de Desenvolvimento CientÃfico e Tecnologico (CNPq), Brazilian Ministry of Science and Technology. KMM MD. Scholarship (april-june/2008) has been provided by Coordenação de Aperfeiçoamento de Pessoal de Nível superior (CAPES). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Common variable immunodeficiency disorder (CVID) is the most frequent symptomatic primary immunodeficiency, characterized by recurrent bacterial infections, hipogamaglobulinemia and impaired antibody responses –. CVID patients usually present recurrent respiratory infections, and elevated incidence of autoimmune, gastrointestinal, lymphoproliferative, and granulomatous diseases –. Although genetic defects associated with CVID have been described, they are rare –.
Over the last 20 years various facets of cellular and immunological dysfunctions in CVID have been described, for example, abnormalities in B cell populations, low frequencies of naive CD4 T cells, and an increase in cellular activation –. A new classification of this disease has recently been proposed according to the B cell phenotype –. However, a significant number of studies have shown alterations in phenotype and function of T cell subpopulations , , , .
T cell functional defects compromise T cell activation and proliferation , . Abnormalities in the secretion of cytokines , –, defects in early T cell receptor (TCR) signaling events , , and impaired expression of activation markers including CD40-ligand, attractin and L-selectin – have been described. In addition, current studies have described a breakdown in mechanism of tolerance in CVID subjects with autoimmunity .
Tolerance to self antigens is an active process that has central and peripheral components . Among T cell subsets, a subgroup of CD4+ T cell, referred to as regulatory T cells (Tregs), has an important role in controlling other immune responses, maintaining peripheral self-tolerance , . Treg cells are characterized by the expression of CD25 and the Forkhead family transcription factor (FOXP3) ,  and play a significant role in down regulating the immune response to a variety of non-self antigens, such as microbial, tumor and alloantigens –.
Tregs are dysfunctional in several human diseases ,  and are a potential target for therapeutic modulation , . In CVID, recent reports describe a decreased frequency of Treg cells , . Here, we investigate the frequency of Tregs in CVID patients, and their relationship to cell activation status.
Materials and Methods
Sixteen patients with a diagnosis of CVID according to the criteria established by the Pan-American Group for Immunodeficiency (PAGID)  and, fourteen healthy controls were enrolled in the study. The patients were recruited at the Division of Clinical Immunology at UNIFESP (Sao Paulo, Brazil). All patients were on regular intravenous immunoglobulin (IVIG) substitution therapy. For each subject, complete blood counts and lymphocyte subsets were performed, and the blood sample was collected immediately before IVIG infusion. Clinical information was collected by questionnaire based on the medical charts. The subjects' characteristics are shown in Table 1. Among patients with autoimmunity two were receiving oral corticosteroids, in a dosage less than 0.5 mg/Kg on alternative days. After approval by the local institutional review board (IRB, Comitê de Ética em Pesquisa da Universidade Federal de São Paulo/UNIFESP), written informed consent was obtained from all participants or their legal representatives.
Peripheral blood mononuclear cells (PBMC) were isolated by density-gradient sedimentation using Ficoll-Paque (Pharmacia Biothec, Upssala, Sweden). Isolated PBMC were then washed twice in Hank's balanced salt solution (Gibco, Grand Island, NY). Cells were cryopreserved in RPMI 1640 (Gibco), supplemented with 20% heat-inactivated fetal bovine serum (FBS; Hyclone Laboratories, Logan UT), 50 U/ml of penicillin (Gibco), 50 µg/ml of streptomycin (Gibco), 10 mM glutamine (Gibco) and 7.5% dimethylsulphoxide (DMSO; sigma, St Louis, MO). Cryopreserved cells from all subjects (patients and controls) were stored in liquid nitrogen for a mean time of 2.5 months (1–5 months) until used in the assays. On the day of assay, PBMC were rapidly thawed in a 37°C water bath and washed in RPMI 1640 supplemented with 10% fetal calf serum, 100 U/ml of penicillin, 100 µg/ml of streptomycin and 20 mM glutamine (R10). Cells were counted, checked for viability and resuspended in R10 at concentration of 10 6cells/ml.
The following monoclonal antibodies were used in the assays: CD3- peridin chlorophyll protein (PerCP) and CD4- allophycocyanincarbocyanin 7 (APC-Cy7), and in addition to: HLA-DR- FITC, CD38- PE, CD8-APC, CCR5- PeCy7 (panel A); OX40-fluorescein isothiocyanate (FITC), CD127- phytoerythrin (PE), FOXP3- allophycocyan (APC), CD25- phycoerithrincarbocyanin (PE-Cy7) (panel B); intracellular staining for cytokines was performed using mouse anti-human Ki-67- FITC mouse anti-human interferon (IFN -γ) -PE-Cy7 Ki-67-FITC, all from BD PharMingen (panel C). Fluorescence-minus-one (FMO) was used for gating strategy .
Thawed PBMC were centrifuged at 1500 g for 15 minutes and transferred into V-bottom 96-well plates (Nunc, Roskilde, DenmarK) in 200 µl of staining buffer [phosphate-buffered saline (PBS) supplemented with 0.1% sodium azide (Sigma) and FBS, pH 7.4–7.6)] with the panel of surface monoclonal antibodies. Cells were incubated at 4°C in darkness for 30 minutes, washed twice and then resuspended in 200 µl of fixation buffer [1% paraformaldehyde (Polyscience, Warrigton, PA) in PBS, pH 7.4–7.6.
For panel B, cells were resuspended in 50 µl of staining buffer with monoclonal antibody FOXP3, then were incubated at 4°C in darkness for 30 min, washed twice and resuspended in 200 µl of 2% of paraformaldehyde (PFA). For panel C, intracellular staining was performed after surface with CD3- PerCP, CD4-APC-Cy7 and CD8-APC. Cells were incubated with 100 µl of 4% fixation buffer and washed with permeabilization buffer (PBS supplemented with 0.1% sodium azide, 1% FBS and 0.1% saponin; Sigma). Each sample was resuspended in 100 µl of permeabilization buffer, incubated for 15 minutes at room temperature in darkness, washed with 100 µl of staining buffer and incubated for 30 minutes at 4°C in darkness with either without antibody (unstained tube) or anti-Ki67–FITC, anti- IFN-γ–PE–CY7 in 50 µl of staining buffer. Cells were washed with 170 µl of staining buffer and resuspended in 100 µl of 1% paraformaldehyde (PFA) for flow cytometry analysis. Samples were acquired on a FACSCanto, using FACSDiva software (BD Biosciences), and analyzed with FlowJo software (Tree Star, San Carlo, CA). Fluorescence voltages were determined using matched unstained cells. Compensation was carried out using CompBeads (BD Biosciences) single stained with CD3-PerCP, CD4-APC-CY7, CD4-PE-CY7, CD3-PE or CD3-APC. Samples were run for a minimum of 300,000 events in a live lymphocyte gate.
Groups were compared using non-parametric models; data are reported as median and interquartile range (IQR). Comparisons among groups were carried out using the Mann Whitney test. Correlations were performed using the Spearman non-parametric test. P - values were considered significant if below 0.05.
CD8+ T lymphocytes are highly activated in patients with CVID
We assessed the characteristics of the lymphocyte subsets in 30 subjects, among CVID patients and healthy controls. Further analysis using a combination of activation markers showed a marked up-regulation of CD38+ and CCR5+ expressing CD4+ T cells in the CVID patients (P = 0.04) (data not shown) and an increased frequency of CD38+ expressing CD8 T cells in CVID patients compared to controls (P = 0.002). Both high co-expression levels of HLA-DR+ (P = 0.008) or CCR5+ (P = 0.021) confirmed the high level of CD8 T cell activation in CVID patients, (Figure 1A–C). Four out of 16 patients had a marked increase in T cell activation. Of these, just one, 24 year old male, presented autoimmune hepatitis, the others had no particular associated pathology.
Circulating Tregs are diminished in patients with CVID
We defined Tregs by a series of gating strategies that best represent this suppressor T cell population (FOXP3+CD25highCD127lowCD4+ T cells) Figure 2. In Figure 3, we show that the frequency of Tregs was markedly reduced in CVID patients, (P = 0.03 and P = 0.003) compared to healthy controls (Figure 2).
(A) The gate is set around the population of lymphocytes; (B) CD3 T cells that were evaluated for (C), FOXP3, CD25; (D) Side scatter and CD3+FOXP3+CD25highCD127low. (E) The gate is set around the population of FOXP3+CD25highCD127lowCD4+ T cells. Fluorescence minus one was used to define the gate used. Arrows indicate the gated population subsequently analyzed.
In CVID group there was a significant lower percentage of FOXP3+CD25highCD127lowCD4+ T cell (P = 0.0034).
Interestingly, stratifying our patients based on autoimmune status did not reveal any differences in Tregs frequency (data not shown). A similar result was observed when we classified CVID patients according to EUROclass  (data not shown). The impact of Tregs and immune activation is well documented , , however in a regression analysis, we did not observe any association between CD4+ or CD8+ T cell activation status and the frequency of Tregs in this cohort (data not shown).
Higher activation was not reflected in higher spontaneous production of IFN-gamma and expression of Ki-67
Presumably due to the high level of T cell activation in CVID, we predicted this would lead to an elevated level of T cell functionality. We thus assessed CD4+ and CD8+ T cell proliferation by measuring Ki-67 nuclear antigen expressed in the G1, S, G2, and M phases, but not in the G0 phase, of the cell cycle. We also assessed for spontaneous IFN-gamma production in the absence of any stimuli in the CVID cohort. No difference was observed in either Ki-67 expression or spontaneous IFN-gamma production compared to controls subjects (data not shown).
Previous reports have demonstrated abnormalities in cellular immunity on CVID pathogenesis that affect activation and proliferation of T cells and, consequently, B cell differentiation and production of antibody , , , . However, there are few studies examining the influence of Tregs in CVID subjects , .
We observed that our patients presented significantly higher levels of CD8+ T cell activation. The percentage of Tregs expressing CD25highFOXP3+ was found to be lower in CVID subjects compared to controls but no significant association between Treg cells and immune activation was observed in these patients.
One of the greatest difficulties in studying Tregs is to select the combination of markers that best describe these subsets and currently the most common markers used to define these cells are CD25high and FOXP3+ –. The absence of IL-7R (CD127) on CD4 T cells has proven to be a reliable delineator of selection for Treg cells with the highest suppressive function –. Our results using these markers are in line with previous reports, showing a reduced number of Treg cells in CVID patients , . It is important to emphasize that these results were observed in a small group of Brazilian patients with different demographic and clinical characteristics to European subjects, which suggests that the proportion of Tregs could be directly related to this immunodeficiency.
Fevang et al. described that CVID patients with splenomegaly had a lower proportion of Tregs compared to others CVID patients , as our study, just included one patient with splenomegaly, this suggests that others clinical complications must be associated with reduced Treg population. One topic for further discussion is whether this low percentage is the cause or consequence of clinical complications observed in CVID patients.
No differences were observed in Tregs frequency when comparing CVID patients with and without AID. Despite preliminary studies having suggested an association between these cells and AID in CVID , it has not been established and can be influenced by other associated clinical complications that affect not only the frequency, but the profile of cytokine secreted by Tregs.
All patients analyzed were being treated with IVIG and, the correlation between Tregs and IVIG has already been described showed and expansion of Tregs in animal models , however we observed a reduced frequency of these cells in CVID subjects. Also, it has been demonstrated that Tregs increases the intracellular expression of TGF-β, IL-10 and FOXP3 following the addition of IVIG , but unfortunately we did not analyze the cytokine secretion by Tregs in our patients.
Furthermore, considering the increase of T cell markers activation in CVID subjects, specially CD8+ T cells, without a significant increase in IFN-g production, we suggest that these cells could have a suppressor function, according to previous reports and this result probably was unaffected by IVIG replacement , . The similar percentage of cells expressing proliferation marker (Ki67) between the two groups could reflect the absence of lymphoproliferative disease in our sample, since contrasting results have been described in the literature , .
To summarize, this paper shows that CD25highFOXP3+CD127low expressing Treg cells were lower in CVID patients, which suggests that Tregs cells can be impaired in CVID, thus having influence on the mechanism pathogenic of this complex immunodeficiency.
Conceived and designed the experiments: KMM KIC LCN DFN EGK BTCC. Performed the experiments: KMM KIC FRB. Analyzed the data: KMM KIC LCN DFN EGK BTCC. Contributed reagents/materials/analysis tools: KMM KIC FRB LCN WB DFN EGK BTCC. Wrote the paper: KMM BTCC.
- 1. Cunningham-Rundles C,Bodian C (1999) Common variable immunodeficiency: clinical and immunological features of 248 patients. Clin Immunol 92: 34–48.
- 2. Di Renzo M,Pasqui AL,Auteri A (2004) Common variable immunodeficiency: a review. Clin Exp Med 3: 211–217.
- 3. de Vries E (2006) Patient-centred screening for primary immunodeficiency: a multi-stage diagnostic protocol designed for non-immunologists. Clin Exp Immunol 145: 204–214.
- 4. Chapel H,Lucas M,Lee M,Bjorkander J,Webster D,et al. (2008) Common variable immunodeficiency disorders: division into distinct clinical phenotypes. Blood 112: 277–286.
- 5. Blanco-Quiros A,Solis-Sanchez P,Garrote-Adrados JA,Arranz-Sanz E (2006) Common variable immunodeficiency. Old questions are getting clearer. Allergol Immunopathol (Madr) 34: 263–275.
- 6. Glocker E,Ehl S,Grimbacher B (2007) Common variable immunodeficiency in children. Curr Opin Pediatr 19: 685–692.
- 7. Quinti I,Soresina A,Spadaro G,Martino S,Donnanno S,et al. (2007) Long-term follow-up and outcome of a large cohort of patients with common variable immunodeficiency. J Clin Immunol 27: 308–316.
- 8. Salzer U,Grimbacher B (2006) Common variable immunodeficiency: The power of co-stimulation. Semin Immunol 18: 337–346.
- 9. Cunningham-Rundles C,Knight AK (2007) Common variable immune deficiency: reviews, continued puzzles, and a new registry. Immunol Res 38: 78–86.
- 10. Serrano D,Becker K,Cunningham-Rundles C,Mayer L (2000) Characterization of the T cell receptor repertoire in patients with common variable immunodeficiency: oligoclonal expansion of CD8(+) T cells. Clin Immunol 97: 248–258.
- 11. Viallard JF,Blanco P,Andre M,Etienne G,Liferman F,et al. (2006) CD8+HLA-DR+ T lymphocytes are increased in common variable immunodeficiency patients with impaired memory B-cell differentiation. Clin Immunol 19: 51–58.
- 12. Giovannetti A,Pierdominici M,Mazzetta F,Marziali M,Renzi C,et al. (2007) Unravelling the complexity of T cell abnormalities in common variable immunodeficiency. J Immunol 178: 3932–3943.
- 13. Zhang L,Radigan L,Salzer U,Behrens TW,Grimbacher B,et al. (2007) Transmembrane activator and calcium-modulating cyclophilin ligand interactor mutations in common variable immunodeficiency: clinical and immunologic outcomes in heterozygotes. J Allergy Clin Immunol 120: 1178–1185.
- 14. Haymore BR,Mikita CP,Tsokos GC (2008) Common variable immune deficiency (CVID) presenting as an autoimmune disease: role of memory B cells. Autoimmun Rev 7: 309–312.
- 15. Warnatz K,Denz A,Drager R,Braun M,Grouth C,et al. (2002) Severe deficiency of switched memory B cells (CD27(+)IgM(-)IgD(-)) in subgroups of patients with common variable immunodeficiency: a new approach to classify a heterogeneous disease. Blood 99: 1544–551.
- 16. Piqueras B,Lavenu-Bombled C,Galicier L,Bergeron–van der Cruyssen F,Mouthon L,et al. (2003) Common variable immunodeficiency patient classification based on impaired B cell memory differentiation correlates with clinical aspects. J Clin Immunol 23: 385–400.
- 17. Wehr C,Kivioja T,Schmitt C,Ferry B,Witte T,et al. (2008) The EUROclass trial: defining subgroups in common variable immunodeficiency. Blood 111: 77–85.
- 18. Kondratenko I,Amlot PL,Webster AD,Farrant J (1997) Lack of specific antibody response in common variable immunodeficiency (CVID) associated with failure in production of antigen-specific memory T cells. MRC Immunodeficiency Group. Clin Exp Immunol 108: 9–13.
- 19. Pons J,Ferrer JM,Martinez-Pomar N,Iglesias-Alzueta J,Matamoros N (2006) Costimulatory molecules and cytokine production by T lymphocytes in common variable immunodeficiency disease. Scand J Immunol 63: 383–389.
- 20. Cunningham-Rundles C (2001) Common variable immunodeficiency. Curr Allergy Asthma Rep 1: 421–429.
- 21. Ferrer JM,Iglesias J,Hernandez M,Matamoros N (1995) Alterations in interleukin secretion (IL-2 and IL-4) by CD4 and CD4 CD45RO cells from common variable immunodeficiency (CVI) patients. Clin Exp Immunol 102: 286–289.
- 22. North ME,Webster AD,Farrant J (1998) Primary defect in CD8+ lymphocytes in the antibody deficiency disease (common variable immunodeficiency): abnormalities in intracellular production of interferon-gamma (IFN-gamma) in CD28+ (‘cytotoxic’) and CD28- (‘suppressor’) CD8+ subsets. Clin Exp Immunol 111: 70–75.
- 23. Holm AM,Aukrust P,Damas JK,Muller F,Halvorsen B,Froland SS (2005) Abnormal interleukin-7 function in common variable immunodeficiency. Blood 105: 2887–2890.
- 24. Fischer MB,Hauber I,Wolf HM,Vogel E,Mannhalter JW,et al. (1994) Impaired TCR signal transduction, but normal antigen presentation, in a patient with common variable immunodeficiency. Br J Haematol 88: 520–526.
- 25. Farrington M,Grosmaire LS,Nonoyama S,Fischer SH,Hollenbaugh D,et al. (1994) CD40 ligand expression is defective in a subset of patients with common variable immunodeficiency. Proc Natl Acad Sci USA 91: 1099–1103.
- 26. Nordoy I,Muller F,Aukrust P,Froland SS (1998) Adhesion molecules in common variable immunodeficiency (CVID) -a decrease in L-selectin-positive T lymphocytes. Clin ExpImmunol 114: 258–263.
- 27. Pozzi N,Gaetaniello L,Martire B,Balestrieri B,Cosentini E,et al. (2001) Defective surface expression of attractin on T cells in patients with common variable immunodeficiency (CVID). Clin Exp Immunol 123: 99–104.
- 28. Lopes-da-Silva S,Rizzo LV (2008) Autoimmunity in common variable immunodeficiency. J Clin Immunol 28: Suppl 1S46–S55.
- 29. Campbell DJ,Ziegler SF (2007) FOXP3 modifies the phenotypic and functional properties of regulatory T cells. Nat Rev Immunol 7: 305–310.
- 30. Bacchetta R,Gambineri E,Ronacarolo MG (2007) Role of regulatory T cell and FOXP3 in human diseases. J Allergy Clin Immunol 120: 227–235.
- 31. Sakaguchi S,Sakaguchi N,Shimizu J,Yamazaki S,Sakihama T,et al. (2001) Immunologic tolerance maintained by CD25+ CD4+ regulatory T cells: their common role in controlling autoimmunity, tumor immunity, and transplantation tolerance. Immunol Rev 182: 18–32.
- 32. Coombes JL,Robinson NJ,Maloy KJ,Uhlig HH,Powrie F (2005) Regulatory T cells and intestinal homeostasis. Immunol Rev 204: 184–194.
- 33. Waldmann H,Chen TC,Graca L,Adams E,Daley S,et al. (2006) Regulatory T cells in transplantation. Semin Immunol 18: 111–119.
- 34. Michaelsson J,Barbosa HM,Jordan KA,Chapman JM,Brunialti MK,et al. (2008) The frequency of CD127low expressing CD4+CD25high T regulatory cells is inversely correlated with human T lymphotrophic virus type-1 (HTLV-1) proviral load in HTLV-1-infection and HTLV-1-associated myelopathy/tropical spastic paraparesis. BMC Immunol 9: 41.
- 35. Taams LS,Palmer DB,Akbar AN,Robinson DS,Brown Z,et al. (2006) Regulatory T cells in human disease and their potential for therapeutic manipulation. Immunology 118: 1–9.
- 36. Fevang B,Yndestad A,Sandberg WJ,Holm AM,Muller F,et al. (2007) Low numbers of regulatory T cells in common variable immunodeficiency: association with chronic inflammation in vivo. Clin Exp Immunol 147: 521–525.
- 37. Conley ME,Notarangelo LD,Etzioni A (1999) Diagnostic criteria for primary immunodeficiencies. Representing PAGID (Pan-American Group for Immunodeficiency) and ESID (European Society for Immunodeficiencies). Clin Immunol 93: 190–197.
- 38. Roederer M (2001) Spectral compensation for flow cytometry: visualization artifacts, limitations, and caveats. Cytometry 45: 194–205.
- 39. Carbone J,Sarmiento E,Micheloud D,Rodriguez-Molina J,Fernandez-Cruz E (2006) Elevated levels of activated CD4 T cells in common variable immunodeficiency: association with clinical findings. Allergol Immunopathol (Madr) 34: 131–135.
- 40. Yu GP,Chiang D,Song SJ,Hoyte EG,Huang J,et al. (2009) Regulatory T cell dysfunction in subjects with common variable immunodeficiency complicated by autoimmune disease. Clin Immunol 131: 240–253.
- 41. Sakaguchi S (2005) Naturally arising FOXP3-expressing CD25+CD4+ regulatory T cells in immunological tolerance to self and non-self. Nat Immunol 6: 345–52.
- 42. Yagi H,Nomura T,Nakamura K,Yamazaki S,Kitawaki T,et al. (2004) Crucial role of FOXP3 in the development and function of human CD25+CD4+ regulatory T cells. Int Immunol 16: 1643–1656.
- 43. Lopes JE,Torgerson TR,Schubert LA,Anover SD,Ocheltree EL,et al. (2006) Analysis of FOXP3 reveals multiple domains required for its function as a transcriptional repressor. J Immunol 177: 3133–3142.
- 44. Eggena MP,Barugahare B,Jones N,Okello M,Mutalya S,et al. (2005) Depletion of regulatory T cells in HIV infection is associated with immune activation. J Immunol 174: 4407–4414.
- 45. Liu W,Putnam AL,Xu-Yu Z,Szot GL,Lee MR,et al. (2006) CD127 expression inversely correlates with FOXP3 and suppressive function of human CD4+ T reg cells. J Exp Med 203: 1701–1711.
- 46. Ndhlovu LC,Loo CP,Spotts G,Nixon DF,Hecht FM (2008) FOXP3 expressing CD127lo CD4+ T cells inversely correlate with CD38+ CD8+ T cell activation levels in primary HIV-1 infection. J Leukoc Biol 83: 254–262.
- 47. Ephrem A,Chamat S,Miquel C,Fisson S,Mouthon L,et al. (2008) Expansion of CD4+CD25+ regulatory T cells by intravenous immunoglobulin: a critical factor in controlling experimental autoimmune encephalomyelitis. Blood 111: 715–722.
- 48. Kessel A,Ammuri H,Peri R,Pavlotzky ER,Blank M,et al. (2007) Intravenous immunoglobulin therapy affects T regulatory cells by increasing their suppressive function. J Immunol 179: 5571–5575.
- 49. Sewell WA,North ME,Cambronero R,Webster AD,Farrant J (1999) In vivo modulation of cytokine synthesis by intravenous immunoglobulin. Clin Exp Immunol 116: 509–515.