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Abnormal phenotypic distribution of regulatory and effector T cells in octogenarian and nonagenarian women

Distribuição fenotípica anormal das células T reguladoras e efetoras em mulheres octogenárias e nonagenárias

Summary

Introduction:

aging is associated with several immunologic changes. Regulatory (Treg) and effector T cells are involved in the pathogenesis of infectious, neoplastic, and autoimmune diseases. Little is known about the effects of aging on the frequency and function of these T cell subpopulations.

Methods:

peripheral blood mononuclear cells (PBMC) were obtained from 26 young (under 44 years old) and 18 elderly (above 80 years old) healthy women. T cell subpopulations were analyzed by flow cytometry.

Results:

elderly individuals had lower frequency of several activated effector T cell phenotypes as compared with young individuals: CD3+CD4+CD25+ (3.82±1.93 versus 9.53±4.49; p<0.0001); CD3+CD4+CD25+CD127+(2.39±1.19 versus 7.26±3.84; p<0.0001); CD3+CD4+CD25+ (0.41±0.22 versus 1.86±0.85, p<0.0001); and CD3+CD4+CD25highCD127+(0.06±0.038 versus 0.94±0.64, p<0.0001). Treg (CD3+CD4+CD25+CD127øFoxp3+) presented lower frequency in elderly individuals as compared to young adults (0.34±0.18 versus 0.76±0.48; p=0.0004) and its frequency was inversely correlated with age in the whole group (r=-0.439; p=0.013). The elderly group showed higher frequency of two undefined CD25øFoxp3+ phenotypes: CD3+CD4+CD25øFoxp3+(15.05±7.34 versus 1.65±1.71; p<0.0001) and CD3+CD4+CD25øCD127øFoxp3+(13.0±5.52 versus 3.51±2.87; p<0.0001).

Conclusions:

the altered proportion of different T cell subsets herein documented in healthy elderly women may be relevant to the understanding of the immunologic behavior and disease susceptibility patterns observed in geriatric patients.

Keywords:
T-lymphocytes; aging; regulators T-lymphocytes

Resumo

Introdução:

o envelhecimento está associado a diversas alterações imunológicas. Células T reguladoras e efetoras estão envolvidas na patogênese de enfermidades infecciosas, neoplásicas e autoimunes. Pouco se sabe acerca dos efeitos da idade sobre a frequência e a função dessas populações celulares.

Métodos:

células mononucleares do sangue periférico foram obtidas de participantes saudáveis (26 com idade inferior a 44 anos e 18 acima de 80 anos). As subpopulações celulares foram analisadas por citometria de fluxo.

Resultados:

o grupo constituído por idosas apresentou menor frequência de vários fenótipos de células T efetoras ativadas em comparação com jovens: CD3+CD4+CD25+ (3,82±1,93 versus 9,53±4,49, p<0,0001); CD3+CD4+ CD25+CD127+ (2,39±1,19 versus7,26±3,84, p<0,0001); CD3+CD4+CD25high(0,41±0,22 versus 1,86±0,85, p<0,0001); CD3+CD4+CD25highCD127+(0,06±0,038 versus 0,94±0,64, p<0,0001). As células T reguladoras CD3+CD4+CD25highCD127øFoxP3+ apresentaram menor frequência em indivíduos idosos em comparação com adultos jovens (0,34±0,18 versus0,76±0,48, p=0,0004) e sua frequência foi inversamente correlacionada com a idade em todo o grupo (r=-0,439; p=0,013). O grupo de idosas apresentou maior frequência de dois fenótipos indefinidos (CD25øFoxP3+), células CD3+CD4+CD25øFoxP3+ (15,05±7,34 versus 1,65±1,71, p<0,0001) e células CD3+CD4+CD25øCD127øFoxP3+(13,0±5,52 versus 3,51±2,87, p<0,0001).

Conclusão:

as proporções alteradas de diferentes subpopulações de células T em idosas saudáveis contribuem para a compreensão dos padrões de comportamento e suscetibilidade a doenças imunológicas evidenciadas em pacientes geriátricos.

Palavras-chave:
linfócitos T; envelhecimento; linfócitos T reguladores

Introduction

Aging is associated with decreased humoral immune response, shortened duration of protective immunity, decline in T cell diversity and thymus function, and decreased ability in T cell response to novel antigens.1Steger MM, Maczek C, Berger P, Grubeck-Loebenstein B. Vaccination against tetanus in the elderly: do recommended vaccination strategies give sufficient protection. Lancet. 1996; 348(9029):762.

Weksler ME, Szabo P. The effect of age on the B-cell repertoire. J Clin Immunol. 2000; 20(4):240-9.
-3Naylor K, Li G, Vallejo AN, Lee W-W, Koetz K, Bryl E, et al. The influence of age on T cell generation and TCR diversity. J Immunol. 2005; 174(11):7446-52. As a consequence, aging is associated with diminished immunity against microorganisms and malignant cells,4Hwang KA, Kim HR, Kang I. Aging and human CD4(+) regulatory T cells. Mech Ageing Dev. 2009; 130(8):509-17. as well as tolerance breakdown and increased frequency of autoimmune diseases.5Urbán L, Bessenyei B, Márka M, Semsei I. On the role of aging in the etiology of autoimmunity. Gerontology. 2002; 48(3):179-84. Naturally occurring regulatory T cells (Treg) are key regulators in the control of immune responses, with significant impact on autoimmunity, tumor immunity, infection, allergy and transplant tolerance.6Cools N, Ponsaerts P, Van Tendeloo VFI, Berneman ZN. Regulatory T cells and human disease. Clin Dev Immunol. 2007; 2007:89195.

Kondelková K, Vokurková D, Krejsek J, Borská L, Fiala Z, Ctirad A. Regulatory T cells (TREG) and their roles in immune system with respect to immunopathological disorders. Acta Medica (Hradec Kralove). 2010; 53(2):73-7.

Safinia N, Sagoo P, Lechler R, Lombardi G. Adoptive regulatory T cell therapy: challenges in clinical transplantation. Curr Opin Organ Transplant. 2010; 15(4):427-34.
-9Sakaguchi S, Sakaguchi N, Asano M, Itoh M, Toda M. Immunologic self-tolerance maintained by activated T cells expressing IL-2 receptor alpha-chains (CD25). Breakdown of a single mechanism of self-tolerance causes various autoimmune diseases. J Immunol.1995; 155(3):1151-64. Among other characteristic features, these cells express the transcription factor Foxp3, high levels of membrane CD25, and low levels of membrane CD127.1010 Hori S, Nomura T, Sakaguchi S. Control of regulatory T cell development by the transcription factor Foxp3. Science 2003; 299(5609):1057-61.

11 Fontenot JD, Gavin MA, Rudensky AY. Foxp3 programs the development and function of CD4 CD25 regulatory T cells. Nat Immunol. 2003; 4(4):330-6.
-1212 Khattri R, Cox T, Yasayko S-A, Ramsdell F. An essential role for Scurfin in CD4 CD25 T regulatory cells. Nat Immunol. 2003; 4(4):337-42. There have been some studies pointing to an increase in the proportion of CD25 regulatory T cells in the peripheral blood of aged BALB/c1313 Sharma S, Dominguez AL, Lustgarten J. High accumulation of T regulatory cells prevents the activation of immune responses in aged animals. J Immunol. 2006; 177(12):8348-55.,1414 Zhao L, Sun L, Wang H, Ma H, Liu G, Zhao Y. Changes of CD4 CD25 Foxp3 regulatory T cells in aged Balb/c mice. J Leukoc Biol. 2007; 81(6):1386-94. and C57BL/6 mice1515 Nishioka T, Shimizu J, Iida R, Yamazaki S, Sakaguchi S. CD4 CD25 Foxp3 T cells and CD4 CD25-Foxp3 T cells in aged mice. J Immunol. 2006; 176(11):6586-93.,1616 Williams-Bey Y, Jiang J, Murasko DM. Expansion of regulatory T cells in aged mice following influenza infection. Mech Ageing Dev. 2011; 132(4):163-70. as well as in elderly people.1717 Gregg R, Smith CM, Clark FJ, Dunnion D, Khan N, Chakraverty R, et al. The number of human peripheral blood CD4 CD25high regulatory T cells increases with age. Clin Exp Immunol. 2005; 140(3):540-6.

18 Gottenberg JE, Lavie F, Abbed K, Gasnault J, Le Nevot E, Delfraissy JF, et al. CD4 CD25high regulatory T cells are not impaired in patients with primary Sjögren's syndrome. J Autoimmun. 2005; 24(3):235-42.

19 Trzonkowski P, Szmit E, Mysliwska J, Mysliwski A. CD4 CD25 T regulatory cells inhibit cytotoxic activity of CTL and NK cells in humans-impact of immunosenescence. Clin Immunol. 2006; 119(3):307-16.

20 Vukmanovic-stejic M, Zhang Y, Cook JE, Fletcher JM, Mcquaid A, Masters JE, et al. Human CD4 CD25hi Foxp3 regulatory T cells are derived by rapid turnover of memory populations in vivo. J Clin Invest. 2006; 116(9):2423-33.
-2121 Lages CS, Suffia I, Velilla PA, Huang B, Warshaw G, Hildeman DA, et al. Functional regulatory T cells accumulate in aged hosts and promote chronic infectious disease reactivation. J Immunol. 2008; 181(3):1835-48. Such imbalance might contribute to the decreased immune functional activity and other peculiarities of the immune system in this age group.4Hwang KA, Kim HR, Kang I. Aging and human CD4(+) regulatory T cells. Mech Ageing Dev. 2009; 130(8):509-17.,5Urbán L, Bessenyei B, Márka M, Semsei I. On the role of aging in the etiology of autoimmunity. Gerontology. 2002; 48(3):179-84.,1919 Trzonkowski P, Szmit E, Mysliwska J, Mysliwski A. CD4 CD25 T regulatory cells inhibit cytotoxic activity of CTL and NK cells in humans-impact of immunosenescence. Clin Immunol. 2006; 119(3):307-16.

20 Vukmanovic-stejic M, Zhang Y, Cook JE, Fletcher JM, Mcquaid A, Masters JE, et al. Human CD4 CD25hi Foxp3 regulatory T cells are derived by rapid turnover of memory populations in vivo. J Clin Invest. 2006; 116(9):2423-33.

21 Lages CS, Suffia I, Velilla PA, Huang B, Warshaw G, Hildeman DA, et al. Functional regulatory T cells accumulate in aged hosts and promote chronic infectious disease reactivation. J Immunol. 2008; 181(3):1835-48.

22 Hunder GG. Clinical features of GCA/PMR. Clin Exp Rheumatol. 2000; 18(4 Suppl 20):S6-8.
-2323 Hasler P, Zouali M. Immune receptor signaling, aging, and autoimmunity. Cell Immunol. 2005; 233(2):102-8. In addition, increased frequency of the CD4 Foxp3 Treg phenotype has been shown in aged C57BL/62424 Chougnet CA, Tripathi P, Lages CS, Raynor J, Sholl A, Fink P, et al. A major role for Bim in regulatory T cell homeostasis. J Immunol. 2011; 186(1):156-63. and in elderly people.2525 Rosenkranz D, Weyer S, Tolosa E, Gaenslen A, Berg D, Leyhe T, et al. Higher frequency of regulatory T cells in the elderly and increased suppressive activity in neurodegeneration. J Neuroimmunol. 2007; 188(1-2):117-27. In contrast, more recent studies have found normal CD4 Foxp3 Treg cell frequency in elderly people.4Hwang KA, Kim HR, Kang I. Aging and human CD4(+) regulatory T cells. Mech Ageing Dev. 2009; 130(8):509-17.,2626 Yan J, Greer JM, Hull R, O'Sullivan JD, Henderson RD, Read SJ, et al. The effect of ageing on human lymphocyte subsets: comparison of males and females. Immun Ageing. 2010; 7:4.,2727 Santner-Nanan B, Seddiki N, Zhu E, Quent V, Kelleher A, Fazekas de St Groth B, et al. Accelerated age-dependent transition of human regulatory T cells to effector memory phenotype. Int Immunol. 2008; 20(3):375-83.

In face of the controversial literature on the frequency of Treg cells in aged people, and considering the evolving paradigm on Treg cell phenotypic characterization,2828 Raynor J, Lages CS, Shehata H, Hildeman DA, Chougnet CA. Homeostasis and function of regulatory T cells in aging. Curr Opin Immunol. 2012; 24(4):482-7.,2929 Fessler J, Ficjan A, Duftner C, Dejaco C. The impact of aging on regulatory T-cells. Front Immunol. 2013; 4:231. we have studied the frequency of Treg cells based on the evaluation of a stringent phenotypic profile (CD3+CD4+CD25highCD127ØFoxp3) in healthy volunteer women over a wide age range. With the understanding that the immune function is ruled by the equilibrium among several cellular phenotypes, we concurrently studied the frequency of activated effector T cells, a major T cell phenotype counteracted by Treg cells. We could not confirm previous reports of increased Treg cell frequency in elderly individuals. Instead, we observed a decline in the frequency of T cells, expressing markers related with cell activation and a higher proportion of two yet undefined Foxp3 CD25Ø phenotypes in elderly women.

Methods

Study population

Eighteen octogenarian and nonagenarian women were recruited from the cohort of healthy elderly individuals at the geriatric division at Universidade Federal de São Paulo, with ages between 80 and 93 (85±64.6 years old). The young adult group (<44 years old) comprised 26 healthy women with age between 19 and 44 years (29.23±6.63 years old), recruited among workers at the Medical School at Universidade Federal de São Paulo. All enrolled individuals were not receiving immunosuppressive medication and had no chronic infection, malignancy or cognitive impairment. In addition, there was no previous or current evidence of allergy or autoimmune diseases. Several of the elderly individuals did receive medication for arterial hypertension, diabetes mellitus, hypercholesterolemia or age-related heart diseases. Informed consent was obtained from all participants and the study was approved by the institution’s ethics committee.

Phenotypic evaluation of peripheral CD3 CD4 T lymphocytes

The phenotype of peripheral CD3+CD4 T cells was analyzed according to the expression of CD25, CD127 and Foxp3 in 44 healthy women, divided according to two age strata: young and elderly women. Peripheral blood mononuclear cells (PBMC) were isolated by density gradient centrifugation in Ficoll Paque™ Plus (GE Healthcare Life Science, Pittsburgh, PA) and cryopreserved in 90% fetal bovine serum (FBS) and 10% dimethyl sulfoxide in liquid nitrogen. For labeling with antibodies, cells were quickly defrosted at 37ºC, centrifuged and re-suspended on fresh RPMI medium with 10% FBS. Next cells were washed in phosphate buffer saline (PBS) and 0.5x106 cells were incubated with FITC-labeled anti-CD127, APC-Cy3-labeled anti-CD3, PerCP-labeled anti-CD4, and PE-Cy7-labeled anti-CD25, according to the manufacturer’s instructions (Becton Dickinson, San Jose, CA). After 30 minutes at 4ºC cells, were washed with Macs® buffer, fixed and permeabilized with Foxp3 fixation/permeabilization buffer (eBioscience, San Diego, CA) and, then, processed for Foxp3 staining using Foxp3 staining kit and APC-labeled anti-Foxp3 (eBioscience, San Diego, CA), according to the manufacturer’s instructions. For establishment of background staining and threshold for genuine expression of each phenotypic marker, we used the fluorescence minus one (FMO) method.3030 Hristov M, Schmitz S, Nauwelaers F, Weber C. A flow cytometric protocol for enumeration of endothelial progenitor cells and monocyte subsets in human blood. J Immunol Methods. 2012; 381(1-2):9-13. Cells were analyzed in a FACSCanto® flow cytometer (Becton Dickinson, San Jose, CA) and the obtained data were analyzed using the FlowJo software (Tree Star Inc, Ashland, OR). Three hundred thousand events were acquired for each sample. Parameters were expressed as mean and standard error (SE). Cell frequency was expressed as the percent frequency of each subpopulation relative to total CD3 cells.

Statistical analysis

Comparison of quantitative parameters between two groups was performed using Student’s t test. Linear regression analysis (Spearman or Pearson correlation test, depending on the distribution pattern of the variable) was performed to determine the relationship between age in years and the frequency of different cell phenotypes. Differences were considered significant if p-values were less than 0.05.

Results

The frequency of activated effector T cells was lower in elderly subjects as compared with young individuals, either when analyzing the CD3+CD4+CD25 phenotype (3.82±1.93% versus 9.53±4.49%; p<0.0001), the CD3+ CD4+CD25+CD127 phenotype (2.39±1.19% versus 7.26±3.84%; p<0.0001), the CD3+CD4+CD25high phenotype (0.41±0.22% versus 1.86±0.85%; p<0.0001) and the CD3+CD4+CD25highCD127 phenotype (0.06±0.038% versus 0.94±0.64%; p<0.0001) (Figure 1 A-D and Table 1). In fact, there was an inverse correlation between the frequency of all these T lymphocyte activated phenotypes and age (Table 1). Next, it was investigated whether the frequency of Treg cells, based on the expression of CD3, CD4, CD25, CD127, and Foxp3, was altered in elderly individuals (>80 years old) compared with young adults (<44 years old). In contrast to what we observed for effector T cells, there was an ambiguous representation of the several T cell phenotypes possibly associated with Treg function. No significant difference between elderly and young women was found in the frequency of phenotypes CD3+CD4+CD25 Foxp3 (1.51±0.95% versus 1.42±0.76%; p=0.74) and CD3+CD4+CD25+CD127ØFoxp3 (1.02±0.75%; versus 1.26±0.60%; p=0.26) (Table 1). However, the CD3+CD4+CD25highFoxp3 and CD3+CD4+CD25highCD127Ø Foxp3 phenotypes, occurred at a decreased frequency in elderly individuals as compared to young people (0,47±0,16% versus 0,85±0,64%; p=0,014 and 0.34±0.18% versus 0.76±0.48%; p=0.004, respectively) (Figure 1E-F, Table 1). In addition, the percentage of this phenotypes (CD3+CD4+CD25highFoxp3 and CD3+CD4+CD25high CD127ØFoxp3) presented an inverse correlation with age among the 26 young and 5 elderly individuals (r=-0.355 p=0.049 and r=-0.439 p=0.013, respectively) (Table 1).

FIGURE 1
Relative frequency of activated effector T cell phenotypes CD3+CD4+CD25+ (A), CD3+CD4+CD25+CD127+ (B), CD3+CD4+CD25High (C), and CD3+CD4+CD25HighCD127+ (D). Relative frequency of Treg cell phenotypesCD3+CD4+CD25HighFoxp3+ (E) and CD3+CD4+CD25HighCD127ØFoxp3+ (F) in elderly (age >80 years) and young women (age <44 years). Sample size varied in some experiments, due to restriction in availability of enough material. Relative frequency expressed as a percentage of CD3+ cells. Horizontal lines represent the mean and standard deviation for each group.
TABLE 1
Relative frequency of different CD3 CD4 T cell phenotypes in elderly and young healthy women

Interestingly, there was a strikingly higher frequency of two CD25Øphenotypes in elderly people as compared with the young group (Figure 2A-B, Table 1). The relative frequency of the CD3+CD4+CD25ØFoxp3 phenotype was 15.05±7.34% in the elderly against 1.65±1.71% in the young group (p<0.0001), and the relative frequency of the CD3+CD4+CD25ØCD127ØFoxp3 phenotype was 13.0±5.52% in the elderly group against 3.51±2.87% in the young adults (p<0.0001). Accordingly, there was a significant correlation between the frequency of these two T cell phenotypes and age among the 44 young and elderly women (r=0.71 and 0.74, respectively, p<0.0001) (Figure 2C-D, Table 1).

FIGURE 2
Relative frequency of CD25ø cell phenotypes CD3+CD4+CD25øFoxp3+ (A) and CD3+CD4+CD25øCD127øFoxp3+ (B) in 18 elderly (age >80 years) and 26 young women (age <44 years). Correlation between age and the frequency of CD25ø cell phenotypes CD3+CD4+CD25øFoxp3+(C) and CD3+CD4+CD25øCD127øFoxp3+(D). Horizontal lines represent the mean and standard deviation for each group. Relative frequency expressed as a percentage of CD3+ cells.

Discussion

The frequency of four activated effector T cell phenotypes was significantly lower in elderly as compared with young women (Figure 1 A-D and Table 1). In addition, there was an inverse correlation between the frequency of these activated T lymphocytes and age (Table 1). Considering the evolving criteria for identification of Treg cells, we investigated several alternative Treg cell-related phenotypes based on the expression of CD3, CD4, CD25, CD127, and Foxp3. We observed an ambiguous representation of the several T cell phenotypes possibly associated with Treg function. No significant difference between elderly and young women was found in the frequency of phenotypes CD3+CD4+CD25+Foxp3 and CD3+CD4+CD25+ CD127ØFoxp3 (Table 1). However, the most stringent Treg cell-related phenotypes CD3+CD4+CD25highFoxp3 and CD3+CD4+CD25highCD127ØFoxp3 occurred at significantly lower frequency in elderly as compared to young individuals (Figure 1 E-F and Table 1). In addition, the percentage of these two phenotypes presented an inverse correlation with age (Table 1). None of the phenotypic approaches pointed to increased Treg cell frequency in elderly individuals. These findings suggest a possible decrease in the production of Treg cells in elderly individuals. Studies from 2005 to 2008 have found an increased frequency of circulating Treg cells in elderly people.1313 Sharma S, Dominguez AL, Lustgarten J. High accumulation of T regulatory cells prevents the activation of immune responses in aged animals. J Immunol. 2006; 177(12):8348-55.

14 Zhao L, Sun L, Wang H, Ma H, Liu G, Zhao Y. Changes of CD4 CD25 Foxp3 regulatory T cells in aged Balb/c mice. J Leukoc Biol. 2007; 81(6):1386-94.
-1515 Nishioka T, Shimizu J, Iida R, Yamazaki S, Sakaguchi S. CD4 CD25 Foxp3 T cells and CD4 CD25-Foxp3 T cells in aged mice. J Immunol. 2006; 176(11):6586-93.,1717 Gregg R, Smith CM, Clark FJ, Dunnion D, Khan N, Chakraverty R, et al. The number of human peripheral blood CD4 CD25high regulatory T cells increases with age. Clin Exp Immunol. 2005; 140(3):540-6.

18 Gottenberg JE, Lavie F, Abbed K, Gasnault J, Le Nevot E, Delfraissy JF, et al. CD4 CD25high regulatory T cells are not impaired in patients with primary Sjögren's syndrome. J Autoimmun. 2005; 24(3):235-42.

19 Trzonkowski P, Szmit E, Mysliwska J, Mysliwski A. CD4 CD25 T regulatory cells inhibit cytotoxic activity of CTL and NK cells in humans-impact of immunosenescence. Clin Immunol. 2006; 119(3):307-16.

20 Vukmanovic-stejic M, Zhang Y, Cook JE, Fletcher JM, Mcquaid A, Masters JE, et al. Human CD4 CD25hi Foxp3 regulatory T cells are derived by rapid turnover of memory populations in vivo. J Clin Invest. 2006; 116(9):2423-33.
-2121 Lages CS, Suffia I, Velilla PA, Huang B, Warshaw G, Hildeman DA, et al. Functional regulatory T cells accumulate in aged hosts and promote chronic infectious disease reactivation. J Immunol. 2008; 181(3):1835-48.,3131 Simone R, Zicca A, Saverino D. The frequency of regulatory CD3 CD8 CD28- CD25 T lymphocytes in human peripheral blood increases with age. J Leukoc Biol. 2008; 84(6):1454-61. The apparent discrepancy of the present findings and earlier studies in the literature may be related to the combination of phenotypic markers used for definition of Treg cells. In fact, these results are more comparable with more recent studies that used phenotypic markers similar to these and showed no increase in Treg cell frequency in elderly individuals.4Hwang KA, Kim HR, Kang I. Aging and human CD4(+) regulatory T cells. Mech Ageing Dev. 2009; 130(8):509-17.,2626 Yan J, Greer JM, Hull R, O'Sullivan JD, Henderson RD, Read SJ, et al. The effect of ageing on human lymphocyte subsets: comparison of males and females. Immun Ageing. 2010; 7:4. Other possible factors contributing to discrepancy in the literature include peculiarities in subject selection and methodological aspects (e.g., frozen versus fresh cells, cell handling protocols, and flow cytometry parameters). In particular, it should be noted that the present study analyzed only females, and it has been shown previously that sexual hormones may influence the frequency of Treg cells.3232 Arruvito L, Sanz M, Banham AH, Fainboim L. Expansion of CD4 CD25 and FOXP3 regulatory t cells during the follicular phase of the menstrual cycle: implications for human reproduction. J Immunol. 2006; 178(4):2572-8. The actual impact of the modest magnitude of decrease in Treg cell frequency observed in this series (average 0.69% to 0.29%) on tolerance maintenance remains to be determined. It should be noticed that such a decrease was observed in successful octogenarians and nonagenarians, and, therefore, should be regarded as a tendency along the normal aging process. However, this may lead to predisposition for surpassing the threshold necessary for immune disequilibrium in some individuals, favoring the appearance of autoantibodies and autoimmune diseases in susceptible individuals. Although B and T lymphocyte response to antigenic stimulus has been shown to decline in aged individuals, paradoxically there is an increase in the frequency of autoantibodies and organ-specific autoimmune diseases in elderly people.3333 Prelog M. Aging of the immune system: a risk factor for autoimmunity? Autoimmun Rev. 2006; 5(2):136-9. This apparent incongruity might be reconciled by the finding of a reduced frequency and function of Treg cells. Therefore, further studies are granted to investigate whether the slight reduction in Treg frequency observed in this study is functionally relevant to tolerance maintenance in elderly individuals.

Interestingly, there was a strikingly higher frequency of two yet undefined CD4+CD25Ø phenotypes (CD3+CD4+CD25ØFoxp3 and CD3+CD4+CD25ØCD127ØFoxp3) in elderly as compared with young women (Figure 2A-B, Table 1). Accordingly, there was a significant correlation between the frequency of these two T cell phenotypes and age (r=0.71 and 0.74, respectively, p<0.0001) (Figure 2C-D). This has not been reported previously in elderly individuals, possibly due to a lack of focus on these particular T cell subsets. However, in previous studies the authors and others have shown these cells to be increased in patients with active systemic lupus erythematosus.3434 Yan B, Liu Y. The nature of increased circulating CD4 CD25 Foxp3 T cells in patients with systemic lupus erythematosus: a novel hypothesis. Open Rheumatol J. 2009; 3:22-4.,3535 Mesquita D, de Melo Cruvinel W, Araujo J, Pucci F, Salmazi K, Kallas E, et al. Systemic lupus erythematosus exhibits a dynamic and continuum spectrum of effector/regulatory T cells. Scand J Rheumatol. 2011; 40(1):41-50. Bonelli et al. have shown that CD3+CD4+CD25ØFoxp3 cells are able to suppress effector T cell proliferation, but not IFN-y production in vitro.3636 Bonelli M, Savitskaya A, Steiner C, Rath E, Smolen JS, Scheinecker C. Phenotypic and functional analysis of CD4 CD25- Foxp3 T cells in patients with systemic lupus erythematosus. J Immunol. 2009; 182(3):1689-95. In view of this, we hypothesize that these cells may represent intermediate or incomplete phenotypes towards Treg or effector T cells. It is possible that the increased frequency of these incomplete phenotypes in elderly people would be a consequence of counteracting mechanisms in a senescent immune system.

In the present study, the authors demonstrated some interesting peculiarities in the frequency of effector and regulatory T CD4 cells in the peripheral blood of healthy octogenarians and nonagenarians. These were characterized by a decrease in the frequency of activated effector T cell phenotypes and of the T cell phenotype, most strictly associated with Treg function (CD3+CD4+CD25highCD127ØFoxp3). Interestingly, there was a strikingly increased frequency of two still undefined CD4+CD25Ø T cell subsets (CD3+CD4+CD25ØFoxp3 and CD3+CD4+CD25ØCD127ØFoxp3). The knowledge obtained about diverse T cell subsets in elderly people should be of practical relevance to the search for means of increasing the efficacy of vaccination, boosting immunity in cancer, and avoiding tolerance breakdown, therefore, delaying the development of immunodeficiency, cancer, and autoimmune inflammatory diseases, respectively, in aged people. The present results in healthy octogenarian and nonagenarian women bring important preliminary information to the understanding of the cellular immune system in healthy elderly subjects.

  • Study conducted at Division of Rheumatology, Universidade Federal de São Paulo (Unifesp), São Paulo, SP, Brazil
  • Financial support: Fapesp: 06/51934-0, 07/51349-2 and CNPq (#476356/2008-3)

References

  • 1
    Steger MM, Maczek C, Berger P, Grubeck-Loebenstein B. Vaccination against tetanus in the elderly: do recommended vaccination strategies give sufficient protection. Lancet. 1996; 348(9029):762.
  • 2
    Weksler ME, Szabo P. The effect of age on the B-cell repertoire. J Clin Immunol. 2000; 20(4):240-9.
  • 3
    Naylor K, Li G, Vallejo AN, Lee W-W, Koetz K, Bryl E, et al. The influence of age on T cell generation and TCR diversity. J Immunol. 2005; 174(11):7446-52.
  • 4
    Hwang KA, Kim HR, Kang I. Aging and human CD4(+) regulatory T cells. Mech Ageing Dev. 2009; 130(8):509-17.
  • 5
    Urbán L, Bessenyei B, Márka M, Semsei I. On the role of aging in the etiology of autoimmunity. Gerontology. 2002; 48(3):179-84.
  • 6
    Cools N, Ponsaerts P, Van Tendeloo VFI, Berneman ZN. Regulatory T cells and human disease. Clin Dev Immunol. 2007; 2007:89195.
  • 7
    Kondelková K, Vokurková D, Krejsek J, Borská L, Fiala Z, Ctirad A. Regulatory T cells (TREG) and their roles in immune system with respect to immunopathological disorders. Acta Medica (Hradec Kralove). 2010; 53(2):73-7.
  • 8
    Safinia N, Sagoo P, Lechler R, Lombardi G. Adoptive regulatory T cell therapy: challenges in clinical transplantation. Curr Opin Organ Transplant. 2010; 15(4):427-34.
  • 9
    Sakaguchi S, Sakaguchi N, Asano M, Itoh M, Toda M. Immunologic self-tolerance maintained by activated T cells expressing IL-2 receptor alpha-chains (CD25). Breakdown of a single mechanism of self-tolerance causes various autoimmune diseases. J Immunol.1995; 155(3):1151-64.
  • 10
    Hori S, Nomura T, Sakaguchi S. Control of regulatory T cell development by the transcription factor Foxp3. Science 2003; 299(5609):1057-61.
  • 11
    Fontenot JD, Gavin MA, Rudensky AY. Foxp3 programs the development and function of CD4 CD25 regulatory T cells. Nat Immunol. 2003; 4(4):330-6.
  • 12
    Khattri R, Cox T, Yasayko S-A, Ramsdell F. An essential role for Scurfin in CD4 CD25 T regulatory cells. Nat Immunol. 2003; 4(4):337-42.
  • 13
    Sharma S, Dominguez AL, Lustgarten J. High accumulation of T regulatory cells prevents the activation of immune responses in aged animals. J Immunol. 2006; 177(12):8348-55.
  • 14
    Zhao L, Sun L, Wang H, Ma H, Liu G, Zhao Y. Changes of CD4 CD25 Foxp3 regulatory T cells in aged Balb/c mice. J Leukoc Biol. 2007; 81(6):1386-94.
  • 15
    Nishioka T, Shimizu J, Iida R, Yamazaki S, Sakaguchi S. CD4 CD25 Foxp3 T cells and CD4 CD25-Foxp3 T cells in aged mice. J Immunol. 2006; 176(11):6586-93.
  • 16
    Williams-Bey Y, Jiang J, Murasko DM. Expansion of regulatory T cells in aged mice following influenza infection. Mech Ageing Dev. 2011; 132(4):163-70.
  • 17
    Gregg R, Smith CM, Clark FJ, Dunnion D, Khan N, Chakraverty R, et al. The number of human peripheral blood CD4 CD25high regulatory T cells increases with age. Clin Exp Immunol. 2005; 140(3):540-6.
  • 18
    Gottenberg JE, Lavie F, Abbed K, Gasnault J, Le Nevot E, Delfraissy JF, et al. CD4 CD25high regulatory T cells are not impaired in patients with primary Sjögren's syndrome. J Autoimmun. 2005; 24(3):235-42.
  • 19
    Trzonkowski P, Szmit E, Mysliwska J, Mysliwski A. CD4 CD25 T regulatory cells inhibit cytotoxic activity of CTL and NK cells in humans-impact of immunosenescence. Clin Immunol. 2006; 119(3):307-16.
  • 20
    Vukmanovic-stejic M, Zhang Y, Cook JE, Fletcher JM, Mcquaid A, Masters JE, et al. Human CD4 CD25hi Foxp3 regulatory T cells are derived by rapid turnover of memory populations in vivo. J Clin Invest. 2006; 116(9):2423-33.
  • 21
    Lages CS, Suffia I, Velilla PA, Huang B, Warshaw G, Hildeman DA, et al. Functional regulatory T cells accumulate in aged hosts and promote chronic infectious disease reactivation. J Immunol. 2008; 181(3):1835-48.
  • 22
    Hunder GG. Clinical features of GCA/PMR. Clin Exp Rheumatol. 2000; 18(4 Suppl 20):S6-8.
  • 23
    Hasler P, Zouali M. Immune receptor signaling, aging, and autoimmunity. Cell Immunol. 2005; 233(2):102-8.
  • 24
    Chougnet CA, Tripathi P, Lages CS, Raynor J, Sholl A, Fink P, et al. A major role for Bim in regulatory T cell homeostasis. J Immunol. 2011; 186(1):156-63.
  • 25
    Rosenkranz D, Weyer S, Tolosa E, Gaenslen A, Berg D, Leyhe T, et al. Higher frequency of regulatory T cells in the elderly and increased suppressive activity in neurodegeneration. J Neuroimmunol. 2007; 188(1-2):117-27.
  • 26
    Yan J, Greer JM, Hull R, O'Sullivan JD, Henderson RD, Read SJ, et al. The effect of ageing on human lymphocyte subsets: comparison of males and females. Immun Ageing. 2010; 7:4.
  • 27
    Santner-Nanan B, Seddiki N, Zhu E, Quent V, Kelleher A, Fazekas de St Groth B, et al. Accelerated age-dependent transition of human regulatory T cells to effector memory phenotype. Int Immunol. 2008; 20(3):375-83.
  • 28
    Raynor J, Lages CS, Shehata H, Hildeman DA, Chougnet CA. Homeostasis and function of regulatory T cells in aging. Curr Opin Immunol. 2012; 24(4):482-7.
  • 29
    Fessler J, Ficjan A, Duftner C, Dejaco C. The impact of aging on regulatory T-cells. Front Immunol. 2013; 4:231.
  • 30
    Hristov M, Schmitz S, Nauwelaers F, Weber C. A flow cytometric protocol for enumeration of endothelial progenitor cells and monocyte subsets in human blood. J Immunol Methods. 2012; 381(1-2):9-13.
  • 31
    Simone R, Zicca A, Saverino D. The frequency of regulatory CD3 CD8 CD28- CD25 T lymphocytes in human peripheral blood increases with age. J Leukoc Biol. 2008; 84(6):1454-61.
  • 32
    Arruvito L, Sanz M, Banham AH, Fainboim L. Expansion of CD4 CD25 and FOXP3 regulatory t cells during the follicular phase of the menstrual cycle: implications for human reproduction. J Immunol. 2006; 178(4):2572-8.
  • 33
    Prelog M. Aging of the immune system: a risk factor for autoimmunity? Autoimmun Rev. 2006; 5(2):136-9.
  • 34
    Yan B, Liu Y. The nature of increased circulating CD4 CD25 Foxp3 T cells in patients with systemic lupus erythematosus: a novel hypothesis. Open Rheumatol J. 2009; 3:22-4.
  • 35
    Mesquita D, de Melo Cruvinel W, Araujo J, Pucci F, Salmazi K, Kallas E, et al. Systemic lupus erythematosus exhibits a dynamic and continuum spectrum of effector/regulatory T cells. Scand J Rheumatol. 2011; 40(1):41-50.
  • 36
    Bonelli M, Savitskaya A, Steiner C, Rath E, Smolen JS, Scheinecker C. Phenotypic and functional analysis of CD4 CD25- Foxp3 T cells in patients with systemic lupus erythematosus. J Immunol. 2009; 182(3):1689-95.

Publication Dates

  • Publication in this collection
    Jul-Aug 2015

History

  • Received
    13 Aug 2014
  • Accepted
    21 Oct 2014
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