Developmental changes of FOXP3-expressing CD4+CD25+ regulatory T cells and their impairment in patients with FOXP3 gene mutations
Introduction
CD4+CD25+ regulatory T (Treg) cells represent a unique lineage of immunoregulatory cells both in humans and animals, and play a central role in the maintenance of immunological self-tolerance [1], [2]. In addition, Treg cells were shown to exert a suppressive function on the immune responses to various self and nonself antigens, alloantigens, external antigens, and tumor antigens [3], [4], [5]. These observations provide new insights into the immunopathogenesis of human diseases, such as autoimmune and allergic diseases, malignances, and complications of bone marrow or organ transplantation.
Most recently, forkhead box P3 (FOXP3) has emerged as a master control gene for the generation and function of Treg cells [6], [7], as illustrated by the demonstration that naturally occurring defects in FOXP3 cause characteristic autoimmune syndrome in mice and humans [8], [9], [10], [11]. Failure to properly regulate CD4+ effector T cells, caused by lack of CD4+CD25+FOXP3+ Treg cells, is directly responsible for the X-linked scurfy syndrome (sf) in mice [12], characterized by early-onset lymphohistiocytic infiltrates of the skin and lymphoid organs, severe runting secondary to chronic diarrhea, and hypergammaglobulinemia. The human orthologue, immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) is a rare inborn error characterized by the early onset of multiple autoimmune diseases [13]. It has been shown that FOXP3 is expressed mainly in a subset of CD4+CD25+ T cells which correlates with the suppressive activity of regulatory T cells [7]. Retroviral gene transfer of FOXP3 converts naive T cells into a phenotype similar to that of naturally occurring Treg cells [6]. Thus, FOXP3 expression is used as a surrogate molecular marker for quantifying Treg cells in the peripheral blood.
Whether Treg cells are exclusively generated in the thymus or may differentiated from peripheral CD4+CD25+ naive T cells is controversial. In vitro activation of human CD4+CD25− T cells through T cell receptor stimulation was shown by some investigators to induce FOXP3 expression and suppressor function similar to freshly isolated CD4+CD25+ T cells [14], [15], but not by others [16]. In the peripheral blood of healthy adults, the majority of Treg cells, identified as CD4+CD25+FOXP3+ T cells, were shown to have the memory CD45RA-CD45RO+ phenotype [17]. Recently, it has been reported that human CD4+CD45RO+CD25hiFOXP3+ T cells are susceptible to apoptosis and have limited self-renewal capacity, suggesting that conversion of CD25-negative to CD25-positive memory T cells in the periphery is one of the pathways for the generation of Treg cells [18]. In contrast, CD4+CD25+FOXP3+ T cells in cord blood appear to essentially show the naive CD45RA+CD45RO− phenotype [19]. However, a small number of Treg cells of the naive phenotype have been identified in the peripheral blood of adults; their frequency seems to decrease with age [20], [21]. In this study, we explored the quantity of circulating Treg cells during childhood development in order to understand better the potential immunopathogenesis of human diseases resulting from dysregulated peripheral tolerance. Using flow cytometric analysis and an anti-FOXP3 monoclonal antibody (mAb), we examined how FOXP3-expressing CD4+CD25+ T cells of the naive and memory phenotypes might change from birth to adulthood. We also analyzed FOXP3 expression in IPEX patients to study the effect of different mutations of FOXP3 gene on the generation of Treg cells.
Section snippets
Subjects
Venous blood samples were obtained from children of various ages and adult volunteers at the Toyama University Hospital. Individuals with allergic or autoimmune diseases, malignancies, hematologic disorders, or acute infections were excluded. Cord blood samples were obtained from the placentas of full-term newborns at Okada Obstetrics and Gynecology Hospital, who did not have hereditary disorders, hematologic abnormalities, or infectious complications. The total number of samples was 147 and
FOXP3-expressing lymphocytes
Flow cytometric analysis using a murine anti-human FOXP3 mAb revealed that FOXP3 expression, at any age, was detectable mainly within the CD4+CD25+ T cell population and only rarely in CD4+CD25− T cells (Fig. 1). Few CD8+ T, B, and NK cells expressed FOXP3 (data not shown). Table 2 summarizes the frequency of CD25+FOXP3+ cells within the CD4+ T cell population at various ages. We found that the percentages of CD25+FOXP3+ cells within CD4+ T cells in cord blood were significantly lower than
Discussion
Treg cells stably expressing FOXP3 are generated in the thymus and released into the circulation as lineage specific CD4+CD25+FOXP3+ T cells. Human CD4+CD25− T cells if activated in vitro have been shown to express FOXP3 and acquire T regulatory activity [14]. However, activation-induced FOXP3 expression is transient and the cells fail to develop suppressor function, suggesting that a sustained high level of FOXP3 expression is required for Treg phenotype and function [25]. Activation of CD4+
Acknowledgments
We thank Masatoshi Okada (Okada Obstetrics and Gynecology Hospital) for kindly collecting cord blood samples, and Chikako Sakai and Hitoshi Moriuchi for excellent technical assistance. This work was supported by Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan, and a grant from Ministry of Health, Labour and Welfare of Japan.
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