CD4⁺CD25⁺ T-cell development is regulated by at least 2 distinct mechanisms

Immunologic tolerance is a feature of the immune system essential for discrimination between self and nonself. Recent data suggest that in addition to clonal deletion and anergy, regulatory T cells play a significant role in the generation and maintenance of tolerance.1-4 The term regulatory T cells is used for a variety of immunoregulatory cells that can be subdivided into a number of subsets based on expression of cell surface proteins and pattern of cytokine production.1-4 Because these subsets of regulatory T cells have been characterized in experimental models using different assays, the interrelationship between the subsets is difficult to understand.1-4 

One of the best-characterized subsets of CD4⁺ regulatory T cells is defined by its constitutive expression of interleukin-2 receptor (IL-2R)–α chain (CD25) (CD4⁺CD25⁺ T cells).3,4 CD4⁺CD25⁺ T cells constitute approximately 10% of peripheral CD4⁺ T cells in nonimmunized naive mice and exhibit a broad spectrum of autoimmunity-preventive activity.5-8CD4⁺CD25⁺ T cells are naturally anergic and, on T-cell receptor (TCR)–mediated activation, potently suppress the proliferation of CD4⁺CD25⁻ T cells by an antigen-nonspecific mechanism.9-11 Molecular mechanisms by which CD4⁺CD25⁺ T cells mediate suppression are unclear but seem to be independent of cytokine production9,10 and dependent on cell contact9,10 and to require constitutive expression of CTLA-4.12 13 

CD4⁺CD25⁺ T cells can be subdivided by the expression pattern of CD45RB^high or CD45RB^low, CD38⁺ or CD38⁻, CD69⁺ or CD69⁻, or CD62L^high or CD62L^low.5,9,11,14 Although these findings suggest that CD4⁺CD25⁺ T cells might be heterogeneous and composed of a mixture of regulatory T cells and activated conventional T cells, it has recently been shown that the anergic and suppressive properties of CD4⁺CD25⁺T cells cannot be subdivided to a smaller subpopulation defined by the expression levels of CD45RB, CD62L, CD38, or CD69.11 14Therefore, CD4⁺CD25⁺ T cells may represent a relatively homogeneous population of regulatory T cells.

Precise signals that promote the development of CD4⁺CD25⁺ T cells remain elusive, but considerable evidence suggests that costimulatory molecules and cytokines play important roles. It has been shown that CD4⁺CD25⁺ T cell levels are severely decreased in mice lacking CD28⁸ or CD40L,15 indicating that signaling by CD28 and by CD40L regulates the number of CD4⁺CD25⁺ T cells in periphery. In addition, CD4⁺CD25⁺ T-cell levels are decreased in mice lacking IL-216 or IL-2R component,17indicating that IL-2 signaling also regulates the number of CD4⁺CD25⁺ T cells.

The CD25⁺ population constitutes approximately 5% of CD4⁺CD8⁻CD3^high mature thymocytes in normal mice (CD4⁺CD25⁺ thymocytes), and the CD4⁺CD25⁺ thymocytes exhibit a similar functional property to that found in peripheral CD4⁺CD25⁺ T cells.18 Moreover, thymectomy on day 3 of life decreases the number of CD4⁺CD25⁺ T cells from the peripheral lymphoid organ.6 Furthermore, CD4⁺CD25⁺ T cells cannot be produced by in vitro culture of CD4⁺CD25⁻ T cells.7,9 14 Taken together, these findings suggest that CD4⁺CD25⁺T cells arise in the thymus and migrate to the periphery.

Recently, Itoh et al18 have shown that CD4⁺CD25⁺ T cells can develop in TCR transgenic mice of wild-type background but not of RAG-2–deficient background. Thus, it is possible that B cells, γδ T cells, or natural killer (NK) T cells, all of which are absent in TCR transgenic RAG-2–deficient mice but are present in TCR transgenic mice, are required for the development of CD4⁺CD25⁺ T cells. It is also possible that the expression of endogenous TCR and the subsequent interaction with self-major histocompatibility complex (MHC) at a certain affinity is required for the development of these cells. However, a great deal of uncertainty remains about differentiation factors and antigen specificity of CD4⁺CD25⁺ T cells.

In the current study, to determine whether positive or negative selection in thymus regulates the development of CD4⁺CD25⁺ T cells, we investigated CD4⁺CD25⁺ T-cell development in mice expressing TCR transgene in positively selecting and negatively selecting MHC backgrounds. We also investigated the requirement of B cells and NK T cells for the development of CD4⁺CD25⁺ T cells. Our results indicate that the size of the CD4⁺CD25⁺ T-cell pool in periphery is regulated at least 2 distinct levels. First, in the thymus, where CD4⁺CD25⁺ T cells are produced, a certain range of TCR–MHC affinity is required for their development. In addition, B cells, but not NK T cells, regulate the expansion and/or survival of CD4⁺CD25⁺ T cells in periphery.

BALB/c and C57BL/6 mice were purchased from Japan SLC (Shizuoka, Japan). Immunoglobulin μ-chain–deficient mice19 and NK T-cell–deficient mice20 of C57BL/6 background were previously described. Ovalbumin-specific DO11.10 (DO10⁺) TCR transgenic mice21 were backcrossed to BALB/c mice for more than 10 generations. BALB/c RAG-2^−/−mice22 were a kind gift from Dr T. Saito (Chiba University). TCR-α^−/− mice23(H-2^b/b) were crossed with DO10⁺ mice (H-2^d/d), and then offspring DO10⁺TCR-α^+/− H-2^d/b mice were crossed with TCR-α^+/− H-2^d/b mice to develop DO10⁺ TCR-α^−/− mice in H-2^d/d, H-2^d/b, or H-2^b/b background. Expressions of DO10⁺ transgene and H-2 haplotypes were determined by fluorescence-activated cell sorter analysis (FACS), as described below. The genotype of TCR-α allele was determined by polymerase chain reaction using the following primer pairs: to detect TCR-α wild-type allele, 5′-aagatcctcggtctcaggacagc-3′ and 5′-ggtaggtggcgttggtctctttg-3′; to detect TCR-α mutant allele, 5′-attcgcagcgcatcgccttctatcg-3′ and 5′-ggtaggtggcgttggtctctttg-3′. Homozygosity for the TCR-α mutant allele was confirmed, using FACS, by the absence of T cells that express endogenous TCR V-α2. Mice were housed in micro-isolator cages under pathogen-free conditions; 8- to 10-week-old mice were used in all experiments.

Cells from thymus and spleen were stained and analyzed on a FACScalibur (Becton Dickinson, San Jose, CA) using CELLQuest software. For direct staining, the following conjugated antibodies were purchased from PharMingen (San Diego, CA): anti-CD3 fluorescein isothiocyanate (FITC) (145-2C11), anti-CD4 FITC, phycoerythrin (PE), PerCP, allophycocyanin (APC) (H129.19), anti-B220 FITC (RA3-6B2), anti-CD8 FITC, APC (53.6.7), anti-CD25 PE (PC61), anti-CD25 FITC (7D4), anti-CD45RB FITC (16A), anti–NK-1.1 PE (PK136), anti-TCR V-α2 FITC (B20.1), anti-TCR V-α11 FITC (RR8-1), anti-TCR Vβ8 FITC (F23.1), anti–H-2K^b PE (AF6-88.5), anti–H-2K^d FITC (SF1-1.1), anti–I-A^b PE (AF6-120.1), and anti–I-A^d FITC (AMS-32.1). KJ1-26 monoclonal antibody, anti-idiotype for DO10 TCR,24 was purified from supernatants of hybridoma cells using protein G columns (Pharmacia, Uppsala, Sweden) and conjugated to FITC or biotin. Before staining, Fc receptors were blocked with anti-CD16/32 antibody (2.4G2; PharMingen).

After cells were stained with anti-CD4 APC and anti-CD25 PE and washed twice with phosphate-buffered saline–1% bovine serum albumin, cells were stained with annexin V–FITC (R&D Systems, Minneapolis, MN) according to the manufacturer's instructions. Cells were then analyzed on a FACScalibur.

Data are summarized as mean ± SD. Statistical analysis of the results was performed by the unpaired t test.P < .05 was considered significant.

It is well established that allelic exclusion of the TCR-β chain is almost perfect, whereas that of the TCR-α chain is incomplete.25 Thus, a considerable fraction of T cells express 2 different TCR-αβ pairs.26 Because it has been shown that CD4⁺CD25⁺ T cells can develop in TCR transgenic mice of wild-type background but not of RAG-2–deficient background,18 it is possible that CD4⁺CD25⁺ T cells preferentially express the endogenous TCR-α chain and that signaling through the endogenous TCR-α chain coupled with the transgenic TCR-β chain is involved in the development of CD4⁺CD25⁺ T cells in TCR transgenic mice. To examine this possibility, we first investigated the frequency of CD4⁺ T cells that express both transgenic TCR (recognized by anti-idiotypic monoclonal antibody, KJ1-26) and one of the endogenous TCR V-α chains, V-α2, in the CD25⁺ or CD25⁻ population in ovalbumin-specific TCR transgenic (DO10⁺) mice. As shown in Figure1A, splenic CD4⁺ T cells that express transgenic TCR and TCR V-α2 chain were found at a higher frequency in CD4⁺CD25⁺ T cells than in CD4⁺CD25⁻ T cells (CD4⁺CD25⁺ T cells 12.0% ± 1.6% versus CD4⁺CD25⁻ T cells 2.5% ± 1.0%, mean ± SD; n = 5; P < .001). Moreover, CD4⁺ T cells that express both transgenic TCR and TCR V-α11 were also found at a higher frequency in CD4⁺CD25⁺ T cells (data not shown). These results suggest that CD4⁺CD25⁺ T cells preferentially express the second TCR-α chain in DO10⁺ mice. For controls, we performed the same analysis for splenocytes in DO10⁺RAG-2–deficient mice. We found that, consistent with the previous report by Itoh et al,18 CD4⁺CD25⁺T cells were almost absent in these mice (Figure 1B) and that no CD4⁺CD25⁻ T cell expressed TCR V-α2 (Figure1B) or TCR V-α11 (data not shown) in these mice.

CD4⁺CD25⁺ T cells preferentially expressed dual TCR-α chains in DO10⁺ mice (Figure 1A). To determine whether dual TCR-α expression is required for the development of CD4⁺CD25⁺ T cells, we investigated the development of CD4⁺CD25⁺ T cells in TCR-α heterozygous mice, in which only one allele of TCR-α was available for the expression. As shown in Figure 2, the number of CD4⁺CD25⁺ T cells in thymus and in spleen was normal in TCR-α heterozygous mice, suggesting that though CD4⁺CD25⁺ T cells preferentially expressed the second TCR-α chain in TCR transgenic mice, dual TCR-α expression was not essential for the development of CD4⁺CD25⁺ T cells.

Because CD4⁺CD25⁺ T cells are absent in DO10⁺ RAG-2^−/− mice (Figure 1B) and because CD4⁺CD25⁺ T cells preferentially express the second TCR-α chain in DO10⁺ mice (Figure 1A), the expression of endogenous TCR-α chain and subsequent interaction with self-MHC at an appropriate affinity may be required for the development of CD4⁺CD25⁺ T cells. To test this possibility, we investigated the development of CD4⁺CD25⁺ T cells in DO10⁺ TCR-α–deficient (TCR-α^−/−) mice (Figure 3). Indeed, the frequency of the CD25⁺ population in CD4⁺CD8⁻ mature thymocytes was severely decreased in DO10⁺ TCR-α^−/− mice with positively selecting H-2^d background compared with that in DO10⁺ TCR-α^+/+ mice (Figure 3B). In contrast, no significant difference was observed in the number of conventional CD4⁺CD25⁻ mature thymocytes between DO10⁺ TCR-α^−/− mice and DO10⁺ TCR-α^+/+ mice (Figure 3B). These results indicate that expression of the endogenous TCR-α chain is required for the development of CD4⁺CD25⁺ T cells in DO10⁺ mice. In addition, they indicate that the appropriate affinity of TCR to self-MHC for the development of CD4⁺CD25⁺ thymocytes is different from that of CD4⁺CD25⁻ thymocytes and that the interaction of TCR with self-MHC at a certain affinity is essential for the development of CD4⁺CD25⁺ thymocytes.

We next analyzed CD4⁺CD25⁺ T-cell development by transgenic TCR in DO10⁺ mice in a negatively selecting H-2^b background.27 28 For this purpose, we investigated DO10⁺ TCR-α^−/− mice to exclude the effect of endogenous TCR-α expression. Interestingly, the number of total thymocytes in DO10⁺ TCR-α^−/− mice was severely decreased in H-2^b background (DO10⁺ TCR-α^−/− H-2^b mice compared with DO10⁺ TCR-α^−/−H-2^d mice; 0.6 ± 0.2 × 10⁷ versus 13.2 ± 3.0 × 10⁷; n = 5 each;P < .001). Most thymocytes were within the CD4⁻CD8⁻ stage (Figure 3A); consequently, the number of CD4⁺CD8⁻ thymocytes was severely decreased in these mice. When gated on the CD4⁺CD8⁻ thymocytes that could escape from negative selection in DO10⁺ TCR-α^−/− mice in H-2^b background, the frequency of the CD25⁺population was still severely decreased (Figure 3B). These results suggest that CD4⁺CD25⁺ T cells could not develop when the affinity between TCR and MHC was so high.

Interestingly, though the number of CD4⁺CD25⁺thymocytes was severely decreased in DO10⁺TCR-α^−/− mice, CD4⁺CD25⁺ T cells were readily observed in the spleens of DO10⁺TCR-α^−/− mice in H-2^d background (Figure3C). The population of CD4⁺CD25⁺ T cells was also approximately 2-fold in the spleens of DO10⁺TCR-α^+/+ mice compared with that of CD4⁺CD25⁺ thymocytes (Figure 3B-C). Most splenic CD4⁺CD25⁺ T cells in DO10⁺TCR-α^−/− mice and DO10⁺TCR-α^+/+ mice were of the CD45RB^lowCD69⁻ phenotype, similar to that in wild-type mice (data not shown). These results suggest that there existed some homeostatic machinery that regulated the size of the CD4⁺CD25⁺ T-cell pool in periphery.

CD4⁺CD25⁺ T cells exist in the spleens of DO10⁺ TCR-α^−/− H-2^d mice (Figure 3C) but not in spleens of DO10⁺RAG-2^−/− mice (Figure 1B). Thus, it is possible that a certain population of cells absent in RAG-2–deficient mice regulates the size of the CD4⁺CD25⁺ T-cell pool in periphery. The possibility that B cells might affect CD4⁺CD25⁺ T-cell development was examined by flow cytometric analyses of CD4⁺CD25⁺ T cells in μ-chain–deficient mice, which lack mature B cells.19Interestingly, a profound decrease of splenic CD4⁺CD25⁺ T cells was observed in μ-chain–deficient mice (Figure 4A). Less than 7% of CD4⁺ T cells in μ-chain–deficient mice expressed CD25 compared with 13% in wild-type mice (n = 5 each) (Figure 4A). Because the absolute number of splenic T cells was decreased in μ-chain–deficient mice, the number of splenic CD4⁺CD25⁺ T cells was significantly decreased in μ-chain–deficient mice (approximately 20% of wild-type levels;P < .001; Figure 4B). Interestingly, in contrast to splenic CD4⁺CD25⁺ T cells, the number of CD25⁺ population in CD4⁺ CD8⁻mature thymocytes was normal in μ-chain–deficient mice (Figure5). Taken together, these results indicate that though the development of CD4⁺CD25⁺ T cells in thymus does not require help from B cells, the size of CD4⁺CD25⁺ T-cell pool in periphery is regulated by B cells. In contrast, the number of splenic CD4⁺CD25⁺ T cells (Figure 4) and CD4⁺CD25⁺ thymocytes (Figure 5) was normal in NK T cell-deficient mice, indicating that NK T cells are not essential for the development of CD4⁺CD25⁺ T cells.

In this study, we show that the size of the peripheral CD4⁺CD25⁺ T-cell pool is regulated by at least 2 distinct mechanisms. First, in the thymus, where CD4⁺CD25⁺ T cells are produced, a certain TCR–MHC affinity is required for the development of CD4⁺CD25⁺ T cells. We found that CD4⁺CD25⁺ T cells preferentially expressed the second TCR-α chain in DO10⁺ TCR transgenic mice (Figure1A) and that the development of CD4⁺CD25⁺thymocytes was severely impaired in DO10⁺TCR-α^−/− mice (Figure 3B), indicating that the expression of the endogenous TCR-α chain is required for the development of CD4⁺CD25⁺ T cells in TCR transgenic mice. Moreover, we found that CD4⁺CD25⁺ thymocytes were severely decreased in DO10⁺ TCR-α^−/− mice in the positively selecting background and the negatively selecting background, whereas CD4⁺CD25⁻ thymocytes effectively developed by transgenic TCR in DO10⁺ TCR-α^−/− mice in the positively selecting background (Figure 3B). Taken together, these results indicate that the appropriate affinity of TCR to self-MHC for the development of CD4⁺CD25⁺ thymocytes is different from that of CD4⁺CD25⁻ thymocytes and that the interaction of TCR with self-MHC at a certain affinity is essential for the development of CD4⁺CD25⁺thymocytes. Second, in the periphery, B cells regulate the expansion and/or survival of the developed CD4⁺CD25⁺ T cells. We found that, in contrast to the thymus, CD4⁺CD25⁺ T cells were readily detected in the spleens of DO10⁺ TCR-α^−/− mice in positively selecting background (Figure 3C), suggesting that a small number of CD4⁺CD25⁺ T cells that developed in the thymus might have expanded in the periphery. In addition, we found that the number of splenic CD4⁺CD25⁺ T cells, but not of CD4⁺CD25⁺ thymocytes, was decreased in B-cell–deficient mice (Figures 4, 5). Therefore, B cells may control the peripheral pool of CD4⁺CD25⁺ T cells, possibly by inducing the expansion or prolonging the survival of these cells.

Although it is accepted that CD4⁺CD25⁺ T cells need activation by TCR for regulatory function, the antigen specificity of CD4⁺CD25⁺ T cells is unknown. We found that CD4⁺CD25⁺ thymocytes were severely decreased in DO10⁺ TCR-α^−/− mice in negatively selecting H-2^b background (Figure 3). Moreover, we found that Mtv-9–induced clonal deletion of TCR V-β5 cells proceeded normally in CD4⁺CD25⁺ T cells in BALB/c mice (data not shown). Furthermore, Papiernik et al29 have shown that Mls-1^a–induced clonal deletion of TCR V-β6 cells is also normal in CD4⁺CD25⁺ T cells. These findings indicate that CD4⁺CD25⁺ T cells are not resistant to clonal deletion in thymus. Recently, an altered negative-selection model of CD4⁺CD25⁺ T-cell development was proposed.3 In this model, the suboptimal stimulation of thymocytes with low-affinity self-antigens would result not in deletion but in a permanent change in TCR signaling. Although we could not identify the TCR–MHC affinity that specifically induced the development of CD4⁺CD25⁺ T cells, identification will extend the understanding of the nature of CD4⁺CD25⁺ T cells.

Although the expression of dual TCR-α chain is not essential for the development of CD4⁺CD25⁺ T cells (Figure 2), CD4⁺CD25⁺ T cells preferentially express the second TCR-α chain in DO10⁺ mice (Figure 1). In addition, though transgenic TCR-α expression is not sufficient for the development of CD4⁺CD25⁺ T cells in DO10⁺ mice (Figure 3), the ligation of transgenic TCR has been shown to be sufficient for the regulatory function of CD4⁺CD25⁺ T cells.10 11 These findings suggest that both TCRs on dual TCR-α–expressing cells are functional for their regulatory activity of CD4⁺CD25⁺ T cells. Therefore, dual TCR-α chain expression may increase the chance for TCR signaling of CD4⁺CD25⁺ T cells.

We found that B cells played a significant role in the regulation of the CD4⁺CD25⁺ T-cell pool in the spleen (Figure4) but not in the development of CD4⁺CD25⁺ T cells in the thymus (Figure 5). Because it has been shown that B7/CD28 interaction8 and CD40/CD40L interaction15regulate the CD4⁺CD25⁺ T-cell pool in periphery, B cells may regulate the CD4⁺CD25⁺T-cell pool through B7/CD28 interaction or CD40/CD40L interaction. Recently, it has been shown that the number of dividing cells is modestly increased in CD4⁺CD25⁺ T cells compared with that in CD4⁺CD25⁻ T cells in periphery.29 Moreover, we found that apoptotic cells, which were assessed as annexin V binding cells, were not increased in CD4⁺CD25⁺ T cells (data not shown). Thus, it is likely that B cells regulate the cell expansion of CD4⁺CD25⁺ T cells in periphery.

Another subset of CD4⁺ regulatory T cells, isolated after T cells were activated with alloantigens in the presence of IL-10, was termed type 1 T regulatory (Tr1) cells.30 Tr1 cells are distinct from classical Th1 or Th2 cells in that they produce large amounts of IL-10 and moderate amounts of transforming growth factor-β.30 Significantly, Tr1 cells suppress immune responses in vitro and in vivo through a mechanism dependent on the production of the immunoregulatory cytokine IL-10.31 In contrast, the regulatory function of CD4⁺CD25⁺T cells is independent of IL-10⁹. Although it is still possible that they are in fact the same subset of regulatory T cells in different stages of differentiation, the observation that CD4⁺CD25⁺ T cells cannot be differentiated from naive CD4⁺CD25⁻ T cells in vitro,7,9,14 whereas Tr1 cells can be differentiated from naive cells,30 favors the hypothesis that CD4⁺CD25⁺ T cells and Tr1 cells are 2 distinct regulatory T cells with similar functions. Moreover, though NK T cells have been shown to be involved in the development of IL-10–dependent regulatory T cells,32 NK T cells were not required for the development of CD4⁺CD25⁺ T cells (Figures 4,5). These observations also support the above hypothesis.

In the past 10 years, a great deal has been learned about the regulation of the CD25 gene. It is known that the CD25 gene has at least 3 important elements for regulation, denoted positively regulatory regions (PRR) I, II, and III.33 NF-κB and SRF bind PRRI, Elf-1 and HMG-I (Y) bind PRRII, and Stat5 and Elf-1 bind PRRIII.33 Although PRRI is essential for mitogen–antigen-mediated induction of the gene and PRRIII is essential for IL-2–induced gene expression, PRRII is essential for transcription in response to either mitogen or IL-2. Thus, the CD25 gene can be controlled not only by activation-dependent signals (NF-κB and Stat5) but also by lineage-specific signals (Elf-1). Consistent with these in vitro findings, we found that the CD4⁺CD25⁺ T cells were decreased in both DO10⁺ TCR-α^−/−mice (Figure 3) and DO10⁺ Stat5a^−/−mice,34 suggesting that both TCR-mediated signaling and Stat5-mediated signaling are required for the development of CD4⁺CD25⁺ T cells in vivo. More detailed mechanisms will be uncovered by promoter analyses of the CD25 gene in CD4⁺CD25⁺ T cells.

In summary, we have shown that the peripheral pool of CD4⁺CD25⁺ T cells is regulated at least 2 distinct levels. First, a certain TCR–MHC affinity is required for the development of CD4⁺CD25⁺ T cells in thymus. Second, B cells are required for the expansion of CD4⁺CD25⁺ T cells in periphery. The identification of TCR–MHC affinity that preferentially induces the development of CD4⁺CD25⁺ T cells will extend our understanding of the nature of CD4⁺CD25⁺ T cells.

We thank Dr T. Saito for BALB/c RAG-2^−/− mice, Dr D. Y. Loh for DO10⁺ mice, and Drs. K. M. Murphy, K. Suzuki, and S.-I. Kagami for valuable discussions.

After our submission of this paper to Blood, Jordan et al reported that selection of CD4⁺CD25⁺ thymocytes required a TCR with high affinity for a self-peptide.35