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International Immunology Advance Access originally published online on February 20, 2007
International Immunology 2007 19(4):455-463; doi:10.1093/intimm/dxm010
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© The Japanese Society for Immunology. 2007. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org

The origin of thymic CD4+CD25+ regulatory T cells and their co-stimulatory requirements are determined after elimination of recirculating peripheral CD4+ cells

Yifan Zhan, Dorothee Bourges, James A. Dromey, Leonard C. Harrison and Andrew M. Lew

Autoimmunity and Transplantation Division, Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, Victoria 3050, Australia

Correspondence to: Y. Zhan; E-mail: zhan{at}wehi.edu.au or A. M. Lew; E-mail: lew{at}wehi.edu.au


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
Studies on the thymic ontogeny of naturally arising CD4+CD25+ regulatory T cells (TR cells) are complicated by the contamination of recirculating cells from the periphery (both activated CD4+ T and TR cells). We investigated TR cells in anti-CD4 antibody transgenic (Tg) (GK) mice that continuously deplete peripheral CD4 T cells but not thymocytes so that the generation of thymic TR cells and their developmental requirement can be accurately assessed. We show that in the thymuses of mice that lack peripheral CD4+ cells, TR cells were present but were fewer in number compared with wild-type (WT) mice. Therefore, we show that peripheral TR cells do re-enter the thymus, comprising 20% of TR cells in the normal thymus. TR cells from both WT and GK mice expressed Foxp3 and GITR, and suppressed the proliferation of CD25–CD4+ T cells. Furthermore, the co-stimulation requirements for TR generation were evaluated in mice with or without peripheral CD4 cells. Splenic TR cells in CD40L–/– mice and CTLA4Ig Tg mice were fewer compared with WT mice. Mice deficient in both co-stimulatory pathways had further reduction in splenic TR cells. Unlike the periphery, the reduction in thymic TR cells was only seen for CD40L–/– but not for CTLA4Ig Tg mice. Therefore, we found that the co-stimulation requirements for the thymic development of TR cells differed from those for peripheral homeostasis.

Keywords: CD4+CD25+ regulatory T cells, costimulation, thymus, tolerance, transcription factor Foxp3


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
CD4+CD25+ regulatory T cells (TR cells) play a major role in the maintenance of self-tolerance and in the control of autoimmune diseases (1, 2). Many studies have demonstrated that TR cells exist in the thymus of both mice and humans (1, 2) and that they are functionally and phenotypically similar to peripheral TR cells (3, 4). More recently, the identification of transcription factor Foxp3 as a specific marker of TR cells allows tracking the ontogeny of TR cells (5–7). A series of studies using a green fluorescent protein reporter system have demonstrated the thymic origin of Foxp3+ TR cells. For example, a small population of Foxp3-expressing double-positive cells occurred in the adult thymus (8), although in neonatal mice Foxp3 induction occurred preferentially at the CD4 single-positive (SP) stage or during transition to this stage (9). However, Foxp3+ TR cells can also be generated from Foxp3–CD25– cells after transfer into thymectomized mice, indicating peripheral differentiation (10, 11).

Peripheral T cells with an activation phenotype have the potential to re-enter the thymus (12–15). Thus, TR cells with their CD25+ phenotype could conceivably re-enter the thymus and thus influence the thymic pool of TR cells. The physiological significance of such thymic re-entry of peripheral T cells remains unclear. In models of allotransplantation, the re-entry of T cells has been proposed to serve as the source of allo-antigens to induce central tolerance (16–18). Beside T cells, other types of cells such as dendritic cells have recently been shown to enter the thymus (19).

An active research area in TR cell biology is the co-stimulation requirement for TR homeostasis. CD28 co-stimulation has been reported to be required in TR development (20–22). As CD28 is also required for the in vitro expansion of TR cells (23), studies on co-stimulation requirement for thymic development could not exclude the effect by recirculating peripheral cells. Relative to CD28 co-stimulation, there are fewer studies addressing the role of CD40L in the generation of TR cells, particularly in thymic development of TR cells. Although there are no reports on the status of thymic TR cells in CD40L–/– mice, CD40–/– mice were found to have reduced percentages of TR cells in the thymus and periphery (24, 25). CD40L–/– non-obese diabetic mice have a slightly reduced peripheral TR population (26). Blocking CD40/CD40L by antibody also reduces the numbers of peripheral TR cells (25). Currently, there are no reports on whether combined deficiency in CD28 and CD40L pathways affects TR development even further.

In this study, we addressed the ontogeny of thymic TR cells using the GK mouse (mouse transgenic for the anti-CD4 antibody, GK1.5) model that transgenically expresses the depleting antibody GK1.5; such mice lack peripheral CD4+ cells while retaining an intact thymic CD4 compartment (27, 28). This allows assessment of thymic TR cell development without any complication from the peripheral pool of CD4+ cells. Furthermore, we evaluated the requirement for CD28- and CD40L-mediated co-stimulation during TR cell development in mice deficient in either one or both pathways, both in the presence or absence of peripheral CD4+ cells so that the contribution of two pathways (singly and combined) in thymic generation and peripheral homeostasis can be delineated.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
Mice
C57BL/6 mice and C57BL/6 mice with mutations in MHC class I:C.H-2bm1 (called bm1) were used as wild-type (WT) control mice. Mice deficient in peripheral CD4+ T cells (GK mice), as a result of the transgenic (Tg) expression of anti-CD4 antibody under the control of the human Cytomegalovirus promoter, and GK/CTLA4Ig Tg mice, with peripheral CD4+ T cell depletion combined with expression of the CTLA4Ig fusion protein (blocks CD28/B7 co-stimulation) under the control of the rat insulin promoter, have been described previously (27, 29, 30). Mice with a disruption of the CD40L gene (CD40L–/– mice) and CD40L–/– mice with the GK transgene (GK Tg/CD40L–/– mice) have both been previously described (30, 31). The CD40L–/– mice have been backcrossed to C57Bl/6 mice for >12 generations. The CTLA4Ig mice were derived on a bm1 background, but in all our studies bm1 as expected behaved identically to C57Bl/6. CTLA4Ig Tg/CD40L–/– mice and GK Tg/CTLA4Ig Tg/CD40L–/– mice were obtained by cross-breeding of the appropriate homozygous Tg mice. This resulted in mice containing blocks in both co-stimulatory pathways, both in the presence and absence of peripheral CD4+ T cells. All lines of mice were bred and maintained at the Walter and Eliza Hall Institute.

Flow cytometric analysis of lymphocytes
Antibodies used in this study to stain cell-surface markers included the following: PE–Cy7 or allophycocyanin (APC)-conjugated anti-mouse CD4 (clone RM4-5, BD PharMingen), APC–Cy7-conjugated anti-mouse CD8 (clone 53-6.7, BD PharMingen), FITC- or PE-conjugated rat anti-mouse CD25 antibody (clone PC61, BD PharMingen) and biotinylated or FITC-conjugated anti-mouse GITR (DTA-1, Hybridoma Facility, Walter and Eliza Hall Institute). To stain cell-surface molecules, cells were incubated with optimally diluted antibody for 30 min on ice. Viable cells (determined by propidium iodide exclusion gating) were then analyzed using a FACSaria (Becton Dickinson, San Jose, CA, USA) in conjunction with CellQuest software (San Carlos, CA, USA). A commercially available kit was used for the detection of intracellular Foxp3 protein (eBioscience). Cells were first stained for cell-surface markers CD4, CD8, CD25 and GITR, as above. After washing, cells were then fixed and permeabilized before incubation with PE-conjugated anti-Foxp3 or isotype control antibody as per the manufacturer's instructions.

T cell proliferation and suppression
Peripheral CD4+CD25– and CD4+CD25+ T cells were purified from spleen or/and lymph nodes of C57BL/6 mice by sorting cells stained with antibodies for cell-surface markers and analyzed using the FACSaria. For the isolation of thymic CD4+CD25+ cells, the cell population was enriched for CD25+ cells using magnetic bead separation magnetic cell sorter. CD25+-enriched thymocytes were then stained for cell-surface CD4 and CD8 using antibodies, in order to obtain CD4+CD25+(CD8–) thymocytes by cell sorting using the FACSaria. For proliferation assays, CD4+CD25– T cells (2 x 104 cells per well) were cultured in 96-well round-bottomed plates in the presence of 8 x 104 antigen-presenting cells (irradiated and T cell depleted) with or without TR cells (2 x 104 CD4+CD25+ T cells) from peripheral or thymic organs. Cells were stimulated for 72 h with 5 µg ml–1 anti-CD3 antibody and pulsed with 1 µCi per well of [3H]thymidine for the final 8 h.

Statistic analysis
Data [mean and standard deviation (SD)] were analyzed by the Student's t-test.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
The majority of thymic TR cells develop de novo but substantial thymic TR cells are derived from the periphery
To assess the development of thymic TR cells without the complication of re-entry by peripheral CD4 cells, we used anti-CD4 antibody Tg GK mice, which completely lack peripheral CD4 cells (27, 28) but retain an intact thymocyte population. Thymic TR cells were defined as CD4+CD8–CD25+ cells. Figure 1(a) shows that CD4-deficient GK mice lack splenic CD4 T cells but have a population of splenic CD8 T cells comparable to WT mice. In contrast, the population of thymic CD4 SP T cells in GK mice was obviously present. The number of CD4 SP thymocytes was slightly smaller in GK mice compared with sex, age-matched non-Tg WT mice. This difference was not statistically significant (P = 0.15; Fig. 1b). The other three main populations (CD4–CD8–, CD4+CD8+ and CD8+ SP) were similar in GK and WT mice. We therefore surmise that the slight reduction of CD4 SP cells is due to a lack of recirculating CD4 cells from the periphery. As for TR cells, ~10% of the total splenic CD4 T cell population are CD25+ in C57BL/6 mice. As expected, this population was absent in GK mice (Fig. 1b). It should be pointed out that there were no CD25+ cells in the CD4– splenic cell population (both CD8+CD4– and CD8–CD4– cells) of these mice (data not shown). Next, the thymic TR cells were compared in C57BL/6 and GK mice. In C57BL/6 mice, the percentage of CD25+GITR+ cells within the CD4 SP population in the thymus (3–4% of CD4 SP thymocytes) was less compared with the percentage of CD25+ cells from the CD4 cell population in the spleen (8–10%) (Fig. 1c). In CD4-deficient GK mice, there was a consistent 20% reduction in the percentage of CD25+ cells from the CD4 SP population in the thymus, when compared with WT mice. Therefore, we conclude that ~20% of the TR in a normal thymus is derived from the periphery.


Figure 1
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  		Fig. 1. TR cells in the spleen and thymus of B6 and GK mice. Spleen cells and thymocytes were prepared from sex- and age-matched B6 and GK mice. Cells were stained for cell-surface markers CD4 (APC), CD8 (APC–Cy7) and CD25 (PE). Spleen cells and thymocytes were plotted for CD4 and CD8 (a), and the percentages of CD4 SP thymocytes from individual mice of B6 and GK mice were shown (b). CD25 expression by spleen cells and gated CD4 SP cells were plotted (c). The numbers in the plots are the mean percentages of the indicated population. Seven similar experiments were performed.

 
Thymic TR cells from GK and WT mice express Foxp3 and GITR
CD25 can be expressed by activated effector T cells as well as TR cells (1, 2). To determine whether the CD25+CD4+ SP cells in the thymuses of these mice belonged to the TR subset, we looked at the expression of Foxp3 and GITR in addition to CD25 expression, because Foxp3 is considered to be a more specific marker for TR cells and GITR is also constitutively expressed at high levels by TR cells. CD4+CD25+ thymocytes expressing similar levels of Foxp3 were present in both WT and GK mice (Fig. 2a). Using Foxp3 as the marker of TR cells, the total numbers of thymic TR cells were compared between B6 and GK mice. The numbers and the percentages of thymic TR cells were significantly fewer in GK mice (Fig. 2b); the cellularity of total thymus and CD4 SP thymocytes was not significantly different between two groups. Using Foxp3 as a TR marker on CD4 SP cells, we also conclude that 20% of TR cells in the thymus are derived from the periphery (Fig. 2b).


Figure 2
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  		Fig. 2. Foxp3 and GITR expression by thymic TR cells and the suppressive activity of thymic TR cells from B6 and GK mice. Thymocytes were stained for cell-surface markers CD4 (PE–Cy7), CD8 (APC–Cy7), CD25 (FITC) and GITR (biotinylated DTA-1/streptavidin–PE) or intracellular mouse Foxp3 (PE). (a) Gated CD4 SP thymocytes from five individual mice each group were plotted for CD25 and Foxp3 or CD25 and GITR expression. The numbers in the plots indicate the percentage of gated population. The means and SD of Foxp3+ (both CD25+ and CD25– cells) and of GITR+CD25+ CD4 SP cells were also indicated. (b) The numbers of thymic TR cells in B6 and GK mice. Total thymocytes, thymic CD4 SP cells as well as the numbers and the percentages of (CD25+ and CD25–) Foxp3+CD4+ SP cells and GITR+CD25+ from five individual mice of each group were plotted. *P < 0.05 compared with B6. Three similar experiments were performed. (c) Suppressive activity of thymic TR cells: CD4+CD25– T cells (2 x 104 cells per well) in the presence of T cell-depleted, irradiated 8 x 104 antigen-presenting cells in 96-well round-bottomed plates were cultured with or without thymic TR (2 x 104 CD4+CD25+ T cells) from B6 and GK mice. Cells were stimulated for 72 h with 5 µg ml–1 anti-CD3 antibody and pulsed with 1 µCi of [3H]thymidine per well for the final 8 h. Mean counts per minute and SD of triplicate cultures from each group are presented. Three similar experiments were performed.

 
Notably, not all CD25+ cells were positive for Foxp3 expression in both mouse strains. Within the Foxp3+ thymocyte population, a substantial proportion of cells was CD25–. On the other hand, nearly all Foxp3+ thymocytes from WT and GK mice expressed high levels of GITR (data not shown). Since GITR expression correlated well with Foxp3 expression by thymic TR cells, CD25 and GITR expression were used in some subsequent experiments as the markers of thymic TR cells.

Thymic CD8–CD4+CD25+ cells suppress the proliferation of non-TR cells
To compare the functional capacity of thymic TR cells which developed in the absence or presence of peripheral CD4 cells, CD4+CD25+(CD8–) thymocytes were isolated from GK and WT mice. They were cultured with or without CD4+CD25– responder spleen cells in the presence of T cell-depleted irradiated spleen cells, and stimulated with soluble anti-CD3 antibody. CD4+CD25+ cells from both GK and WT mice clearly suppressed the proliferation of CD4+CD25– cells in response to anti-CD3 antibodies (Fig. 2c). The suppressive activity of these TR cells from GK mice was comparable to WT mice. As expected, isolated CD4+CD25+ cells, from both GK and WT mice, did not proliferate in response to TCR ligation by anti-CD3 antibody (data not shown).

Both CD28 and CD40L co-stimulation are required for homeostasis of TR cells in the spleen
To evaluate the impact of co-stimulation deficiency on TR cells, we enumerated the splenic TR cells in WT (C57BL/6 or bm1), CD40L–/–, CTLA4Ig Tg and CD40L–/–/CTLA4Ig Tg mice. All mice were on a C57BL/6 or bm1 background. WT C57Bl/6 mice and bm1 mice behaved similarly (as expected, given their near identical genetic background). The numbers and percentages of CD4+ splenocytes were very similar in all groups of mice (data not shown). However, compared with WT mice, CD40L–/– mice and CTLA4Ig Tg mice had a significant decrease (50%) in the percentage of Foxp3+CD4+ cells from total CD4+ cells (Fig. 3a and b). The total numbers of splenic TR cells were also 50% fewer in CD40L–/– and CTLA4Ig Tg mice. Consistently, CD40L–/– mice had a slightly lower percentage of TR cells compared with CTL4Ig Tg mice. Furthermore, the size of the splenic TR cell population in CD40L–/–/CTLA4Ig Tg mice was further reduced to 30% of WT (Fig. 3). Similar data were obtained when we used the co-expression of GITR and CD25 to define TR cells in spleen. These data revealed a non-overlapping role for both the CD40L and CD28 co-stimulatory pathways in the maintenance of the peripheral TR cell pool. As expected, CD25+GITR+ cells were not detected in GK mice, even in the splenic CD4– cell population (data not shown). There was also no population of Foxp3+ TR cells in CD4– spleen cells in all types of mice studied (data not shown).


Figure 3
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  		Fig. 3. Evaluation of the co-stimulation requirement for the peripheral TR pool. Spleen cells from WT, CD40L–/–, CTLA4Ig Tg and CD40L–/–/CTLA4Ig Tg mice were prepared and stained for cell-surface markers CD4, CD8 and CD25 and then stained for intracellular Foxp3. (a) Plots show the profile of Foxp3 on gated CD4 cells. The numbers in the plots indicate the cell percentage of the corresponding quadrant. (b) The numbers and (c) the mean percentages (lower) of (CD25+ and CD25–) Foxp3+CD4+ cells in spleen. *P < 0.05 and **P < 0.01 compared with WT mice.

 
CD28 and CD40L co-stimulation impact differentially on development of thymic TR cells
To assess the impact of co-stimulation on thymic TR cell development, we enumerated the thymic TR cells in WT, CD40L–/–, CTLA4Ig Tg and CD40L–/–/CTLA4Ig Tg mice. When CD4 SP thymocytes were analyzed for CD25 and GITR expression, it was clear that the percentages of CD25+GITR+ cells in CD40L–/– mice were significantly lower compared with that of WT mice (47%; P < 0.01) (Fig. 4a). However, the percentage of CD25+GITR+ population in CD4 SP thymocytes of CTLA4Ig Tg mice was not significantly different from that of WT cells (unlike the spleen; Fig. 2). In agreement with the finding that CTLA4Ig has minimal impact on the thymic CD25+GITR+ population, CD40L–/–/CTLA4Ig Tg mice had similar levels of CD25+GITR+ cells as those in CD40L–/– mice. Again, the above findings were confirmed using Foxp3 as the marker of TR cells (Fig. 4b).


Figure 4
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  		Fig. 4. Evaluation of co-stimulation requirements for thymic TR development. (a) Thymocytes from WT, CD40L–/–, CTLA4Ig Tg and CD40L–/–/CTLA4Ig Tg mice were prepared and stained for CD4 (APC), CD8 (APC–Cy7), CD25 (PE) and GITR (FITC). The left panel shows the profile of CD25 and GITR on gated CD4 SP thymocytes. The numbers in the plots are the percentages of the indicated population. The right panel shows the mean percentage and SD of CD25+GITR+ cells within thymic CD4 SP cells for each type of mice. Five similar experiments were performed. P values for specific comparison are indicated (**P < 0.001) compared with WT mice. (b) Thymocytes from the above four groups of mice were stained for cell-surface markers CD4, CD8 and CD25 and then stained for intracellular Foxp3. The left panel shows the profile of CD25 and Foxp3 on gated CD4 SP thymocytes. The numbers in the plots indicate the cell percentage of corresponding quadrant. The right panel shows the mean percentage and SD of Foxp3+ cells within thymic CD4 SP cells from three to four mice of each type. **P < 0.001. (c) The absolute numbers of total, CD4 SP and TR cells in the thymus were enumerated. Plots show the mean and SD of three individual mice in each group. *P < 0.05 and **P < 0.01 compared with WT mice.

 
Whereas the percentages of thymic TR cells were consistently and significantly different between CD40L–/– mice and WT mice, the difference in absolute numbers of thymic TR cells between the two groups was less striking (Fig. 4c, right panel).

As co-stimulation influences the homeostasis of peripheral TR cells and the thymic TR pool might be subjected to the influence of peripheral TR pool, we evaluated the impact of co-stimulation on thymic TR cell development in mice without peripheral CD4 cells (GK mice). Similar to CD40L–/– mice, GK/CD40L–/– mice had a slightly larger population of CD4 SP thymocytes compared with GK mice (Fig. 5a). Out of total CD4 SP thymocytes, the percentage of CD25+GITR+ cells was also lower in GK/CD40L–/– mice than in GK mice (Fig. 5b and c). As the percentage of CD25+GITR+ cells in GK mice was consistently and significantly lower than in WT mice (Fig. 2), there was no significant difference in the percentage of CD25+GITR+ cells between CD40L–/– mice and GK/CD40L–/– mice. Similarly, there was no difference in the percentage of CD25+GITR+ cells between CTLA4Ig Tg and GK/CTLA4Ig Tg mice (data not shown).


Figure 5
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  		Fig. 5. Evaluation of co-stimulation requirement for thymic TR development without peripheral CD4 cells. Thymocytes from WT, CD40L–/–, CTLA4Ig Tg and CD40L–/–/CTLA4Ig Tg mice with or without the GK transgene were stained as in Fig. 4(a). For clarity, only data from WT and CD40L–/– mice with or without GK transgene are shown. (a) The CD4 and CD8 profiles of thymoytes, (b) the profile of CD25 and GITR on gated CD4 SP thymocytes. (c) The mean percentages of CD25+GITR+CD4 SP thymocytes of 5–10 mice. **P < 0.01 for the indicated comparison.

 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
The origin of TR cells has been subject to extensive investigation but remains to be fully elucidated (32–34). Using GK mice (27), we observed that TR cells can be detected in these mice but the size of the population was consistently smaller (~20%) than that of mice with intact peripheral CD4 cells. Moreover, the percentage of TR cells within CD4 SP thymocytes was also consistently lower in mice without peripheral CD4+ cells. The most parsimonious interpretation for the difference between mice with or without peripheral CD4 cells is that some peripheral TR cells might re-enter thymus and thus influence the size of thymic pool of TR cells. Thymic re-entry of peripheral CD4 T cells (although not TR cells specifically until this report) has been demonstrated previously (12–15, 35). Cells with an activated phenotype preferentially re-enter thymus (14), although naive T cells can also re-enter thymus (15).

Although the exact mechanism of thymic re-entry is not entirely known, it is likely to involve chemokine/adhesion molecules (36). Indeed, in a cell transfer model, re-entry of antigen-experienced rat T cells (CD45RC–) into thymus has been found to be dependent on the alpha 4 integrin–vascular cell adhesion molecule-1 interaction (37). Recirculation may be a nuisance in determining the development of thymic TR cells de novo, but there is little clue as to its physiological significance. In models of allotransplantation, the re-entry of T cells has been proposed to serve as the source of allo-antigens to induce central tolerance (16–18). Also, dendritic cells have recently been shown to enter thymus (19) and what role such cells have in positive or negative selection in the thymus remains moot.

We do not believe that the development of CD4 T cells is grossly altered in GK mice, even though a large bolus injection of anti-CD4 or -CD8 antibody has been reported to influence CD4 and TCR expression on thymocytes and so influence CD4 T cell development (38). However, in GK mice, thymocyte expression of CD4, TCR, CD24 and CD5 was indistinguishable from WT mice (27) (Y. Zhan; unpublished data).

We contend that the substantial cohort of thymic TR cells in GK mice represent those that are generated de novo in the thymus, and we have been able to quantify that this comprises 80% of TR cells in the thymus. This also confirms the thymic origin of TR cells as suggested by others (1). For example, the appearance of thymic TR cells precedes that of peripheral TR cells and indeed contributes to the peripheral pool (7, 39). Also, these TR cells from GK mice function similarly to TR cells from WT mice.

The requirements for the generation of TR cells have been actively investigated in recent years, partly to try to produce enough cells for clinical application. One requirement is the TCR engagement, as drawn from animal models that were Tg for both TCR and cognate antigen (22, 39, 40). Another requirement for the generation of TR cells is co-stimulation. In general, both CD28 and CD40L pathways have a profound effect on the peripheral homeostasis of TR cells. This can occur at different levels: differentiation from non-TR cells (generation), survival and expansion (25, 41). In our study with CD40L–/– mice and CTLA4Ig Tg mice, we observed a reduction both in the percentages and total numbers of splenic TR cells, reinforcing that both pathways are involved in TR homeostasis. Furthermore, we show here that concurrent inhibition of both pathways further reduced the numbers of TR cells in the periphery. This also poses the question whether concurrent stimulation of CD28 and CD40L pathways might enhance the expansion of TR cells.

In CD40L–/– mice, the percentages of thymic TR cells (regardless of using CD25+GITR+ or Foxp3+ to identify TR cells) were significantly lower than that of WT mice. It agrees with the previously reported data derived from CD40–/– mice (25). However, the total numbers of thymic TR cells in CD40L–/– mice were not always significantly different from that of WT mice. That is because CD40L–/– mice had more thymocytes and CD4 SP thymocytes (both the percentage and absolute number). This may reflect the multiple impacts of co-stimulation on development of T cells. It is unknown why CD40L–/– mice had more thymocytes, although several studies with the CD40L–/– mice have reported that deletion of self-reactive T cells is impaired in several models of negative selection (42, 43). Interestingly, in the study with CD40–/– mice (25), the authors state that there was a decreased number of thymic TR cells in CD40–/– mice; however, only the percentages of thymic TR cells were shown. Similarly, in several studies addressing the role of CD28 in the generation of thymic TR cells, data were expressed as the percentages of TR out of CD4 SP thymocytes and therefore it is difficult to estimate the total numbers of thymic TR cells (21, 41). In a TCR Tg model, a modest reduction in the numbers, compared with the percentages, of thymic TR cells was found in B7-deficient mice (40).

A paradox regarding on the contribution of co-stimulation to TR cells raised from our study is that blocking CD28 co-stimulation in CTLA4Ig Tg mice has a profound effect on splenic TR cells (both the number and percentage) but has no detectable influence on thymic TR cells. This is at odds with several studies showing that CD28 deficiency and CD80/86 double deficiency reduced the percentages of thymic TR cells as well as peripheral TR cells (21–23, 40, 41). One partial explanation is that CTLA4 and CD28 have differential binding kinetics to CD80 and CD86. In humans, the amount of CTLA4Ig required to inhibit a CD80-mediated cellular response was ~100-fold lower than for a CD86-mediated response (44). Furthermore, CD80 is the major ligand for CTLA4 while CD86 is the main ligand for CD28 during formation of the immune synapse (45). Consistent with the data from CD28–/– mice, deficiency in CD86 but not CD80 has a more profound effect on TR generation (41, 46). Perhaps, in the thymus of the CTLA4Ig mice, CD80 may be differentially affected more than CD86 and that CD86 is more important in TR generation in the thymus.

Several molecules have been used in this study to define TR cells. Clearly, they are not always co-expressed. CD4+CD25+ cells contained Foxp3+ and Foxp3– cells while Foxp3+ cells contained CD25+ and CD25– cells. On the other hand, nearly all Foxp3+ cells express high levels of GITR. It remains moot what is the relationship between all these subsets. CD25–Foxp3+GITR+ cells could be induced from immature thymocytes by in vitro TCR and CD28 stimulation and they did not obtain potent regulatory function (22). Thus, these cells are proposed as the precursors of TR cells. We also found that CD4+CD25–GITR+ thymocytes do not potently suppress the proliferation of CD4+CD25– T cells (data not shown). In the periphery, mature CD4+CD25– cells can be converted into CD4+CD25+ cells that regulate immune responses (11, 47, 48). Under certain conditions, CD4+CD25– cells can directly regulate immune responses (49). Overall, given that the current protocol of detection of intracellular Foxp3 requires fixed cells and prevents the functional evaluation, both CD25 and GITR would still be the useful molecular markers to identify TR cells for function valuation. The avail of Foxp3-GFP reporter mouse will advance the functional evaluation of TR cells.

In conclusion, the current study presents a clear demonstration of the origin of thymic TR cells using a mouse model lacking the peripheral CD4 T cell compartment. In such a model, the impact of co-stimulation on the generation of thymic TR cells and the maintenance of the peripheral pool of TR cells were able to be delineated. As the influence of co-stimulation on peripheral TR cells is profound, the influence of co-stimulation on thymic TR cells is more subtle and is more complicated by the fact that co-stimulation deficiency (especially CD40L) affects other aspects of thymocyte development.


   Acknowledgements
 
We thank Danielle Cooper for excellent assistance with mouse maintenance. We thank Sarah Londrigan and Margo Honeyman for critically reading the manuscript. This work was supported by National Health and Medical Research Council of Australia and Juvenile Diabetes Research Foundation.


   Abbreviations
 
APC, allophycocyanin
SD, standard deviation
SP, single positive
Tg, transgenic
TR cell, CD4+CD25+ regulatory T cell
WT, wild type

   Notes
 
Transmitting editor: A. Cooke

Received 6 September 2006, accepted 18 January 2007.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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