SEARCH

SEARCH BY CITATION

Keywords:

  • Autoreactivity;
  • Foxp3;
  • Regulatory T cells

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgments
  8. Conflict of interest
  9. References
  10. Supporting Information

In the periphery, Foxp3 expression is considered sufficient to maintain natural regulatory CD4+ T-cell suppressive function. In this study, we challenge this model. Indeed, in mouse chimeras in which major histocompatibility complex (MHC) class II expression is restricted to the thymus, peripheral regulatory CD4+ T cells lack suppressive activity. In addition, regulatory CD4+ T cells recovered 5 days after transfer into recipient mice lacking expression of MHC class II molecules (self-deprived) are unable to inhibit the proliferative response of conventional CD4+ T cells both in vitro and in vivo. Disruption of TCR/MHC class II interactions rapidly leads to alterations in the regulatory CD4+ T-cell phenotype, the ability to respond to stimulation and to produce interleukin-10, and the transcriptional signature. Interestingly, self-deprivation does not affect Foxp3 expression indicating that in regulatory CD4+ T cells, self-recognition induces unique transcriptional and functional features that do not rely on Foxp3 expression.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgments
  8. Conflict of interest
  9. References
  10. Supporting Information

Naturally occurring regulatory CD4+ T (Treg) cells are important for the maintenance of self-tolerance in the periphery. In particular, they are key players in the prevention of various autoimmune and inflammatory disorders. Natural Treg cells arise in the thymus where T-cell receptor (TCR) signals lead to interleukin (IL)-2 sensitivity enhancement in developing thymocytes. Then, IL-2 signaling induces Foxp3 expression that, in turn, strengthens Treg-cell lineage stability [[1, 2]]. Foxp3 expression is then important to maintain a distinct transcriptional program required for their suppressive function [[3-5]].

image

Figure 1. Major histocompatibility complex (MHC) IIΔ/Δ-CD3KO chimeras lack functional Treg cells in the periphery. CD3KO mice were lethally irradiated and their immune system reconstituted with wild-type (WT) (WT-CD3KO chimeras) or MHC IIΔ/Δ (MHC IIΔ/Δ-CD3KO chimeras) bone marrow(BM) cells. Chimeras were analyzed 28 days after BM-cell transfer. (A) Diagram illustrating the experimental model. (B) CD4+ CD25+ (Treg) cells were purified from the periphery or the thymus of chimeras and WT mice. CD4+ CD25 (Tconv) cells were purified from lymph nodes of WT mice, labeled with carboxyfluorescein diacetate succinimidyl ester(CFSE), and cultured alone (–) or together with the indicated Treg cells at a 1/1 Treg/Tconv cell ratio. CFSE fluorescence histograms of Tconv cells (CFSE+) are shown 64 h after the beginning of culture. Histograms in the absence of anti-CD3 stimulation are also shown (left, filled histograms). Values correspond to the average number of cell divisions undergone by Tconv cells in response to anti-CD3 stimulation during the culture period. The histograms shown were generated from one experiment but are representative of three individual experiments.

Download figure to PowerPoint

image

Figure 2. Self-deprived Treg cells are not functional in vitro. T cells purified from the periphery (lymph nodes + spleen) of WT mice were injected into CD3KO mice (CD3KO-MHC II+ recipients) or into CD3KO-MHC IIΔ/Δ mice (CD3KO-MHC IIΔ/Δ recipients). Five days later, the suppressive capacities of Treg cells purified from the periphery of recipient mice were analyzed. (A) Diagram illustrating the experimental model. (B) Tconv cells were purified from lymph nodes of WT mice, labeled with CFSE, and cultured alone (–) or together with the indicated Treg cells in the presence of soluble anti-CD3 and antigen-presenting cells (APCs). Forward Scatter (FSC), CD25, and CD69 histograms of Tconv cells (CFSE+) are shown 16 h after the beginning of culture for a 1/1 Treg/Tconv ratio. (C) FSC, CD25, and CFSE histograms of Tconv cells (CFSE+) are shown 64 h after the beginning of culture for a 1/1 Treg/Tconv ratio. Histograms in the absence of anti-CD3 stimulation are shown as controls (left, filled histograms). (D) Inhibition indexes (inhibition of Tconv-cell CD25 expression was calculated after a culture period of 16 h; inhibition of Tconv-cell proliferation was estimated 64 h after the initiation of culture) are shown for various Treg/Tconv-cell ratios (2, 1, 1/2, 1/4, 1/8). (E) The same inhibition indexes are shown for a 1/1 Treg/Tconv-cell ratio as means ± SEM values of six independent experiments. (F) Treg cells from the indicated mice were stimulated for 2 days with soluble anti-CD3 and APCs in the presence or absence of interleukin (IL)-2. Then, they were tested for their suppressive abilities. Data are shown as inhibition indexes of Tconv-cell proliferation after a culture period of 64 h are shown for various Treg/Tconv-cell ratios (2, 1, 1/2, 1/4, 1/8, 1/16, 1/32) and are representative of two individual experiments. **p < 0.01, ***p < 0.001, Student's unpaired t-test.

Download figure to PowerPoint

Recent studies have clearly established that the TCR has an instructive role in inducing commitment of developing thymocytes into the Treg-cell lineage [[6, 7]]. More precisely, Treg-cell development would be instructed by TCRs with high avidity for self-peptides bound to major histocompatibility complex (MHC) class II molecules (self). Indeed, the proportion of Treg cells is increased when TCR transgenic T cells are forced to see their cognate antigens in the thymus [[1, 2, 8, 9]]. This model is further supported by the observation that there is a limited amount of overlap (10–20%) between the TCR sequences expressed within the conventional CD4+ T (Tconv) cell and the Treg-cell repertoire [[10]]. Interestingly, overlapping is more important when the Treg-cell repertoire was compared with the repertoire of pathogenic autoreactive effector T cells [[11]].

After migrating to the periphery, Treg cells still interact with self. Indeed, based on autoimmune ovarian disease and prostatitis models, Tung and colleagues [[12, 13]] have determined that continuous interactions with self are required to allow Treg cells to accumulate in the draining lymph-nodes. More recently, Lathrop et al. [[14]] have confirmed that the Treg-cell TCR repertoire varies by anatomical location in the periphery. Finally, Darrasse-Jèze et al. [[15]] have obtained interesting data showing that MHC class II-expressing dendritic cells are required to maintain Treg-cell numbers in the periphery. Altogether, these data strongly suggest that natural Treg cells are submitted to continuous interactions with self in the periphery.

In this study, we investigated whether self-deprivation (induced in our experimental settings by the nonexpression of MHC class II molecules in the periphery) would alter peripheral Treg-cell suppressive capacities. By using two complementary mouse experimental models, we show that self-deprived Treg cells lack suppressive activity. Interestingly, self-deprivation does not affect Foxp3 expression indicating that in Treg cells, self-recognition induces unique transcriptional and functional features that do not rely on Foxp3 expression.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgments
  8. Conflict of interest
  9. References
  10. Supporting Information

Self-deprived Treg cells are not functional

To assess the role of interactions with self in the suppressive capacities of peripheral Treg cells, we first studied mouse bone-marrow (BM) chimeras in which MHC class II expression was restricted to the thymus (Fig. 1A). When MHC class II expressing BM cells were injected, the resulting chimeras (WT-CD3KO chimeras) displayed a pattern of MHC class II molecule expression similar to that of unmanipulated wild-type (WT) mice. In chimeras generated by injecting BM cells that did not express MHC class II molecules into recipient mice (MHC IIΔ/Δ-CD3KO chimeras), MHC class II molecule expression was mostly restricted to radioresistant thymic epithelial cells [[16]]. In these chimeras, CD4+ T cells, notably Treg cells, were produced in the thymus even more efficiently than in WT-CD3KO chimeras, due to the lack of efficient thymic negative selection by BM-derived antigen-presenting cells (APCs) in the thymic medulla [[16]]. Four weeks after BM cell transfer, the proportion of peripheral CD4+ T cells expressing Foxp3 was more important in WT-CD3KO chimeras than in MHC IIΔ/Δ-CD3KO chimeras (26.3 ± 0.8% in WT-CD3KO chimeras versus 16.8% ± 1.0 in MHC IIΔ/Δ-CD3KO chimeras).

We compared the in vitro suppressive capacities of thymic and peripheral Treg cells from both types of chimeras. Thymic Treg cells from MHC IIΔ/Δ-CD3KO chimeras inhibited Tconv-cell proliferation to a similar extent than thymic Treg cells from WT-CD3KO chimeras or from WT mice (Fig. 1B). By contrast, peripheral Treg cells from MHC IIΔ/Δ-CD3KO chimeras failed to efficiently suppress the proliferative response of Tconv cells to anti-CD3 stimulation. Thus, in MHC IIΔ/Δ-CD3KO chimeras, Treg cells are losing their functional characteristics when migrating from the thymus (MHC II+) to the periphery (MHC II).

Then, we transferred large numbers of total T cells from the periphery of WT mice into CD3ε−/− recipient mice lacking or not MHC class II molecule expression (CD3KO-MHC IIΔ/Δ or CD3KO-MHC II+ recipient mice, respectively; Fig. 2A). Five days later, peripheral Treg cells were purified and their suppressive capacities tested in vitro (Fig. 2B–F).

As soon as 16 h after the beginning of culture, Treg cells from MHC II+ recipient mice or from WT mice inhibited the expression of late-activation markers by Tconv cells (increase in cell size and expression of CD25; Fig. 2B). By contrast, Treg cells from CD3KO-MHC IIΔ/Δ recipient mice were not able to suppress the activation of Tconv cells in response to anti-CD3 stimulation in vitro. These results were further confirmed when the suppressive capacities of Treg cells from CD3KO-MHC IIΔ/Δ and CD3KO-MHC II+ recipient mice were studied 64 h after the beginning of the coculture (Fig. 2C). Indeed, at that time point, Treg cells from CD3KO-MHC IIΔ/Δ recipient mice failed to efficiently suppress the proliferative response of Tconv cells to anti-CD3 stimulation. The lack of efficient inhibition of Tconv-cell activation and proliferation by Treg cells from CD3KO-MHC IIΔ/Δ recipient mice was found to apply for various Treg/Tconv-cell ratios (Fig. 2D). These results were found to be highly statistically significant when data from six independent experiments were pooled (Fig. 2E). In the above experiments, Treg cells were purified as CD25+ CD4+ T cells using magnetic beads (see Materials and methods and Supporting Information Fig. 1A). To exclude the possibility that contaminants in Treg cells purified from CD3KO-MHC IIΔ/Δ recipient mice (activated CD25+ Foxp3 CD4+ T cells for example) might explain our results, we repeated the suppression assay using Foxp3-GFP (where GFP is green fluorescent protein) mice and isolating Treg cells by flow cytometry sorting. Using this protocol, whatever the origin of Treg cells, purity was above 99% (Supporting Information Fig. 1B). Highly purified Treg cells from CD3KO-MHC IIΔ/Δ recipient mice were still not able to suppress the response of Tconv cells in response to anti-CD3 stimulation in vitro (Supporting Information Fig. 1C). Interestingly, whatever their origin and suppressive capacities, Treg cells exhibited a stable phenotype, still expressing Foxp3 and CD25 after 3 days of coculture (Supporting Information Fig. 1D).

We injected large numbers of T cells in order to fill the periphery and to limit lymphopenia-induced proliferation (LIP) of injected T cells. Indeed, we have previously shown that when more than 50 × 106 CD4+ T cells were transferred into T-cell deficient recipient mice, their LIP was largely reduced [[17]]. However, although limited, lymphopenia-induced Treg-cell activation and proliferation still existed to a certain extent in CD3KO-MHC II+ recipient mice and led to improvement of their suppressive capacities as shown by their enhanced capacities to control Tconv-cell proliferation when compared with Treg cells purified directly from WT mice (Fig. 2D and E, Supporting Information Fig. 1).

The nonfunctionality of Treg cells from CD3KO-MHC IIΔ/Δ recipient mice was not due to increased cell death of these cells in our culture assay. Indeed, for all Treg/Tconv-cell ratios tested, 16 h after the beginning of culture, only slight differences in the proportion of Treg cells can be observed in the culture wells whatever the origin of Treg cells (Supporting Information Fig. 2).

To test whether this loss of function was definitive or could be reversed, Treg cells from both types of recipient mice were precultured for 2 days in the presence of anti-CD3 and APC, with or without IL-2, before testing their suppressive capacities (Fig. 2F). After preactivating them in the presence or absence of IL-2, Treg cells from CD3KO-MHC IIΔ/Δ recipient mice were as efficient as Treg cells from CD3KO-MHC II+ recipient mice in suppressing the response of Tconv cells to anti-CD3 stimulation (Fig. 2F). Moreover, Treg cells from CD3KO-MHC IIΔ/Δ recipient mice did not recover their suppressive capacities when precultured for 2 days with IL-2 alone (Supporting Information Fig. 3). TCR signaling is thus necessary and sufficient to reverse the loss of suppressive function observed when Treg cells cannot interact with self.

Altogether, our data suggest that continuous interactions with self are required for maintaining the suppressive capacities of Treg cells. However, in CD3KO-MHC IIΔ/Δ recipient mice, CD4+ Tconv cells are also not receiving any TCR signals, which may result in environmental changes such as diminished in vivo IL-2 levels that may explain our data. To exclude this possibility, CD3KO-MHC IIΔ/Δ recipient mice were injected daily with 2 × 105 IU of IL-2 (Fig. 3). As described previously [[18]], WT mice injected 3 consecutive days with 2 × 105 IU of IL-2 exhibited increased proportion of Treg cells in the periphery and their Treg cells expressed significantly higher CD25 surface amounts but unchanged Foxp3 levels when compared with those of Treg cells from untreated mice (Fig. 3A). Treg cells from IL-2-treated CD3KO-MHC IIΔ/Δ recipient mice were still completely inefficient in inhibiting CD25 expression on CD4+ Tconv cells 16 h after the beginning of culture, and were only able to inhibit slightly the proliferation of CD4+ Tconv cells 64 h after the beginning of culture (Fig. 3B). In CD3KO-MHC IIΔ/Δ recipient mice, increased production of IL-2 by injected CD8+ Tconv cells may compensate for “self-depriving” CD4+ Tconv cells (Fig. 3C). Moreover, as described previously [[19]], peripheral CD4+ Treg cells from MHC IIΔ/Δ mice (that are presumably selected on MHC class I molecules) were fully suppressive in vitro in spite of the virtual absence of CD4+ Tconv cells in these mice (Fig. 3D). Thus, in contrast with self-deprived Treg cells, CD4+ Tconv-cell deprived Treg cells are functional.

image

Figure 3. Decreased IL-2 production by Tconv cells does not account for self-deprived Treg-cell loss of suppressive function. (A) WT mice were treated 3 days with daily i.p. injections of 200,000 IU of recombinant human IL-2. Twenty-four h after the last injection, peripheral cells of treated and control mice were recovered and stained for CD4, CD8α, CD25, and Foxp3 expression. The proportion of Foxp3-expressing cells among CD4+ CD8α cells was estimated. Foxp3 and CD25 fluorescence histograms of peripheral CD4+ CD8α Foxp3+ from IL-2-treated (IL-2+) or -untreated (IL-2 –) WT mice are shown. Each symbol represents a single mouse (left). The histograms shown in the right part were generated from the data for one mouse, but are representative of six mice from two individual experiments. (B) CD3KO-MHC IIΔ/Δ recipient mice were daily injected or not with IL-2. Treg cells were purified from the periphery of CD3KO-MHC II+, CD3KO-MHC IIΔ/Δ, and IL-2-treated CD3KO-MHC IIΔ/Δ recipient mice. Tconv cells were purified from lymph nodes of WT mice, labeled with CFSE, and cultured alone or together with the indicated Treg cells at various Treg/Tconv-cell ratios, in the presence of soluble anti-CD3 and APCs. Data are shown as inhibition indexes for various Treg/Tconv-cell ratios (2, 1, 1/2, 1/4, 1/8) and are representative of two independent experiments. (C) Peripheral cells from CD3KO-MHC II+, CD3KO-MHC IIΔ/Δ recipient mice and WT mice were cultured for 2 h in the presence of phorbol myristate acetate(PMA), ionomycin, and brefeldin A. They were then stained for the surface expression of CD25, CD4, and CD8α, and finally for intracellular IL-2. Representative FSC/IL-2 fluorescence dot-plots are shown. Percentages of IL-2-producing cells among CD4 CD8α+ T cells are expressed as means ± SEM values of six mice for two independent experiments. **p < 0.01, ***p < 0.001, Student's unpaired t-test. (D) Treg cells were purified from the periphery of MHC IIΔ/Δ and WT mice. Tconv cells were purified from lymph nodes of WT mice, labeled with CFSE, and cultured alone or together with the indicated Treg cells in the presence of soluble anti-CD3 and APCs. Data are shown as inhibition indexes for various Treg/Tconv-cell ratios (2, 1, 1/2, 1/4, 1/8) and are representative of two independent experiments.

Download figure to PowerPoint

Finally, we tested whether self-deprived Treg cells were also lacking suppressive capacities in vivo. CD4+ Tconv cells (CD45.1) were injected alone or together with Treg cells (CD45.2) from CD3KO-MHC II+, CD3KO-MHC IIΔ/Δ recipient mice or WT mice at a 1/1 ratio (Fig. 4A). Two weeks later, absolute numbers of CD4+ T cells recovered from the periphery of injected mice were estimated. As previously described [[20]], with such a ratio, control Treg cells significantly inhibited the initial expansion of CD4+ Tconv cells (Fig. 4B). In lines with our in vitro data, Treg cells from CD3KO-MHC II+ recipient mice were more efficient than Treg cells purified directly from WT mice in inhibiting Tconv-cell proliferation in vivo. Interestingly, no significant difference was observed whether CD4+ Tconv cells were injected alone or together with Treg cells from CD3KO-MHC IIΔ/Δ recipient mice (Fig. 4B). This loss of function did not correlate with decreased numbers of Treg cells (Fig. 4C). Thus, self-deprived Treg cells are not functional both in vitro and in vivo.

image

Figure 4. Self-deprived Treg cells are not functional in vivo. Purified lymph node CD4+ T cells from C57BL/6 CD45.1 mice were stained for CD44 and CD25 expression and naive CD4+ Tconv cells were sorted by flow cytometry. Naive CD4+ Tconv cells (CD45.1) were injected alone or together with Treg cells (CD45.2) from CD3KO-MHC II+, CD3KO-MHC IIΔ/Δ recipient mice or WT mice. Two weeks later, secondary recipient mice were sacrificed and peripheral cells were stained for CD4, CD8α, T-cell receptor(TCR)-β, CD45.1, CD45.2, and Foxp3 expression. (A) Diagram illustrating the experimental model. (B) The absolute numbers of CD45.1+ CD4+ T cells in the periphery (pooled lymph node and spleen cells) were estimated. (C) The absolute numbers of CD45.2+ Foxp3+ CD4+ T cells in the periphery (pooled lymph node and spleen cells) were estimated. (B, C) Data are expressed as means ± SEM of n = 10 mice per group pooled from two individual experiments. *p < 0.05, **p < 0.01, ***p < 0.001, Student's unpaired t-test.

Download figure to PowerPoint

Increased response to TCR stimulation of Treg cells deprived of self-recognition in vivo

Treg cells require antigen stimulation via their TCR to exert their suppressive function in vitro [[21, 22]]. Several studies have demonstrated that TCR contacts with self amplify naive T-cell responsiveness to foreign antigens [[23]]. Thus, Treg cells recovered from CD3KO-MHC IIΔ/Δ recipient mice may be less responsive to anti-CD3 stimulation than Treg cells from WT or CD3KO-MHC II+ recipient mice and this may explain their severely impaired suppressive capacities in vitro. To test this hypothesis, we compared the response of Treg cells from CD3KO-MHC II+, CD3KO-MHC IIΔ/Δ recipient mice or WT mice to anti-CD3 stimulation (Fig. 5). As already described [[24]], we found that, after TCR ligation, Treg cells mobilized intracellular calcium stores less efficiently than Tconv cells (Fig. 5A). Surprisingly, Treg cells from CD3KO-MHC IIΔ/Δ recipient mice displayed markedly increased TCR-induced calcium flux in comparison with Treg cells from CD3KO-MHC II+ recipient mice or WT mice (Fig. 5A and B). Accordingly, a higher proportion of Treg cells from CD3KO-MHC IIΔ/Δ recipient mice upregulated CD69 in response to anti-CD3 stimulation and resulting CD69+ cells expressed higher surface amounts of this activation marker when compared with that of Treg cells from CD3KO-MHC II+ recipient mice or WT mice (Fig. 5C and D). Such an increased expression of CD69 by self-deprived Treg cells in response to stimulation was also observed when Treg cells were cocultured with Tconv cells (at a 1/1 ratio, after 16 h of culture, % CD69+ cells among Treg cells = 43 ± 10 for CD3KO-MHC II+ recipient mice, 79 ± 6 for CD3KO-MHC IIΔ/Δ recipient mice, and 61 ± 8 for WT mice).

image

Figure 5. Efficient response of self-deprived Treg cells to TCR stimulation. (A) Example of calcium (Ca) mobilization after anti-CD3 stimulation (+150 s, arrow) in CD4+ CD25+ Treg cells and Tconv cells from WT mice. (B) Example of the average Ca response measured in Treg cells from CD3KO-MHC II+ or CD3KO-MHC IIΔ/Δ recipient mice. (C) Purified Treg cells and Tconv cells were stimulated in the presence of soluble anti-CD3 and APCs. CD69 fluorescence histograms are shown 16 h after the beginning of culture. Histograms in the absence of anti-CD3 stimulation are shown as controls (filled histograms). (D) The percentage of CD69+ cells among Treg cells or Tconv cells was estimated after 16 h of culture. (E) Purified CFSE-labeled Treg cells and Tconv cells were stimulated in the presence of soluble anti-CD3 and APCs. After 48 h of culture, cells were stained for CD4, CD25, and Foxp3 expression, and the CFSE dilution induced by stimulation was estimated. The average number of cell divisions for each subset was then calculated. For Treg cells, analysis was restricted to Foxp3-expressing cells. (F) CFSE histograms are shown 48 h after the beginning of culture (For Treg cells, analysis was restricted to Foxp3-expressing cells). Histograms in the absence of anti-CD3 stimulation are shown as controls (filled histograms). (A–C, F) The histograms shown were generated from the data for one mouse, but are representative of mice from three individual experiments. (D, E) Results are expressed as means ± SEM values of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, Student's unpaired t-test.

Download figure to PowerPoint

Finally, 2 days after the beginning of culture, Treg cells from CD3KO-MHC IIΔ/Δ recipient mice (analysis was restricted to Foxp3-expressing cells) proliferated to a similar extent than Tconv cells (Fig. 5E and F). Although responding to anti-CD3 stimulation more efficiently than did Treg cells from WT mice, Treg cells from CD3KO-MHC II+ recipient mice cycled significantly less than self-deprived Treg cells (Fig. 5E).

Thus, in contrast with naive T cells, self-deprivation did not lead to a defect in Treg-cell ability to be activated by TCR signals. On the contrary, their response was found to be augmented and comparable with the response of Tconv cells in terms of calcium mobilization, CD69 upregulation and proliferation.

Self-deprivation alters the phenotype of Treg cells and their ability to produce IL-10

We compared the phenotype of self-deprived Treg cells with the phenotype of Treg cells from CD3KO-MHC II+ recipient mice or WT mice. Numerous molecules are differentially expressed by Treg cells and Tconv cells (Fig. 6A, Supporting Information Fig. 4A). Expression of several of them including CD39, CD103, glucocorticoid-induced tumor necrosis factor receptor (GITR), and CTLA-4 was found to be unaffected by self-deprivation (Supporting Information Fig. 4B). In contrast, self-deprived Treg cells overexpressed PDL1 and CD25 when compared with Treg cells from CD3KO-MHC II+ recipient mice or WT mice. CD25 upregulation was even more pronounced 10 days after transfer (Fig. 6B). Interestingly, Treg cells from CD3KO-MHC IIΔ/Δ recipient mice exhibited lower levels of CD73 than Treg cells from CD3KO-MHC II+ recipient mice or WT mice. CD73 is an ectoenzyme that catalyzes the generation of adenosine and its activity has been associated with Treg-cell suppressive capacities in vitro [[25]]. CD73 surface amounts on self-deprived Treg cells decreased over time. Indeed, CD73 surface expression on Treg cells was nearly completely lost 10 days after transfer into MHC II deficient mice (Fig. 6B). Other molecules have been shown to be involved in the in vitro Treg-cell suppressive capacities such as galectin-1, IL-35 (EBI3 + IL-12p35), transforming growth factor (TGF)-β, and granzyme B [[25]]. Expression of these molecules was not modified (or augmented) by self-deprivation (Supporting Information Fig. 4C).

image

Figure 6. Altered phenotype and cytokine production of self-deprived Treg cells. (A) Foxp3, CD25, CD73, and PDL1 fluorescence histograms of peripheral CD4+ CD8α Foxp3+ (Treg cells; solid line histogram) and CD4+ CD8α Foxp3 (Tconv cells; filled histogram) cells are shown for WT mice. (B) The expression of these markers is shown for peripheral Treg cells from CD3KO-MHC II+ (dotted line histogram) and CD3KO-MHC IIΔ/Δ (bold line histogram) recipient mice 5 and 10 days after transfer as well as from WT mice (filled histogram). The histograms shown were generated from the data for one mouse, but are representative of three individual experiments with three mice per group. (C) Peripheral cells were cultured for 2 h in the presence of PMA, ionomycin, and brefeldin A. They were then stained for the surface expression of CD25, CD4, and CD8α, and finally for intracellular IL-10. Percentages of cytokine-producing cells among Treg cells (CD25+) are shown for CD3KO-MHC II+ and CD3KO-MHC IIΔ/Δ recipient mice 5 days after transfer, and WT mice. Data obtained with Tconv cells (CD25) purified from the periphery of WT mice are also shown. Data are mean ± SEM values of nine mice pooled from three independent experiments. (D) The transcript levels of IL-10 were analyzed by qRT-PCR in the indicated CD4+ T-cell subsets (5 days after transfer for recipient mice) and are shown as mean ± SEM values of relative expression. *p < 0.05, **p < 0.01, ***p < 0.001, Student's unpaired t-test.

Download figure to PowerPoint

It has been shown that decreased Foxp3 expression in the periphery causes defective suppressive function of Treg cells and their conversion into effector cells, which contribute to, rather than inhibit, autoimmune diseases [[26, 27]]. Here, we found that Foxp3 expression was not affected by self-deprivation. Indeed, Treg cells from CD3KO-MHC II+, CD3KO-MHC IIΔ/Δ recipient mice as well as from WT mice expressed similar amounts of Foxp3 (Fig. 6B). Thus, self-deprivation leads to multiple alterations in the phenotype of Treg cells with up- or downregulation of key suppressor molecules without affecting Foxp3 expression.

Then, we assessed the ability of Treg cells from CD3KO-MHC II+ and CD3KO-MHC IIΔ/Δ recipient mice to produce the anti-inflammatory cytokine, IL-10 (Fig. 6C). Interestingly, Treg cells from CD3KO-MHC IIΔ/Δ recipient mice produced less IL-10 than Treg cells from CD3KO-MHC II+ recipient mice or WT mice. Similar differences were observed at the mRNA level (Fig. 6D). Thus, lack of self-recognition events alters both the phenotype and cytokine production of Treg cells.

Self-deprivation alters Treg-cell transcriptional signature

To further compare self-deprived Treg cells with the fully functional Treg cells from CD3KO-MHC II competent mice, we obtained Affymetrix gene expression profiles from CD4+ CD25+ TCR+ cells directly isolated from the periphery of CD3KO-MHC II+ or CD3KO-MHC IIΔ/Δ recipient mice by flow cytometry sorting (Fig. 7). A total of 563 Affymetrix targets (representing 547 genes) were significantly differentially expressed (at a 1.5-fold cutoff) between the two types of Treg cells (263 overexpressed and 300 underexpressed in Treg cells from CD3KO-MHC IIΔ/Δ recipient mice, respectively, Supporting Information Fig. 5A).

image

Figure 7. Gene expression profiling of self-deprived Treg cells. Treg cells were isolated by fluorescence-activated cell sorter(FACS) from the periphery of CD3KO-MHC II+ and CD3KO-MHC IIΔ/Δ recipient mice, 5 days after transfer. Total mRNA was isolated, amplified, biotin labeled, purified, and hybridized to Affymetrix mouse genome arrays. (A) Expression pattern of Affymetrix targets differentially expressed (± 1.5-fold change) between Treg cells from CD3KO-MHC IIΔ/Δ and CD3KO-MHC II+ recipient mice that have been identified as genes of the Treg-cell transcriptional signature by Hill et al. [[28]]. The Z-score normalized induction (red) or repression (blue) is shown for each Affymetrix target. (B) Overlapping between Foxp3-dependent genes of the Treg-cell signature and the self-deprived Treg-cell signature. (C) The transcript levels of a panel of the genes presented in (A) were analyzed by qRT-PCR in the indicated CD4+ T-cell subsets. Mean ± SEM values of relative expression are shown for indicated genes and were calculated from six mice per group pooled from two independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, Student's unpaired t-test. (D) Scatter plot of the changes induced by self-deprivation versus the correlation values to Foxp3 for genes of the common Treg-cell signature. Red dots correspond to genes significantly differentially expressed between Treg cells from CD3KO-MHC IIΔ/Δ and CD3KO-MHC II+ recipient mice.

Download figure to PowerPoint

Hill et al. have recently published a list of 603 Affymetrix targets (corresponding to 490 genes) defined as representing the peripheral Treg-cell transcriptional signature [[28]]. Comparison of our gene list with this Treg-cell signature revealed an overlap of 50 genes (14 genes downregulated in Treg cells when compared with Tconv cells; 36 upregulated; Fig. 7A and B). Interestingly, among the 14 genes defined by Hill et al. as downregulated in Treg cells, 11 (79%) were upregulated in Treg cells from CD3KO-MHC IIΔ/Δ recipient mice. Similarly, 32 of the 36 genes (89%) that are normally upregulated in Treg cells were downregulated in self-deprived Treg cells. These results were confirmed by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) (Fig. 7C, Supporting Information Fig. 5B). Indeed, all of the 11 genes normally downregulated in Treg cells when compared with Tconv cells, and found to be more transcribed in CD3KO-MHC IIΔ/Δ than in CD3KO-MHC II+ recipient mice were upregulated in self-deprived Treg cells nearly to the levels observed in Tconv cells from WT mice. Gene expression profiles were also obtained from peripheral Treg cells of MHC IIΔ/Δ-CD3KO and WT-CD3KO chimeras. Interestingly, microarray analysis of Treg cells from chimeras and adoptive transfers revealed overlapping results (Pearson's correlation: p < 0.0001; Supporting Information Fig. 5C). In particular, the expression of several genes of the Treg-cell signature (such as pde3b, atp8b4, and klrd1) was affected in both experimental models by self-deprivation. Thus, part of the Treg-cell transcriptional signature is abolished by self-deprivation.

The expression of approximately one-third of the 490 genes of the Treg-cell signature correlates with Foxp3 expression or exhibits the strong negative correlation expected for genes repressed by Foxp3 (Pearson's correlation coefficient > 0.5 or < –0.5 as calculated in [[28]]). Interestingly, the transcription of only nine of these 141 genes was affected by self-deprivation (Fig. 7B). More precisely, we did not find any clear correlation between the Treg-cell genes affected by self-deprivation and Foxp3 (Fig. 7D). Similar results were obtained in the BM chimeras experimental model (Supporting Information Fig. 5D). Accordingly, the expression of several genes of the Treg-cell signature known to be strongly correlated with Foxp3 (such as Foxp3 itself) was unaffected by self-deprivation (Fig. 7D, Supporting Information Fig. 5D). Thus, continuous interactions with self induce unique transcriptional and functional signatures in Treg cells that do not rely on Foxp3 expression.

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgments
  8. Conflict of interest
  9. References
  10. Supporting Information

In the periphery, Foxp3 expression is required to maintain Treg-cell suppressive capacities. Indeed, decreased Foxp3 expression in the periphery causes defective suppressive function of Treg cells and their conversion into effector cells, which contribute to, rather than inhibit, autoimmune diseases [[26, 27, 29]]. Recent data suggest strongly that IL-2 may play a role in the maintenance of peripheral Treg-cell suppressive capacities by promoting sustained expression of Foxp3 [[18, 30, 31]]. In the present article, we show that continuous interactions with self are required for maintaining Treg-cell suppressive function in the periphery. Indeed, peripheral Treg cells from mouse chimeras in which MHC class II expression is restricted to the thymus lack suppressive activity. Similarly, Treg cells recovered 5 days after transfer into recipient mice lacking expression of MHC class II molecules are unable to inhibit the proliferative response of Tconv cells to anti-CD3 stimulation. By contrast to IL-2, TCR-dependent stabilization of peripheral Treg-cell suppressive function does not appear to rely on Foxp3 expression. Indeed, we found that Foxp3 expression is unaffected by self-deprivation. Moreover, among the genes of the Treg-cell signature in which expression is altered by self-deprivation, very few are known to be directly or indirectly controlled by Foxp3. In many aspects, self-deprived Treg cells share more functional and phenotypic similarities with Tconv cells than non-“self-deprived” Treg cells do. Indeed, several genes of the Treg-cell signature are similarly expressed by Tconv cells and self-deprived Treg cells. Moreover, self-deprived Treg cells mobilize intracellular calcium stores and upregulate CD69 as efficiently as Tconv cells do in response to anti-CD3 stimulation. Finally, the ability of self-deprived Treg cells to produce the anti-inflammatory cytokine, IL-10, is similar to that observed for Tconv cells. Thus, continuous interactions with self induce unique transcriptional and functional signatures in Treg cells that do not rely on Foxp3 expression.

Altogether, our data suggest strongly that Foxp3 expression, although necessary, is not sufficient to maintain the suppressive function of peripheral Treg cells. Such a conclusion may seem contradictory to previous data showing that ectopic Foxp3 expression is sufficient to induce suppressive capacities in Tconv cells [[3-5]]. In two of these studies, naive T cells were stimulated with anti-CD3 and IL-2 and transduced with a retrovirus expressing Foxp3 [[3, 5]]. Infected cells were thus receiving strong TCR signals while acquiring Foxp3 expression. In the last study, Khattri et al. used Foxp3 transgenic mice [[4]]. All T cells from these mice expressed Foxp3 and showed suppressor activity in vitro. Interestingly, although T cells from Foxp3 transgenic mice expressed higher amounts of Foxp3 than WT Treg cells, they were less efficient than the latter cells to control the proliferation of naive T cells in response to anti-CD3 stimulation. Thus, suppressor activity is not strictly correlated to Foxp3 expression level. One explanation could be that most T cells from Foxp3 transgenic mice are in fact naive T cells forced to express Foxp3, and that, as all naive T cells, they have only a limited affinity for self and subsequently receive only weak TCR signals.

Interruption of Tconv-cell contact with self-peptide MHC ligands leads to a rapid decline on signaling and response sensitivity to foreign stimuli [[23]]. In the present article, we show that in contrast with Tconv cells, self-deprivation does not lead to a defect in Treg-cell ability to be activated by TCR signals. On the contrary, their response was found to be augmented in terms of calcium mobilization, CD69 upregulation and proliferation. Recent data show that Treg cells deficient for the expression or expressing inactive forms of key molecules of the TCR signaling pathway exhibit defective suppressive function in vitro [[32-36]]. Nevertheless, in these studies, it was not possible to determine precisely at which step TCR signals were important. Indeed, it is now well established that engagement of their TCR during the in vitro assay is required for allowing Treg cells to suppress the activation of responder T cells [[21, 22]]. Thus, the defective in vitro suppressive function of Treg cells with an impaired TCR signaling pathway may result either from inefficient integration of TCR signals resulting from continuous interactions with self in vivo, or from a defective response to anti-CD3 stimulation in vitro. Our experimental model allows us to discriminate between these two possibilities. Indeed, in the present article, we show that self-deprived Treg cells, although they respond well to anti-CD3 stimulation, lack suppressive function in vitro.

In the thymus, strong TCR signals lead to IL-2 sensitivity enhancement in developing thymocytes. Then, IL-2 signaling induces Foxp3 expression that, in turn, strengthens Treg-cell lineage stability [[1, 2]]. These three successive events are now well recognized to be important steps of thymic Treg-cell development. Then, Foxp3 expression is considered as sufficient to maintain natural Treg-cell suppressive function in the periphery. Recent data suggest that IL-2 is important to stabilize Foxp3 expression in peripheral Treg cells [[18, 30, 31]]. Our study places on firm ground the importance of continuous interactions with self in maintaining Treg-cell suppressive capacities in the periphery. Thus, the three actors leading to Treg-cell generation in the thymus still act in concert in the periphery to allow maintenance of their suppressive function.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgments
  8. Conflict of interest
  9. References
  10. Supporting Information

Mice

C57BL/6 mice were obtained from Harlan. C57BL/6 CD45.1 mice, C57BL/6 MHC IIΔ/Δ mice [[37, 38]], C57BL/6 CD3ε−/− mice (CD3KO [[39]]), and CD3ε/MHC IIΔ/Δ double-deficient mice (CD3KO-MHC IIΔ/Δ mice [[40]]) were maintained in our own animal facilities (Cochin Institute, Paris, France) under specific pathogen-free conditions in agreement with current European legislation on animal care, housing, and scientific experimentation. C57BL/6 Foxp3-GFP mice were obtained from Dr. B. Malissen [[34]]. All experiments were performed in compliance with French Ministry of Agriculture regulations for animal experimentation (number 75–562).

Adoptive transfer of BM cells

BM chimeras were generated as previously described [[16]].

Adoptive transfer of T cells

Peripheral cells were incubated on ice with anti-CD11b (Mac-1) and anti-CD19 (1D3) antibodies, and then with magnetic beads coupled to anti-rat immunoglobulin (Dynal Biotech). Purified T-cell subsets were usually 90–95% pure. We injected 100 × 106 purified T cells i.v. into each recipient mouse.

IL-2 treatment

When indicated, mice were treated with daily i.p. injections of 200,000 IU of recombinant human IL-2 (Proleukin, Novartis).

In vitro suppression assay

Thymic and peripheral CD4+ T cells were purified as previously described [[40]] and labeled with phycoerythrin (PE)-conjugated anti-CD25 antibodies (clone PC61, BD Biosciences). CD25+ T cells were then positively selected using magnetic-activated cell sorting (MACS) anti-PE microbeads (Miltenyi Biotech). After magnetic bead purification based on CD25, the percentage of Foxp3-expressing cells among CD4+ T cells was around 95% for WT mice, 90% for WT-CD3KO and MHC IIΔ/Δ-CD3KO chimeras, and at least 75% for CD3KO-MHC II+ and CD3KO-MHC IIΔ/Δ recipient mice. CD4+ CD25 cells purified from WT mice were labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE) (Molecular Probes). A total of 5 × 104 CD4+ CD25 CFSE-labeled cells were then cultured alone or together with various numbers of CD4+ CD25+ cells purified from WT mice, BM chimeras, CD3KO-MHC IIΔ/Δ or CD3KO-MHC II+ recipient mice, in the presence of soluble anti-CD3 antibodies (145–2C11; 0.2 μg/mL) and APCs (25 × 104 irradiated splenocytes from CD3KO-MHC IIΔ/Δ mice).

Preculture assays consisted in culture of various numbers of CD4+ CD25+ cells for 2 days in the presence of soluble anti-CD3 antibodies (0.2 μg/mL), 25 × 104 APC (irradiated splenocytes from CD3KO-MHC IIΔ/Δ mice), and in the presence or absence of human recombinant IL-2. Culture medium was then washed away and 5 × 104 CD4+ CD25 CFSE-labeled cells were added per well with soluble anti-CD3 antibodies and 15 × 104 APCs. In all protocols, cells were recovered, stained, and analyzed by flow cytometry, 16–64 h after the beginning of culture.

In vivo suppression assay

Purified lymph node CD4+ T cells from C57BL/6 CD45.1 mice were stained for CD44 and CD25 expression and naive CD4+ Tconv cells, flow cytometry sorted as CD44−/low CD25 cells. A total of 1.5 × 105 naive CD4+ Tconv cells (CD45.1) were injected alone or together with 1.5 × 105 Treg cells (CD45.2) from the indicated mice.

Flow cytometry

Cell surface and intracellular staining were performed as previously described [[40]].

In vitro Treg-cell activation

For calcium measurements, T cells were loaded with 0.5 μM Fura-2/AM (Molecular Probes) for 15 min at 37°C. T cells were stimulated with anti-CD3 antibody (145–2C11; 10 μg/mL). Images were acquired at 37°C every 5 s on a Nikon microscope, with a 20× objective. Cells were excited alternatively at 350 and 380 nm and emissions at 510 nm were used to measure Ca variations with Metafluor software (Molecular devices). Ca levels are represented as a 350/380 fluorescence intensity ratio normalized to the ratio at t0. For studying CD69 expression and proliferation in response to stimulation, Treg cells were cultured alone in the presence of soluble anti-CD3 antibodies (0.2 μg/mL) and 25 × 104 APCs.

Microarray

Treg cells from CD3KO-MHC II+ and CD3KO-MHC IIΔ/Δ recipients as well as from WT-CD3KO and MHC IIΔ/Δ-CD3KO chimeras were enriched as described above and flow cytometry sorted as CD4+ TCRβ+ CD25+ cells. Total RNA was extracted using the RNeasy Mini kit (QIAGEN). A total of 100 ng of total RNA was reversed transcribed following the Genechip Whole transcript (WT) Sense Target labeling assay kit (Affymetrix). The cDNA obtained was then purified, fragmented, and hybridized to GeneChip® murine Gene (Affymetrix) at 45°C for 17 h. Statistical analysis was then performed with MEV software (TIGR, Rockville MD, USA). Data discussed in this publication have been deposited in the Gene Expression Omnibus at http://www.ncbi.nlm.nih.gov/geo/ (accession number GSE27153).

qRT-PCR analysis

Total RNA was isolated from flow cytometry sorted cells as described above and reverse transcribed with SuperScript™ III Reverse Transcriptase (Invitrogen) using 100 ng of Random Hexamers. Quantitative PCR analysis was performed using Power SYBR® Green PCR Master Mix (Applied Biosystems) and a real-time PCR system (ABI7300; Applied Biosystems) according to standard PCR conditions. For quantitative calculations, results were normalized to hprt expression. Primers used are listed in Supporting Information Table 1.

Calculations

The average number of cell divisions in response to anti-CD3 stimulation was calculated as follows. First, we estimated the CFSE dilution factor (f) due to stimulation: f = CFSE mean fluorescence intensity (MFI) in absence of stimulation divided by CFSE MFI in presence of stimulation. Then, as the intracellular amount of CFSE is halved during each cell cycle, the average number of cell divisions (A) was calculated with the following formula: A = LOG2(f). Inhibition indexes were calculated as follows:

Proliferation Inhibition = 100 × ([A(CD4+ Treg cells = 0) – A(CD4+ Treg cells = +))/A(CD4+ Treg cells = 0) in which A is the average number of cell divisions in response to anti-CD3 stimulation calculated as explained above.

CD25 Expression Inhibition = 100 × (%CD25+ among CD4+ Tconv cells (CD4+ Treg cells = 0) – %CD25+ among CD4+ Tconv cells (CD4+ Treg cells = +))/%CD25+ among CD4+ Tconv cells (CD4+ Treg cells = 0).

Statistics

Data are expressed as mean ± SEM, and the significance of differences between two series of results was assessed using the Student's unpaired t-test. Values of p < 0.05 were considered significant (*p < 0.05; **p < 0.01; ***p < 0.001).

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgments
  8. Conflict of interest
  9. References
  10. Supporting Information

We thank A. Trautmann, A. Le Bon, B. Martin, D. Fradin, and F. Lepault for comments on the manuscript, L. Stouvenel and M. Desousa for their invaluable help in cell sorting. Authors are indebt to B. Malissen for providing Foxp3-GFP mice. This work was supported by a grant from the “Ligue contre le Cancer” and by a grant from the “Association pour la Recherche contre le Cancer.” A. Pommier was supported by a Ph.D. fellowship from the “Association pour la Recherche sur le Cancer.”

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgments
  8. Conflict of interest
  9. References
  10. Supporting Information
  • 1
    Burchill, M. A., Yang, J., Vang, K. B., Moon, J. J., Chu, H. H., Lio, C. W., Vegoe, A. L. et al., Linked T cell receptor and cytokine signaling govern the development of the regulatory T cell repertoire. Immunity 2008. 28: 112121.
  • 2
    Lio, C. W. and Hsieh, C. S., A two-step process for thymic regulatory T cell development. Immunity 2008. 28: 100111.
  • 3
    Hori, S., Nomura, T. and Sakaguchi, S., Control of regulatory T cell development by the transcription factor Foxp3. Science 2003. 299: 10571061.
  • 4
    Khattri, R., Cox, T., Yasayko, S. A. and Ramsdell, F., An essential role for Scurfin in CD4+CD25+ T regulatory cells. Nat. Immunol. 2003. 4: 337342.
  • 5
    Fontenot, J. D., Gavin, M. A. and Rudensky, A. Y., Foxp3 programs the development and function of CD4+CD25 +regulatory T cells. Nat. Immunol. 2003. 4: 330336.
  • 6
    Bautista, J. L., Lio, C. W., Lathrop, S. K., Forbush, K., Liang, Y., Luo, J., Rudensky, A. Y. et al., Intraclonal competition limits the fate determination of regulatory T cells in the thymus. Nat. Immunol. 2009. 10: 610617.
  • 7
    Leung, M. W., Shen, S. and Lafaille, J. J., TCR-dependent differentiation of thymic Foxp3+ cells is limited to small clonal sizes. J. Exp. Med. 2009. 206: 21212130.
  • 8
    Jordan, M. S., Boesteanu, A., Reed, A. J., Petrone, A. L., Holenbeck, A. E., Lerman, M. A., Naji, A. et al., Thymic selection of CD4+CD25+ regulatory T cells induced by an agonist self-peptide. Nat. Immunol. 2001. 2: 301306.
  • 9
    Apostolou, I., Sarukhan, A., Klein, L. and von Boehmer, H., Origin of regulatory T cells with known specificity for antigen. Nat. Immunol. 2002. 3: 756763.
  • 10
    Hsieh, C. S., Liang, Y., Tyznik, A. J., Self, S. G., Liggitt, D. and Rudensky, A. Y., Recognition of the peripheral self by naturally arising CD25+ CD4+ T cell receptors. Immunity 2004. 21: 267277.
  • 11
    Hsieh, C. S., Zheng, Y., Liang, Y., Fontenot, J. D. and Rudensky, A. Y., An intersection between the self-reactive regulatory and nonregulatory T cell receptor repertoires. Nat. Immunol. 2006. 7: 401410.
  • 12
    Samy, E. T., Parker, L. A., Sharp, C. P. and Tung, K. S., Continuous control of autoimmune disease by antigen-dependent polyclonal CD4+CD25+ regulatory T cells in the regional lymph node. J. Exp. Med. 2005. 202: 771781.
  • 13
    Setiady, Y. Y., Ohno, K., Samy, E. T., Bagavant, H., Qiao, H., Sharp, C., She, J. X. et al., Physiologic self antigens rapidly capacitate autoimmune disease-specific polyclonal CD4+CD25+ regulatory T cells. Blood 2006. 107: 10561062.
  • 14
    Lathrop, S. K., Santacruz, N. A., Pham, D., Luo, J. and Hsieh, C. S., Antigen-specific peripheral shaping of the natural regulatory T cell population. J. Exp. Med. 2008. 205: 31053117.
  • 15
    Darrasse-Jeze, G., Deroubaix, S., Mouquet, H., Victora, G. D., Eisenreich, T., Yao, K. H., Masilamani, R. F. et al., Feedback control of regulatory T cell homeostasis by dendritic cells in vivo. J. Exp. Med. 2009. 206: 18531862.
  • 16
    Poitrasson-Riviere, M., Bienvenu, B., Le Campion, A., Becourt, C., Martin, B. and Lucas, B., Regulatory CD4+ T cells are crucial for preventing CD8+ T cell-mediated autoimmunity. J. Immunol. 2008. 180: 72947304.
  • 17
    Martin, B., Becourt, C., Bienvenu, B. and Lucas, B., Self-recognition is crucial for maintaining the peripheral CD4+ T-cell pool in a nonlymphopenic environment. Blood 2006. 108: 270277.
  • 18
    Grinberg-Bleyer, Y., Baeyens, A., You, S., Elhage, R., Fourcade, G., Gregoire, S., Cagnard, N. et al., IL-2 reverses established type 1 diabetes in NOD mice by a local effect on pancreatic regulatory T cells. J. Exp. Med. 2010. 207: 18711878.
  • 19
    Bochtler, P., Wahl, C., Schirmbeck, R. and Reimann, J., Functional adaptive CD4 Foxp3 T cells develop in MHC class II-deficient mice. J. Immunol. 2006. 177: 83078314.
  • 20
    Annacker, O., Pimenta-Araujo, R., Burlen-Defranoux, O., Barbosa, T. C., Cumano, A. and Bandeira, A., CD25 +CD4+ T cells regulate the expansion of peripheral CD4 T cells through the production of IL-10. J. Immunol. 2001. 166: 30083018.
  • 21
    Takahashi, T., Kuniyasu, Y., Toda, M., Sakaguchi, N., Itoh, M., Iwata, M., Shimizu, J. et al., Immunologic self-tolerance maintained by CD25+CD4+ naturally anergic and suppressive T cells: induction of autoimmune disease by breaking their anergic/suppressive state. Int. Immunol. 1998. 10: 19691980.
  • 22
    Thornton, A. M. and Shevach, E. M., CD4+CD25+ immunoregulatory T cells suppress polyclonal T cell activation in vitro by inhibiting interleukin 2 production. J. Exp. Med. 1998. 188: 287296.
  • 23
    Garbi, N., Hammerling, G. J., Probst, H. C. and van den Broek, M., Tonic T cell signalling and T cell tolerance as opposite effects of self-recognition on dendritic cells. Curr. Opin. Immunol. 2010. 22: 601608.
  • 24
    Gavin, M. A., Clarke, S. R., Negrou, E., Gallegos, A. and Rudensky, A., Homeostasis and anergy of CD4(+)CD25(+) suppressor T cells in vivo. Nat. Immunol. 2002. 3: 3341.
  • 25
    Tang, Q. and Bluestone, J. A., The Foxp3+ regulatory T cell: a jack of all trades, master of regulation. Nat. Immunol. 2008. 9: 239244.
  • 26
    Wan, Y. Y. and Flavell, R. A., Regulatory T-cell functions are subverted and converted owing to attenuated Foxp3 expression. Nature 2007. 445: 766770.
  • 27
    Zhou, X., Bailey-Bucktrout, S. L., Jeker, L. T., Penaranda, C., Martinez-Llordella, M., Ashby, M., Nakayama, M. et al., Instability of the transcription factor Foxp3 leads to the generation of pathogenic memory T cells in vivo. Nat. Immunol. 2009. 10: 10001007.
  • 28
    Hill, J. A., Feuerer, M., Tash, K., Haxhinasto, S., Perez, J., Melamed, R., Mathis, D. et al., Foxp3 transcription-factor-dependent and -independent regulation of the regulatory T cell transcriptional signature. Immunity 2007. 27: 786800.
  • 29
    Komatsu, N., Mariotti-Ferrandiz, M. E., Wang, Y., Malissen, B., Waldmann, H. and Hori, S., Heterogeneity of natural Foxp3+ T cells: a committed regulatory T-cell lineage and an uncommitted minor population retaining plasticity. Proc. Natl. Acad. Sci. U S A 2009. 106: 19031908.
  • 30
    Yu, A., Zhu, L., Altman, N. H. and Malek, T. R., A low interleukin-2 receptor signaling threshold supports the development and homeostasis of T regulatory cells. Immunity 2009. 30: 204217.
  • 31
    Duarte, J. H., Zelenay, S., Bergman, M. L., Martins, A. C. and Demengeot, J., Natural Treg cells spontaneously differentiate into pathogenic helper cells in lymphopenic conditions. Eur. J. Immunol. 2009. 39: 948955.
  • 32
    Kim, J. K., Klinger, M., Benjamin, J., Xiao, Y., Erle, D. J., Littman, D. R. and Killeen, N., Impact of the TCR signal on regulatory T cell homeostasis, function, and trafficking. PLoS One 2009. 4: e6580.
  • 33
    Tanaka, S., Maeda, S., Hashimoto, M., Fujimori, C., Ito, Y., Teradaira, S., Hirota, K. et al., Graded attenuation of TCR signaling elicits distinct autoimmune diseases by altering thymic T cell selection and regulatory T cell function. J. Immunol. 2010. 185: 22952305.
  • 34
    Wang, Y., Kissenpfennig, A., Mingueneau, M., Richelme, S., Perrin, P., Chevrier, S., Genton, C. et al., Th2 lymphoproliferative disorder of LatY136F mutant mice unfolds independently of TCR-MHC engagement and is insensitive to the action of Foxp3+ regulatory T cells. J. Immunol. 2008. 180: 15651575.
  • 35
    Patton, D. T., Garden, O. A., Pearce, W. P., Clough, L. E., Monk, C. R., Leung, E., Rowan, W. C. et al., Cutting edge: the phosphoinositide 3-kinase p110 delta is critical for the function of CD4+CD25+Foxp3+ regulatory T cells. J. Immunol. 2006. 177: 65986602.
  • 36
    Fu, G., Chen, Y., Yu, M., Podd, A., Schuman, J., He, Y., Di, L. et al., Phospholipase C{gamma}1 is essential for T cell development, activation, and tolerance. J. Exp. Med. 2010. 207: 309318.
  • 37
    Madsen, L., Labrecque, N., Engberg, J., Dierich, A., Svejgaard, A., Benoist, C., Mathis, D. et al., Mice lacking all conventional MHC class II genes. Proc. Natl. Acad. Sci. U S A 1999. 96: 1033810343.
  • 38
    Martin, B., Bourgeois, C., Dautigny, N. and Lucas, B., On the role of MHC class II molecules in the survival and lymphopenia-induced proliferation of peripheral CD4+ T cells. Proc. Natl. Acad. Sci. USA 2003. 100: 60216026.
  • 39
    Malissen, M., Gillet, A., Ardouin, L., Bouvier, G., Trucy, J., Ferrier, P., Vivier, E. et al., Altered T cell development in mice with a targeted mutation of the CD3-e gene. EMBO J. 1995. 14: 46414650.
  • 40
    Le Campion, A., Gagnerault, M. C., Auffray, C., Becourt, C., Poitrasson-Riviere, M., Lallemand, E., Bienvenu, B. et al., Lymphopenia-induced spontaneous T-cell proliferation as a cofactor for autoimmune disease development. Blood 2009. 114: 17841793.
Abbreviations
LIP

lymphopenia-induced proliferation

qRT-PCR

quantitative reverse transcriptase PCR

self-deprivation

lack of MHC class II expression

Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgments
  8. Conflict of interest
  9. References
  10. Supporting Information

Disclaimer: Supplementary materials have been peer-reviewed but not copyedited.

FilenameFormatSizeDescription
eji2247-sup-0001-s1.pdf978K

Figure 1. Highly purified self-deprived Treg cells are not functional in vitro.

Figure 2. Unaltered survival of self-deprived Treg cells in culture.

Figure 3. Interleukin (IL)-2 is not sufficient to restore the suppressive function of self-deprived Treg cells.

Figure 4. Phenotypic analysis of self-deprived Treg cells.

Figure 5. Self-deprived Treg cells from MHC IIΔ/Δ-CD3KO chimeras exhibit altered transcriptional signature.

Table 1. Primers

Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.