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The Journal of Immunology, 2007, 178: 3474-3482.
Copyright © 2007 by The American Association of Immunologists, Inc.

Regulatory T Cells Are Resistant to Apoptosis via TCR but Not P2X71

Simon R. J. Taylor*, Denis R. Alexander{dagger}, Joanne C. Cooper{dagger}, Christopher F. Higgins* and James I. Elliott2,*

* Medical Research Council Clinical Sciences Centre, Faculty of Medicine, Imperial College, Hammersmith Hospital Campus, Du Cane Road, London, U.K.; and {dagger} Laboratory of Lymphocyte Signalling and Development, The Babraham Institute, Babraham, Cambridge, U.K.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Regulatory T cells (Tregs) are relatively autoreactive yet, paradoxically, have been found to display normal sensitivity to thymic deletion. The relationship between self-avidity, apoptosis, and the selection of Tregs therefore remains unclear. We show that thymic Tregs develop efficiently, even at low self-avidity, and are moderately resistant to apoptosis in comparison to conventional thymocytes. Consistent with this, although conventional self-reactive T cell populations undergo chronic peripheral deletion, self-reactive Tregs are largely spared removal. Similarly, the distribution of Tregs among peripheral CD4+ cells exhibits a linear inverse relationship with CD45RB expression, indicating relative apoptosis resistance of Tregs in chronic responses to environmental Ags. We also show that appropriate controls for CD45RB levels are important for comparisons of Treg and conventional T cell activity. When thus controlled, and contrary to previous reports, Tregs exhibit normal sensitivity to cell death through TCR-independent stimuli, such as the purinergic receptor, P2X7. Finally, although absence of CD45 in gene-targeted mice results in profound T cell hyporesponsiveness, there is little or no effect on thymic Treg frequency. In summary, the data support a model in which signal strength plays little part in Treg lineage specification, though moderate resistance of self-reactive Tregs to apoptosis may result in progressive biasing of peripheral Treg TCRs toward autoreactivity in comparison to those of conventional T cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Regulatory CD4+ T cells expressing the forkhead transcription factor FoxP3 are crucial for self-tolerance (1, 2, 3, 4, 5) while themselves being disproportionately self-reactive (6, 7). Although there appears to be an overlap between regulatory T cells (Treg)3 and conventional repertoires (6, 7, 8, 9, 10), the means by which distinct but overlapping specificities of regulatory and conventional T cells develop within the thymus is unclear. According to the one hypothesis, thymocytes are directed into the Treg lineage by interaction with MHC class II with avidity just below that required for deletion. In favor of this, Tregs develop in TCR-transgenic mice which are MLR (RAG) sufficient, but not RAG deficient, other than in the presence of agonist ligand (11). By contrast, others have argued that the overrepresentation of Tregs in the presence of agonist ligands may reflect a relative resistance to apoptosis (12). According to this hypothesis, an apparent requirement for endogenous TCR chains or cognate peptide for Treg development reflects bias from the use of conventional T cell-derived TCRs in transgenic mice and their abnormal thymic architecture, perhaps disrupting niches required for Treg development. Most reports, however, have found Tregs to display normal sensitivity to thymic deletion (7, 9). Thus, paradoxically, although Tregs appear to be relatively autoreactive they also appear to exhibit normal susceptibility to deletion by self-Ags. Additionally, although it would be expected that if Tregs preferentially use only a subset of TCRs (those with relatively high self-avidity), they should express a relatively restricted TCR repertoire, this does not appear to be the case. Indeed, recent reports indicate that TCR diversity of the Treg population is equivalent to or greater than that of conventional T cells (7, 10, 13). The role of self-avidity as a biasing factor in conventional/regulatory T cell lineage determination therefore remains unclear.

Although many early studies used CD25 as a Treg marker, the availability of Abs to FoxP3 allows more accurate identification. We therefore studied the influence of self-avidity on T cell selection into the FoxP3+ Treg lineage. To study the effect of relatively high self-avidity on Treg selection, we took advantage of the fact that mouse mammary tumor virus superantigens (Mtv sags) are encoded in the murine genome and provide a useful means of following T cell selection in unmanipulated animals in a TCRV beta-specific manner (14, 15, 16). In the presence of H2-E, the vast majority of Mtv sag-reactive cells are deleted. However, as H2-A appears to present sags relatively poorly, thymic deletion of Mtv sag-reactive CD4+ T cells is less pronounced in mice lacking H2-E, with many escaping into the periphery, where they are chronically removed over several weeks or months (17). Such CD4+ H2-A/Mtv sag-reactive T cells represent a population selected relatively close to the border with negative selection. Therefore, if Tregs are selected by interaction with MHC/Ag with avidity near the borderline with deletion and/or are resistant to deletion, thymic Mtv sag-reactive populations should be markedly enriched for FoxP3+ cells. Similarly, if Tregs are relatively resistant to deletion, any enrichment for FoxP3+ cells in thymic Mtv sag-reactive populations should be accentuated in the periphery of mice lacking H2-E.

To complement the study of relatively autoreactive Tregs, we also studied the selection of CD4+V{alpha}3.2+ cells into the FoxP3+ lineage as these cells interact poorly with H2-A (18) and are selected with avidity relatively close to the border between positive selection and neglect. Hence, if selection into the Treg repertoire is favored by moderately strong interaction with MHC, few CD4+ V{alpha}3.2+ cells should express FoxP3.

Our analysis supports a model in which positive selection of conventional and Treg thymocytes occurs with similar efficiency, even at low avidity for self-MHC. However, moderate resistance to TCR-stimulated apoptosis of both the more self-reactive thymocytes and peripheral lymphocytes skews the repertoire toward self-reactivity. Consistent with this hypothesis, we show that successive rounds of in vivo T cell proliferation and cell death in response to environmental Ags (as indicated by decreasing cell surface expression of CD45RB (19)) are associated with a linear increase in the proportion of Tregs, consistent with relative apoptosis resistance. One consequence of the latter observation is that to properly compare Treg and conventional T cell activities, it is important to control for cell surface expression of CD45RB. For example, it has recently been suggested that a heightened sensitivity of Tregs to cell death mediated via the purinergic receptor, P2X7, underlies autoimmune phenomena following infection (20). However, we show that there is no difference in the susceptibility to P2X7-induced cell death of Tregs (which are predominantly CD45RBlow) and conventional cells when CD45RB expression is adequately controlled, though both populations are more sensitive than CD45RBhigh (conventional) cells.

Finally, we show that Treg development is similar in parental and CD45-deficient thymocytes, despite the marked hyporesponsiveness of the latter to TCR-mediated signals. Thus, we find that signal strength plays only a limited role in thymic Treg development, dependent on moderate resistance to TCR-mediated apoptosis of the Treg population. Given the probability that many intermediate affinity self-reactive T cells escape thymic deletion (17, 21), the bias of self-reactive TCRs toward the Treg population may reflect the relative resistance of peripheral autoreactive Tregs to apoptosis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

C57BL/10 and C57BL/6 mice were purchased from Harlan Sprague Dawley. Mice were between 6 and 12 wk of age and maintained under barrier conditions in the Biological Services Unit at Imperial College, Hammersmith Campus. Mice lacking CD45 have been described elsewhere (22) and were compared with age-matched nontransgenic mice from the same breeding program at the Babraham Institute (Babraham, Cambridge, U.K.). Mice lacking P2X7 have been described elsewhere (23) and were compared with age-matched nontransgenic C57BL/6 mice. All home office and local ethical guidelines for the care of laboratory animals were followed.

Flow cytometry

Cells in DMEM (Sigma-Aldrich) were stained with CD4APC, CD4CYCHROME, CD4PE, CD4FITC CD8CYCHROME, CD4PERCP, CD25PE, CD45RBBIO, CD45RBPE, CD45RBFITC, CD62LFITC V{alpha}2PE, V{alpha}3.2PE, Vbeta3PE, Vbeta5PE, Vbeta8.1/2PE, Vbeta10FITC, Vbeta11FITC, and Vbeta12FITC Abs (BD Biosciences), as indicated. Cells were stained with CD45RBBIO, washed, and labeled with streptavidinPERCP (BD Biosciences). Cells were then stained with anti-FoxP3FITC or anti-FoxP3APC Abs in accordance with the manufacturer’s instructions (Insight Biotechnology). Forward light scatter (FSC) was used as a measure of the volume of spherical cells (24), its sensitivity being greatest when light is collected over an angle of <10 degrees (25) as in the FACSCalibur. Data were acquired on a FACSCalibur machine and analyzed using CellQuest (BD Biosciences).

P2X7 receptor and calcium ionophore stimulation

Cells were stained with CD4PERCP, CD45RBFITC, and CD25PE, equilibrated with annexin VCY5 (AV; BD Biosciences) to assess cell surface phosphatidylserine (PS) exposure, and analyzed by flow cytometry on a FACSCalibur machine using CellQuest (BD Biosciences) or FlowJo (Tree Star) software. Baseline fluorescence was established for ~1 min before addition of 2',3'-O-(4-benzoylbenzoyl)-adenosine 5'-triphosphate (BzATP; Sigma-Aldrich) or 2.5 µM calcium ionophore (calcimycin, A23187; Sigma-Aldrich). Cells were monitored for PS exposure continuously in real time.

All results are representative of at least three independent experiments.

Statistics

Significance was assessed by Student’s t test or ANOVA.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
FoxP3+ cells are overrepresented in Mtv sag-reactive and V{alpha}3.2+ populations in the periphery of B10 mice

Because FoxP3 is a nuclear Ag, we first verified that the anti-FoxP3 Ab distinguishes distinct populations (Fig. 1A). As expected, background staining was negligible and Tregs were restricted to the CD45RBlow population (Fig. 1B). To compare the TCR repertoire of Tregs with that of conventional T cells, lymphocytes were stained with anti-FoxP3 and anti-TCR Vbeta or V{alpha} Abs. To internally control experiments, relative usage of TCRs within the Treg population was calculated as the percentage of cells bearing a given Vbeta or V{alpha} chain expressing FoxP3 divided by that percentage of FoxP3+ cells in the remaining CD4+ cells in the same tube (thus in Fig. 1C; 45.0% of CD4+Vbeta5+ cells and 16.5% of CD4+Vbeta5– cells express FoxP3; ratio = 45/16.5 = 2.7).


Figure 1
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  		FIGURE 1. FoxP3 expression in lymphocyte subpopulations. Lymph node cells from B10 mice were labeled with anti-CD4CYCHROME, anti-CD45RBPE along with either anti-FoxP3FITC (solid line) or an irrelevant FITC-conjugated control Ab (dashed line). FoxP3 labeled a discrete population of CD4+ lymphocytes (A) confined to the CD45RBlow subset (B). C, Lymph node cells from B10 mice were labeled with anti-CD4CYCHROME, anti-CD45RBPCP, anti-Vbeta5.1/2PE, and anti-FoxP3FITC. In this sample, 45% of CD4+Vbeta5.1/2+ cells and 16.5% of CD4+Vbeta5– cells expressed FoxP3 (a ratio of 2.7; see text).

 
In B10 mice (lacking H2-E), the predominant Mtv sag-reactive populations are those CD4+ cells expressing Vbeta5+, Vbeta11+, or Vbeta12+, with many such cells escaping thymic deletion, but undergoing chronic deletion in the periphery (17). The affinity of these TCRVbeta regions for Mtv sag is variable (26), and consistent with a lower avidity of Vbeta11+ cells, peripheral deletion of these cells is much less than that of Vbeta5+ and Vbeta12+ populations (17). We studied the frequency of sag-reactive cells among CD4+ peripheral lymphocytes of young adult mice. Consistent with the hypothesis that Tregs are disproportionately self-reactive in the periphery (6), FoxP3+ cells were overrepresented in the Mtv sag-reactive (Vbeta5+, Vbeta11+, or Vbeta12+) populations (Fig. 2A). To determine whether the disproportionate Treg representation among Mtv sag-reactive T cells reflects peripheral effects or thymic selection, we performed an equivalent analysis in the thymic CD4+CD8– population (Fig. 2B). FoxP3+ cells were less abundant in the thymus (5.1%) than the periphery (13.3%) (see below). Vbeta5+ and Vbeta12+ Mtv sag-reactive populations were enriched for thymic Tregs though to a lesser extent than in the periphery. Consistent with this, the weakly Mtv sag-reactive Vbeta11+ population, which in the periphery was marginally enriched with Tregs, contained only normal, or relatively low, Treg numbers in the thymus. Thus, Vbeta5+ and Vbeta12+ populations, which are partially deleted in the thymus, are overrepresented in the thymic Treg population, and extrathymic events appear to accentuate this in the periphery. Among Vbeta11+ cells (with somewhat lower Mtv sag-reactivity), enrichment for FoxP3+ cells is found in the peripheral Treg population only. Thymocytes bearing Mtv sag-unreactive TCRbeta-chains (8.1/2 and 10) appear slightly underrepresented in the Treg population, but this is likely at least in part to be a consequence of the calculations used, as overrepresentation of Tregs in some populations (with Mtv sag-reactive beta-chains) necessarily causes statistical underrepresentation of Tregs in others.


Figure 2
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  		FIGURE 2. Relative frequency of Tregs in V region-specific lymphocyte populations in B10 mice. A, Lymph node cells from B10 mice were labeled with CD4, CD45RB, and FoxP3-specific Abs in combination with TCR V region-specific Abs as indicated. The relative frequency of Tregs in CD4+ populations expressing specific TCR V regions was calculated as in Fig. 1. The percentage of V region-expressing cells in the CD4+ population was: V{alpha}2, 13.7 + 0.6; V{alpha}3.2, 0.8 + 0.04; Vbeta5, 3.8 + 0.2; Vbeta8.1/2, 12.8 + 1.0; Vbeta10, 3.7 + 0.1; Vbeta11, 2.5 + 0.2; and Vbeta12, 3.2 + 0.3. B, Thymocytes from B10 mice were labeled with CD4, CD8, and FoxP3-specific Abs along with TCR V region-specific Abs as indicated. The relative frequency of Treg cells in CD4+CD8– populations expressing specific TCR V regions was calculated as in Fig. 1. The percentage of V-region expressing cells in the CD4+SP population was: V{alpha}2, 13.0 + 0.4; V{alpha}3.2, 1.3 + 0.2; Vbeta5, 3.4 + 0.2; Vbeta8.1/2, 15.5 + 1.0; Vbeta10, 2.6 + 0.3; Vbeta11, 3.9 + 0.2; and Vbeta12, 2.1 + 0.2. Tregs are overrepresented within Vbeta5+ and 12+ Mtv sag-reactive populations in thymus and periphery, but only in the periphery within the V{alpha}3.2+ and weakly Mtv-reactive Vbeta11+ populations.

 
The relative resistance of Tregs to deletion is illustrated by the comparative fates of FoxP3+ and FoxP3– Vbeta5+ cells in the periphery (Fig. 3). Thus, while as expected, FoxP3– Vbeta5+ cells decrease slightly as a proportion of the CD4+ population due to peripheral, Mtv sag-dependent deletion (17), the total frequency of CD4+Vbeta5+ cells increases due to the increased numbers of FoxP3+ cells within the Vbeta5+ pool. Hence, FoxP3+ Vbeta5+ cells largely escape Mtv sag-mediated deletion and indeed may proliferate in the periphery.


Figure 3
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  		FIGURE 3. Tregs preferentially survive in populations declining in the periphery. A, Samples described in Fig. 2 were assessed for overall frequency of Vbeta5.1/2+ cells in the CD4+ population (left pair), or of FoxP3–Vbeta5.1/2+ cells in the CD4+FoxP3– population (right pair) in thymus (•) and lymph node cells ({circ}). Although the frequency of Vbeta5.1/2+ cells increases slightly in the periphery compared with the thymus, this expansion is entirely due to FoxP3+ cells. In the FoxP3– population (right) the use of Vbeta5.1/2 declines due to deletion through interaction with Mtv sags. B, Samples described in Fig. 2 were assessed for frequency of V{alpha}3.2+ cells in the thymus (•) and lymph node cells ({circ}). The frequency of V{alpha}3.2+ cells decreases as a proportion of the CD4+ in the periphery. C, Representative histograms of FoxP3 expression within V{alpha}3.2+ population in thymus (top) and lymph node (bottom). D, Samples described in Fig. 2 were assessed for the frequency of FoxP3+ cells within CD4+SP and CD4+V{alpha}3.2+ populations within the thymus and lymph node. Tregs are slightly underrepresented in the thymic V{alpha}3.2+ population (4.1% c.f. 5.1% FoxP3+ cells in total CD4+SP population; p < 0.01; n = 12), but overrepresented in the lymph node V{alpha}3.2+ population (16.5% c.f. 13.3% FoxP3+ cells in total CD4+ population; p < 0.01; n = 11).

 
We also studied the recruitment of V{alpha}3.2+ cells into the Treg lineage, as cells bearing this receptor interact inefficiently with H2-Ab and, therefore, are selected at the borderline with thymic neglect (18). Thus, if Treg selection requires relatively high self-avidity, few V{alpha}3.2+ cells should express FoxP3. FoxP3 is expressed by about 4.1% of thymic CD4+V{alpha}3.2+ cells, but ~16.5% of peripheral CD4+V{alpha}3.2+ cells. Hence, Tregs are slightly underrepresented in the thymic V{alpha}3.2+ population but overrepresented in the peripheral V{alpha}3.2+ population (Fig. 3). Importantly, however, the thymic FoxP3+ frequency among V{alpha}3.2+ cells is similar to those in the Vbeta8.1/2+, Vbeta10+, and V{alpha}2+ populations, none of which is poorly selected, and is even very similar to that in the Vbeta11+ population, which possesses significant Mtv sag-reactivity.

The apparent increase in the representation of FoxP3+ cells within the V{alpha}3.2+ population between thymus and periphery (4.1–16.5%; Fig. 3B) is greater than the increase in average FoxP3 representation (5.1–13.3%). However, as with Mtv sag-reactive cells, the overall usage of V{alpha}3.2 within the CD4+ population decreases between the thymus and periphery, the simplest explanation being that as the peripheral maintenance of T cells requires efficient recognition of MHC (27), V{alpha}3.2+ cells survive comparatively poorly. The large relative increase in Treg frequency within the peripheral CD4+V{alpha}3.2+ population therefore suggests that FoxP3 imparts resistance not only to apoptosis resulting from chronic stimulation, but also to that resulting from peripheral neglect.

Progressive differentiation and deletion of T cells is associated with increasing Treg frequency

Although our data indicate that peripheral Tregs are somewhat resistant to deletion, previous reports have yielded mixed results, with some studies suggesting Tregs are relatively apoptosis sensitive (20, 28, 29, 30, 31, 32, 33). To investigate this further and exclude the possibility that the response to superantigens does not reflect that to conventional Ags, we took advantage of the fact that as CD4+ populations undergo repeated cycles of stimulation, division, and death in vivo in response to environmental (or conventional self (21)) Ags they undergo a progressive loss of the differentiation marker CD45RB (19). We reasoned that if Tregs are abnormally sensitive to TCR-mediated apoptosis, their frequency should fall in response to chronic lymphocyte stimulation, and consequently FoxP3+ cells would be relatively scarce among cells expressing the lowest levels of CD45RB (these having undergone most rounds of stimulation). In striking contrast, however, Treg frequency as a proportion of the CD4+ population increases linearly with decreasing CD45RB expression (within the CD45RBlow population, Fig. 4, A–C). Moreover, decreasing expression of CD45RB is associated with a steady fall in real numbers of conventional T cells, but not (or to a much lesser extent) Tregs (Fig. 4D). However, consistent with the suggestion that decreasing expression of CD45RB on Tregs (as on conventional T cells) is progressive and associated with evidence of increased levels of prior activation, CD45RB levels on both populations are inversely correlated with the proportion of cells having shed CD62L (Fig. 4, E–G). Thus, although ~80% of CD4+ single-positive (SP) thymic Tregs express high levels of CD62L (data not shown), this decreases to 50% of CD45RBint Tregs, and progressively falls to around 10% of Tregs expressing the lowest levels of cell surface CD45RB. Hence, our data show that Treg distribution in the CD45RBlow subset is not uniform and indicates that as peripheral T cells go through successive rounds of stimulation, apoptosis of Tregs is substantially lower than that of conventional T cells.


Figure 4
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  		FIGURE 4. The frequency of FoxP3+ Tregs is inversely proportional to CD45RB expression levels within the CD45RBlow population. A, Lymph node cells from C57BL/10 mice were labeled with CD4, CD45RB, and FoxP3-specific Abs. A, A dot plot of FoxP3 expression plotted against CD45RB for cells staining positive for CD4. Five gates defining different expression levels of CD45RB are shown. Overlaid histograms of FoxP3 expression from six lymph nodes for the five gates defined in A are shown in B and the percentage of FoxP3+ cells is indicated (mean ± SD). Thirty gates similar to those in A were drawn, based on expression levels of CD45RB in one lymph node, and a histogram of the frequency of FoxP3+ cells plotted against CD45RB expression is shown in C. D, Histograms of number of conventional T cells and Tregs within the gates shown in A (mean ± SD). E, Lymph node cells from C57BL/10 mice were labeled with CD4, CD45RB, CD62L, and FoxP3-specific Abs. Panels show contour plots of CD62L vs CD45RB expression within FoxP3+ Tregs and FoxP3– conventional CD4+ T cells. A representative CD45RB expression gate used to derive a histogram of CD62L expression (F) is shown in the Treg panel. Eleven gates similar to those in E based on expression levels of CD45RB were drawn, and a histogram of the frequency of CD62Llow cells plotted against CD45RB expression is shown in G. {diamondsuit}, Conventional T cells; {diamond}, Tregs. H, A comparison of TCR V region-specific responsiveness to stimulation of T cells by P2X7. Lymph node cells were labeled with anti CD4APC and either anti-Vbeta5PE or –V{alpha}2PE. Cells were then equilibrated with AVFITC and the rate of PS exposure following stimulation of P2X7 with 150 µM BzATP at 30 s was monitored by flow cytometry. The panel compares the percentage of Vbeta5+ and Vbeta5– cells in the same tube (68.3% ± 3.9 vs 59.2% ± 2.6, mean ± SD; n = 6; p = 2 x 10–5 by t test), or V{alpha}2+ cells and V{alpha}2– cells (56.4% ± 2.6 vs 60.6% ± 2.9; n = 6; p = 0.002) in the same tube having exposed PS at 200 s.

 
Tregs show normal sensitivity to P2X7-stimulated cell death

As noted above, several studies have reported that Tregs are abnormally sensitive to apoptosis in response to various stimuli and express increased levels of mRNA for proapoptotic genes (20, 30, 31, 32). However, because CD45RBlow cells in general also appear to possess elevated mRNA for proapoptotic genes and to be relatively sensitive to apoptosis (19), apparent Treg apoptosis sensitivity may simply reflect the CD45RBlow phenotype of these cells.

To study this, we reassessed the important suggestion that CD25+ Tregs are highly sensitive to cell death stimulated through the purinergic receptor, P2X7 (20). It was suggested that such sensitivity may play a significant role in the breakdown of self-tolerance during infection. Although P2X7-stimulated cell death is caspase independent, it appears to have many features of classic apoptotic cell death, such as early exposure of PS and cell shrinkage (34, 35). As reported previously (20), CD25+ Tregs indeed showed markedly higher sensitivity to P2X7 stimulation than did conventional CD4+ cells as indicated by PS translocation. However, CD25+ Tregs and CD25– conventional cells bearing equivalent levels of CD45RB were of equal sensitivity (Fig. 5). A close relationship between the level of CD45RB and the rate of P2X7-induced PS exposure was apparent even within the CD25+ Treg population. Hence, when subdivided into CD45RBlow and CD45RBint fractions, those Tregs with lower CD45RB expression were more responsive to stimulation (Fig. 5C). This is consistent with the suggestion that progressive loss of CD45RB within the Treg population is associated with altered reactivity. As around 85–90% of CD25+ cells in unmanipulated mice in our hands express FoxP3 (data not shown), CD25 expression in this system is a good approximation to the FoxP3+ Treg subset. The data therefore show that though Tregs are indeed more sensitive than CD45RBhigh T cells to P2X7-induced cell death (20), this is a reflection of differentiation state and not Treg status per se.


Figure 5
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  		FIGURE 5. CD25+ Tregs and CD25– cells of equivalent CD45RB density are equally sensitive to P2X7 or calcium ionophore-induced cell death but more sensitive than naive T cells. Lymph node cells from C57BL/10 mice were labeled with CD4, CD25, and CD45RB-specific Abs (A) and equilibrated with AVCY5. B, Density plots of the rate of PS exposure (as indicated by AV binding) following stimulation of the P2X7 receptor (arrows) with (i) 150 µM (ii) and 75 µM BzATP by populations gated as in A; CD4+ CD25+CD45RBlow (Tregs, left), CD25– (conventional CD4+, middle), and CD4+ CD25–CD45RBlow (differentiated conventional, right). C, Density plots of the rate of PS exposure following stimulation of the P2X7 receptor (arrows) with 75 µM BzATP by CD25+ Tregs subdivided on the basis of CD45RB expression into (i) CD45RBlow and (ii) CD45RBint populations. D, Plot of cell size (as indicated by forward scatter of light (FSC units)) against time following stimulation with 150 µM BzATP by CD45RBint/low (left) and CD45RBhigh cells. The panels show overlays of the results of three consecutive experiments. In these experiments, CD45RBint/low exposed PS, but little or no response was found within the CD45RBhigh population (data not shown). Thus, PS translocation was associated with early cell shrinkage. E, Histogram of the percentage of cells with exposed PS following stimulation of cells stained as above, but treated with 2.5 µM calcium ionophore (cal, arrow) in place of BzATP. Red line, CD4+CD25+CD45RBlow Tregs; blue line, CD4+CD25–CD45RBlow differentiated conventional T cells; and green line, naive CD4+CD45RBhigh cells.

 
As Vbeta5+ cells are somewhat overrepresented in the (CD45RBlow) Treg population, and V{alpha}2+ cells slightly underrepresented, a prediction of our hypothesis is that the proportions of Vbeta5+ and V{alpha}2+ cells exhibiting sensitivity to P2X7 stimulation should be slightly above and below average, respectively. Indeed, this was borne out by a comparison of the rate of P2X7-stimulated PS exposure by TCR V region-labeled cells and controls within the same tube (Fig. 4H).

To investigate whether or not CD45RB expression levels correlate with different rates of sensitivity to apoptosis in a different model, we examined rates of response to calcium ionophore, a nonspecific stimulator of caspase-independent apoptosis (36, 37). As following P2X7 stimulation, the rates of ionophore-induced apoptosis were equivalent in CD45RBlow Treg and CD45RBlow conventional populations and both were greater than that of CD45RBhigh conventional T cells (Fig. 5E). Hence, in studies comparing the activity of murine Tregs and conventional T cells, it is important to control for differentiation state as defined by CD45RB expression.

The selection of Tregs in P2X7–/– and CD45–/– mice

Mice carrying targeted mutations in specific genes are an important tool in the analysis of the requirements for Treg selection and maintenance, and indeed it has been reported that Tregs are increased in frequency in mice lacking P2X7 (20). To ascertain whether the reported increase in Treg frequency might reflect the lack of a control for differentiation state, we compared FoxP3 staining in P2X7–/– and parental mice after normalization for CD45RB expression. However, at least in our colony, there is no increase in Treg frequencies in P2X7–/– mice either before or after normalization for the differentiation state (Fig. 6A). This result is consistent with our finding that Tregs per se are not hypersensitive to P2X7 stimulation.


Figure 6
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  		FIGURE 6. Treg frequencies in mice lacking P2X7 or CD45. A, Lymph node cells from parental mice (C57BL/6) or those lacking P2X7 were labeled with anti-CD4FITC, anti-CD45RBPE, and FoxP3APC Abs. Upper panel, A representative lymphocyte stain of CD4 and CD45RB from a parental mouse along with a gate used to compare frequencies of FoxP3 cells between mice after normalization for CD45RB levels. Lower panel, An overlay of FoxP3 staining in cells (gated as in the upper panel) from parental (solid line) and P2X7–/– (dotted line) mice. Treg frequencies within the CD4+ population were equivalent in the parental and P2X7–/– mice (n = 3) both before (11.6 ± 0.8 vs 11.5 ± 0.6%; mean ± SD) or after (20.8 ± 3.5 vs 16.2 ± 2.5%) CD45RB normalization. B, Thymocytes and mesenteric lymph node cells from mice lacking CD45(–/–) and age-matched parental controls (+/+) from the same breeding colony were labeled with anti-CD4FITC, anti-CD8PE, and FoxP3APC Abs. The mean frequency (±SD, n = 6) of FoxP3+ cells as a percentage of CD4+8– (thymus) and CD4+ (lymph node) populations is shown. *, Significant difference, p < 0.01.

 
CD45 is a transmembrane tyrosine phosphatase that positively regulates TCR signaling (38). Mice lacking CD45 possess greatly decreased numbers of single-positive thymocytes and peripheral T cells (39), and these are markedly hypo- or nonresponsive to TCR stimulation. Although expression of CD45RB in wild-type mice progressively decreases with Ag-experience in vivo, that of CD45RO, the predominant isoform in murine CD4+ cells (but for which there is no specific Ab), increases (40). We therefore studied Treg selection in CD45–/– mice for two reasons. First, the inverse correlation between CD45RB expression and Treg frequency suggested a possible causal link between CD45 levels and Treg survival dependent on altered threshold of TCR signaling. Second, if thymic Treg selection is dependent on relatively high signal strength through the TCR, Treg frequency would be expected to be greatly reduced in CD45–/– thymi. Thus, both in the thymus and periphery, CD45 might be an important regulator of Treg frequency. However, despite the reduced signaling in CD45–/– thymocytes and peripheral T cells, frequencies of Tregs were not significantly altered in CD4+CD8– thymocytes and were increased in CD45–/– lymph nodes (Fig. 6B). These data are consistent with the hypothesis that the selection of Tregs and conventional thymocytes occurs at equivalent signaling strengths. Given the results above, the comparatively high peripheral Treg frequencies in CD45–/– mice most likely reflects the relative apoptosis resistance of FoxP3+ lymphocytes in the face of the poor overall survival of CD45–/– cells. Interestingly, frequencies of thymic Tregs were significantly lower in C57BL/6 parental controls (2.2%) than in C57BL/10 mice (5%—above). Thus, while Treg frequencies are under genetic control (41), modulation of thymocyte signal strength has little effect.


   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
The rules governing the selection of Tregs are unclear, and in particular whether or not positive selection of Tregs requires TCRs conferring relatively high avidity for self-MHC at the border with negative selection. One difficulty in the study of Treg development is the fact that many studies use highly manipulated systems, typically using TCR transgenes and/or the transfer of cells into lymphopenic recipients, and the extent to which results reflect experimental artifact is unclear. We have therefore studied unmanipulated mice and asked whether TCR usage by thymic and peripheral Tregs indicates an influence of the self avidity on Treg generation.

We took advantage of the fact that the avidity of normal murine TCRs for self-MHC is heterogeneous and is Vbeta and V{alpha} chain dependent. Notably, T cell development is profoundly influenced by reactivity to endogenous Mtv sags that delete T cells in a TCR-Vbeta-specific manner (14, 15, 16) in the thymus and, in the absence of H2-E, also chronically in the periphery (17). Because surviving T cells bearing Mtv sag-reactive TCRs (in mice lacking H2-E) are selected relatively close to the borderline with negative selection, if high avidity is a prerequisite for Treg selection, these populations would be expected to be markedly enriched for FoxP3+ cells. Our data show that in the thymus of B10 mice, although some CD4+ populations bearing Mtv sag-reactive TCRbeta chains contain relatively high frequencies of Tregs, the bias is quite small and indeed expression of Vbeta11+ (which is weakly Mtv sag-reactive) did not confer any thymic Treg predisposition. In the periphery, however, all Mtv sag-reactive populations were enriched for Tregs. Since Mtv sag populations in the absence of H2-E are chronically deleted in the periphery, the simplest explanation for the relative increase in Treg frequency between thymus and periphery is that Tregs are relatively apoptosis resistant. It is also possible that some proliferation of Mtv-reactive Tregs occurs. For example, the Mtv-sag reactive Vbeta5+ population as a proportion of CD4+ cells increases slightly between thymus and periphery, but the expansion is entirely due to an increase in Vbeta5+ Tregs, with conventional Vbeta5+ T cells declining in frequency. The data therefore contrast with reports that Tregs and conventional T cells are equally sensitive to (superantigen-dependent or peptide-dependent) deletion (7, 9). The simplest explanation for this discrepancy is that the thymic resistance to apoptosis is modest and may be missed in studies in which strong deleting agonists are studied. Indeed, we have found that few Mtv sag-reactive Tregs escape into the periphery in mice expressing H2-E+, and though deletion is slightly less than that of conventional cells (data not shown), because few lymphocytes survive, differences are somewhat harder to measure. Together, our data indicate that the Treg resistance to thymic deletion is relative but not absolute. Furthermore, the skewing of autoreactive TCRs toward the Treg population progressively increases in the periphery as self-reactive conventional T cells are chronically purged from the repertoire.

In parallel with studying Treg development in Mtv sag-reactive populations, we also studied those selected at the borderline of neglect, as represented by thymocytes expressing V{alpha}3.2, which are selected with very poor efficiency into the CD4+8– population (18). Within the V{alpha}3.2+CD4+ population, Tregs were only slightly underrepresented in the thymus (and indeed at very similar levels to levels in Vbeta8.1/2+, Vbeta10+, Vbeta11+, and V{alpha}2+ populations) but were overrepresented in the periphery. As with Mtv sag-reactive cells, conventional V{alpha}3.2+ cells chronically decline in frequency in the periphery, although presumably in this instance as a result of neglect, not stimulation. Notably, the patterns of thymic selection of cells with weak avidity (V{alpha}3.2+), and at least one population with relatively strong avidity (Vbeta11+), are very similar. The data therefore indicate that MHC avidity plays little role in Treg generation, with the slight overrepresentation of Tregs in thymic Vbeta5+ and Vbeta12+ populations likely to reflect preferential survival of Tregs in the face of partial Mtv sag-mediated deletion.

A complementary explanation for the finding that the relative frequencies of Vbeta5+, 11+, and 12+ cells increase in the periphery is that although weak superantigen stimulation induces deletion of conventional T cells, it causes Tregs to proliferate. Indeed, proliferation of Tregs to signals that kill conventional T cells would clearly itself be a form of apoptosis resistance. It has been reported, for example, that CD25+ cells (largely, but not exclusively, Tregs) can proliferate in response to transgenically expressed Ag in conditions in which CD25– cells are deleted (29). Indeed, some proliferation of Tregs in response to weak endogenous superantigen is consistent with our observation that the frequency of CD4+ Vbeta5+ T cells increases slightly between the thymus and periphery, entirely due to an increase in the Vbeta5+ Treg population. However, the progressive increase in Treg frequencies we report occurs following both strong (Mtv sag-reactive) and suboptimal (V{alpha}3.2+) interactions. It appears unlikely that proliferation underlies the expansion of V{alpha}3.2+ Tregs. Hence, the most consistent explanation for our data is that Tregs are relatively resistant to deletion associated with weak (V{alpha}3.2) or strong (Mtv sag-reactive) stimulation of T cells. Although it remains possible that low-level stimulation by superantigen stimulates the conversion of come conventional cells into Tregs (42), the evidence suggests that, at least in mice, acquisition of Treg phenotype in the periphery is likely to be a rare event (1, 2, 7, 13, 43, 44). The frequency of conventional T cell to Treg conversion following transfer of cells into lymphopenic hosts (42) therefore is probably not representative of normal murine immune responses. Conventional T cell to Treg conversion also appears an unlikely explanation for the relative expansion of Tregs in the low self-avidity V{alpha}3.2+ population.

The results above indicate that peripheral Tregs are relatively resistant to superantigen-induced apoptosis. Although it is possible such Treg responses to superantigens differ from those to conventional Ags, studying the latter in vivo generally requires highly manipulated systems using cell transfer into lymphopenic hosts (that might promote conventional T cell to Treg conversion (42)). To circumvent this, we used the fact that many peripheral T cells, even in the absence of deliberate immunization, are stimulated by environmental (and perhaps conventional self (21)) Ags. Importantly, as T cells undergo chronic stimulation and consequent division in vivo, many die to maintain constant lymphocyte numbers and the remainder exhibit progressive loss of the marker CD45RB with each round of division (19). Consequently, those cells with the lowest expression of CD45RB have resisted activation-induced cell death in response to environmental Ags. Our data show that declining levels of CD45RB are associated with a linear increase in the proportion of FoxP3+ cells in the CD4+ population. This appears to reflect a greater decline in absolute numbers of conventional T cells than Tregs with falling levels of CD45RB (Fig. 4D). Although it is not possible to infer that populations matched for CD45RB expression levels have undergone the same number of cell divisions, together, the simplest explanation of our data is that Tregs are relatively resistant to apoptosis resulting from chronic stimulation by environmental Ags. Indeed, as by several criteria Tregs appear chronically activated (45, 46), relative resistance to activation-induced cell death may be necessary for continued function.

That Treg representation increases linearly with decreasing CD45RB expression has one further important implication. Hence, studies on apoptosis of peripheral Tregs have yielded inconsistent results and indeed several have suggested they are highly sensitive to apoptosis induced through TCR, CD95, or the purinergic receptor P2X7 and that they express high levels of apoptotic genes (20, 30, 31). However, as similar observations have been made of CD45RBlow cells in general (19), we hypothesized that the apparent high-apoptosis sensitivity of Tregs in some studies might reflect the fact that the activity of Tregs, which are CD45RBlow, was compared with that of conventional cells, which are predominantly CD45RBhigh. Thus, any differences might reflect the differentiation state and not Treg phenotype per se. That this is true, at least in some cases, is shown by the equivalent sensitivity to P2X7-induced and calcium ionophore-induced death of CD25highCD45RBlow (Treg) and CD25–CD45RBlow (conventional) cells, and the relatively high sensitivity of both when compared with conventional CD45RBhigh cells. Hence, the differentiation state, as evidenced by level of CD45RB, is an important factor in the design of controls for Treg activity. In contrast to a previous report (20), we do not find P2X7–/– mice possess increased Treg frequencies.

Finally, we studied Treg formation in CD45–/– mice for two reasons. First, the inverse relationship between CD45RB expression and Treg frequency indicated a possible direct role for CD45 (a positive regulator of TCR signaling (38)) in Treg development. Second, given that TCR signaling is reduced in CD45–/– thymocytes and T cells, comparing Treg frequencies with those in parental mice tests the hypothesis that TCR signal strength is a key determinant of Treg lineage specification. Our results show that although, as expected, total numbers of thymocytes and T cells were severely reduced in CD45–/– mice, the proportion of Tregs was unaffected among thymocytes, and increased among peripheral CD4+ cells. The simplest explanation of these data is that (as argued from the somewhat analogous selection of low self avidity V{alpha}3.2+ cells) strength of TCR signaling has little impact on Treg thymocyte generation, while in the periphery Treg expression is relatively resistant to neglect-induced apoptosis.

In summary, our data are consistent with a model whereby, if a window of signaling strength predisposing to Treg lineage-commitment exists, its effect appears to be small. Moderate Treg resistance to apoptosis molds the thymic T cell repertoire, leading to a slight concentration of autoreactive TCRs within the Treg population. In the periphery, chronic exposure to self and environmental Ags is associated with Ag-induced cell death of conventional T lymphocytes to which Tregs appear resistant. Consequently, the weak bias of autoreactive TCRs within the thymic Treg repertoire increases over time in the periphery. Finally, differentiation status as indicated by CD45RB expression level should be controlled for when comparing the activity of Tregs and conventional T cells. When so controlled, and contrary to previous reports (20), Tregs show no heightened sensitivity to cell death stimulated through the P2X7 receptor.


   Acknowledgments
 
We thank Drs. F. Tam (Department of Renal Medicine, Imperial College, London, U.K.), I. Chessell, and J. Hatcher (GlaxoSmithKline, Harlow, U.K.) for the use of P2X7-deficient mice, and Dr. J. Dyson for helpful discussions and the use of a panel of anti-TCR Abs.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was funded by the Medical Research Council, U.K. Back

2 Address correspondence and reprint requests to Dr. James Elliott, Membrane Transport Biology Group, Medical Research Council Clinical Sciences Centre, Du Cane Road, London, U.K. E-mail address: james.elliott{at}csc.mrc.ac.uk Back

3 Abbreviations used in this paper: Treg, regulatory T cell; Mtv sags, mouse mammary tumor virus superantigens; FSC, forward light scatter; PS, phosphatidylserine; SP, single positive. Back

Received for publication September 7, 2006. Accepted for publication January 3, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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