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Lymphocyte Population Biology Unit, Unité de Recherche Associée, Centre National de la Recherche Scientifique, Institut Pasteur, Paris, France
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Abstract |
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endows these cells with the capacity to explore the IL-2 resource, ensuring their presence while keeping their number tied to the number of CD4+ T cells that produce IL-2. We show that such a homeostatic mechanism allows the increased expansion of T cells without causing disease. The indexing of Treg cells to the number of activated IL-2-producing cells may constitute a feedback mechanism that controls T cell expansion during immune responses, thus preventing autoimmune or lymphoproliferative diseases. The present study highlights that maintenance of proportions between different lymphocyte subsets may also be critical for the immune system and are under strict homeostatic control. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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We recently demonstrated that peripheral CD4+CD25+ natural regulatory T lymphocytes play a major, suppressive role in peripheral CD4+ T cell homeostasis (5). Comprising 5–10% of the peripheral CD4+ T cell pool in humans and mice (6), this subpopulation originates in the thymus and represents a specific lineage whose development is dependent on the expression of the transcription factor scurfin encoded by the foxp3 gene (7, 8, 9). One of the most studied proprieties of these cells is their ability to prevent the development of autoimmune-like diseases that arise when naive T cells are transferred into lymphopenic mice (6, 10, 11, 12). Our group and others (5, 13) have demonstrated that cotransfer of CD4+CD25+ regulatory T (Treg)4 cells in this model modifies the size of the peripheral T cell pool according to their fraction in the transferred populations (5). Mechanisms ensuring the presence and maintaining the relative fraction of Treg cells in the peripheral T cell pool are thus vital; the homeostasis of CD4+CD25+ Treg cells controls overall CD4+ T cell homeostasis (5).
A role for IL-2 and IL-2R in the homeostasis of CD4+CD25+ Treg cells is suggested by lymphoproliferative disorders that occur in mice genetically deficient in IL-2 (14, 15), CD25 (IL-2R, the high-affinity receptor subunit) (16), or IL-2R
(the low-affinity subunit) (17, 18). Although the role for the IL-2R in CD4+CD25+ Treg cell biology is established (5, 19, 20, 21, 22, 23), the exact role of IL-2 itself is still an open question. IL-2 might be involved in thymus development, peripheral expansion, survival, and/or function of Treg cells. With respect to development, CD4+CD25+ Treg cells are greatly reduced in the thymus of IL-2–/– mice (19). However, IL-2 does not seem to be essential for the generation of Treg cells because the few peripheral CD4+CD25+ cells found in IL-2–/– mice are capable of suppression, a hallmark of Treg function (5, 21). This result suggests that IL-2 may instead be critically important for the survival or expansion of CD4+CD25+ Treg cells in the periphery (5, 20, 24).
We now report direct studies of CD4+CD25+ Treg cell homeostasis "in vivo." We show that the expression of the high-affinity IL-2R provides CD4+CD25+ Treg cells with an advantage in the exploitation of a niche defined by the IL-2 resource. We also show that the CD4+CD25+ Treg cell subpopulation is dependent on IL-2 produced by other T cells. Because the Treg cells control the expansion of CD4+ T cells, the two populations reach equilibrium in which CD4+CD25+ Treg cells make up 15–20% of the non-naive peripheral CD4+ T cell pool. Because this equilibrium is independent of the absolute size of the T cell pool, this represents a novel homeostatic mechanism that maintains the ratio of the different subpopulations stable independently of their absolute numbers. Thus, peripheral homeostatic regulation relies not only on the isolated control of subpopulations, but also on a mechanism that indexes the control of different subpopulations to each other. This mechanism of indexation, probably by acting at the later contraction phase of the immune response, may ensure that T cell expansion remains under control, thus preventing deregulation and self-aggression.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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C57BL/6.Ly5.2 mice from Iffa-Credo; B6.CD3
–/–, B6.IL-2R–/–, B6.IL-2–/–, B6.TCR–/–, B6.Rag2–/–, and C57BL/6.Ly5.1 mice from the Centre de Devélopment des Tecniques Avancées-Centre National de la Recherche Scientifique (Orléans, France); and B6.IL-2–/–IL-2R–/–, B6.Rag2–/–IL-2–/–, a gift from J. Di Santo (Institut Pasteur, Paris, France); B6.Rag2–/–IL7Tg, a gift from R. Ceredig (University of Basel, Basel, Switzerland); and B6.CD3–/–IL-2–/– bred in our own breeding facilities at the Pasteur Institute were age (6–12 wk) and sex matched.
Cell sorting and cell transfers
Lymph node (LN) cells from the Ly5.2 and Ly5.1 donor mice were enriched for CD4+ T cells using a MPC6 magnetic cell sorting (Dynal Biotech) or by auto-MACS (Miltenyi Biotec). Briefly, cells were incubated with rat Abs directed to mouse B220 (RA3-6B2), CD11b, and CD8 (53-6.7), all from BD Pharmingen, followed by sheep anti-rat Ig Dynabeads (Dynal Biotech) or goat anti-rat Ig MACS beads (Miltenyi Biotec). After selection >90% of the remaining population was CD4+. These cells labeled with combinations of anti-CD4 (L3T4/RM4-5), anti-CD45RB, anti-CD25 (7D4), and anti-CD45.1 Abs were sorted on a MoFlo cytometer (DakoCytomation). The purity of the sorted populations varied from 96 to 99.9%. Nonirradiated B6.CD3–/– hosts were injected i.v. with the purified CD4 T cell populations alone or mixed at different cell ratios. By using mice differing by Ly5 allotypes, we were able to discriminate the cells from the different donors. Host mice were sacrificed at different times after cell transfer. Spleen cell and inguinal and mesenteric LN cell suspensions were prepared and the number and phenotype of the cells from each donor population were evaluated. The total peripheral T cells showed in the results represent the number of cells recovered in the host’s spleen added to twice the number of cells recovered from the host’s inguinal and mesenteric LNs.
Bone marrow (BM) chimeras
Host 8-wk-old Rag2–/– B6 or Rag2–/–IL-2–/– B6 mice were lethally irradiated (900 rad) with a 137Ce source and received i.v. 4 x 106 T cell-depleted (<0.05%) BM cells from different donor mice, mixed at different ratios. T cell depletion was done on an auto-MACS (Miltenyi Biotec) magnetic sorter after incubating the BM cells with anti-CD3 PE Abs followed by anti-PE MACS beads. By using donor and host mice that differ according to Ly5 allotype markers, we were able to discriminate between the T cells originating from the different donors.
Flow cytometry analysis
The following mAbs were used: anti-CD45.1, anti-CD45.2, anti-CD3 (145-2C11), anti-CD4 (L3T4/RM4-5), anti-CD69 (H1.2F3), anti-CD25 (7D4), anti CD45RB, and anti-TCR (H57) from BD Pharmingen, anti-CD44 (IM781), anti-CD62L (MEL14) from Caltag Laboratories, anti-CD25 (Southern Biotechnology Associates), and anti-foxp3 (FJK 16-s; eBioscience). Four-color staining was preformed with the appropriate combinations of FITC, PE, TriColor, PerCP, biotin, and allophycocyanin-coupled Abs. Biotin-coupled Abs were secondary labeled with allophycocyanin-coupled, TriColor-coupled (Caltag Laboratories), or PerCP-coupled (BD Biosciences) streptavidin. Dead cells were excluded during analysis according to their light-scattering characteristics. All acquisitions and data were performed with a FACSCalibur (BD Biosciences) interfaced to the Macintosh CellQuest software; analysis was performed also with FloJo software.
Real-time quantitative RT-PCR
RNA was extracted from sorted populations using TRIzol (Invitrogen Life Technologies), and cDNA was prepared using standard procedures. The expression of foxp3 and hypoxanthine phosphoribosyltransferase (HPRT) was determined by quantitative real-time PCR, using an Applied Biosystems 7000 Sequence Detection System and the SYBR Green PCR Master Mix. Primers were: 5'-CTTCCCAGAGTTCTTCCACA-3' and 5'-TTGGCTCCTCTTCTTGCGAA-3' (foxp3) and 5'-GGTGGAGATGATCTCTCAAC-3' and 5'-CTGTACTGCTTAACCAGGGA-3' (HPRT). Expression was normalized to the levels of HPRT in each sample.
Statistical analysis
Sample mean results were compared using the unpaired Students’ t test. When variances were found to be statistically different, Welch’s correction was used. Samples were considered statistically different for values of p < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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We have shown that CD4+CD25+ T cells are capable of expansion after adoptive transfer into lymphopenic (CD3–/–) hosts and that some of these cells lose CD25+ expression after transfer. Although only those cells retaining CD25 expression were capable of suppressing naive CD4+CD25– T cell expansion upon secondary transfer (5), we wished to confirm that this ability correlated with a molecularly defined Treg cell identity, namely, high foxp3 mRNA expression. After 1 year in CD3–/– hosts, CD4+ T cells expressing CD25 also express high levels of foxp3 mRNA (Fig. 1A). Importantly, when CD4+CD25+ T cells were transferred into CD3–/– hosts the vast majority of CD25+ cells recovered in the hosts were foxp3+, whereas <5% of the CD25– cells retained foxp3 expression (Fig. 1B). Thus, the number of cells that retain CD25 expression after expansion closely represents the expansion of the transferred natural CD4+ Treg cells.
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CD4+CD25+ T cells occupy a specific niche in the peripheral CD4+ T cell pool
During lymphopenia-driven proliferation (LDP), the expansion of the injected cells toward a fixed steady-state regardless of the starting conditions suggests that the different populations are not simply proliferating at their own predetermined rates, but rather are expanding until available niches are filled. The ability of CD4+CD25+ T cells to repopulate their own pool and the constancy of their numbers and proportions led us to consider the possibility that these cells occupy a specific niche in the peripheral CD4+ T cell pool. To test this hypothesis, we conducted additional experiments in which the fraction of cells competent to become Treg cells, those with an advantage for the hypothesized niche, was varied in the precommitted state. Using irradiated Rag2–/– mice as hosts, we constructed BM chimeras by mixing BM from CD25–/– B6 mice and BM from wild-type (WT) B6Ly5.1 mice in different proportions. These were diluted in TCR–/– BM cells to maintain a constant fraction of competent T cell precursors (25). We obtained chimeras in which the fraction of the peripheral T cell pool competent to generate CD25+ cells, i.e., the Ly5.1+ fraction, ranged from 10 to 100% (Fig. 2A, top), yet the fraction of CD4+CD25+ T cells was remarkably constant, around 10% (Fig. 2A, bottom). We also found that the final number of CD4+CD25+ T cells generated in chimeras with a limited number of WT precursors was the same as in chimeras reconstituted with 100% WT BM cells or in nonmanipulated WT mice (data not shown).
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IL-2 is an essential resource for CD4+CD25+ foxp3+ Treg cells
We have shown in BM reconstitution experiments that IL-2–/– BM is able to generate a peripheral compartment of CD4+CD25+ T cells when in the presence of CD25–/– T cells. We have also shown that these BM chimeras were protected from autoimmune manifestations and death (5). It remained to be established whether these protective IL-2–/–CD4+CD25+ cells belong to the naturally arising foxp3+ CD25+ regulatory CD4+ T cell lineage. To verify this question, we prepared mixed BM chimeras using BM from CD25–/– and from IL-2–/– Ly5.1 donors. Eight weeks after reconstitution, we compared foxp3 mRNA expression in purified CD3+CD4+CD25+Ly5.1+ (IL-2–/–) T cells in regulatory (CD4+CD25+) and nonregulatory (CD4+CD25–CD45Rbhigh and CD4+CD25–CD45RBlow) T cells from WT and IL-2–/– mice (Fig. 3). We found that although CD25+ cells from IL-2–/– mice showed lower expression of foxp3, the IL-2–/–CD25+ cells from the mixed CD25–/– plus IL-2–/– BM chimeras expressed foxp3 levels similar to levels found in WT CD4+CD25+ T cells (Fig. 3). These results confirm that IL-2 or IL-2R signaling is required for the generation and/or maintenance of foxp3 expressing CD4+CD25+ Treg cells. They strongly suggest that IL-2 is an essential resource for the foxp3+ CD4+CD25+ Treg cells and that the expression of the high-affinity form of the IL-2R reflects their capacity to exploit the IL-2 resource.
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Although our findings demonstrated that IL-2 is an essential resource for CD4+CD25+ Treg cells, the cell type providing IL-2 remained to be identified. To study this query, we established four groups of mixed BM chimeras in Rag2–/–IL-2–/– hosts to control the source of IL-2 (Fig. 4A). In the first group, reconstituted with BM cells from IL-2–/– and TCR–/– donors, the only source of IL-2 are the non-T cells derived from the IL-2+ TCR–/– BM donors, which cannot generate T cells. In a second group reconstituted with BM cells from IL-2–/– and CD25–/– donors the CD25–/– BM-derived cells, including T cells, are genetically capable of producing IL-2 (IL-2+ T cells). In a third group, reconstituted with BM cells from IL-2–/– and CD25–/–IL-2–/– donors, no IL-2 is available because the T cells from CD25–/– origin are not able to produce IL-2. In the fourth group, no IL-2 source was available, since the host IL-2–/– mice received only BM cells from IL-2–/– donors. We found that the only group protected from death contained a T cell source of IL-2 (Fig. 4A). Moreover, 7 wk after reconstitution, only mice containing IL-2+ T cells had developed a normal proportion of CD4+CD25+ T cells (Fig. 4B). Mice receiving IL-2+ TCR–/– BM (with IL-2–/– BM) also contained a small population of CD4+CD25+ T cells, but these mice were not rescued; therefore, the levels of IL-2 produced by non-T TCR–/– BM-derived cells were not sufficient to develop a fully functional population of Treg cells. Rag2–/–IL-2–/– hosts reconstituted with only IL-2–/– BM cells also developed inflammatory bowel disease (IBD) leading to death.
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T cells represent the major source of the IL-2 required for meaningful homeostasis of CD4+CD25+ Treg cells and for fulfillment of their regulatory role. Activated-memory CD4+ T cells are key actors in the survival and expansion of CD4+CD25+ Treg cells
Our observations led us to ask which T cells provided the IL-2 necessary for tight regulation of the CD4+CD25+ T cell subset. It is generally accepted that CD4+CD25+ T cells are unable to produce IL-2 (26, 27) so we investigated whether these cells rely on IL-2 produced by naive CD4+CD25–45RBhigh or activated/memory CD4+CD25–CD45RBlow cells. For this purpose we cotransferred sorted CD4+CD25+ T cells along with freshly sorted naive cells from either WT or IL-2–/– donors into Rag2–/–IL-2–/– hosts (Fig. 5A). We found that expansion of both WT and IL-2–/– naive T cells was suppressed, suggesting that the cotransferred CD4+CD25+ cells were benefiting from some source of IL-2 perhaps from a few CD4+CD25low foxp3– T cells (28) contaminating the CD4+CD25+ population. To verify this possibility, we created IL-2–/–CD4+CD25+ cells bearing the allotype marker Ly5.1, and cotransferred them with CD4+CD25–CD45RBlow cells from WT or from IL-2–/– donors into T cell-deficient IL-2–/– hosts (Fig. 5B). We found that IL-2–/–CD4+CD25+ Ly5.1 T cells expanded significantly when cotransferred with IL-2+ CD4+ T cells, whereas when transferred alone or along with IL-2–/– CD4+ T cells they failed to expand (Fig. 5B). These findings indicate that IL-2 produced by the activated/memory CD4+ T cells strongly influenced the expansion and survival of CD4+CD25+ foxp3+ Treg cells.
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Because the number and proportion of CD4+CD25+ T cells recovered in BM chimeras or after transfer into lymphopenic hosts (Figs. 1 and 2) is relatively constant, and because both number and proportion are reduced in cotransfer experiments when IL-2 is lacking (Fig. 5, B and C) we hypothesized that the numbers of CD4+CD25+ T cells might be tied to the numbers of T cells capable of producing IL-2. To evaluate this possibility more directly, we cotransferred purified CD4+CD25+ T cells from IL-2+ donors with naive T cells from IL-2+ or IL-2–/– mice and determined the proportions of the resulting subsets. We found that the fraction and the number of CD4+CD25+ cells were higher when the naive cells were capable of secreting IL-2 (Fig. 6, A and B, black histogram). Further support for the link between the number of CD4+CD25+ cells and the number of T cells that can produce IL-2 was obtained when we correlated the fraction of CD4+CD25+ T cells to the total number of IL-2+ T cells. We found that in two different experimental conditions the fraction of CD4+CD25+ cells remained the same (Fig. 6B, dotted histograms), suggesting that the number of CD4+CD25+ T cells represents a relatively constant fraction of the number of IL-2+CD4+ T cells.
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15% of the activated CD4+CD45RBlow pool, i.e., the cells that we have shown represent the major source of IL-2. Taken together, these experiments suggest that the number of CD4+CD25+ T cells is homeostatically maintained as a steady fraction of the number of activated (IL-2-producing) CD4+ T cells. Such control of the number of CD4+CD25+ cells by IL-2-producing T cells has implications for immune health. It predicts that increases in the overall CD4+ T cell pool would be allowed without resulting in autoimmune disease as long as a steady proportion of CD4+CD25+ cells was kept. We have searched for situations in which we could increase the total CD4+ T cell number while keeping T cell autoimmunity in check. Because IL-7 promotes peripheral survival and expansion of naive CD4+ T cells (29, 30) and IL7Tg mice show increased numbers of peripheral T cells (31), we wished to probe the relationship between total CD4+ T cell number and the fraction of CD4+CD25+ cells in these mice. We transferred naive CD4+ T cells into Rag2–/– or Rag2–/–IL7Tg hosts, with or without CD4+CD25+ Treg cells. Both hosts receiving only naive cells developed visible features of IBD and wasting; disease appeared earlier and was more severe in the Rag2–/–IL7Tg hosts (data not shown). Disease was prevented in both hosts by the cotransfer of CD4+CD25+ Treg cells even though the number of cells from naive CD4+ T cell origin was 5- to 10-fold higher in Rag2–/–IL7Tg hosts than in Rag2–/– hosts and despite a similar total number of T cells as unprotected Rag2–/– hosts injected with naive CD4+ T cells only (Fig. 7A). In fact, the number of CD4+CD25+ T cells was also higher in the Rag2–/–IL7Tg hosts (Fig. 7B) resulting in the maintenance of the proportion of CD25+ cells in the CD4+ T cell pool of the protected hosts (Fig. 7C). Thus, significant increases of peripheral T cell numbers, including potentially autoreactive cells, do not necessarily induce disease if the number CD4+CD25+ Treg cells increases proportionally. Overall these experiments lead us to conclude that the peripheral homeostasis of CD4+CD25+ Treg cells is indexed to the number of activated T cells, even when the total number of CD4+ T cells is dramatically altered.
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Discussion |
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–/– (17), or in scurfy and foxp3–/– (9) mice. The reintroduction of CD4+CD25+ cells in these mice is sufficient to rescue not only the autoimmune disease, but also the normal physiological numbers and proportions of lymphocyte subpopulations (5, 9, 15, 20). We have now investigated homeostasis mechanisms affecting CD4+CD25+ Treg cells. We have previously demonstrated that CD4+CD25+ T cells expand upon transfer into lymphopenic hosts and found that although some of the progeny cells retain CD25 expression, others do not (5). We have shown that only those continuing to express CD25 retain the suppressive properties of Treg cells (5). We now show that these cells express Foxp3 and retain increased levels of foxp3 mRNA 1 year after transfer, confirming that they are indeed the Treg progeny of the transferred Treg cells and suggesting that a stable homeostasis of CD4+CD25+ foxp3+ Treg cells is achieved. These observations suggest a mechanism for controlling Treg cell numbers and led us to ask more detailed questions about the expansion capacity of CD4+CD25+ Treg cells during LDP.
We found that the number of cells expressing CD25 at equilibrium is remarkably stable and is independent of the number of cells initially transferred (from 104 to 3 x 105). During LDP their number is also independent of the presence of other populations and of the ratios at which the mixed cell populations were cotransferred. These observations suggest that CD4+CD25+ Treg cells occupy a specific niche whose dimensions are under homeostatic control. Additional experiments support this notion. Adoptive transfer of a limited number of CD25+ T cells rescues CD25–/– BM chimeras (5), neonatal Foxp3sf mice (9), and IL-2R–/– mice (20, 33), as it reconstitutes a normal CD4+CD25+ Treg pool that persists indefinitely without new thymus output. More importantly, in the current experiments, by using mixed BM chimeras we show that similar numbers and proportions of peripheral CD4+CD25+ Treg cells are recovered regardless of the fraction of precursor BM cells competent to generate CD4+CD25+ T cells (Fig. 2). The following question was to identify the resources and mechanisms determining the size of CD4+CD25+ Treg niche.
A major role for IL-2 in CD4+CD25+ Treg biology is reflected in the expression of CD25 (IL-2R) by the vast majority of CD4+ foxp3+ Treg cells (28), but the exact role has been uncertain (5, 19, 20, 21, 22, 23). IL-2 could play a role in the generation, peripheral survival, and expansion or to the effector function of CD4+CD25+ Treg cells. The presence of a small fraction of CD4+CD25+ Treg cells, which express foxp3 mRNA, in the peripheral pool of IL-2–/– mice (5, 21), shows there is no absolute IL-2 requirement for CD4+CD25+ Treg generation. So what is the role of IL-2 in CD4+CD25+ Treg cells? Our current findings argue firmly against a major role for IL-2 consumption as the mechanism of suppression by CD4+CD25+ Treg cells (34, 35, 36); we show that expansion of naive CD4 T cells does not require IL-2 or IL-2R, and importantly, that CD4+CD25+ Treg cells suppression is not mediated by IL-2 because Treg cells can suppress equally well the proliferation of naive T cells from either WT, IL-2–/–, or CD25–/– donors. Instead, our results demonstrate that IL-2 is essential for the peripheral survival and expansion of CD4+CD25+ Treg cells. First, CD4+CD25+ cells transferred into IL-2-free mice fail to engraft (Fig. 5). Second, establishment of a sizeable population of Treg cells able to rescue IL-2–/– BM chimeras is strictly dependent on IL-2 produced by other T cells. Third, IL-2–/–CD4+CD25+ T cells transferred into IL-2–/– lymphopenic hosts do not expand unless cotransferred with activated T cells from IL-2+ donors. These direct studies indicate that IL-2 is strictly required for the expansion of CD4+CD25+ Treg cells, a conclusion consistent with prior studies in which the anti-IL-2 administration shrunk the peripheral CD4+CD25+ Treg pool in WT mice (27). However, this latter study also claims that IL-2 is not required for CD4+CD25+ Treg expansion in lymphopenic hosts. This conclusion does not take into account that CD25+ T cell populations may produce their own IL-2, as we demonstrate in this study by comparing the fate of WT and IL-2–/–CD4+CD25+ T cells. Further support for the peripheral role of IL-2 comes from recent studies using foxp3gfp knock-in mice (24). The recent observation that significant numbers of CD4+ foxp3+ cells are unable to prevent fatal lymphoproliferative disease in CD25–/– mice (our unpublished observations) suggests that IL-2 and IL-2R are also required for the suppressive effector functions of the Treg cells.
The observation that CD4+CD25+ T cell numbers and proportions are remarkably stable did not explain how CD4+CD25+ Treg homeostasis is achieved or the "modus operandi" of IL-2. Parsimony suggested that the expression of high-affinity IL-2R and the dependence on IL-2 for survival were critical, linked clues to understanding how this small subpopulation of T cells maintains its homeostasis. It has been suggested that competition is a major determinant of the life span of individual lymphocytes (4). Thus, a specific cell has a higher probability of survival if it possesses superior competitive fitness, that is, a superior ability to exploit resources common to competitors, or a unique ability to specialize, thereby permitting the exploitation of alternative resources. In either case, success depends on whether a cell’s particular characteristics provide an advantage in the use of a survival-enhancing resource. In the current context we believe the expression of high levels of the high-affinity IL-2R specializes the CD4+CD25+ Treg cells for exploitation of the IL-2 resource. Reconstitution of B6.Rag2–/– mice with a mixture of precursors differing only in their potential to exploit IL-2 (B6.Ly5.1 vs B6.Ly5.2.CD25–/–) leads to a CD4+ T cell pool where the proportions of derived cells diverge from the ratios of injected precursors: Ly5.1 CD4+ T cell population has a distinct advantage in the seeding of the peripheral CD4+ T cell pool over the Ly5.2 CD4+ cells from CD25–/– donors that cannot efficiently use IL-2. Thus, the specialized expression of CD25 by Treg cells permits them to occupy a niche defined by the IL-2 resource, and allows them to avoid direct competition with other CD4+ T cells lacking the high-affinity IL-2R. This explains how the immune system ensures the presence of this subpopulation of cells, but it fails to explain the constancy on their relative proportion.
Indeed, the situation appears to be more complex than simple direct competition and the occupation of a specialized niche. We show that in steady-state conditions, T cells, in particular activated CD4+ T cells, represent the major source of the IL-2 required for the establishment of a functional population of Treg cells, a finding wholly consistent with other studies (27, 37). Thus, the homeostatic control of the CD4+CD25+ Treg cells is not encapsulated. Rather, it is dependent on other CD4+ T cell populations. For example, we showed that the fraction of CD4+CD25+ Treg cells was proportional to the fraction of IL-2+ cells present in mixed BM chimeras; we reduced the size of the CD4+CD25+ Treg pool by diluting the peripheral pool of IL-2+ T cells with IL-2–/– CD4+ T cells, and we increased it by driving proliferation of naive CD4+ T cells with excess IL-7. We deduce that the size of the Treg niche corresponds to available quantities of IL-2; this means that the number of CD4+CD25+ Treg cells is tied, or indexed, to the number of CD4+ T cells that can make IL-2. This explains why the relative proportion of the two populations is stably maintained. Unlike the independent segregation of the naive and memory pools (2, 3) where alterations in the size of one pool do not affect the other, the indexing of CD4+CD25+ Treg cells to the CD4+ T cells that may produce IL-2 creates an interdependence; increases or decreases in numbers of the latter will be reflected in the expansion or contraction of the former, thereby maintaining the relative proportions.
The interdependence also constitutes a feedback mechanism that controls activated T cells while allowing immune responses to occur. In the initial stages of an immune response the presence of Ag perturbs the immune system equilibrium and incites proliferation of Ag-specific cells. As the number of IL-2-producing cells (T and non-T) and the IL-2 concentrations increase, Treg cells respond with proliferation and eventually reestablish steady state. This mechanism might have evolved to control peripheral cross-reactive or autoreactive T cell clones while allowing controlled increases in overall peripheral T cell number. Indeed, we found that we could expand CD4+CD25– naive T cells 5- to 10-fold in B6.Rag2–/–IL7Tg hosts without signs of IBD because CD4+CD25+ Treg cells expanded proportionately. This experiment is important because it demonstrates that it is not the augmented T cell proliferation and number that causes disease in this model, but rather the disruption of Treg indexing.
This study highlights the qualitative dimension of peripheral lymphocyte homeostasis and emphasizes that not all homeostatic control mechanisms have as their target the maintenance of numbers. As demonstrated in this study, the maintenance of proportions is also critical for immune system equilibrium. Our findings also predict a novel, central role for IL-2 in homeostasis: indexing of CD4+CD25+ Treg cells to the number of IL-2-producing cells ensures that peripheral T cell proliferation during an immune response is controlled and does not degenerate into autoimmune or lymphoproliferative diseases.
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Acknowledgments |
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Disclosures |
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Footnotes |
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1 This work was supported by the Ligue Nationale Contre le Cancer, Association pour la Recherche Contre le Cancer, Agence Nationale de la Recherche Contre le SIDA, Association Française des Myopathies, Centre National de la Recherche Scientifique, and the Institut Pasteur. A.R.M.A. was supported by the Fundaçao para a Ciencia e Tecnologia, Lisboa, Portugal. 
2 Current address: Institute for Research in Biomedicine, Via Vincenzo Vela 6, CH-6500 Bellinzona, Switzerland.
3 Address correspondence and reprint requests to Dr. Antonio A. Freitas, Lymphocyte Population Biology Unit, Unité de Recherche Associaé, Centre National de la Recherche Scientifique 1961, Institut Pasteur, 28 Rue du Dr. Roux, 75015 Paris, France or Dr. Afonso R. M. Almeida at the current address: Institute for Research in Biomedicine, Via Vincenzo Vela 6, CH-6500, Bellinzona, Switzerland. E-mail addresses: afreitas{at}pasteur.fr and almeida{at}irb.unisi.ch
4 Abbreviations used in this paper: Treg, regulatory T; LDP, lymphopenia-driven proliferation; LN, lymph node; HPRT, hypoxanthine phosphoribosyltransferase; BM, bone marrow; WT, wild type; IBD, inflammatory bowel syndrome.
Received for publication December 12, 2005. Accepted for publication April 20, 2006.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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and IL-2 shape a population of regulatory cells that controls CD4+ T cell numbers. J. Immunol. 169: 4850-4860. -chains (CD25): breakdown of a single mechanism of self-tolerance causes various autoimmune diseases. J. Immunol. 155: 1151-1164. [Abstract] chain regulates the size and content of the peripheral lymphoid compartment. Immunity 3: 521-530. [Medline]. Science 268: 1472-1476. chain, resistance to clonal deletion and IL-2 dependency. Int. Immunol. 10: 371-378. -deficient mice: implications for the nonredundant function of IL-2. Immunity 17: 167-178. [Medline]Related articles in The JI:
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  A. M. Wolf, K. Eller, R. Zeiser, C. Durr, U. V. Gerlach, M. Sixt, L. Markut, G. Gastl, A. R. Rosenkranz, and D. Wolf The Sphingosine 1-Phosphate Receptor Agonist FTY720 Potently Inhibits Regulatory T Cell Proliferation In Vitro and In Vivo J. Immunol., September 15, 2009; 183(6): 3751 - 3760. [Abstract] [Full Text] [PDF]
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 A. Le Campion, M.-C. Gagnerault, C. Auffray, C. Becourt, M. Poitrasson-Riviere, E. Lallemand, B. Bienvenu, B. Martin, F. Lepault, and B. Lucas Lymphopenia-induced spontaneous T-cell proliferation as a cofactor for autoimmune disease development Blood, August 27, 2009; 114(9): 1784 - 1793. [Abstract] [Full Text] [PDF]
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L. Myers, R. J. Messer, A. B. Carmody, and K. J. Hasenkrug Tissue-Specific Abundance of Regulatory T Cells Correlates with CD8+ T Cell Dysfunction and Chronic Retrovirus Loads J. Immunol., August 1, 2009; 183(3): 1636 - 1643. [Abstract] [Full Text] [PDF]
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 S Mori and P Levin A brief review of potential mechanisms of immune reconstitution inflammatory syndrome in HIV following antiretroviral therapy Int J STD AIDS, July 1, 2009; 20(7): 447 - 452. [Abstract] [Full Text] [PDF]
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W. E. O'Gorman, H. Dooms, S. H. Thorne, W. F. Kuswanto, E. F. Simonds, P. O. Krutzik, G. P. Nolan, and A. K. Abbas The Initial Phase of an Immune Response Functions to Activate Regulatory T Cells J. Immunol., July 1, 2009; 183(1): 332 - 339. [Abstract] [Full Text] [PDF]
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R. W. van Olffen, N. Koning, K. P. J. M. van Gisbergen, F. M. Wensveen, R. M. Hoek, L. Boon, J. Hamann, R. A. W. van Lier, and M. A. Nolte GITR Triggering Induces Expansion of Both Effector and Regulatory CD4+ T Cells In Vivo J. Immunol., June 15, 2009; 182(12): 7490 - 7500. [Abstract] [Full Text] [PDF]
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J. P. Monteiro, J. Farache, A. C. Mercadante, J. A. Mignaco, M. Bonamino, and A. Bonomo Pathogenic Effector T Cell Enrichment Overcomes Regulatory T Cell Control and Generates Autoimmune Gastritis J. Immunol., November 1, 2008; 181(9): 5895 - 5903. [Abstract] [Full Text] [PDF]
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C. J. Winstead, J. M. Fraser, and A. Khoruts Regulatory CD4+CD25+Foxp3+ T Cells Selectively Inhibit the Spontaneous Form of Lymphopenia-Induced Proliferation of Naive T Cells J. Immunol., June 1, 2008; 180(11): 7305 - 7317. [Abstract] [Full Text] [PDF]
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M.-E. Blais, S. Brochu, M. Giroux, M.-P. Belanger, G. Dulude, R.-P. Sekaly, and C. Perreault Why T Cells of Thymic Versus Extrathymic Origin Are Functionally Different J. Immunol., February 15, 2008; 180(4): 2299 - 2312. [Abstract] [Full Text] [PDF]
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M. Giroux, E. Yurchenko, J. St.-Pierre, C. A. Piccirillo, and C. Perreault T Regulatory Cells Control Numbers of NK Cells and CD8{alpha}+ Immature Dendritic Cells in the Lymph Node Paracortex J. Immunol., October 1, 2007; 179(7): 4492 - 4502. [Abstract] [Full Text] [PDF]
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V. Sanchez-Guajardo, C. Tanchot, J. T. O'Malley, M. H. Kaplan, S. Garcia, and A. A. Freitas Agonist-Driven Development of CD4+CD25+Foxp3+ Regulatory T Cells Requires a Second Signal Mediated by Stat6 J. Immunol., June 15, 2007; 178(12): 7550 - 7556. [Abstract] [Full Text] [PDF]
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P. Bostik, E. S. Noble, S. T. Stephenson, F. Villinger, and A. A. Ansari CD4+ T Cells from Simian Immunodeficiency Virus Disease-Resistant Sooty Mangabeys Produce More IL-2 Than Cells from Disease-Susceptible Species: Involvement of p300 and CREB at the Proximal IL-2 Promoter in IL-2 Up-Regulation J. Immunol., June 15, 2007; 178(12): 7720 - 7729. [Abstract] [Full Text] [PDF]
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 N. Komatsu and S. Hori Full restoration of peripheral Foxp3+ regulatory T cell pool by radioresistant host cells in scurfy bone marrow chimeras PNAS, May 22, 2007; 104(21): 8959 - 8964. [Abstract] [Full Text] [PDF]
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K. N. Couper, D. G. Blount, J. B. de Souza, I. Suffia, Y. Belkaid, and E. M. Riley Incomplete Depletion and Rapid Regeneration of Foxp3+ Regulatory T Cells Following Anti-CD25 Treatment in Malaria-Infected Mice J. Immunol., April 1, 2007; 178(7): 4136 - 4146. [Abstract] [Full Text] [PDF]
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P. Hoffmann, R. Eder, T. J. Boeld, K. Doser, B. Piseshka, R. Andreesen, and M. Edinger Only the CD45RA+ subpopulation of CD4+CD25high T cells gives rise to homogeneous regulatory T-cell lines upon in vitro expansion Blood, December 15, 2006; 108(13): 4260 - 4267. [Abstract] [Full Text] [PDF]
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 A. R. M. Almeida, B. Zaragoza, and A. A. Freitas Competition controls the rate of transition between the peripheral pools of CD4+CD25- and CD4+CD25+ T cells Int. Immunol., November 1, 2006; 18(11): 1607 - 1613. [Abstract] [Full Text] [PDF]
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) in the transferred populations and the proportion (mean ± SE) of CD25+ T cells (
) recovered in the host mice are shown.
) provides a source of IL-2+ BM-derived cells that excludes only the 
), all surviving mice from the other groups displayed signs of autoimmune disease, (hunched back, diarrhea, and skin lesions), indicating that 
