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Keywords:

  • Autoimmunity;
  • Bone marrow chimeras;
  • Regulatory T cells

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Methods
  7. Acknowledgements
  8. Supporting Information

Sublethally irradiated, immunodeficient, C57BL/6 RAG-2 gene-deleted recipient mice reconstituted with T cell-depleted bone marrow (BM) grafts frequently developed diarrhea, lost weight and showed signs of autoimmunity, dying between 4 and 7 weeks after reconstitution. Mice died despite evidence of efficient donor-derived hemato-lymphoid reconstitution, and disease was associated with the presence of IgG anti-nuclear antibodies. Autoimmunity was initiated by T cells, but could be prevented by transfer of naturally arising regulatory T cells. In contrast, lethally irradiated, BM-reconstituted immunocompetent, C57BL/6 mice survived without signs of autoimmunity. Survival of immunocompetent mice was shown to be due to the presence of residual, extra-thymically located, radio-resistant, functional regulatory T cells. The importance of regulatory T cells was further shown by the reduced survival of immunocompetent BM recipients whose CD25+ T cells had been depleted prior to bone marrow transplantation. The implications of these results in the context of syngeneic graft-versus-host disease following BM transplantation are discussed.

See accompanying commentary: http://dx.doi.org/10.1002/eji.200636571

Abbreviation:
IBD:

inflammatory bowel disease

Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Methods
  7. Acknowledgements
  8. Supporting Information

Reconstitution of the lympho-hemopoietic system by bone marrow (BM) transplantation is a frequently used treatment modality for various hematological abnormalities, including anemia, leukemia and lymphoma 1, and more recently fulminant autoimmunity 2. Although this is frequently carried out with BM from haplo-disparate donors, even in situations where the BM graft is haplo-identical, patients can develop syngeneic graft-versus-host disease (GVH) 35. Disease can be acute, starting at day 8 4 or more chronic starting at about 5 weeks 3 following BM transplantation, a time when lymphoid reconstitution has begun, but when the patient is still relatively lymphopenic. Many of the features of syngeneic chronic GVH, including diarrhea, weight loss, cutaneous and lymphocytic infiltrations in multiple organs and the presence of autoantibodies are also seen in systemic autoimmune diseases such as inflammatory bowel disease (IBD), rheumatoid arthritis, systemic lupus erythematosus and systemic sclerosis 6.

Recent studies have implicated so-called naturally arising regulatory T cells (Treg) as key components controlling autoimmunity 79. Treg were first described as a population of CD5highCD4+ cells that upon transfer could protect mice from autoimmune disease caused by neonatal thymectomy 10. Later, it was shown that during mouse ontogeny, Treg appeared shortly after birth and that CD25, the alpha chain of the IL-2R complex, could be used as a surrogate marker for these cells 8, 11. More recently, the transcription factor FoxP3 has been shown to be critically associated with Treg function 12, and indeed a knock-in transgenic mouse line containing a GFP-FoxP3 construct indicated that FoxP3 may be used as a lineage marker for Treg 13. Many phenotypic features of Treg, namely spontaneous CD25 expression, down-regulation of CD4, CD3 and TCRβ transcripts and surface antigen expression are similar to those of activated T cells 1416. The repertoire of TCR expressed by Treg is generally thought to be broad; however, it would seem that their TCR have a relatively high affinity for self antigens 17. Thus, their CD5high phenotype may have protected them from negative selection in the thymus 18. Functionally, Treg inhibit the proliferation of naïve responder T cells in vitro 19. Their main functional role in vivo 20 appears to be in preventing the activation and reducing the expansion of activated T cells 21. This inhibition of T cell expansion can be seen as advantageous in situations of autoimmunity, but may be disadvantageous in situations of lymphopenia-induced proliferation or anti-tumor immunity 22, 23. It is therefore rather paradoxical that autoimmunity is controlled in a dominant fashion by a population of T cells which is itself intrinsically autoreactive 7.

In the mouse, syngeneic BM transplantation of immunocompetent recipients rarely results in disease. In contrast, in man, syngeneic GVH has been reported to develop with a cumulative incidence of 18% among syngeneic hematopoietic cell transplant recipients 3. This difference could be due to the relatively faster kinetics of reconstitution in mice, which is of the order of weeks compared with months in man. However, in analyzing the reconstitution potential of sublethally irradiated immunodeficient recipient mice with in vitro-generated, T cell-restricted, BM-derived progenitor cells, recipient mice frequently died between 4 and 6 weeks after reconstitution (A. Rolink, unpublished observations). Mice died despite evidence indicating that the T cell compartment was being well reconstituted by donor-derived cells. In this report, we describe results of experiments where the outcome of syngeneic BM transplantation in sublethally irradiated immunodeficient C57BL/6 RAG-2–/– was compared with that in immunocompetent wild-type (WT) C57BL/6 recipients. Whereas immunocompetent mice survived, immunodeficient mice succumbed to autoimmunity with diarrhea and weight loss beginning at 3 weeks following BM transplantation. Disease was associated with IgG anti-nuclear antibodies. Autoimmunity was initiated by T cells but could be prevented by co-transferring naturally arising Treg with the BM inoculum. Survival of immunocompetent recipients was shown to be due to the presence of residual, extrathymically located, radio-resistant, functional Treg in the lethally irradiated host. Moreover, depletion of Treg in WT mice prior to BM transplantation resulted in a phenotype similar to that of immunodeficient hosts, although with milder clinical symptoms. The implications of these results in the context of BM transplantation are discussed.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Methods
  7. Acknowledgements
  8. Supporting Information

RAG-2–/– mice show symptoms of autoimmune disease after transplantation of syngeneic RAG proficient BM

When sublethally irradiated C57BL/6 Ly5.2 RAG-2–/– mice were reconstituted with C57BL/6 WT Ly5.1 T cell-depleted BM, and despite successful reconstitution of T lymphopoiesis in the thymus and B lymphopoiesis in the BM, most animals died between 4 and 7 weeks later. Fig. 1A shows the survival curve of a group of 25 RAG-2–/– mice from five individual experiments reconstituted with T cell-depleted BM from WT syngeneic donors. Until about 33 days, 100% mice survived, but from then on there was a progressive decrease in survival with only 20% of mice surviving beyond 50 days. Prior to day 33, mice began having diarrhea and when groups of five to six mice were individually weighed following BM transplantation (Fig. 1B), there was progressive weight loss from day 21 with mice loosing approximately 40% of their initial body weight by day 42. Thus, diarrhea and weight loss preceded death of the mice.

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Figure 1. (A) Survival curve of C57BL/6.Ly5.1[RIGHTWARDS ARROW]C57BL/6.RAG-2–/–.Ly5.2 BM chimeras. Sublethally irradiated Ly5.2 RAG2–/– mice were transplanted with T cell-depleted bone marrow from Ly5.1 C57BL/6 mice. Shown is the pooled survival curve of 25 mice from four different experiments. Survival did not differ between individual experiments. (B) Weight loss among BM chimeras. Shown is the kinetic of mean weight loss in one group of six BM chimeras. Mice were weighed every 3 days over a 6-week observation period. (C) Widespread lymphocytic infiltration in non-lymphoid organs of immunodeficient mice after bone marrow transplantation. The upper picture is a photograph of H+E-stained 3-μm paraffin sections of the lower third of the colon from BL/6[RIGHTWARDS ARROW]RAG2–/– BM chimeras (original magnification ×100). The lower picture is a photograph of H+E-stained 3-μm frozen sections of liver from BL/6[RIGHTWARDS ARROW]RAG2–/– BM chimeras (original magnification ×400).

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Inspection of the intestines from mice with diarrhea showed that the lower third of the colon was dilated with thickening of the wall. Histological analysis of this region (Fig. 1C, upper panel) showed characteristic features of IBD 24, namely epithelial hyperplasia, extensive lymphocytic infiltration of the lamina propria, crypt abscesses and destruction of the mucous membrane with prominent ulceration. Histological analysis of other organs, for example the liver (Fig. 1C, lower panel), also showed evidence of lymphocytic infiltration. Moreover, the architecture of secondary lymphoid organs was disturbed, in that discrete lymphoid follicles were absent.

Flow cytometric analysis of LN cells from mice with diarrhea was carried out. Results obtained (Fig. 2) showed that, whereas there was clear evidence of B cell (left cytogram) and T cell (middle cytogram) reconstitution, further analysis of gated CD4+ cells (right cytogram) showed that many had up-regulated CD69 and CD25 expression. Two color analysis of gated CD4+ T cells showed that ∼19% were CD69+CD25, 18% CD69+CD25+ and 12% CD69CD25+. Thus, CD4+ T cells from affected mice showed an activated phenotype.

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Figure 2. Lymphocyte reconstitution in sublethally irradiated C57BL/6.Ly5.1[RIGHTWARDS ARROW]C57BL/6.RAG-2–/–.Ly5.2 BM chimeras. Shown are two color cytogram displays of LN cells from BM chimeras stained with the indicated mAb. In each panel, quadrants are placed so that 100% unstained cells were contained in the lower left quadrant. In the upper right panel, the % positive cells in each quadrant is indicated. The left panel shows B cell reconstitution, the middle panel that of CD4 and CD8 T cell subpopulations and the right panel expression of CD69 and CD25 on gated CD4+ T cells. See text for details.

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Onset of disease in RAG-2–/– recipient mice is mediated by T cells

In RAG-2–/– mice reconstituted with syngeneic WT BM, onset of diarrhea and weight loss coincided with a state of relative lymphopenia during the initial phase of lymphocyte reconstitution. In addition, CD4+ T cells from affected mice showed evidence of extensive activation. Therefore, to see whether T cells were responsible for initiating disease, RAG-2–/– mice were reconstituted with BM from donors incapable of reconstituting the T cell compartment, namely Ly5.1 CD3ϵ–/– mice. In six Ly5.2 RAG-2–/– mice reconstituted with Ly5.1 CD3ϵ–/– BM and in which the BM B cell compartment was fully reconstituted with Ly5.1 B cells, none developed signs of diarrhea or weight loss, with all mice surviving until 6 months following reconstitution (not shown). Thus, T cells were responsible for initiating disease in BM-transplanted mice.

Co-transplantation of Treg protects RAG-2–/– recipient mice from disease

RAG-2–/– mice reconstituted with syngeneic BM manifested signs of IBD and this was initiated by T cells. Previous experiments have shown that naturally arising Treg could protect lymphopenic mice from IBD 25. Therefore, to see if naturally arising Treg could protect BM-transplanted mice from IBD, Ly5.2 RAG-2–/– recipient mice were reconstituted with a mixture of T cell-depleted Ly5.2 BM together with 1 × 105 to 3 × 105 sorted Ly5.1 CD4+CD25+ LN cells. In three groups of experimental mice, no diarrhea or weight loss was noted. Gross inspection of the intestines showed them to be normal and histological analysis of the lower third of the colon confirmed this (Supplementary Figure) showing a completely normal picture. Histological analysis of other organs from these mice showed the total absence of lymphocytic infiltration. Moreover, secondary lymphoid organs showed a normal architecture. However, relatively large numbers of germinal centers were present in the spleen (data not shown). This finding strongly suggests that, although not showing signs of disease, an immune response was ongoing in these mice.

The phenotype of the transferred Ly5.1+ CD4+CD25+ T cells as well as that of the cohort of T cells derived from the BM donor was analyzed by three-color flow cytometry. Fig. 3 shows the results of a typical experiment of spleen cells 7 weeks after BM transplantation. Thus, 0.2% of gated lymphocytes expressed Ly5.1, the Ly5 allotype of the injected CD4+CD25+ LN T cells (Fig. 3A, histogram). Further analysis of gated Ly5.1+ spleen cells (right cytogram displays) showed that they were practically all CD4+CD8 cells (right upper cytogram) with the vast majority (80%, lower right cytogram) retaining the CD25+ phenotype. Thus, the transferred Ly5.1+ cells were not contaminated by cells capable of reconstituting the host thymus, and they persisted in recipient mice for up to 10 weeks after BM transplantation (not shown). When gated on Ly5.1-negative (namely Ly5.2+) cells, the spleen contained the expected subpopulations of donor-derived CD4+ and CD8+ T cells, with in this case a high CD4/CD8 T cell ratio (∼4) (left upper cytogram). However, the proportion of CD25+ cells among the CD4+ subpopulation was relatively low (∼7%, left lower cytogram). As is the case for naturally arising CD25+ Treg, donor-derived CD4+CD25+ cells had a slightly decreased CD4 expression 14. Additional staining of LN cells from such chimeras with CD4, CD25 and CD69 showed that co-transplantation of Treg prevented, to a large extent, the activation of BM donor-derived T cells. Thus, only 11.6% of BM donor CD4 cells expressed the early activation marker CD69 (Fig. 3) compared with 36.6% in the chimera without co-transferred Treg (Fig. 2). Moreover, most gated CD25+ cells were now CD69 (Fig. 3B), confirming that they were not simply activated CD25+ T cells. Thus, co-transfer of naturally arising CD25+ Treg to BM-transplanted mice inhibits the activation of BM-derived T cells and also protected mice from IBD.

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Figure 3. (A) CD4+CD25+ T cells co-injected into recipient mice survive for up to 9 weeks following BM reconstitution. Sublethally-irradiated (500 rad) Ly5.2.RAG2–/– mice were reconstituted with T cell-depleted Ly5.2.B6 WT BM together with sorted Ly5.1+CD4+CD25+ T cells. The top histogram is of spleen cells from one of four similar experiments stained with FITC-labeled anti-Ly5.1mAb. and shows the presence of residual Ly5.1+CD4+CD25+ Treg at this time. Cytogram displays show the two color staining profiles of gated donor-derived (left, Ly5.1) and injected Ly5.1+CD4+CD25+ (right) T cells stained for CD4 and CD8 (upper cytograms) or CD4 and CD25 (lower cytograms). Figures in the upper right quadrant of each panel show the % of positive cells in each quadrant. See text for details. (B) Donor-derived CD4+ T cells retain a naive phenotype. The two-color cytogram shows the CD69 versus CD25 staining profile of B6 WT-derived CD4 cells in C57BL/6.Ly5.2[RIGHTWARDS ARROW]C57BL/6.RAG-2–/–.Ly5.2 BM chimeras co-injected with sorted Ly5.1.CD4+CD25+ T cells.

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Co-transplantation of Treg prevents the formation of autoantibodies in RAG-2–/– recipients

Given the fact that BM-transplanted mice showed signs of systemic autoimmunity, we decided to investigate whether the serum from such mice contained autoantibodies. For this, frozen sections of kidneys from RAG-2–/– mice were incubated with serum from BM-transplanted RAG-2–/– mice reconstituted with or without Treg and the presence of autoantibodies determined by indirect immunofluorescence. As shown in Fig. 4, whereas the serum of BM-transplanted mice contained readily detectable anti-nuclear antibodies, they were undetectable in BM recipients co-transferred with Treg.

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Figure 4. Co-injection of Treg inhibits the formation of antinuclear autoantibodies in BM chimeras. Shown are the titers of anti-nuclear autoantibodies, detected by indirect immunofluorescence as described, in sera from BM chimeras co-injected (left, BM+Treg) or not (right, BM) with sorted CD4+CD25+ Treg.

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Normal BL/6 mice do not show symptoms of autoimmunity after BM transplantation

Autoimmunity was only seen when RAG-2–/– recipients were reconstituted with T cell-depleted BM. Normal BL/6 mice reconstituted with T cell-depleted BM did not suffer from IBD. To see whether radio-resistant, host-derived, naturally arising CD25+ Treg were responsible for protecting mice from IBD, a series of chimeras was established in which normal Ly5.1+ BL/6 mice were reconstituted with T cell-depleted Ly5.2+ BL/6 BM alone. As expected, no diarrhea or weight loss was seen in normal BL/6 recipients. Again, histological analysis of intestines and other organs showed no abnormality (not shown). Flow cytometric analysis of LN cells at 6 weeks following BM transplantation showed the presence of a significant (14%) population of host-derived (Ly5.1+) cells (Fig. 5A, histogram) and further analysis of these gated host-derived LN cells (right cytograms) showed that most (93.5%) were T cells, being 74% CD4+ and 19.5% CD8+. Further analysis of the CD4+ subpopulation (lower right cytogram) showed that 18.7% (13.9/74.5) were CD25+ cells, the latter again having a slightly reduced CD4 expression as is characteristic of Treg. Parallel analysis of the donor-derived (Ly5.1-negative) population showed that, as expected, the majority (67%, lower left quadrant) expressed neither CD4 nor CD8 and were mostly CD19+ B cells. Indeed, after 6 weeks, analysis of the thymus and BM in such chimeras showed that 98.8 ± 0.7% (n=5) of thymocytes and 100% BM CD19+ cells were donor derived. This result showed that with this irradiation dose, the progenitor T and BM B cell lineage was fully reconstituted with donor-derived cells and that there was no detectable population of surviving host-derived B cells (not shown). Donor-derived T cells (23.4% CD4 and 9% CD8) had a normal CD4:CD8 ratio and additional analysis of the CD4+ cells (lower cytogram) showed a distinct population of 7.5% CD25+ cells.

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Figure 5. (A) Radio-resistant, host-derived CD4+CD25+ regulatory T cells protect WT recipients from autoimmune disease. The top histogram is of LN cells from C57BL/6.Ly5.2[RIGHTWARDS ARROW]C57BL/6. Ly5.1 BM chimeras stained with FITC-labeled anti-Ly5.1 mAb. and shows the presence of 14% residual, host-derived, cells. Below are cytogram displays of cells stained for CD4 and CD8 (upper cytograms) or CD4 and CD25 (lower cytograms) and gated for either donor (Ly5.1, left) or host (Ly5.1+right) derived cells. C57BL/6 Ly5.1+ mice were irradiated with 9.5 Gy before being reconstituted with T cell-depleted BM cells from B6 Ly5.2+ mice. (B) Host-derived radio-resistant CD4+CD25+ T cells retain Treg function. Shown are the [3H]thymidine counts from cultures containing Ly5.1+CD4+CD25 responder naive T cells cultured either alone (left column) or together with sorted Ly5.2+CD4+CD25+ host-derived cells from C57BL/6.Ly5.[RIGHTWARDS ARROW]C57BL/6. Ly5.2 BM chimeras at the indicated ratios (Ly5.2CD4+CD25+ : Ly5.1CD4+CD25-). Cells were cultured as described in Methods for 72 h with anti-CD3 mAb and irradiated spleen cells as APC. Each column represents the mean of [3H]thymidine incorporation from triplicates wells.

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To show that radio-resistant, host CD4+CD25+ T cells possessed regulatory function, we carried out a classical T cell co-culture assay 19 using naïve Ly5.1+ CD4+CD25 responder T cells and as potential inhibitory cells host-derived Ly5.2+CD4+CD25+ T cells from Ly5.1[RIGHTWARDS ARROW]Ly5.2 BM chimeras 6 weeks after BM transplantation. Results obtained (Fig. 5B) show clearly that when stimulated with anti-CD3ϵ mAb in the presence of irradiated APC, naïve T cells alone proliferated normally (left light gray bar) but that addition of Ly5.2+ CD4+CD25+ host-derived T cells from chimeras inhibited this proliferation (right dark gray hatched bars). Thus, the host-derived CD4+CD25+ subpopulation that survived lethal irradiation possessed classical T regulatory activity.

Compared with donor-derived cells, the host-derived Ly5.1+CD4+ subpopulation was relatively enriched in CD25+ cells (Fig. 5A, bottom cytograms). This suggested that CD25+ T cells might preferentially survive a lethal dose of irradiation. The possibility also existed that host-derived CD4+CD25+ T cells were the progeny of the transient, host-derived, cohort of thymocytes generated following irradiation and BM reconstitution 12. To determine whether the thymus was required for the appearance of these peripheral CD4+CD25+ cells, recipient adult mice were first thymectomized and 1 week later, irradiated and reconstituted with T cell-deficient RAG-2–/– BM. In these thymectomized mice, any surviving T cells must have been in an extrathymic location at the time of irradiation. Flow cytometric analysis of LN cells from such a chimera at 5 weeks following reconstitution showed that about 3% were CD4+ TCRβ+ T cells and about 30% of these expressed CD25 (Fig. 6). However, it should be noted that the number of host-derived T cells in thymectomized chimeras was dramatically lower than in euthymic controls. Thus, the majority of host peripheral T cells in BM chimeras are the progeny of the cohort of thymocytes generated following irradiation and BM reconstitution.

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Figure 6. Preferential survival of CD4+CD25+ host T cells following irradiation. Shown is a cytogram display of LN cells from a 6-week-old BL/6 mouse that had been thymectomized, body-irradiated 1 week later with 4.5 Gy, reconstituted with BM from B6.RAG2–/– donors and stained with the indicated markers. As shown, about 35% (0.84/2.96) of surviving host-derived CD4 cells expressed CD25.

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Treg-depleted BL/6 mice suffer from IBD following BM transplantation

To check whether among radio-resistant cells, Treg were the ones responsible for protecting the mice against IBD, we depleted mice of Treg prior to irradiation and BM reconstitution. It has been previously shown that injection of anti-CD25 mAb in vivo depletes the Treg compartment 26, and several groups have used this approach to boost anti-tumor immune responses 27. Anti-CD25-mediated depletion might not eliminate all Treg 13, 28; nevertheless, so far it is the only method of eliminating the majority of Treg in vivo. Therefore, groups of five Ly5.2+BL/6 mice received either a single 0.5 mg dose of PC61 anti-CD25 mAb intravenously or PBS as control. After 5 days, mice were lethally irradiated (8 Gy) and subsequently reconstituted with T cell-depleted Ly5.1+BL/6 WT BM. At 3 weeks after reconstitution, mice started losing weight, and by 6 weeks anti-CD25-treated mice had lost 34% of their initial body weight (Fig. 7), accompanied by extensive diarrhea. Of the five mice, one died after 4 weeks and the other four were killed 7 weeks after transplantation according to Institutional guidelines due to their deteriorating health. Flow cytometry analysis with an anti-CD25 mAb recognizing a CD25 epitope distinct from that recognized by PC61, the CD25 mAb used for in vivo depletion, showed that CD25+ cells had been reduced by between 92.5% and 99.9%. In addition, in the absence of CD25+ cells, surviving CD4+CD25 cells were in a more activated state as shown by enrichment for CD69+ cells (data not shown). However, clinical parameters were not as severe as in RAG2–/– recipients presumably because, as shown by others, some Treg activity still remains following anti-CD25 mAb depletion 13, 28.

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Figure 7. Mice depleted of Treg suffer weight loss after BM reconstitution. Ly5.2+BL/6 mice were lethally irradiated with 9.5 Gy and then transplanted with T cell-depleted bone marrow cells from Ly5.1+C57BL/6 mice. Bone marrow transplanted BL/6 mice were weighted every 7 days for 6 weeks. The above figure represents the mean weight variation from one group of five mice.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Methods
  7. Acknowledgements
  8. Supporting Information

BM transplantation, whether by haplo-identical or non-identical grafts, is a frequently used clinical procedure for the reconstitution of the immune system following chemotherapeutic and irradiation treatment 29. One of the major complications of such a procedure is the appearance of GVH especially in cases were patients are transplanted with BM from haplo-disparate donors. However, and thus far for largely unknown reasons, a similar type of GVH is also seen even when a haplo-identical graft is used. Clinically, this is marked by cutaneous, hepatic and gastrointestinal infiltrations 1. In this study, we have investigated this phenomenon in a mouse model of syngeneic BM transplantation. These studies were initiated because lethally or sublethally irradiated immunodeficient RAG-2–/– mice reconstituted with either T cell-depleted BM or in vitro-generated T cell-committed lymphoid progenitors developed diarrhea and weight loss, frequently dying 4 to 7 weeks after reconstitution 30. Here, we show that so-called naturally arising Treg, either transferred together with the BM graft, or derived from endogenous radio-resistant T cells of the host, protect recipient mice from developing autoimmunity. In corollary, depletion of Treg in WT mice prior to BM transplantation resulted in the appearance a relatively mild, non-lethal, disease.

Reconstituted mice developed diarrhea and weight lost. Histological analysis of the lower third of the colon showed the classical signs of IBD, namely epithelial hyperplasia, extensive lymphocytic infiltration of the lamina propria, crypt abscesses and destruction of the mucous membrane with prominent ulceration (Fig. 1). Initiation of this disease was T cell-dependent as shown by absence of disease in mice reconstituted with CD3ϵ–/– BM (Fig. 2). Transfer of naturally arising Treg prevented the appearance of IBD in this model (Fig. 2). Autoimmunity was only seen when RAG-2–/– recipients were reconstituted with T cell-depleted BM and onset of IBD could be prevented by transferred naturally arising CD25+Treg. However, WT BL/6 mice reconstituted with BM did not suffer from IBD. One clear difference between RAG-2–/– and BL/6 mice is the presence of a mature T cells in the latter prior to irradiation. It is known that some thymocyte progenitors 31 and peripheral T cells 32 can survive a lethal dose of irradiation. Indeed, it was shown some time ago that mitogen-activation of normal mouse peripheral T or B cells rendered them more resistant to irradiation in vitro 32. In addition, it as been recently reported that recipient CD4+ T cells surviving irradiation can regulate chronic GVH disease in a B10.D2 (H-2d)[RIGHTWARDS ARROW]BALB/c (H-2d) MHC-compatible, multiple minor histocompatibility antigen-incompatible BM transplantation model 33.

Functional tests indicated that host-derived CD4+CD25+ cells inhibited the proliferation of anti-CD3ϵ-activated naïve responder T cells in vitro, thereby satisfying the criterion that they were genuine Treg 19. Moreover, depletion of CD25+ cells in lethally irradiated BL/6 WT mice prior to reconstitution led to development of IBD as observed when using immunodeficient hosts, although to a milder extent. Interestingly, naturally arising CD25+ Treg share many features with activated T cells, namely down-regulation of CD4, CD3 and TCRβ as well as expression of CD25 14, 16. In addition, transcriptome analysis has shown that Treg contain abundant transcripts of pro-survival genes, for example GITR 34, and more recently Bcl-xL has been suggested to be up-regulated in Treg 35. They are thought to be constantly encountering their cognate self-antigen in the periphery and therefore to be in a semi-activated state 17. However, by comparing thymectomized mice transferred with RAG–/– BM with WT recipients transplanted with WT BM, we could show that most of the host radio-resistant Treg were probably derived from the thymus.

Anderson et al.33 recently reported that radio-resistant Treg could prevent chronic GVH in a mouse model of BM transplantation across multiple minor histocompatibility loci [B10.D2 (H-2d)[RIGHTWARDS ARROW]BALB/c (H-2d)]. Thus, BALB/c RAG2–/– mice transplanted with BM from B10.D2 develop a severe GVH, whereas the same recipients transplanted with BM from WT BALB/c, used as controls, stayed healthy. Unlike our present results, the authors did not describe any signs of syngeneic GVH. However, it should be noted that the mice in their experiments were treated with antibiotics throughout the time course of the experiment. When we treated our RAG2–/– BM recipients with antibiotics, we observed milder disease with more animals surviving. These findings strongly suggest that the antigenic load, and more specifically the gut flora, seem to play important roles in determining disease onset and evolution in this experiment setting. In this context, it is worthwhile noting that mice prone to IBD do not develop this disease when kept under germ-free conditions 3639. We conclude that the syngeneic GVH described in the present paper might be strongly influenced by antigen-driven T cell responses. This is substantiated by the activated phenotype of T cells in RAG2–/– recipients. The fact that Treg can prevent both the activated phenotype and disease onset might suggest that they are controlling the strength of this immune response.

Another parameter worthy of consideration is the age of the recipient mice. No sign of disease was observed in sublethally irradiated neonatal RAG2–/– BL/6 mice transplanted with WT syngeneic BM. At least two mutually non-exclusive explanations could explain this. Firstly, the antigenic load in neonatal mice may be dramatically lower than in adults and secondly the neonatal thymus might be more efficient at generating Treg.

The T cell-induced autoimmune disease seen in immunodeficient mice reconstituted with syngeneic BM resembles, in many ways, the so-called syngeneic GVH disease seen in a clinical setting. In our mouse model, Treg of either host or donor origin play a key role in controlling both the severity of IBD and the appearance of autoantibodies. Using reconstitution of the lympho-hemopoietic system, after BM transplantation, development of autoimmunity is clearly dependent upon the number of Treg. This leads us to propose that infusion of such cells into BM transplant recipients should be considered as a treatment modality for the prevention of syngeneic GVH.

Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Methods
  7. Acknowledgements
  8. Supporting Information

Mice

Ly5.1 and Ly5.2 C57BL/6 (BL/6), Ly5.1 BL/6 CD3ϵ gene deleted (CD3ϵ–/–) 40 and Ly5.2 BL/6 RAG-2–/–41 mice were maintained in our own animal SPF facilities. Male and female mice were used at 8 weeks of age and all experiments carried out according to Institutional guidelines. Adult thymectomy was performed on 6-week-old C57BL/6 mice and mice were used 7 weeks later and absence of residual thymus tissue verified at the time of autopsy.

Reagents and antibodies

The following mAb were purchased from PharMingen (San Diego, USA): anti-CD25FITC (7D4), anti-CD62LFITC (MEL-14), FITC or biotin-conjugated anti-CD8α (53–6.7), anti-CD69PE (H1.2F3), anti-TCRβPE (H57–597). Anti-CD4PE (RM4–5) antibody was purchased from eBioscience (San Diego, USA). The anti-Ly5.1FITC (A20) and anti-Ly5.2FITC (104) antibodies were produced and labeled in our laboratory according to standard techniques. To reveal biotin-labeled antibody, streptavidin-allophycocyanin (Becton Dickinson, San Diego, USA) was used. Anti-CD25 mAb PC61 was purified from hybridoma culture supernatant by standard procedures.

Flow cytometric analysis and sorting

Single-cell suspensions of thymus, spleen and lymph nodes were prepared in PBS supplemented with 2% FBS and 0.2% sodium azide as described previously 30. Cells were adjusted to 20 × 106 –10 × 106 cells/mL and 0.5 × 106 –1 × 106 cells incubated for 30 min at 4°C with the indicated reagents at saturating concentrations as previously described. Stained cells resuspended in PBS 2% FBS 0.2% azide containing propidium iodide (PI) were analyzed using a FACSCalibur (Becton Dickinson) and data analyzed using CellQuest (Becton Dickinson). Viable lymphoid cells were defined by a combination of FSC, SSC and PI fluorescence. Stained cells were sorted on a on a FACSAria (Becton Dickinson).

BM transplantation and adoptive transfer of Treg

Bone marrow cell suspensions from three to five BL/6 donor mice were prepared by flushing femurs and tibias with PBS using a 23-g needle. After red blood cell lysis, T cells were depleted by re-suspending cells in a mixture of rat IgM anti-CD90 (AT83) anti-CD4 (RL172) and anti-CD8α (31 M) mAb hybridoma supernatants and incubated for 20 min at 4°C 42. Following a washing step, antibody-coated cells were lysed by adding rabbit complement (Low-Tox, Cedarlane, Canada) dissolved in serum-free Dulbecco's modified Eagle's medium (DMEM). After incubation for 45 min at 37°C, cells were washed and resuspended in DMEM prior to injection. To obtain naturally arising Treg, spleen cell suspensions from Ly5.2+ BL/6 mice were stained with anti-CD4PE (RM4–5) and anti-CD25FITC (7D4) antibodies and CD4+CD25+ cells sorted on a FACSAria. The purity of sorted cells was always > 98%.

For in vivo Treg depletion, mice received one injection of anti-CD25 mAb (PC61; 0.5 mg/injection i.v.) 5 days prior to irradiation and BM transplantation.

Viable BM and sorted cells were counted and resuspended in DMEM. Mice were reconstituted with 200 μL containing 3 × 106 T cell-depleted BM cells alone or together with 1 × 105–3 × 105 sorted CD4+CD25+ cells. Recipient mice were γ-irradiated using a Cobalt source (Gammacell 40, Atomic Energy of Canada, Ltd) 4 h prior to reconstitution. RAG-2–/– mice were irradiated with 4–5 Gy and BL/6 mice with 8–9.5 Gy. Chimeric mice were weighed once a week for up to 12 weeks and analyzed between 4 and 12 weeks. The origin and composition of lymphoid cells was determined by means of the Ly5.1 and Ly5.2 markers.

Functional Treg assay

The ability of CD4+CD25+ T cells from chimeras to inhibit the in vitro proliferation of naïve T cells was carried out as previously described 19. Briefly, CD4+CD25 naïve T cells and CD4+CD25+ isolated from chimeras were purified by sorting. APC were T cell-depleted, irradiated syngeneic spleen cells from BL/6 mice. Control cultures contained 2.5 × 104 CD4+CD25 naïve responder T cells, 5 × 104 APC and 0.1 μg anti-CD3ϵ mAb. To test for inhibition, 2.5 × 104 CD4+CD25+, obtained from the indicated chimeric mice were added. Cells were cultured in round-bottom 96-well plates for 72 h and 1 μCi/well [3H]thymidine was added for the last 8 h prior to harvesting.

Immunohistochemistry and histological staining

Organs were snap frozen in Tissue-Tek OCT compound (Sakura Finetechnical, Tokyo, Japan) and 5-μm sections cut on a cryostat. Sections were then fixed for 10 min in acetone and stored at −20°C. For staining, sections were covered with antibody solution at saturating concentrations and incubated for 30 min at room temperature. For the second step, sections were washed in PBS and incubated with a Neutralite Avidin-TXRD/PBS (Southern Biotech) solution for 15 min at room temperature. Primary antibodies used included anti-CD90FITC, anti-IgMCy5 (M41) and biotinylated peanut agglutinin (Vector laboratories). Slides were washed; one to two drops of a 1:1 mixture of PBS and glycerin were placed onto the slide and covered with a coverslip 43. A 10× or 5× objective was used for magnification. Intestines were fixed in 4% paraformaldehyde, embedded in paraffin, cut in 3-µm sections and stained with hematoxylin/eosin (H+E).

For detection of serum anti-nuclear antibodies, snap-frozen sections of kidneys from RAG-2–/– mice were incubated with sera diluted 1:10 to 1:10 240 and bound antibodies revealed with a 1:50 dilution of FITC-labeled, mouse IgG subclass-specific secondary antibodies (Jackson ImmunoResearch Laboratories). The pattern and titer of anti-nuclear antibodies were assessed using an incident-light fluorescence microscope (Zeiss axioskope) and serum titer defined as the highest dilution showing specific nuclear staining.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Methods
  7. Acknowledgements
  8. Supporting Information

Antonius G. Rolink is holder of the chair in Immunology endowed by F. Hoffman-La Roche Ltd., Basel. This work was supported by a grant from the European Community awarded to the EuroThymaide consortium (Contract LSHB-CT-2003–503410). R. Ceredig thanks INSERM for support. The authors declare they have no competing financial interests.

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Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Methods
  7. Acknowledgements
  8. Supporting Information

Supporting information for this article is available on the WWW under http://www.wiley-vch.de/contents/jc_2040/2006/36434_s.pdf or from the author.

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