Transplantation of retrovirally transduced bone marrow prevents autoimmune disease in aged mice by peripheral tolerance mechanisms

Transplantation of bone marrow (BM) engineered to express self-antigen has been shown to protect 100% of young mice from myelin oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE), with thymic clonal deletion as a tolerance mechanism. Here, we asked whether aged mice can also be tolerised following transplantation with self-antigen-engineered BM and whether castration-induced thymus regrowth can enhance this outcomes. Then, 50% of aged mice were protected from EAE regardless of castration-induced thymus regrowth. EAE-free and diseased mice demonstrated MOG-specific lymphocyte proliferation and antibody production regardless of castration-induced thymus regrowth, consistent with lack of intrathymic deletion of self-antigen-reactive T cells. Although low chimerism levels (?相似文献   

Deletion of alloantigen-reactive thymocytes as a mechanism of adult tolerance induction following intrathymic antigen administration

Direct injection of foreign antigen into the adult thymus is a potent route of antigen delivery for the induction of tolerance in vivo. In this report, we demonstrate that tolerance to C57BL/10 (H2^b/BL10) alloantigens can be induced in CBA/Ca (H2^k/CBA) mice by intrathymic (IT) administration of BL10 spleen leukocytes coincident with transient peripheral immunomodulation of CD4⁺ T cells using a depleting anti-CD4 monoclonal antibody. T cell receptor (TCR) transgenic mice (BM3.6; H2^k) expressing a CD8-independent TCR specific for H2K^b were used as recipients to facilitate investigation of the mechanisms responsible for tolerance induction by allowing visualization of events in the thymus following IT injection. IT administration of 5 × 10⁷ BL10 spleen leukocytes and concomitant transient peripheral T cell depletion in BM3.6 mice resulted in a substantial H2K^b-specific deletion of transgenic-TCR⁺ (tg-TCR) thymocytes which was dependent on the level of tg-TCR expression. IT deletion and the failure to export CD8⁺ T cells to the peripheral lymphoid organs correlated with the induction of tolerance to H2K^b; TCR transgenic mice that had received IT injection of BL10 splenocytes and peripheral T cell depletion accepted a H2K^b+ cardiac allograft indefinitely. Analysis of tolerant BM3.6 mice revealed that there were low numbers of CD8⁺ T cells in the periphery giving rise to a substantially reduced reactivity in vitro despite the fact that no donor cells or IT deletion were observed in the thymi of the majority of tolerant mice. These results demonstrate for the first time that IT injection of foreign alloantigen into an adult thymus results in the deletion of thymocytes expressing a TCR specific for the injected alloantigen and suggest that this is an important mechanism of tolerance induction following IT injection of alloantigen in vivo. Furthermore, analysis of tolerant TCR-transgenic mice suggests that IT deletion is not required for the maintenance of tolerance, and that peripheral mechanisms enforce continued hyporesponsiveness to H2K^b following transplantation.     

Thymic function in young/old chimeras: substantial thymic T cell regenerative capacity despite irreversible age-associated thymic involution

Age-associated thymic involution results in a diminished capacity to regenerate T cell populations, although the magnitude of this effect is unknown. In this report, thymic function was studied in aged vs.young adult mice after lethal irradiation and administration of T cell-depleted bone marrow (BM) from young mice. Abnormalities observed in aged thymi (reduced thymocyte numbers, histologic abnormalities) were not reversed by administration of young BM via bone marrow transplantation (BMT), but agend thymi displayed a normal thymocyte subset distribution and appropriately deleted Mls-reactive T cells after BMT. Aged BMT recipients regenerated significantly reduced numbers of splenic T cells compared to young recipients and showed increased peripheral expansion of thymic emigrants since a higher proportion of BM-derived T cells expressed a memory phenotype in aged vs.young BMT recipients. Because peripheral expansion of thymic emigrants could substantially increase the number of thymic progeny present in the spleen, we sought to measure thymic T cell regenerative capacity after BMT in a setting devoid of peripheral expansion. To do this, TCR-transgenic (Tg⁺) T cell-depleted BM was administered to aged and young recipients lacking antigen specific for the Tg⁺ TCR. Aged recipients regenerated approximately 50 % of the TCR Tg⁺ cells regenerated in young BMT recipients, providing evidence that even very aged thymi retain the capacity to regenerate significant numbers of mature T cell progeny. Therefore, thymic function is reduced with aged but it is not lost, suggesting that therapeutic approaches to enhance thymic function may be successful even in very aged hosts.     

Inhibition of Alloantigen‐Primed Memory CD4+ and CD8+ T Cells by Hematopoietic Chimerism in Mice

Donor‐reactive memory T cells present a special hurdle in transplantation. Although hematopoietic chimerism is effective for inducing donor‐specific tolerance, the effects on memory T cells are unclear. Here, we induced stable chimerism and tolerance in mice (Tolerance group, n = 6) by donor‐specific transfusion (DST) plus anti‐CD154 monoclonal antibody (mAb), avoiding the toxic myeloablative conditioning treatment to assist bone marrow transplantation (DST/aCD154&BMTx). We then transferred memory CD4⁺ or CD8⁺ T cells from donor antigen primed mice to the tolerance‐induced recipients 4 days after heart transplantation (Tol/CD4⁺ Tm group and Tol/CD8⁺ Tm group, n = 6, respectively), but neither of these memory T‐cell subsets had an effect on the permanent graft survival (median survival time > 100 days). The unaltered rate of memory T cells in spleen and anergy to donor antigen in vitro demonstrated that these memory T cells were well controlled. The chimerism‐promoting protocol DST/aCD154&BMTx produced an immune environment that included high levels of regulatory T cells (Tregs), microchimerism and TGF‐β, all of which may act in suppressing the donor‐reactive memory CD4⁺ or CD8⁺ T cells. These findings have potentially important implications for designing approaches to suppressing memory T cells for success of transplantation.     

Neonatally tolerant rats actively eliminate donor-specific lymphocytes despite persistent chimerism

Rats from the allotype-marked PVG-RT7^b and PVG-RT1^u-RT7^b strains were injected at birth with semi-allogeneic F₁ bone marrow (BM) cells from athymic nude rats (PVG-rnu/rnu x PVG-RT1^u-rnu/rnu) to induce neonatal tolerance. As adults, 97% of the animals accepted donor-specific allogeneic skin grafts and a majority (65%) of rats were chimeric, expressing the major histocompatibility complex class I and allotype marker of the donor strain. Similar results were obtained when PVG-RT1^u-RT7^b rats were injected at birth with fully allogeneic PVG-rnu/rnu nude BM cells: as adults, 94% accepted donor-specific skin allografts and 76% of recipients were chimeric. Donor-derived CD4T cells, CD8T cells and B cells were found in low numbers (< 2%) in peripheral blood of rats made tolerant by F₁ BM cells. A large proportion of T cells bore the phenotype of recent thymic emigrants, suggesting that they were newly produced. All the evidence was consistent with clonal deletion tolerance, induced centrally within the thymus. The thymus was chimeric and thymocytes failed to respond in vitro to alloantigens of the donor-specific haplotype; donor-specific skin allografts survived indefinitely on athymic nude recipients reconstituted with CD4⁺CD8^? thymocytes or peripheral CD4T cells from tolerant animals. The chimeric state was interesting, since the PVG and PVG-RT1^u rat strains contain a natural killer (NK) cell system that rapidly eliminates (within 24 h) intravenously injected allogeneic or semi-allogeneic lymphocytes – a phenomenon known as allogeneic lymphocyte cytotoxicity or ALC. When neonatal tolerant rats were tested, the ALC index (a measure of cell killing) was unchanged in nonchimeric tolerant rats and significantly altered (reduced killing), but not abolished in chimeric animals. Hence, the injection of allogeneic BM cells which induced specific tolerance in the T cell population failed to tolerize the NK cell system, allowing the constant killing of newly produced donor-derived lymphocytes and putting at risk the very survival of the allogeneic BM cells. This has interesting implications for clinical transplantation.     

Antibody‐Mediated T‐Cell Reduction or Increased Levels of Chimerism Overcome Resistance to Cyclophosphamide‐Induced Tolerance in NKT‐Deficient Mice

We reported that invariant NKT‐cell knockout (iNKT KO) mice are resistant to the induction of intrathymic chimerism and clonal deletion in the cyclophosphamide (CP)‐induced tolerance system (CPS). However, another report shows that clonal deletion with chimerism may be intact in iNKT KO recipients in a bone marrow transplantation model. We also reported that pretreatment with anti‐Thy1.2 mAb, which reduces the number of T cells and iNKT cells, promotes allograft tolerance across H‐2 barriers in the CPS. In this study, we evaluated the efficacy of T‐cell depletion in the CPS, and the relationship between the role played by iNKT cells in central tolerance and mixed chimerism. BALB/c (H‐2^d) wild‐type, or iNKT KO (Jα18^?/?) mice were pretreated with 20–100 μg of anti‐Thy1.2 mAb and given 10⁸ donor DBA/2 (H‐2^d) spleen cells on Day 0, and 200 mg/kg CP on Day 2. Pretreatment with T‐cell depletion resulted in higher levels of mixed chimerism, increased intrathymic clonal deletion of donor‐reactive cells, and the induction of skin graft tolerance in iNKT KO recipients in CPS. This suggests that the high levels of mixed chimerism overcame the resistance to CP‐induced tolerance in iNKT KO mice. Consistently, the enhancement of mixed chimerism by injection of tolerant donor spleen cells (SC) rendered iNKT KO recipients susceptible to CP‐induced tolerance. These results suggest that iNKT‐cell‐mediated immunoregulation of central tolerance is evident at low levels of peripheral mixed chimerism in the CPS.     

Loss of central and peripheral CD8+ T-cell tolerance to HFE in mouse models of human familial hemochromatosis

HFE, an MHC class Ib molecule that controls iron metabolism, can be directly targeted by cytotoxic TCR αβ T lymphocytes. Transgenic DBA/2 mice expressing, in a Rag 2 KO context, an αβ TCR that directly recognizes mouse HFE (mHFE) were created to further explore the interface of HFE with the immune system. TCR‐transgenic mHfe WT mice deleted mHFE‐reactive T cells in the thymus, but a fraction of reprogrammed cells were able to escape deletion. In contrast, TCR‐transgenic mice deprived of mHFE molecules (mHfe KO mice) or expressing a C282→Y mutated mHFE molecule – the most frequent mutation associated with human hereditary hemochromatosis – positively selected mHFE‐reactive CD8⁺ T lymphocytes and were not tolerant toward mHFE. By engrafting these mice with DBA/2 WT (mHFE⁺) skin, it was established, as suspected on the basis of similar engraftments performed on DBA/2 mHfe KO mice, that mHFE behaves as an autonomous skin‐associated histocompatibility antigen, even for mHFE‐C282→Y mutated mice. By contrast, infusion of DBA/2 mHFE⁺ mice with naïve mHFE‐reactive transgenic CD8⁺ T lymphocytes did not induce GVHD. Thus, tolerance toward HFE in mHfe WT mice can be acquired at either thymic or peripheral levels but is disrupted in mice reproducing human familial hemochromatosis.     

CD4+ T‐cell development in a mouse expressing a transgenic TCR derived from a Treg

CD4⁺Foxp3⁺ Treg maintain peripheral tolerance and influence immune responses to foreign antigens. The thymus is an important source of Treg, but controversy exists as to whether T cells are selected into the Treg lineage based on signals received through TCR specific for self‐peptides. To examine the specificity of TCR expressed by Treg and its effect on CD4⁺ T‐cell development, we generated Treg‐TCR transgenic mice. Deletion of >90% of CD4⁺ T cells in RAG‐sufficient mice, and nearly 100% deletion in RAG^?/? mice expressing this TCR indicate that the TCR is specific for an unknown, naturally expressed peptide in the thymus. Deletion occurs late in development, suggesting this peptide is presented by APC in the thymic medulla. These studies are the first to describe the effects of expressing a Treg‐TCR on CD4⁺ T‐cell development. The implications of our data for models of Treg selection are discussed.     

Tolerance induction by clonal deletion of CD4+8+ thymocytes in vitro does not require dedicated antigen-presenting cells

The cellular requirements of T cell tolerance induction in the thymus by clonal deletion was investigated by using an in vitro assay: thymocytes from mice expressing a transgenic TcR specific for lymphocytic choriomeningitis virus (LCMV) and H-2D^b were co-cultured with various H-2^b cell types as antigen-presenting cells in the presence of the antigenic LCMV peptide. The results revealed that all cell lines examined including embryonic and transformed fibroblasts, melanoma cells, cortical thymic epithelial cells, lymphomas and neuronal cells induced an antigen dose-dependent deletion of CD4⁺8⁺ thymocytes. Similarly, highly enriched accessory cell populations from thymus and spleen (macrophages, dendritic and cortical epithelial cells, i.e. thymic nurse cells) could induce antigen-specific depletion of immature CD4⁺8⁺ thymocytes. Depending on the cell type examined micromolar to picomolar concentration of LCMV peptide were required to induce deletion. The effectiveness of deletion by the different cell types did not correlate with their major histocompatibility class I expression level; it was, however, influenced by the presence of ICAM-1 adhesion molecules.     

An MHC-linked locus modulates thymic differentiation of CD4+CD25+Foxp3+ regulatory T lymphocytes

CD4+CD25+Foxp3+ regulatory T lymphocytes are crucial for maintenance of immunological tolerance to self and innocuous non-self, are known to modulate immunity to tumors and infectious agents and can induce transplantation tolerance. Surprisingly, only a single genetic polymorphism is known to modulate regulatory T cell (Treg) development in the thymus, leading to a lethal autoimmune disorder. Here, we show that considerably different levels of Tregs are found in the thymi of distinct common laboratory mouse strains. We demonstrate that distinct levels of phenotypically and functionally identical Tregs develop with similar kinetics in the studied mice, that the responsible locus acts in a thymocyte-intrinsic manner and that levels of thymic Foxp3+ Tregs correlate to those found in the periphery. Using several congenic mouse strains, we mapped one of the at least two genetic loci capable of quantitatively modulating thymic Treg development to a 相似文献   

Transplantation of autoimmune regulator-encoding bone marrow cells delays the onset of experimental autoimmune encephalomyelitis

The autoimmune regulator (AIRE) promotes "promiscuous" expression of tissue-restricted antigens (TRA) in thymic medullary epithelial cells to facilitate thymic deletion of autoreactive T-cells. Here, we show that AIRE-deficient mice showed an earlier development of myelin oligonucleotide glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE). To determine the outcome of ectopic Aire expression, we used a retroviral transduction system to over-express Aire in vitro, in cell lines and in bone marrow (BM). In the cell lines that included those of thymic medullary and dendritic cell origin, ectopically expressed Aire variably promoted expression of TRA including Mog and Ins2 (proII) autoantigens associated, respectively, with the autoimmune diseases multiple sclerosis and type 1 diabetes. BM chimeras generated from BM transduced with a retrovirus encoding Aire displayed elevated levels of Mog and Ins2 expression in thymus and spleen. Following induction of EAE with MOG(35-55), transplanted mice displayed significant delay in the onset of EAE compared with control mice. To our knowledge, this is the first example showing that in vivo ectopic expression of AIRE can modulate TRA expression and alter autoimmune disease development.     

Intrathymic Tolerance Induction: Determination of Tolerance to Class II Major Histocompatibility Complex Antigens in Maturing T Lymphocytes by a Bone Marrow-Derived Non-Lymphoid Thymus Cell

Two new experimental approaches were established to analyse the influence of the thymus on tolerance induction to major histocompatibility complex (MHC) antigens: The aim of the first experiment was to perform successful transplantation of adult allogeneic thymus tissue into nude mice, an attempt that has been unsuccessful in the past. Tolerance for the MHC genotype of a prospective thymus graft recipient (A) was induced in mice of strain B by injection of (A X B) splenocytes during the neonatal period. Adult thymic tissue obtained from these allogeneic donors (B) were grafted into the nude mice of strain A. The allogeneic thymus was accepted by the nude mice and immunoreconstitution was achieved. Subsequently the recipients developed tolerance to the MHC antigens of the allogeneic thymus donor as proved by mixed lymphocyte cultures and the acceptance of skin grafts. The second experiment was designed to determine which Ia-positive thymic compartment participates in conferring tolerance to MHC antigens in maturing T lymphocytes. Chimaeric thymus grafts were created by transplantation of neonatal thymus (A) into allogeneic nude mice (B) for a period of 8 weeks. The graft was populated with host bone marrow-derived Ia antigen-positive cells. The chimaeric thymuses consisting of type A epithelium but populated with both type A and B lymphocytes and non-lymphoid cells (i.e. Ia-positive macrophages and dendritic cells), were newly transplanted into nude mice of strain A. The engraftment lead to immunological reconstitution and the nude mice acquired tolerance to the MHC antigens expressed by the allogeneic Ia-positive cells populating the chimaeric graft. Irradiation of the chimaeric thymus prior to transplantation allowed transplantation of chimaeric thymus devoid of living thymocytes but still populated with functionally intact Ia-positive non-lymphoid cells. Transplantation of irradiated chimaeric thymuses resulted in immunoreconstitution and induced exactly the same allotolerance pattern as described above. The results demonstrate that not thymus epithelial cells but a bone-marrow-derived non-lymphoid thymus cell, most likely the Ia-antigen-positive thymic macrophage of dendritic cell, is responsible for the induction of tolerance to MHC antigens in developing T lymphocytes.     

Self-encounters of the third kind: lymph node stroma promotes tolerance to peripheral tissue antigens

Multiple mechanisms have evolved to maintain tolerance among CD8⁺ T cells to innocuous antigens that arise in cutaneous and mucosal tissues. In the thymus, medullary thymic epithelial cells directly present peripheral tissue antigens (PTAs) and incite the deletion of self-reactive thymocytes. Cross-presentation of PTAs by functionally immature, CD8α⁺ dendritic cells can lead to the deletion of self-reactive CD8⁺ T cells in secondary lymphoid organs. A third mechanism of deletional tolerance has recently been uncovered in which lymph node-resident stromal cells of non-hematopoietic origin present endogenously expressed PTAs to circulating CD8⁺ T cells. Emerging data suggest that lymph node stroma is a unique niche for controlling self-reactive T cells.     

Indirect presentation in the thymus limits naive and regulatory T‐cell differentiation by promoting deletion of self‐reactive thymocytes

Acquisition of T‐cell central tolerance involves distinct pathways of self‐antigen presentation to thymocytes. One pathway termed indirect presentation requires a self‐antigen transfer step from thymic epithelial cells (TECs) to bone marrow‐derived cells before the self‐antigen is presented to thymocytes. The role of indirect presentation in central tolerance is context‐dependent, potentially due to variation in self‐antigen expression, processing and presentation in the thymus. Here, we report experiments in mice in which TECs expressed a membrane‐bound transgenic self‐antigen, hen egg lysozyme (HEL), from either the insulin (insHEL) or thyroglobulin (thyroHEL) promoter. Intrathymic HEL expression was less abundant and more confined to the medulla in insHEL mice compared with thyroHEL mice. When indirect presentation was impaired by generating mice lacking MHC class II expression in bone marrow‐derived antigen‐presenting cells, insHEL‐mediated thymocyte deletion was abolished, whereas thyroHEL‐mediated deletion occurred at a later stage of thymocyte development and Foxp3⁺ regulatory T‐cell differentiation increased. Indirect presentation increased the strength of T‐cell receptor signalling that both self‐antigens induced in thymocytes, as assessed by Helios expression. Hence, indirect presentation limits the differentiation of naive and regulatory T cells by promoting deletion of self‐reactive thymocytes.     

Development of T cells in SCID mice grafted with fetal thymus from AKR mice or F344 rats

To examine the development of T cells within an allogeneic or xenogeneic environment, we engrafted the fetal thymus from AKR mice or F344 rats under the kidney capsule of SCID mice (mTG and rTG mice). T lymphopoiesis developed in SCID mice 2 months after transplantation, although the ratio of CD4/CD8 in both experimental groups was different from that of normal control. T cells in mTG mice did not show in vitro proliferation or cytotoxicity against either host-type C.B-17 (H-2^d) or donor-type AKR (H-2^k) cells, while they exerted potent activities against third-party BIO (H-2^b) cells. In contrast, T cells in rTG mice exhibited proliferation against both host-type C.B-17 and donor-type F344 rat cells. Consistently, graft-vs.-host disease symptoms developed in these mice and histological examination showed impressive infiltration of lymphocytes into the skin or into the mucosal layers of the stomach. Activated state of T cells in rTG mice was also evidenced by the positive expression of interleukin-2 receptor. Taken together, fetal thymus appears to contain progenitor cells which are sufficient for in vivo reconstitution of T lymphopoiesis, but species-specific environment is important for the induction of tolerance. In mTG mice, Vβ6⁺ T cells reactive to donor Mls^a determinants and Vβ3⁺ T cells reactive to host Mls^c determinants were deleted, suggesting that tolerance was regulated mainly by clonal deletion. By contrast, Vβ11⁺ T cells reactive to Mls^f determinants were not deleted possibly due to the lack of their ligands.     

Monocyte chemoattractant protein 1 (MCP‐1/CCL2) contributes to thymus atrophy in acute myeloid leukemia

Recent studies on acute myelogenous leukemia (AML) patients have revealed the existence of T‐cell immunodeficiencies, characterized by peripheral T lymphocytes that are unable to interact with blasts, reduced thymic emigrants and oligoclonal restricted repertoires. These observations suggest that there is a profound thymic dysregulation, which is difficult to study in AML patients. Using the C1498 AML mouse model, we demonstrated that leukemia development was associated with thymus atrophy, which was defined by abnormal organ weight and reduced cellularity. In addition, we observed a dramatic loss of peripheral CD4⁺ and CD8⁺ T‐cell numbers with increased frequencies of CD4⁺FoxP3⁺ regulatory and activated/memory T cells. Investigating the mechanisms leading to this atrophy, we observed a significant accumulation of the monocyte chemoattractant protein 1 (MCP‐1/CCL2) in thymi of leukemic mice. Treatment of AML‐bearing animals with a blocking anti‐CCL2 antibody revealed a lower tumor burden, augmented antileukemic T‐cell responses, and improved survival rate compared to nontreated mice. These results were not observed when neutralization of CCL2 was performed in thymectomized mice. Altogether, we show that the CCL2 protein participates in thymic atrophy in AML mice, and this could have important implications for future immunotherapeutic strategies.     

Mouse and human CD8+ CD28low regulatory T lymphocytes differentiate in the thymus

Regulatory T (Treg) lymphocytes play a central role in the control of immune responses and so maintain immune tolerance and homeostasis. In mice, expression of the CD8 co‐receptor and low levels of the co‐stimulatory molecule CD28 characterizes a Treg cell population that exerts potent suppressive function in vitro and efficiently controls experimental immunopathology in vivo. It has remained unclear if CD8⁺ CD28^low Treg cells develop in the thymus or represent a population of chronically activated conventional T cells differentiating into Treg cells in the periphery, as suggested by their CD28^low phenotype. We demonstrate that functional CD8⁺ CD28^low Treg cells are present in the thymus and that these cells develop locally and are not recirculating from the periphery. Differentiation of CD8⁺ CD28^low Treg cells requires MHC class I expression on radioresistant but not on haematopoietic thymic stromal cells. In contrast to other Treg cells, CD8⁺ CD28^low Treg cells develop simultaneously with CD8⁺ CD28^high conventional T cells. We also identified a novel homologous naive CD8⁺ CD28^low T‐cell population with immunosuppressive properties in human blood and thymus. Combined, our data demonstrate that CD8⁺ CD28^low cells can develop in the thymus of mice and suggest that the same is true in humans.     

Regulatory CD4+ CD25+ T cells prevent thymic dysfunction in experimental chronic colitis

Chronic colitis in T-cell deficient Tg epsilon26 mice develops due to a dysfunction of the thymus which generates colitogenic T cells after bone marrow (BM) transplantation. Regulatory CD4+ CD25+ T cells have been shown to prevent colitis in this model by normalizing the peripheral T-cell pool. We tested the hypothesis that T-cell normalization takes place in the thymus. Tg epsilon26 mice were transplanted with BM (BM-->Tg epsilon26 mice) and consequently received either CD4+ CD25+ or CD4+ CD25- cells from syngenic wild type mice. Furthermore, untransplanted Tg epsilon26 mice received CD4+ CD25+ or CD4+ CD25- cells or complete mesenteric lymph node cells. Transfer of regulatory. CD4+ CD25+ cells normalized the total number of thymocytes and the percentage and number of double positive CD4+ CD8+ cells in transplanted mice while percentage of single positive CD4+ and CD8+ thymocytes in BM-->Tg epsilon26 mice was reduced upon CD4+ CD25+ transfer. Timing of CD4+ CD25+ cell injection was important as transfer later than 7 days after BM transplantation failed to prevent abnormal thymic T-cell distribution in BM-->Tg epsilon26 mice. Isolated CD4+ CD25+ cell transfer without preceding BM transplantation failed to reconstitute thymic architecture. Differences of thymic cell composition could not be exclusively explained by presence or absence of colitis, respectively, because 19 days after BM transplantation when both groups showed no histological signs of colitis, animals transferred with CD4+ CD25+ T cells had a significantly higher percentage and number of CD4+ CD25+ thymocytes and CD4+ Foxp3+ cells than BM-->Tg epsilon26 mice. In conclusion, early CD4+ CD25+ cotransfer prevents thymic dysfunction which underlies immune-mediated bowel inflammation in BM-->Tg epsilon26 mice.