Recipient Thymus Research Articles

Intrathymic delivery of plasmid-encoding endoplasmic reticulum signal-sequence-deleted MHC class I alloantigen can induce long-term allograft survival

Abstract Intrathymic (IT) delivery of donor alloantigen is a potent strategy to induce operational tolerance. In this study we determined whether this effect was dependent on direct aliorecognition of the tolerogen. Ten microgrammes of plasmid, encoding either the wildtype major histocompatibility complex (MHC) class I molecule Kb or a truncated form in which the signal sequence for translocation into the endoplasmic reticulum was deleted, preventing cell surface expression and direct allorecognition of the tolerogen, was administered intrathymically to CBA.Ca (H2k) recipients. In addition, recipients were treated with anti-CD4 antibody (YTA3.1) at the time of IT injection and underwent transplantation 28 days later with a fully mismatched C57BL/10 (H2b) cardiac allograft. Wildtype, as well as truncated Kb genes, were able to induce long-term survival of the cardiac allografts, in contrast to empty control plasmid. Reversetranscriptase PCR showed expression of the Kb genes for up to 28 days in thymus and spleen of pretreated recipients. These data show that direct allorecognition of the tolerogen was not required for the induction of long-term allograft survival following the introduction of plasmid-encoded MHC alloantigen into the thymus.

Intrathymic delivery of plasmid-encoding endoplasmic reticulum signal-sequence-deleted MHC class I alloantigen can induce long-term allograft survival.

Intrathymic (IT) delivery of donor alloantigen is a potent strategy to induce operational tolerance. In this study we determined whether this effect was dependent on direct allorecognition of the tolerogen. Ten microgrammes of plasmid, encoding either the wildtype major histocompatibility complex (MHC) class I molecule K(b) or a truncated form in which the signal sequence for translocation into the endoplasmic reticulum was deleted, preventing cell surface expression and direct allorecognition of the tolerogen, was administered intrathymically to CBA.Ca (H2(k)) recipients. In addition, recipients were treated with anti-CD4 antibody (YTA3.1) at the time of IT injection and underwent transplantation 28 days later with a fully mismatched C57BL/10 (H2(b)) cardiac allograft. Wildtype, as well as truncated K(b) genes, were able to induce long-term survival of the cardiac allografts, in contrast to empty control plasmid. Reverse-transcriptase PCR showed expression of the K(b) genes for up to 28 days in thymus and spleen of pretreated recipients. These data show that direct allorecognition of the tolerogen was not required for the induction of long-term allograft survival following the introduction of plasmid-encoded MHC alloantigen into the thymus.

Isolation and tracking of a rare lymphoid progenitor cell which facilitates bone marrow transplantation in mice

Bone marrow cells are composed of pluripotent stem cells to terminally differentiated cells, with a wide variety of abundance of each cell type. In the past, many of the cell types within this heterogeneous population have been characterized either by expression of specific proteins or using functional markers. In spite of promising results obtained with the latter method, various cell types within bone marrow have not been well characterized due to the low abundance of a specific cell type. Considering the demand for a reliable technique to enrich cell types, a wide variety of approaches, ranging from simple nylon wool columns to high-speed cell sorting, have evolved. Only limited success has been obtained with approaches ranging from the detection of MHC antigen to positron emission tomography to track the ontogeny of specific bone marrow-derived cells in studies of syngeneic or allogeneic transplantation. The present study describes a relatively simple method to enrich and track a rare bone marrow cell (facilitating cell, FC), which can facilitate allogeneic bone marrow stem cell transplantation in mice. The isolation technique is comprised of enrichment of FC by magnetic activated cell sorting (MACS) system followed by purification through high-speed cell sorter. An initial inoculation of 30,000 FC obtained from male mice was detected in the thymus, spleen, and bone marrow of allogeneic female recipients, by using 32P-labeled dCTP in a specific PCR for Y-chromosome. This technique may improve the efficiency of isolation of other rare cells from the bone marrow.

PERIPHERAL CD4 T CELL TOLERANCE INDUCED BY GENE THERAPY

P938 Aims: Reconstitution of lethally irradiated B10.AKM (H-2Kk) mice with syngeneic bone marrow cells infected with retroviruses carrying the allogeneic MHC class I gene H-2Kb resulted in stable and lifelong expression of Kb on bone marrow-derived cells. More importantly, these mice were specifically tolerant to H-2Kb skin grafts while still retaining the ability to reject third party skin grafts. Using CD8 transgenic mice, we discovered that alloreactive CD8 T cells underwent negative selection in the thymus, leading to the absence of these potentially alloreactive T cells in the periphery. In this study, we utilize Tg361 CD4 transgenic mice (CBA/Ca (H-2k)) expressing a TCR specific for the MHC class I gene H-2Kb to determine the fate of potentially alloreactive CD4 T cells using our gene therapy protocol. Methods: Tg361 CD4 TCR transgenic mice which express an alloreactive TCR on CD4 T cells specific for Kb were utilized to determine the mechanism by which genetic engineering of bone marrow induces donor specific CD4 T cell tolerance after bone marrow transplantation. Lethally irradiated B10.AKM mice were reconstituted with a 6:1 ratio of mock or Kb-transduced B10.AKM bone marrow to Tg361 bone marrow. Starting at four weeks after bone marrow transplantation, mice were analyzed for H-2Kb expression as well as for the development of Tg361 CD4 T cells. Results: Lethally irradiated recipients that received CD4 transgenic bone marrow in combination with autologous bone marrow transduced with virus encoding H-2Kb displayed stable and long-term expression of Kb on bone marrow-derived cells. Alloreactive CD4 transgenic T cells were readily detectable in the peripheral blood, spleen and thymus of chimeric recipients; although their levels were 2-fold lower than mock-transduced bone marrow recipients. Despite the presence of potentially alloreactive CD4 T cells in the periphery, chimeric recipients were specifically tolerant to H-2Kb expressing skin grafts. Interestingly, chimeric mice which received Kb-transduced and CD4 transgenic bone marrow expressed CD25 on 20-30% of Tg361-derived CD4 T cells, compared to only 6% of Tg361-derived CD4 T cells in mock transduced controls. Conclusions: We conclude that genetic engineering of bone marrow induces donor specific CD4 T cell tolerance through deletional and non-deletional mechanisms. We further hypothesize that expression of Kb on bone marrow derived cells induces CD4 T cells capable of inhibiting alloreactive T cells.

Intrathymic injection of anti-Fas monoclonal antibody prolongs murine non-vascularized cardiac allograft survival.

Fas is a pro-apoptotic molecule involved in activation-induced cell-death of T lymphocytes, central and peripheral tolerance and immune privilege. We sought to determine the role of Fas in the thymus after organ transplantation. Heterotopic non-vascularized heart transplants from C3H mice were placed in C57Bl/6 recipients. A hamster anti-mouse anti-Fas mAb (JO2) was injected in the thymus of allograft recipients at the time of transplant. Results were compared with intrathymic injections of hamster IgG (HIgG), anti-FasL, as well as surgical thymectomy or intraperitoneal or intravenous injections of JO2 as controls. Intrathymic injection of 5 microg JO2 induced massive thymocyte apoptosis and enhanced allograft survival compared to HIgG (median graft survival 16 days vs 12.5 days, respectively, P=0.01). The effect was receptor and ligand specific. Intraperitoneal or intravenous injections of JO2 did not prolong graft survival. Thymocyte apoptosis was confirmed in vitro, in vivo and in situ. In the thymus, double positive immature CD4+8+ thymocytes were most susceptible to Fas-induced apoptosis. Our data shows that specific modulation of Fas pathways in the thymus at the time of transplant improves modestly but significantly murine heterotopic non-vascularized cardiac allograft survival and is associated with apoptosis of immature CD4+8+ thymocytes.

CD25+ immunoregulatory T-cells of donor origin suppress alloreactivity after BMT.

We have previously identified donor-derived Thy1+ alphabeta T-cell receptor (TCR)+ CD4+ CD8- regulatory T-cells that suppress GVH reactivity induced by donor leukocyte infusion (DLI) after BMT. These cells develop in the recipient thymus and may play a role in the maintenance of donor-host tolerance after allogeneic BMT. In the present study, we sought to further characterize the T-cells responsible for the regulatory cell activity in our model. Lethally irradiated recipient AKR mice (H-2k) received transplants of BM from CD25-deficient (-/-) C57BL/6 mice (H-2b). Recipients of CD25-deficient BM developed more severe GVHD after DLI than did recipients of normal BM, a result that indirectly suggests that CD4+ CD25+ regulatory T-cells are important to the suppression of GVH reactivity after allogeneic BMT. GVHD was accompanied by mortality, body weight loss, and elevated percentages of T-cells from the DLI in the peripheral blood in mice that received CD25-deficient BM compared to mice that received normal BM. Both CD40-CD40L and CD28-B7 costimulatory pathways have been implicated in the generation of CD25+ regulatory T-cells. Therefore, we tested whether deficiency in either of these pathways affected the activity of donor BM-derived regulatory T-cells. The absence of CD40L did not affect the regulatory T-cells (ie, recipient mice were still protected from DLI-induced GVHD). In contrast, use of marrow from CD28-deficient mice resulted in complete loss of suppression of GVH reactivity. Thus, CD28 but not CD40L was critical for the generation and/or activation of immunoregulatory T-cells that suppressed GVHD induced by DLI. Together, the results of these experiments suggest that CD4+ CD25+ regulatory T-cells suppress GVH reactivity after BMT and that CD28 expression is indispensable for the generation of these cells.

Immunologic mechanisms in tolerance produced in mice with nonradiation-based lymphoablation and donor-specific bone marrow.

These experiments evaluate the mechanisms associated with tolerance in mice treated with sirolimus, antilymphocyte serum (ALS), and donor-specific bone marrow (BM). Tolerance to fully MHC-incompatible skin allografts was induced as follows: C57Bl/10 (H2b) recipients received 0.5 mL of rabbit anti-mouse polyclonal ALS on days -1, +2, and +5 relative to B10.A (H2k) donor skin grafting on day 0. Sirolimus was given in a single dose (24 mg/kg intraperitoneally) on day 6. Freshly harvested B10.A (H2k) donor-specific BM was administered at a dose of 25 x 10(6) (ALS/BM25/sirolimus) or 150 x 10(6) (ALS/BM150/sirolimus) cells intravenously on day 7. Skin allograft survival was correlated with the recipient's immunologic status. Recipients were assayed for suppressor cell activity (mixed lymphocyte coculture assays), clonal deletion (T-cell receptor Vbeta11 assay), peripheral and thymic chimerism (flow cytometry and reverse transcriptase polymerase chain reaction), and anergy (response to exogenous interleukin 2). Mice treated with ALS/BM25/sirolimus showed specifically prolonged but not indefinite allograft survival (median survival time 116 days). Allograft survival correlated with donor-specific clonal deletion and the presence of donor class II mRNA in the recipient's thymus. Mice given the ALS/BM150/sirolimus protocol showed indefinite donor-specific tolerance. Tolerance could not be broken with the administration of high doses of interleukin 2. Splenocytes taken from mice 14 days after tolerance induction inhibited donor-specific and third-party mixed lymphocyte culture proliferation in a dose-dependent fashion. This suppression could be ablated by depleting splenocytes of cells of donor origin before use in coculture. Clonal deletion was detectable 30 days after tolerance induction in mice treated with ALS/BM150/sirolimus and was maintained indefinitely. Induction of tolerance by ALS, BM, and sirolimus results in a state of donor-specific tolerance, and multilineage chimerism evolves that is permanent and associated with clonal deletion of alloreactive T cells.

Different contributions of thymopoiesis and homeostasis-driven proliferation to the reconstitution of naive and memory T cell compartments.

Following transfer into lymphopenic hosts, naive CD8 T cells proliferate and acquire memory phenotype. Although the acquired phenotype is stable in recombination activating gene-1-deficient (RAG-/-) recipients, in sublethally irradiated mice naive CD8 T cells of donor origin gradually accumulate. The naive cells have been attributed to phenotypic reversion of homeostatic memory cells, implying instability of memory phenotype and restoration of the naive T cell compartment by homeostasis-driven proliferation. We show here that (i) the accumulation of naive CD8 T cells of donor origin only occurs in recipients that have been irradiated and have an intact thymus; (ii) the apparent reversion of memory to naive cells actually results from de novo T cell development of hematopoietic stem cells, present in the donor spleen or lymph node cell populations, in the thymus of irradiated recipients; and (iii) the number of homeostatic memory cells generated in both RAG-/- and irradiated hosts reaches a plateau value and their phenotype is stably maintained even after retransfer into nonirradiated normal mice for 30 days. These findings demonstrate that homeostatic memory T cells do not revert to naive cells. After severe T cell depletion homeostasis-driven proliferation restores only the memory T cell compartment, whereas thymopoiesis is required for the reconstitution of the naive T cell compartment.

T Cell Developmental Defects in ‘Viable Motheaten’ Mice Deficient in SHP-1 Protein-Tyrosine Phosphatase. Developmental Defects are Corrected in vitro in the Presence of Normal Hematopoietic-Origin Stromal Cells and in vivo by Exogenous IL-7

Defects in the gene that encodes SHP-1 protein tyrosine phosphatase result in multiple hematopoietic abnormalities and generalized autoimmunity in viable motheaten (mev) mice. These mice also exhibit early thymic involution and abnormalities in T cell development. Here, we describe the use of fetal thymic organ culture (FTOC) and bone marrow adoptive transfer to study the effects of SHP-1 deficiency on thymocyte development. Chimeric FTOC established with normal bone marrow placed onto deoxyguanosine-treated fetal thymic lobes or onto scid fetal thymic lobes generated T cells. Bone marrow from SHP-1-deficient mev/ mev mice generated decreased numbers of T cells in chimeric FTOC established using deoxyguanosine-treated thymi but generated normal numbers in chimeric FTOC established using scid thymi. However, scid fetal thymi seeded with mev/mev bone marrow also exhibited morphological abnormalities and contained elevated numbers of macrophages. Addition of IL-7 to mev/ mev bone marrow-seededscid FTOC led to increased cell numbers, particularly of macrophages. Intrathymic injection of IL-7 partially restored the ability of progenitor cells in mev/ mev bone marrow to populate the thymus of adoptive recipients. We conclude that abnormal T cell development in mev/mev mice may in part be due to defects in the ability of bone marrow-derived accessory cells to provide bioavailable IL-7 to developing thymocytes.

Thymic re-entry of mature activated T cells and increased negative selection in vascularized allograft recipients.

Transplantation tolerance is a dynamic state that involves several homeostatic mechanisms intrinsic to the host. One of these mechanisms is activation-induced T cell death (AICD). However, it is unclear where AICD takes place during alloreactive responses. Since activated T cells can re-enter the thymus, we hypothesized that mature T cells activated by an allograft could be deleted upon re-entry into the thymus. To test this hypothesis, we used wild-type or 2C TCR transgenic mice receiving syngeneic or allogeneic heterotopic, vascularized heart grafts. First, we demonstrated that ex vivo CFSE-labelled T cells re-entered the thymus when transferred into allograft recipients but not when transferred into isograft recipients. Next, we compared the changes in cell subset numbers and incidence of apoptosis in the thymi and spleens of allograft or isograft recipients. Seven days after transplantation, at a time in which all the allografts were undergoing rejection, cells expressing donor-MHC class II molecules had migrated to the thymus and to the spleen. In the thymus of allograft recipients, overall cellularity was significantly reduced by 40% and associated with an increase in the number of double negative (CD4-CD8-) thymocytes and a decrease in double positive (CD4+CD8+) thymocytes, consistent with increased negative selection of thymocytes. Additionally, thymi of allograft recipients showed an increase in the number of recently activated, mature T cells (TCRhi, CD25+, CD44+) and a significant increase in the number of apoptotic cells, especially in the thymic medulla, that involved mature T cells as indicated by the TCRhi, CD44+, CD4 or CD8 single positive phenotype. Spleens of allograft recipients were increased in size and cellularity but did not show any of the changes in cell subsets seen in the thymi. Our data show that after allografting there is an increase in apoptotic cell death that is associated with negative selection of developing thymocytes as well as of alloreactive mature T cells that have re-entered the thymus upon activation in the periphery. This may occur upon migration of graft-derived antigen-presenting cells to the thymus.

Thymic microchimerism correlates with the outcome of tolerance-inducing protocols for solid organ transplantation.

This study found that pretransplant infusion of donor peripheral blood leukocytes, either total leukocytes (peripheral blood leukocytes) or peripheral blood mononuclear cells (PBMC), under appropriate immunomodulating conditions was more effective than donor bone marrow (BM) in prolonging the survival of rats that received kidney grafts. A higher percentage of MHCII(+) cells was found in donor PBMC than in BM cells, and depletion of MHCII(+) cells from donor PBMC abolished their tolerogenic potential. By the analysis of microchimerism in rats infused with donor cells and killed at different time points thereafter, the better tolerogenic potential of leukocyte infusion related to a higher capability of these cells to engraft the recipient thymus. PCR analysis on OX6-immunopurified cells revealed the presence of donor MHCII(+) cells in the thymus of these animals. The role of intrathymic microchimerism was reinforced by findings that thymectomy at the time of transplant prevented tolerance induction by donor leukocytes. Donor DNA was found in the thymus of most long-term graft animals that survived, but in none of those that rejected their grafts. The presence of intrathymic microchimerism correlated with graft survival, and microchimerism in other tissues was irrelevant. PCR analysis of DNA from thymic cell subpopulations revealed the presence of donor MHCII(+) cells in the thymus of long-term surviving animals. Thus, in rats, donor leukocyte infusion is better than donor BM for inducing graft tolerance, defined by long-term graft survival, donor-specific T cell hyporesponsiveness, and reduced interferon gamma production. This effect appears to occur through migration of donor MHCII(+) cells in the host thymus.

CD8+T Cells Induce Medullary Thymic Epithelium and CD4+CD8+CD25+TCRβ−Thymocytes in SCID Mice

During T-cell development the transition in the thymus of CD4-CD8- double negative (DN) progenitor T cells into CD4+CD8+ double positive (DP) cells is dependent on the expression of a T-cell receptor (TCR)-beta-chain protein. In this study purified peripheral CD4+ and CD8+ T lymphocytes from the C.B-17 strain of mice were adoptively transferred into syngeneic, neonatal SCID mice, where donor cells resided at constant numbers in thymus from 2 weeks until 10 weeks post cell transfer. In the recipient thymus the CD8+ donor cells outnumbered the CD4+ cells by a factor of three to five and both subsets contained a large fraction of activated cells. During the late phase of treatment, CD8+ T cells induced high numbers of DP thymocytes in the SCID mice, a process accompanied by the maturation of medullary epithelial cells. Such thymic development in the SCID mouse was inhibited by coresiding CD4+ donor T cells. These results indicate a regulatory role by mature peripheral T cells on medullary epithelial growth and thymocyte development in the treated SCID mice.

Characterization of thymus-seeding precursor cells from mouse bone marrow

AbstractThe nature of the cells that seed the thymus of an irradiated recipient after intravenous (IV) transfer of bone marrow (BM) cells was investigated using 2 approaches. First, direct entry of a small number of donor BM cells into the thymus was tracked using a Ly-5 marker. Second, secondary IV transfer of the seeded thymus cells into a secondary recipient was used as an assay for precursor activity. A range of cell types was found to enter the recipient thymus initially, including B-lineage cells and myeloid cells, but T precursors were undetectable by flow cytometry over the first few days. Although all cells initially entering the thymus proliferated, no sustained thymus reconstitution was seen until day 4, when recognizable T-lineage precursors began to appear. The secondary transfer assays revealed the presence of lymphoid precursors in the recipient thymus, including T, NKT, NK, and B precursor activity, with a notable early burst of B-lineage generative capacity. There was no evidence of sustained myeloid precursor or multipotent stem cell activity, even though these were seen if BM cells were injected directly into the recipient thymus rather than introduced into the bloodstream. It is concluded that even though many cell types may initially enter an irradiated thymus, the thymus acts as a sieve, allowing lymphoid precursors, but not multipotent stem cells, to seed the environmental niches that permit selected precursor cell development and thymus reconstitution.

Mechanisms of acquired thymic tolerance: induction of transplant tolerance by adoptive transfer of in vivo allomhc peptide activated syngeneic T cells.

Our most recent observation that i.v. injection of Wistar-Furth (WF) major histocompatibility complex Class I peptide 5 (P5)-pulsed self-myeloid or lymphoid dendritic cells (DC) induces transplantation tolerance suggests that adoptive transfer of in vivo allopeptide-primed host T cells might induce acquired tolerance through their interaction with thymic DC. To examine this hypothesis, host myeloid DC cultured in rat granulocyte/macrophage colony stimulating factor and interleukin 4 were pulsed in vitro with P5 and injected intravenously into syngeneic ACI rats. The T cells primed to P5 via the indirect pathway of allorecognition were harvested 7 days later and administered by either intravenously or intrathymically into syngeneic ACI recipients of WF cardiac allografts. Syngeneic T cells obtained from the spleen of P5-primed rats had a high mixed lymphocyte reaction proliferative response to P5 presented by self-DC. I.v. administration of 2x107 P5-primed alloreactive purified host splenic T cells alone on day -7 significantly (P<0.001) prolonged cardiac allograft survival from 10.5+/-1.0 days to 18.6+/-1.8 days in the WF-to-ACI rat combination. I.v. injection of P5-activated host T cells combined with 0.5 ml antilymphocyte serum (ALS)-transient immunosuppression on day -7 led to 100% donor-specific permanent graft survival (>200 days). Thymectomy before i.v. injection of P5-activated syngeneic T cells led to acute graft rejection, suggesting that the homing of in vivo activated T cells to the host thymus might play a role in the induction of tolerance. To further define the role of the recipient thymus in this model, we examined the effects of intrathymic (i.t.) injection of P5-primed alloreactive T cells on graft survival and found that i.t. administration of the P5-primed T cells on day -7 alone significantly prolonged graft survival (15.0+/-0.7 days) and when combined with 0.5 ml ALS led to donor-specific permanent graft survival. The long-term unresponsive recipients permanently (>100 days) accepted second-set donor-specific cardiac allografts but not third-party (Lewis) grafts. These findings demonstrate that the adoptive transfer of splenic T cells primed to an indirectly presented donor peptide induces transplantation tolerance in a transiently immunosuppressed secondary syngeneic recipient. Our data suggest that the interaction of thymic DC with activated peripheral T cells induces alloantigen (Ag)-specific T-cell tolerance by either inactivation or deletion of alloreactive T cells in the thymus. This observation provides the first formal evidence that the interaction between thymic DC and activated peripheral T cells that continuously circulate through the thymus plays an important role in the induction and maintenance of Ag-specific tolerance.

Cells that are not our own: the benefits and secrets of chimerism

Each of us has a characteristic set of tissue markers defining our identity; transplantation demonstrates that non-self tissues are frequently rejected. However, a general principle that we always reject non-self cannot universally be applied: following solid organ transplantation, leukocytes migrate into the new host and frequently establish a stable mixture, or microchimerism. This state is usually associated with improved graft survival. Two new studies shed light on chimerism of lymphocytes and endothelial cells in transplantation. Anderson and Matzinger 1 Anderson C.C Matzinger P Immunity or tolerance: opposite outcomes of microchimerism from skin grafts. Nat. Med. 2001; 7: 80-87 Crossref PubMed Scopus (107) Google Scholar have explored the phenomenon of microchimerism using skin grafts in mice; skin donors were selected for deficiencies of T cells, B cells or both. It was observed that chimerism and acceptance of skin grafts was only demonstrable in young recipients; some mice which differed significantly from the donors showed acceptance or tolerance to donor leukocytes but not the skin graft, which was rejected. In this model the chimeric cells were donor T memory cells, blood-borne passengers from the graft which were found to migrate to the recipient's thymus. If present at levels of one passenger cell for every 1000 recipient thymocytes, all CD8 cells specific for donor antigens were deleted, and tolerance developed to the skin graft. In mature hosts, although the chimeric cells persisted, no tolerance was established, and the antigens on the passenger cells resulted in rejection. The authors discuss a series of practical clinical applications that result from these findings: they referred to chimerism as a double-edged sword, which might be exploited to good effect in patients. Lagaaij and colleagues 2 Lagaaij E.L et al. Endothelial cell chimerism after renal transplantation and vascular rejection. Lancet. 2001; 357: 33-37 Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar reviewed biopsies from 26 renal transplants, 13 of which showed no evidence of rejection, using immunohistochemical staining for ABO antigens and class 1 MHC antigens, as well as in situ hybridization for X and Y antigens. Their techniques were developed in order to identify the origins of endothelial cells lining the blood vessels of the grafts. It was found that there was a strong correlation between the presence of host endothelial cells in the biopsies and rejection. In the seven biopsies collected from kidneys with evidence of vascular rejection, six had more than a third of their endothelium replaced by host or recipient cells: these proportions were lower in kidneys showing signs of interstitial rejection. Endothelial cell chimerism was therefore associated with rejection in renal grafts.

A novel application of cyclosporine A in nonmyeloablative pretransplant host conditioning for allogeneic BMT

The treatment of mice with anti-CD4 and anti-CD8 monoclonal antibodies (mAbs) on day −5, plus 3 Gy whole body irradiation (WBI) and 7 Gy thymic irradiation (TI) on day 0, allows fully major-histocompatibility-complex–mismatched allogeneic bone marrow engraftment and the induction of immunologic tolerance. TI is required in this model to overcome alloreactivity and possibly to make “space” in the recipient thymus so that lasting central tolerance can be achieved. In addition to suppressing mature T cells in the periphery, Cyclosporine A (CYA) and glucocorticoids have a powerful influence on the thymus. In this study, we evaluated whether the administration of CYA to recipient mice for 12 days prior to bone marrow transplant (BMT), of glucocorticosteroids on the day of BMT, or a combination of both, could create space and overcome alloresistance in the thymus by specifically depleting immature and mature thymocytes prior to BMT. High levels of multilineage donor hematopoietic repopulation and specific transplantation tolerance were achieved in mice treated from days −15 to −3 with CYA (20 mg/kg/d subcutaneously), anti-CD4/CD8 mAbs on day −5, followed by 3 Gy WBI and 15 × 106 allogeneic bone marrow cells on day 0. Vβ analysis suggested a central deletional tolerance mechanism. The same treatment without CYA pretreatment allowed only transient chimerism, without tolerance. Corticosteroid treatment abolished the engraftment-promoting and tolerance-inducing effects of CYA. These results demonstrate a novel pretransplantation-only application of CYA, which facilitates allogeneic marrow engraftment with minimal conditioning, by creating thymic space and/or overcoming intrathymic alloresistance.

Partial deletion of donor T cells in the recipient thymus in fully xenogeneic mouse-to-rat chimeras

Partial deletion of donor T cells in the recipient thymus in fully xenogeneic mouse-to-rat chimeras

A novel application of cyclosporine A in nonmyeloablative pretransplant host conditioning for allogeneic BMT

AbstractThe treatment of mice with anti-CD4 and anti-CD8 monoclonal antibodies (mAbs) on day −5, plus 3 Gy whole body irradiation (WBI) and 7 Gy thymic irradiation (TI) on day 0, allows fully major-histocompatibility-complex–mismatched allogeneic bone marrow engraftment and the induction of immunologic tolerance. TI is required in this model to overcome alloreactivity and possibly to make “space” in the recipient thymus so that lasting central tolerance can be achieved. In addition to suppressing mature T cells in the periphery, Cyclosporine A (CYA) and glucocorticoids have a powerful influence on the thymus. In this study, we evaluated whether the administration of CYA to recipient mice for 12 days prior to bone marrow transplant (BMT), of glucocorticosteroids on the day of BMT, or a combination of both, could create space and overcome alloresistance in the thymus by specifically depleting immature and mature thymocytes prior to BMT. High levels of multilineage donor hematopoietic repopulation and specific transplantation tolerance were achieved in mice treated from days −15 to −3 with CYA (20 mg/kg/d subcutaneously), anti-CD4/CD8 mAbs on day −5, followed by 3 Gy WBI and 15 × 106 allogeneic bone marrow cells on day 0. Vβ analysis suggested a central deletional tolerance mechanism. The same treatment without CYA pretreatment allowed only transient chimerism, without tolerance. Corticosteroid treatment abolished the engraftment-promoting and tolerance-inducing effects of CYA. These results demonstrate a novel pretransplantation-only application of CYA, which facilitates allogeneic marrow engraftment with minimal conditioning, by creating thymic space and/or overcoming intrathymic alloresistance.

Evidence for migration of donor bone marrow stromal cells into recipient thymus after bone marrow transplantation plus bone grafts: A role of stromal cells in positive selection.

Intrathymic T-cell differentiation is characterized by two selection events: positive and negative selection. It has been shown that thymic epithelial cells in the cortex are involved in the positive selection, while macrophages and dendritic cells, derived from hemopoietic stem cells, are involved in the negative selection. Here we investigate whether donor-derived bone marrow stromal cells can migrate into the thymus and participate there in positive selection after bone marrow transplantation plus bone grafts (to recruit bone marrow stromal cells). Allogeneic bone marrow transplantation with or without bone grafts was carried out in the [C57BL/6-->C3H] combination. Fluorescence-activated cell sorter analyses of recipient thymic adherent cells showed that donor-type bone marrow stromal cells exist in the thymus of mice that received bone marrow plus bone grafts but not in the mice that received bone marrow cells alone. Histological examination using confocal microscopy also confirmed the existence of donor-type stromal cells in the thymus of mice that received bone marrow cells plus bones. Both T-cell proliferation and plaque-forming cell assays indicated that the T cells of such mice show donor-type major histocompatibility complex-restriction. These findings strongly suggest that stromal cells can migrate from the bone marrow to the thymus, where they participate in the positive selection of thymocytes.

Soluble donor MHC class I gene transfer to thymus promotes allograft survival in a high-responder heart transplant model

Abstract Thymic selection of self and non-self-reactive lymphocytes is a process that may be targeted to induce donor-specific immunologic unresponsiveness in organ transplantation. In the present study, gene transfer was used to preexpose the recipient thymus to soluble donor-specific MHC class I molecules prior to heart transplantation in the high-responder ACI (RTla) to Lewis (RT11) rat strain combination. Specifically, cultured Lewis hepatocytes were transfected with DNA encoding a secreted form of the donor allo-MHC class I antigen, RTl.Aa. Seven days prior to ACI heart transplantation, genetically altered recipient-strain hepatocytes were injected into the thymus of Lewis recipients which also received a dose of antilymphocyte serum (ALS). Results showed that treatment with both ALS and soluble donor MHC-expressing hepatocytes prolonged transplant survival time by twofold, compared to injection of control hepatocytes and ALS. Therefore, intrathymic gene therapy delivery of soluble donor MHC molecules may be useful for promoting allograft survival in heart transplantation.