Prevention of autoimmune hearing loss in MRL/lpr mice by bone marrow transplantation

We examined the effects of bone marrow transplantation (BMT) on immune-mediated inner ear diseases in MRL/Mp-lpr/lpr (MRL/lpr) mice, which manifest not only lupus nephritis but also sensorineural hearing loss (SNHL) at the age of 20 weeks. These mice were treated with cyclophosphamide (CY) and irradiation (5 Gy x 2), followed by the transplantation of bones plus bone marrow cells from allogeneic normal C57BL/6 mice at the age of 12 weeks. Hematolymphoid cells were reconstituted with donor-derived cells 3 months after BMT. Thus-treated MRL/lpr mice showed neither lupus nephritis nor SNHL even 24 weeks after BMT. No pathological findings were observed in either glomeruli or cochleae. These findings suggest that BMT can be used to prevent the development of autoimmune SNHL in MRL/lpr mice.     

Rationale for bone marrow transplantation in the treatment of autoimmune diseases.

Transplantation of normal bone marrow from C3H/HeN nu/nu (H-2k) mice into young MRL/MP-lpr/lpr (MRL/l; H-2k) mice (less than 1.5 mo) prevented the development of autoimmune diseases and characteristic thymic abnormalities in the recipient mice. When female MRL/1 (greater than 2 mo) or male BXSB (H-2b) mice (9 mo) with autoimmune diseases and lymphadenopathy were lethally irradiated and then reconstituted with allogeneic bone marrow cells from young BALB/c nu/nu (H-2d) mice (less than 2 mo), the recipients survived for more than 3 mo after the bone marrow transplantation and showed no graft-versus-host reaction. Histopathological study revealed that lymphadenopathy disappeared and that all evidence of autoimmune disease either was prevented from developing or was completely corrected even after its development in such mice. All abnormal T-cell functions were restored to normal. The newly developed T cells were found to be tolerant of both bone marrow donor-type (BALB/c) and host-type (MRL/1 or BXSB) major histocompatibility complex (MHC) determinants. Therefore, T-cell dysfunction in autoimmune-prone mice can be associated with both the involutionary changes that occur in the thymus of the autoimmune-prone mice and also to abnormalities that reside in the stem cells. However, normal stem cells from BALB/c nu/nu donors can differentiate into normal functional T cells even in mice whose thymus had undergone considerable involution, as in the case of BXSB or MRL/1 mice in the present studies. These findings suggest that marrow transplantation may be a strategy ultimately to be considered as an approach to treatment of life-threatening autoimmune diseases in humans. T-cell dysfunction in autoimmune-prone mice previously attributed to involutionary changes that occur in the thymus of these mice may instead be attributed to abnormalities that basically reside in the stem cells of the autoimmune-prone mice.     

Alloantigen recognition is critical for CD8 T cell-mediated graft anti-tumor activity against murine BCL1 lymphoma after myeloablative bone marrow transplantation

The goal of the current study was to determine whether whole bone marrow cells or splenic CD8(+) T cells from C57BL/6 (H-2(b)) donor mice, which are tolerant to BALB/c (H-2(d)) alloantigens, are capable of mediating graft anti-tumor activity against a BALB/c B-cell lymphoma after injection into irradiated BALB/c hosts. The experimental results show that high doses of splenic CD8(+) T cells mixed with T cell-depleted bone marrow cells from C57BL/6 non-tolerant (normal) donors eliminate the BCL(1) B-cell lymphoma cells and induce lethal graft-versus-host disease (GVHD). CD8(+) T cells from tolerant donors simultaneously lose both their ability to induce GVHD and their anti-tumor activity. Whole bone marrow cell transplants from normal donors eliminated BCL(1) tumor cells without inducing GVHD, and bone marrow cells from tolerant donors failed to eliminate the tumor cells. The infused BCL(1) tumor cells expressed an immunogenic tumor-specific idiotype antigen disparate from host alloantigens, indicating that recognition of the tumor-specific antigen alone was insufficient to elicit graft anti-tumor activity from unimmunized allotolerant donor splenic CD8(+) T cells or whole bone marrow cells. We conclude that CD8(+) T cells from unimmunized normal donor mice require alloantigen recognition to mediate their anti-tumor activity following allogeneic BMT.     

Intra-bone marrow injection of allogeneic bone marrow cells: a powerful new strategy for treatment of intractable autoimmune diseases in MRL/lpr mice

Intractable autoimmune diseases in chimeric resistant MRL/lpr mice were treated by a new bone marrow transplantation (BMT) method consisting of fractionated irradiation, 5.5 Gy x 2, followed by intra-bone marrow (IBM) injection of whole bone marrow cells (BMCs) from allogeneic normal C57BL/6 (B6) mice (5.5 Gy x 2 + IBM). In MRL/lpr mice treated with this method, the number of donor-derived cells in the bone marrow, spleen, and liver rapidly increased (almost 100% donor-derived cells by 14 days after the treatment), and the number of donor-derived hemopoietic progenitor cells concomitantly increased. Furthermore, donor-derived stromal cells were clearly detected in the cultured bone pieces from MRL/lpr mice treated with 5.5 Gy x 2 + IBM. All the recipients thus treated survived more than 1 year (> 60 weeks after birth) and remained free from autoimmune diseases. Autoantibodies decreased to almost normal levels, and abnormal T cells (Thy1.2(+)/B220(+)/CD4(-)/CD8(-)) disappeared. Hematolymphoid cells were reconstituted with donor-derived cells, and newly developed T cells were tolerant to both donor (B6)-type and host (MRL/lpr)-type major histocompatibility complex determinants. Successful cooperation was achieved among T cells, B cells, and antigen-presenting cells when evaluated by in vitro antisheep red blood cell responses. These findings clearly indicate that this new strategy (IBM-BMT) creates the appropriate hemopoietic environment for the early recovery of hemopoiesis and donor cell engraftment, resulting in the complete amelioration of intractable autoimmune diseases in chimeric resistant MRL/lpr mice without recourse to immunosuppressants. This strategy would therefore be suitable for human therapy.     

Treatment of autoimmune diseases by hematopoietic stem cell transplantation

Remarkable advances have been made in bone marrow transplantation (BMT), which now has become a powerful strategy for the treatment of leukemia, aplastic anemia, congenital immunodeficiency disorders, and autoimmune diseases. Using various animal models, allogeneic BMT has been found to be useful in the treatment of autoimmune diseases. In MRL/lpr mice, which are radiosensitive (<8.5 Gy) and are an animal model for autoimmune disorders, conventional BMT resulted in only transient effects; the manifestations of the autoimmune diseases recurred 3 months after BMT. However, the combination of BMT plus bone grafts (to recruit donor stromal cells) was capable of preventing the recurrence of autoimmune diseases in MRL/lpr mice. This strategy was found to be ineffective in the treatment of MRL/lpr mice that had developed autoimmune diseases, because these mice were more sensitive to the effects of radiation after the onset of lupus nephritis due to uremic enterocolitis. We have recently discovered a safer strategy for treatment of autoimmune diseases, which includes fractionated irradiation (5.5 Gy x 2) (day -1) followed by portal venous injection (day 0) plus intravenous injection (day 5) of donor unfractionated bone marrow cells. We successfully treated autoimmune diseases in MRL/lpr mice using this strategy; 100% of MRL/lpr mice treated in this fashion survive >1 year after treatment. We identified the mechanisms underlying the components of this approach and have found that stromal cells play a crucial role in successful BMT. In this review, the conditions essential for successful allogeneic BMT are discussed.     

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.     

Treatment of intractable autoimmune diseases in MRL/lpr mice using a new strategy for allogeneic bone marrow transplantation

A new bone marrow transplantation (BMT) method for treating severe autoimmune diseases in chimeric resistant MRL/lpr mice is presented. The method consists of fractionated irradiation (5.5 Gy x 2), followed by portal venous (PV) injection of whole bone marrow cells (BMCs) from allogeneic normal C57BL/6 (B6) mice and intravenous (IV) injection of whole B6 BMCs 5 days after the PV injection (abbreviated as 5.5 Gy x 2 + PV + IV). All recipients survived more than 1 year after this treatment (more than 64 weeks after birth). Abnormal T cells (Thy1.2(+)/B220(+)/CD3(+)/CD4(-)/CD8(-)) present in MRL/lpr mice before the treatment disappear, and hematolymphoid cells are reconstituted with donor-derived cells. The treated mice are free from autoimmune diseases. Levels of autoantibodies (IgG/IgM anti-ssDNA antibodies and IgG/IgM rheumatoid factors) decrease to normal levels. Successful cooperation is achieved among T cells, B cells, and antigen-presenting cells (APCs) of the treated MRL/lpr mice when evaluated by in vitro anti-SRBC responses. Newly developed T cells are tolerant to both donor (B6)-type and host (MRL/lpr)-type major histocompatibility complex (MHC) determinants. These findings clearly indicate that severe autoimmune diseases in MRL/lpr mice are completely ameliorated by the treatment without recourse to immunosuppressants, and that the treated MRL/lpr mice show normal immune functions, strongly suggesting that this strategy would be applicable to humans. (Blood. 2000;95:1862-1868)     

Alloreactivity following in utero transplantation of cytokine-stimulated hematopoietic stem cells: the role of recipient CD4(-) cells

OBJECTIVE: We have previously reported immunity to donor antigens following in utero transplantation (IUT) of cytokine-stimulated allogeneic hematopoietic stem cells (sca(+)/lin(-)) (day 9 of gestation). Transplanted mice showed accelerated rejection of donor skin grafts and high anti-donor cytotoxic response, a finding not seen in the control mice. This was accompanied by an enhancement of Th1 over Th2 cytokine production and persistent donor microchimerism. In order to assess the role of the thymus in allograft rejection, prenatal transplants were performed under similar experimental conditions at a later gestational age, when the thymus is more developed (day 13). MATERIALS AND METHODS: Cytokine-stimulated stem cell factor (SCF) and granulocyte colony-stimulating factor (G-CSF)-purified sca(+)/lin(-) cells of C57BL/6 (H-2b, 1E(+)) background were injected into MHC-mismatched BALB/c (H-2d, 1E(-)) fetal mouse recipients at day 13 of gestation. Chimerism was determined by highly sensitive (0.001%) semiquantitative polymerase chain reaction (PCR). Mixed lymphocyte reaction (MLR) and cytotoxic T-cell assay (CTL) were used to evaluate tolerance vs immunity. Cytokine levels were quantified in MLR supernatants using ELISA assay. The percent of T cells was determined by flow cytometry (FACS) and CD4/CD8 ratio calculated. Postnatal boosts (transplants without conditioning) were performed at 6 months of age to enhance donor chimerism and test the degree of tolerance. RESULTS: When assayed at 4 months of age, donor-type cells were not detected in the spleen or in the peripheral blood of BALB/c mice inoculated with C57BL/6 sca(+)/lin(-) cells. Transplanted but not control animals demonstrated high anti-donor MLR but not CTL responses. The increase of MLR reactivity was correlated with high levels of IL-2. Furthermore, transplanted mice showed higher resistance to postnatal boosts with allogeneic bone marrow (BM) cells, when compared to the control mice. The later resistance was accompanied by the expansion of host-type CD4 cells. CONCLUSION: These data demonstrate that transplantation of cytokine-stimulated sca(+)/lin(-) allogeneic cells at 13 days of fetal development leads to the allosensitization, characterized by an enhancement of MLR alloreactivity and by the rejection of postnatal boosts (transplants without conditioning). Host-type CD4 cells might play a central role in this rejection. These findings indicate that the late injection of allogeneic cells may result in the development of allosensitization with subsequent donor graft rejection. Precise conditions for the development of tolerance must be established before prenatal transplants in humans with conditions other than SCID can be done.     

Long-term observations of autoimmune-prone mice treated for autoimmune disease by allogeneic bone marrow transplantation.

Long-term effects of allogeneic bone marrow transplantation (ABMT) across major histocompatibility complex barriers were studied in (NZB x NZW)F1 (B/W), BXSB, and MRL/Mr-lpr-lpr (MRL/lpr) mice with established autoimmune disease at the time of ABMT. In the BXSB or B/W mice, ABMT cured all aspects of autoimmune disease. Glomerular damage, revealed by histological study was dramatically improved. Serological abnormalities and immunologic functions also were normalized. Correction of autoimmune disease and advanced renal disease in BXSB and B/W mice regularly lasted greater than 5-6 mo and even 1 yr after ABMT. In the MRL/lpr mice, however, autoimmune and renal disease at first improved but then recurred after ABMT, apparently because of intolerance of mice for high doses of irradiation and a high degree of resistance of recipient stem cells to irradiation. In this model, H-2 typing revealed that by the time of relapse, immunocompetent cells of the chimeric mice had been replaced by host (MRL/lpr; H-2k) cells. B220+ Ly-1+ cells, present in increased numbers in untreated MRL/lpr mice, initially returned to normal levels after ABMT but then reappeared in the MRL/lpr mice that had received marrow from donors having few such lymphocytes. Thus, our results show that MRL/lpr mice possess abnormal radioresistant stem cells and provide impressive evidence that the origin of autoimmune diseases in this strain, as in the several other strains studied, residues in abnormalities present in stem cells.     

Successful liver allografts in mice by combination with allogeneic bone marrow transplantation.

Successful liver allografts were established by combination with allogeneic bone marrow transplantation. When liver tissue of BALB/c (H-2d) or C57BL/6J (H-2b) mice was minced and grafted under the kidney capsules of C3H/HeN (H-2k) mice, it was rejected. However, when C3H/HeN mice were irradiated and reconstituted with T-cell-depleted BALB/c or BALB/c nu/nu bone marrow cells, or with fetal liver cells of BALB/c mice, they accepted both donor (stem-cell)-type (BALB/c) and host (thymus)-type (C3H/HeN) liver tissue. Assays for both mixed-lymphocyte reaction and induction of cytotoxic T lymphocytes revealed that the newly developed T cells were tolerant of both donor (stem-cell)-type and host (thymus)-type major histocompatibility complex determinants. We propose that liver allografts combined with bone marrow transplantation should be considered as a viable therapy for patients with liver disease such as liver cirrhosis and hepatoma.     

Donor origin of multipotent adult progenitor cells in radiation chimeras

Multipotent adult progenitor cells (MAPCs) are bone marrow-derived stem cells that have extensive in vitro expansion capacity and can differentiate in vivo and in vitro into tissue cells of all 3 germinal layers: ectoderm, mesoderm, and endoderm. The origin of MAPCs within bone marrow is unknown. MAPCs are believed to be derived from the bone marrow stroma compartment as they are isolated within the adherent cell component. Numerous studies of bone marrow chimeras in the human and the mouse point to a host origin of bone marrow stromal cells. Mesenchymal stem cells (MSCs), which coexist with stromal cells, have also been proven to be of host origin after allogeneic bone marrow transplantation in numerous studies. We report here that following syngeneic bone marrow transplants into lethally irradiated C57BL6 mice, MAPCs are of donor origin.     

New strategies for BMT,organ transplantation,and regeneration therapy

Bone marrow transplantation (BMT) is becoming a powerful strategy for the treatment of hematologic disorders, congenital immunodeficiencies, metabolic disorders and also autoimmune diseases. We have previously found using various animal models for spontaneous autoimmune diseases, that allogeneic bone marrow transplantation (allo BMT) can be used to prevent and treat various autoimmune diseases. In addition, we have found that autoimmune diseases are stem cell disorders. However, in MRL/lpr mice, which are radiosensitive (<8.5 Gy), we found that conventional BMT had only a transient effect on autoimmune diseases, which were found to recur. Therefore, we concentrated on discovering new strategies to prevent and treat autoimmune diseases in the radiosensitive and chimeric-resistant MRL/lpr mouse. Using MRL/lpr mice, we established a new method for allo BMT. In this method, whole bone marrow cells (BMCs), containing a small number of T cells and mesenchymal stem cells (MSCs), were directly injected into the bone marrow cavity (intra-bone marrow [IBM]-BMT). MRL/lpr mice treated with IBM-BMT survived more than 2 years without showing the symptoms of autoimmune diseases. To apply this BMT method to humans, we have also established a new method for BMC harvesting using cynomolgus monkeys. In this method, BMCs are harvested from the long bones using a "Perfusion Method" (PM) and the whole BMCs (including MSCs) are then injected directly into the IBM. We believe that this new method will become a powerful strategy for the treatment of various intractable diseases, including age-associated diseases such as osteoporosis.     

New Strategies for BMT,Organ Transplantation,and Regeneration Therapy

Bone marrow transplantation (BMT) is becoming a powerful strategy for the treatment of hematologic disorders, congenital immunodeficiencies, metabolic disorders and also autoimmune diseases.

We have previously found using various animal models for spontaneous autoimmune diseases, that allogeneic bone marrow transplantation (allo BMT) can be used to prevent and treat various autoimmune diseases. In addition, we have found that autoimmune diseases are stem cell disorders. However, in MRL/lpr mice, which are radiosensitive (<8.5 Gy), we found that conventional BMT had only a transient effect on autoimmune diseases, which were found to recur. Therefore, we concentrated on discovering new strategies to prevent and treat autoimmune diseases in the radiosensitive and chimeric-resistant MRL/lpr mouse.

Using MRL/lpr mice, we established a new method for allo BMT. In this method, whole bone marrow cells (BMCs), containing a small number of T cells and mesenchymal stem cells (MSCs), were directly injected into the bone marrow cavity (intra-bone marrow [IBM]-BMT). MRL/lpr mice treated with IBM-BMT survived more than 2 years without showing the symptoms of autoimmune diseases.

To apply this BMT method to humans, we have also established a new method for BMC harvesting using cynomolgus monkeys. In this method, BMCs are harvested from the long bones using a "Perfusion Method" (PM) and the whole BMCs (including MSCs) are then injected directly into the IBM. We believe that this new method will become a powerful strategy for the treatment of various intractable diseases, including age-associated diseases such as osteoporosis.     

Specific tolerance across a discordant xenogeneic transplantation barrier.

Successful induction of tolerance across disparate (discordant) species barriers could overcome the organ shortage that presently limits clinical transplantation. We demonstrate here that xenogeneic swine thymic transplants can induce tolerance to swine antigens in mice, while positively selecting functional host CD4+ T cells. Immunologically normal C57BL/10 mice were thymectomized and depleted of T and natural killer cells; then they received transplants of fetal pig thymus and liver fragments. Mature mouse CD4+ T cells developed in the pig thymus grafts and migrated to the periphery. Swine grafts grew markedly and no anti-pig IgG response was produced. Mixed lymphocyte reactions confirmed that the new T cells were functional and were tolerant to pig antigens.     

Transplantation of wheat germ agglutinin-positive hematopoietic cells to prevent or induce systemic autoimmune disease.

Hematopoietic stem cell defects are thought to be involved in the etiopathogenesis of systemic autoimmune disease. Positively selected, stem cell-enriched populations of wheat germ agglutinin-positive (WGA+) low-density bone marrow and fetal liver cells from normal and autoimmune-prone mice were used to determine whether reciprocal transplantation of stem cells between normal and autoimmune-prone mice inhibits or causes development of autoimmune disease. NZB recipients of DBA/2 stem cell populations analyzed greater than 100 days after bone marrow or fetal liver cell transplantation showed decreased levels of anti-DNA antibodies and decreased glomerular lesions when compared with nontreated NZB mice or NZB recipients of NZB stem cell preparations. Female DBA/2 recipients of WGA+ NZB bone marrow cell or fetal liver cell transplants exhibited elevated serum autoantibody levels and developed glomerular lesions characteristic of NZB mice when analyzed greater than 100 days after transplantation. These pathological disturbances were not observed in DBA/2 recipients of DBA/2 stem cell preparations. The data indicate that WGA+ stem cells from autoimmune-prone NZB mice contain the genetic defects responsible for the development of systemic autoimmune disease.     

Hemopoietic stem cell transplantation using mouse bone marrow and spleen cells fractionated by lectins.

Mouse bone marrow and spleen cells were fractionated with the aid of soybean agglutinin and peanut agglutinin. A test for spleen colony-forming units in the isolated fractions showed that the hemopoietic stem cells are agglutinated by both of these lectins. The capacity of the agglutinated fractions to reconstitute lethally irradiated allogeneic mice was investigated. A sequential fractionation of splenocytes from SWR donors by soybean agglutinin and peanut agglutinin, or a single fractionation by soybean agglutinin of splenocytes from BALB/c donors, afforded a cell fraction that successfully reconstituted lethally irradiated (BALB/c X C57BL/6)F1 mice, without complications due to graft-versus-host reaction.     

Addition of a second, different allogeneic graft accelerates white cell and platelet engraftment after T-cell-depleted bone marrow transplantation

Significant delays in engraftment and lymphoid recovery are the 2 major challenges in cord blood transplantation. The cause for this delay is presumed to be the low numbers of hematopoietic precursors found in one unit of cord blood. One approach to increase the stem cell doses could be to combine cord blood units from different donors differing at the major histocompatibility complex (MHC). As a first step toward this goal, the kinetics of hematologic engraftment and immune reconstitution were compared between 1 unit (2.5 x 10(6) cells) of T-cell-depleted bone marrow cells from a single donor (C57BL/6 [H2(b)] or SJL/J [H2(s)]) and 2 units from different donors (C57BL/6 + SJL/J) after transplantation into lethally irradiated (8.5 Gy) BALB/c recipients (H2(d)). Addition of T-cell-depleted bone marrow from an MHC-mismatched allogeneic donor doubled the white blood counts compared with recipients of one single unit on days +10 and +14. Similar effects were also observed on platelets. The beneficial effect of additional cells on peripheral T-cell counts were first observed on day +14. Cells both from donors (C57BL/6 and/or SJL/J) and recipients (BALB/c) contributed to myeloid and lymphoid reconstitution. The chimeras containing cells from 3 strains of mice were able to mount a recall immune response. Our data suggest that combining stem cells from MHC-mismatched allogeneic donors is feasible, that it has beneficial effects on myeloid engraftment and T-cell phenotypic recovery, and that the long-term stable mixed chimeras are immunologically normal following T-cell-depleted bone marrow transplantation.     

Treatment of autoimmune diseases in mice by a new method for allogeneic bone marrow transplantation

Remarkable advances have been made in bone marrow transplantation (BMT), which has become a powerful strategy for the treatment of leukemia, aplastic anemia, congenital immunodeficiency, and also autoimmune disease. Using various animal models, allogeneic (allo) BMT has been found to be useful in the treatment of various autoimmune diseases. In MRL/lpr mice, which are radiosensitive (<8.5 Gy) and are an animal model for autoimmune diseases, conventional BMT resulted in only transient effects; the manifestations of the autoimmune diseases recurred 3 months after BMT. Using MRL/lpr mice, we have very recently established a new strategy for allo BMT. We injected bone marrow cells (BMC) directly into the bone marrow cavity (intrabone marrow [IBM] injection) of recipients that had received fractionated irradiation. This 'IBM-BMT' was found to be effective in treating autoimmune diseases in radiation-sensitive and chimeric-resistant MRL/lpr mice. In addition, this strategy was found to be applicable for the transplantation of organs. We believe that these strategies for BMT and organ transplantation herald a new era in transplantation.     

Studies of immunologic tolerance to host minor histocompatibility antigens following allogeneic bone marrow transplantation in mice

We showed previously that transplantation of 10(7) unmanipulated C57BL/6 marrow cells to irradiated LP mice yields healthy (B6-LP) chimeras showing no signs of rejection or graft-versus-host disease (GVHD). The aim of this work was to gain more insight into the mechanism(s) responsible for tolerance to host minor histocompatibility antigens following allogeneic bone marrow transplantation (BMT). (B6-LP) chimeras showed very good immune reconstitution when studied in vitro for proliferative response to mitogens and alloantigens and generation of T cell cytotoxic activity. In co-culture experiments their spleen cells showed no natural suppressor activity. When used as cell donors, their capacity to initiate GVHD in four strains of mice presenting H-2 differences was normal when compared to C57BL/6 donors. However, they provoked no GVHD in the three strains of H-2 compatible mice studied. Re-irradiated (B6-LP) chimeras rapidly died of GVHD following injection of C57BL/6 marrow + spleen cells. (B6-LP.R111) chimera cells appeared tolerant to LP minor antigens presented in the context of H-2r or H-2b. No anamnestic anti-idiotypic suppressor response was noted when stable (B6-LP) chimeras were stimulated with naive C57BL/6 cells. These findings suggest that in BMT chimeras transplanted across minor histocompatibility barriers: (1) both host and donor-derived antigen-presenting cells can present host antigens to donor T cells whose numbers in the marrow inoculum will determine if GVHD or tolerance will ensue, (2) GVHD can be triggered by only a limited number of 'dominant' minor antigens, and (3) we found no evidence for the presence of natural suppressors, veto cells or anti-idiotypic suppressor T cells.     

Organ-specific and systemic autoimmune diseases originate from defects in hematopoietic stem cells.

Transplantation of bone marrow cells from nonobese diabetic (NOD) mice, a model for type 1 diabetes mellitus, to C3H/HeN mice, which express I-E alpha molecules and have aspartic acid at residue 57 of the I-A beta chain, induced insulitis followed by overt diabetes in the recipient C3H/HeN mice more than 40 weeks after bone marrow transplantation. When cyclosporin A, which perturbs T-cell functions, was injected intraperitoneally into [NOD----C3H/HeN] chimeric mice daily for 1 month, the chimeric mice developed insulitis and overt diabetes within 20 weeks following bone marrow transplantation. Transplantation of bone marrow cells from (NZW x BXSB)F1 mice, which develop lupus nephritis, myocardial infarction, and idiopathic thrombocytopenic purpura, into C3H/HeN or C57BL/6J mice induced in the recipient strains both lupus nephritis and idiopathic thrombocytopenic purpura more than 3 months after transplantation. Transplantation of a stem-cell-enriched population from (NZW x BXSB)F1 mice into normal mice also induced autoimmune disease in the recipients. These results indicate that both systemic autoimmune disease and organ-specific autoimmune disease originate from defects that reside within the stem cells; the thymus and environmental factors such as sex hormones appear to act only as accelerating factors.