Triple opportunistic pulmonary cavitary disease after cord blood transplantation.

Opportunistic infectious diseases in patients are variable and depend on the host as well as the type of immunosuppression. Cord blood transplant recipients appear to be particularly vulnerable to infectious complications. Sequential or concurrent opportunistic infectious diseases can be particularly difficult to manage and have increased mortality. We present a young patient, status post cord blood transplantation for acute myelogenous leukemia, who developed a large pulmonary mass-like infection with Aspergillus, cytomegalovirus, and Mycobacterium avium complex. Radiological, surgical, and pathological features are described.

Characterization of a newly discovered T-cell receptor beta-chain heterodimer expressed on a CD8+ bone marrow subpopulation that promotes allogeneic stem cell engraftment.

The facilitating cell is a rare CD8+ bone marrow subpopulation that can enhance allogeneic hematopoietic stem cell engraftment across complete major histocompatibility complex barriers without inducing acute graft-versus-host disease. Here we describe a CD3epsilon-associated complex on the facilitating cell surface that consists of the T-cell receptor beta-chain disulfide-linked to a previously unknown 33-kilodalton glycoprotein. Provisionally called FCp33, this glycoprotein does not represent any of the known protein chains or surrogates associated with CD3-T-cell receptor beta. Expression of this CD3-T-cell receptor beta-FCp33 complex directly correlates with the facilitating cell's functional ability to enhance allogeneic stem cell engraftment in vivo.

The abrogation of allosensitization following the induction of mixed allogeneic chimerism.

The association of preformed anti-donor Abs with the hyperacute rejection of bone marrow and solid organ allografts and the persistence of the anti-donor immune response secondary to immunologic memory make allosensitization an absolute contraindication to transplantation. Mixed allogeneic (A + B-->A) bone marrow chimerism has been demonstrated to confer donor-specific tolerance in nonsensitized recipients, but has not been evaluated in the setting of allosensitization. The current study documents that despite significant anti-donor sensitization, mixed allogeneic engraftment is possible and provides a marked advantage over fully allogeneic (B-->A) models. Moreover, the acceptance of donor skin grafts and loss of circulating anti-donor Abs suggest that allosensitization can be abrogated with the induction of stable mixed allogeneic chimerism.

Positive and negative selection of alphabetaTCR+ T cells in thymectomized adult radiation bone marrow chimeras.

BACKGROUND: The mature T-cell repertoire is characterized by the negative selection of potentially autoreactive T cells and the positive selection of T cells restricted to antigen-recognition in the context of self-MHC molecules. It is currently believed that the thymus is critical for these selection events. Although alpha(beta)T cell receptor (TCR)+ T cells have been reported in thymectomized recipients, whether this represents clonal expansion of residual T cells or de novo generation of new T cells in the absence of a thymus has not been definitively evaluated. METHODS: In the current study, development of the T cell repertoire was evaluated in adult radiation bone marrow chimeras prepared after complete surgical thymectomy. RESULTS: CD4+ and CD8+ T cells were present and exhibited donor-specific TCR-Vbeta expression and self-tolerance, indicative of negative selection. Positive selection was confirmed with the demonstration of host MHC restriction and the presence of donor-derived CD8+ T cells after the transplantation of marrow from Class I deficient donors into normal recipients. CONCLUSIONS: These data provide evidence, for the first time, that the development of a functional T-cell repertoire can occur in adult recipients without the thymic microenvironment.

Mixed hematopoietic chimerism induces donor-specific tolerance for lung allografts in rodents.

Mixed hematopoietic chimerism is a state in which bone marrow hematopoietic stem cells from two genetically different animals coexist. We investigated whether mixed hematopoietic chimerism, resulting from the transplantation of host and donor bone marrow into a lethally irradiated rat, would confer donor-specific tolerance to lung allografts. Recipient rats (Fisher or or Wistar Furth [WF]) were irradiated (1,100 cGy) and reconstituted with a mixture of T-cell-depleted syngeneic plus allogeneic bone marrow. After mixed chimerism was documented by the presence of donor- and host-derived cells in the peripheral blood 4 wk after bone marrow reconstitution, mixed chimeras underwent orthotopic left lung transplantation with donor-specific and third-party lung allografts. No immunosuppressive agents were administered after lung transplantation. All donor-specific lung allografts were accepted by mixed chimeras (n = 40), while all third-party grafts (n = 7) were rejected within 10 d, a time course similar to that for grafts transplanted into naive recipients (n = 14). Radiation control recipients (n = 7) who did not develop mixed chimerism because the donor bone marrow had failed to engraft, also rejected donor-specific grafts within 10 d. We conclude that mixed hematopoietic chimerism induces donor-specific transplantation tolerance to lung allografts.

A partial conditioning strategy for achieving mixed chimerism in the rat: tacrolimus and anti-lymphocyte serum substantially reduce the minimum radiation dose for engraftment.

Development of partial conditioning strategies to achieve reliable engraftment of allogeneic bone marrow with minimum recipient morbidity could extend the therapeutic application of bone marrow transplantation (BMT) to enzyme deficiency states, hemoglobinopathies, autoimmune diseases, and the induction of tolerance for solid organ and cellular allografts. In this study we describe a nonmyeloablative rat BMT model and examine the effect of clinically available immunosuppressants on the minimum amount of total body irradiation (TBI) required for allogeneic engraftment. Donor ACI marrow was depleted of T cells using immunomagnetic beads and transplanted to major histocompatibility complex- and minor antigen-mismatched Wistar Furth (WF) rats (ACI --> WF) conditioned with varying doses of TBI. Recipients conditioned with TBI alone required myeloablation with 1000 cGy for reliable allogeneic marrow engraftment. Administration to WF recipients of a single dose of anti-lymphocyte serum (ALS) 5 days prior to BMT together with a limited course of tacrolimus (1 mg/kg/day) resulted in engraftment of ACI bone marrow at only 500 cGy TBI. ACI --> WF recipients were stable mixed chimeras (mean donor chimerism 49% at 330 days post-BMT). Chimerism was multilineage. All recipient animals were free of graft-versus-host disease. These results suggest that a nonmyeloablative conditioning strategy based on low-dose TBI and a limited course of tacrolimus plus ALS can produce long-term mixed multilineage chimerism.

Mixed allogeneic chimerism prevents obstructive airway disease in a rat heterotopic tracheal transplant model.

BACKGROUND: Mixed bone marrow chimerism reliably produces donor-specific transplantation tolerance for a variety of solid organ and cellular grafts. We used a rat heterotopic tracheal transplant model for chronic rejection to investigate whether mixed chimerism could successfully prevent obstructive airway disease. METHODS: Mixed allogeneic chimeras were prepared by reconstituting lethally irradiated Wistar-Furth (WF) recipients with a mixture of 5 x 10(6) T-cell-depleted syngeneic (WF) and 100 x 10(6) T-cell-depleted allogeneic (ACI) bone marrow cells (ACI + WF --> WF). Mixed chimerism was present in all animals 28 days after bone marrow transplantation. Donor-specific, syngeneic, or major histocompatibility complex (MHC)-disparate allogeneic tracheas were implanted in recipient's omentum and removed for histologic analysis 30 to 150 days after transplantation. RESULTS: At 30 days after implantation, median luminal obstruction grades (0=none, 4=complete) of syngeneic and allogeneic tracheas were 0 and 4, respectively. Donor-specific (ACI) tracheas implanted in chimeric (ACI + WF --> WF) recipients were remarkably free of obstruction (median luminal obstruction grade=0 at 150 days) and had excellent preservation of respiratory epithelium. Third-party F344 tracheas implanted in chimeric recipients developed progressive luminal obstruction (grade 2 at 30 days, grade 3 at 90 days). CONCLUSIONS: Mixed allogeneic chimerism induces donor-specific tolerance and prevents development of the characteristic fibroproliferative obstructive lesion of bronchiolitis obliterans in a rat heterotopic tracheal transplant model. Excellent preservation of tracheal structure and morphology was achieved across major and minor histocompatibility barriers.

Addition of a bone marrow "facilitating cell" population increases stem cell-derived cobblestone area formation in impaired long-term bone marrow culture stroma.

Treatment of mouse bone marrow (BM) with rabbit anti-mouse brain serum (RAMBS) plus complement (C') depletes several cell types, including T cells and facilitating cells (FCs), that is, cells that facilitate engraftment of sorted allogeneic stem cells (SCs) in vivo. In the present study, treatment of BM with RAMBS+C' resulted in the depletion of approximately half of the late cobblestone area (CA)-forming stem cells as assayed on irradiated long-term bone marrow culture (LTBMC) stroma. In addition, LTBMC of RAMBS+C'-treated BM produced functionally impaired stroma with reduced ability to support CA formation by nontreated exogenous SCs. This stromal impairment was not due to depletion of TCRalphabeta T cells in the BM, because BM cultures from TCR alpha-chain knockout mice supported normal numbers of exogenous CAs. Because CD8+/TCR- cells are enriched for FCs, we tested the effect of adding these cells back to the treated BM prior to culture. The sorted FCs alone did not produce CAs, but did improve the ability of the impaired stroma to support late CA formation by sorted SCs. These studies provide a new model for dissecting the roles of different cellular components of BM in producing functional stroma that supports CA formation by SCs, and show that the number of CAs formed depends on the "quality" of the stroma as well as the number of SCs seeded. These findings further suggest that CD8+/TCR- BM cells may be important for the establishment of functional stroma.

Antiviral cytotoxic activity across a species barrier in mixed xenogeneic chimeras: functional restriction to host MHC.

Reconstitution of lethally irradiated mice with a mixture of mouse and rat bone marrow cells (mouse + rat-->mouse) results in mixed xenogeneic chimerism and donor-specific tolerance. The current study demonstrates that mouse and rat T lymphocytes that have developed in xenogeneic chimeras are restricted to Ag presentation by mouse, but not rat, APC. Restriction to host Ags results in functional immunocompetence with generation of antiviral cytotoxic activity in vivo, within and across species barriers. These data demonstrate for the first time that the host thymus is sufficient to support development and positive selection of functional cross-species T lymphocytes. The superior immunocompetence, as compared with fully xenogeneic (rat-->mouse) chimeras, may prove to be of significant benefit in the clinical application of xenotransplantation to solid organ transplantation and immune reconstitution for AIDS.

In vivo depletion of host CD4+ and CD8+ cells permits engraftment of bone marrow stem cells and tolerance induction with minimal conditioning.

BACKGROUND: Solid organ transplantation has become the preferred approach for the treatment of end-stage organ failure. However, the toxicity associated with the nonspecific immunosuppression essential to graft survival is substantial. Bone marrow transplantation (BMT) can overcome these limitations by the induction of donor-specific tolerance. The morbidity and mortality associated with fully ablative conditioning used to achieve engraftment has prevented the clinical application of BMT for induction of tolerance for solid organ transplantation. Although it was previously believed that fully ablative conditioning was essential to achieve engraftment, it has recently become apparent that partial conditioning may be sufficient to achieve chimerism and tolerance. The focus of this study was to characterize which cells in the host microenvironment must be eliminated for engraftment of MHC-disparate bone marrow to be achieved. METHODS: C57BL/10SnJ mice were depleted of CD4+, CD8+, or both cell types with monoclonal antibodies before irradiation with 300 centigray (cGy) and transplantation of 15 x 10(6) allogeneic (B10.BR) bone marrow cells. Two days after transplantation the animals were treated with 200 mg/kg cyclophosphamide. Animals were typed for chimerism at 28 days and monthly thereafter. RESULTS: The combination of CD4+ and CD8+ depletion resulted in multilineage engraftment in 76.5% of the animals at a level of 57.1 +/- 17.7%. The depletion of CD4+ cells alone was not sufficient to allow engraftment, whereas depletion of CD8+ cells alone was. CONCLUSIONS: T cells in the recipient's marrow space play an important role in hindering allogeneic engraftment in the mouse. The T-cell subset responsible appears to be CD8+ cells rather than CD4+ cells.

Influence of panel-reactive antibody on survival and rejection after lung transplantation.

BACKGROUND: Panel-reactive antibody (PRA) is commonly used before thoracic organ transplantation to estimate a potential recipient's degree of humoral sensitization. METHODS: To assess the influence of an elevated PRA on survival and the incidence of rejection in pulmonary transplantation, the records of 247 patients that underwent single or double lung transplantation were reviewed. RESULTS: Twenty-one of 247 patients (8.5%) had PRA values greater than 10%. Survival of this population was not significantly different from that of patients with low PRA levels: 74% (low PRA) vs 65% (elevated PRA) at 1 year and 58% in both groups at 3 years. The acute rejection rates (episodes/first 100 days) for the elevated and low PRA groups were 2.1 and 1.9, respectively (p = NS). Obliterative bronchiolitis developed in 38.9% of the high and 31.2% of the low PRA groups (p = NS). Six of 247 patients had a retrospective positive lymphocytotoxic cross-match result; three had PRA values greater than 10%. Patients with a positive cross-match result experienced similar survival and incidence of rejection as the remainder of the population. Among 957 patients evaluated for lung transplantation, 16 (1.7%) had a PRA (with dithiothreitol) greater than 15%. All had a history of pregnancy, blood transfusion, connective tissue disease, or previous transplantation. CONCLUSIONS: Humoral sensitization is uncommon in the lung transplantation population. A modestly elevated PRA does not predict survival or the development of acute rejection or bronchiolitis obliterans. PRA testing before lung transplantation should be reserved for those patients with specific risk factors for humoral sensitization.

Mechanism for cotolerance in nonlethally conditioned mixed chimeras: negative selection of the Vbeta T-cell receptor repertoire by both host and donor bone marrow-derived cells.

Bone marrow (BM) chimeras prepared by complete recipient ablation (A-->B) exhibit donor-specific tolerance, yet survival is often limited by graft-versus-host disease (GVHD). Negative selection of potentially donor-reactive T cells, as assessed by relative T-cell receptor (TCR)-Vbeta expression, is dependent on donor BM-derived deleting ligands. Mixed chimerism and tolerance for both donor and host antigens can be achieved using partial recipient myeloablation with 500 cGy total-body irradiation (TBI) before transplantation followed by cyclophosphamide (CyP) on day +2. To examine the influence of residual host elements on negative selection, the peripheral TCR-Vbeta repertoire was analyzed in partially ablated C57BL/10SnJ (B10) recipients reconstituted with BM from major histocompatibility complex (MHC)-disparate B10.BR/SgSnJ or MHC, Hh-1 and Mls-disparate BALB/cByJ donors, which delete Vbeta5+ and 11+ or Vbeta3+, 5+, and 11+ TCR subsets, respectively. As in myeloblated recipients, donor-reactive subfamilies were deleted in B10.BR-->B10 and BALB/c-->B10 chimeras, suggesting that donor I-E and minor lymphocyte-stimulating (Mls) antigens contribute to the deleting ligands in the nonmyeloablated host. In striking contrast to completely ablated B10-->B10.BR chimeras, partially ablated recipients showed intramedullary I-E expression in the thymus and deleted host-reactive Vbeta5+ and Vbeta11+ subfamilies. These data demonstrate that efficient clonal deletion occurs after partial myeloablation and that both donor and host ligands contribute to TCR repertoire selection.

Durable mixed allogeneic chimerism and tolerance by a nonlethal radiation-based cytoreductive approach.

For over 40 years, the association between hemopoietic chimerism and donor-specific tolerance for allografts has been recognized. However, toxicity associated with lethal conditioning has prevented the clinical application of bone marrow (BM) chimerism to induce tolerance. We previously demonstrated that engraftment could be achieved with less than total recipient myeloablation (700 cGy) and that the incidence of engraftment correlated with the dose of total body irradiation (TBI). Administration of cyclophosphamide (CyP) on Day +2 reduced the minimum TBI dose sufficient to permit engraftment to 500 cGy. In the current study, addition of antilymphocyte globulin (ALG) to the TBI/CyP-based conditioning approach reduced the radiation required for engraftment to < or = 300 cGy. B10 (H-2b) mice conditioned with ALG on day -3, 300 cGy of TBI with transplantation of B10.BR (H-2k) or BALB/c (H-2d) BM on day 0, and CyP on day +2 exhibited evidence of donor chimerism (49.6 +/- 3.7% and 38.2 +/- 2.4%, respectively) in 97% of recipients. ALG eliminated CD4+ and CD8+ cells and decreased NK1.1+ cells in the peripheral circulation at the time of transplantation. Moreover, T and NK cells in the host BM were significantly decreased compared with cells of recipients conditioned with TBI alone. CyP delayed repopulation of host thymocytes, providing time for the establishment of donor chimerism before production of mature T cells. Chimeric animals exhibited stable multilineage chimerism and donor-specific tolerance to skin grafts and in in vitro assays. This model may provide a clinically acceptable approach for the induction of donor-specific transplantation tolerance.

Facilitating cells enable engraftment of purified fetal liver stem cells in allogeneic recipients.

It has been reported that while stem cells purified from adult bone marrow engraft in syngeneic recipients, they fail to engraft in allogeneic recipients. We have recently shown that the addition of as few as 30,000 facilitating cells (CD8+/CD3+/CD45R+/Thy 1.2+/TCR-), a unique bone marrow-derived population that does not possess stem cell properties, results in the permanent engraftment of stem cells in a major histocompatibility complex (MHC)-disparate allogeneic host. It has been suggested that fetal hematopoietic tissue may be a source of stem cells with enhanced proliferative and self-renewal properties compared with adult bone marrow. We were interested, therefore, in whether fetal stem cells demonstrated a superior capacity to engraft in allogeneic recipients. In this study, we have examined the engraftment properties of mouse fetal liver cells in syngeneic and allogeneic recipients. Transplantation of unmodified fetal liver cells into allogeneic recipients results in stable multilineage chimerism with donor-specific tolerance, indicating that the pluripotent hematopoietic stem cell is present in fetal liver and is capable of engraftment in allogeneic adult recipients. Similarly, 2000 to 3000 sorted fetal liver stem cells (Sca+/c-kit+/Lin-) successfully reconstituted lethally irradiated syngeneic adults and adults differing only in minor histocompatibility antigens. Two thousand to 10,000 fetal stem cells failed to rescue lethally irradiated allogeneic recipients, but the addition of 30,000 MHC-matched purified facilitating cells to the fetal stem cell inoculum resulted in sustained engraftment with multilineage production. These results, which parallel our earlier work with stem cells derived from adult bone marrow, indicate that the pluripotent fetal stem cell behaves in a fashion similar to that of adult stem cells with regard to allogeneic transplantation.

Chimerism and thoracic organ transplantation.

Current nonspecific immunosuppression for thoracic organ transplantation is limited by infection, end-organ toxicity, malignancy, and failure to completely control rejection. Donor-specific transplantation tolerance after bone marrow transplantation and the creation of mixed chimerism is a promising means for achieving drug-free allograft acceptance. This review explores bone marrow transplantation as a method for tolerance induction, the superior clinical characteristics of mixed chimerism, and recent developments that enhance marrow engraftment, minimize graft-versus host disease, and avoid lethal conditioning of the recipient. The importance of microchimerism in clinical transplantation and clinical trials aimed at augmentation of this phenomenon are reviewed.

A nonlethal conditioning approach to achieve durable multilineage mixed chimerism and tolerance across major, minor, and hematopoietic histocompatibility barriers.

Reconstitution of lethally irradiated mice with a mixture of syngeneic and allogeneic (A+B-->A) bone marrow results in multilineage mixed allogeneic chimerism, donor-specific transplantation tolerance, superior immunocompetence and resistance to graft-vs-host disease. However, the morbidity and mortality associated with lethal irradiation would be a major limitation to the clinical application of chimerism to induce tolerance for solid organ grafts or treat other nonmalignant hematologic diseases. We report here that durable multilineage mixed allogeneic chimerism and donor-specific transplantation tolerance for skin and primarily vascularized allografts can be achieved across multiple histocompatibility barriers using a nonmyeloablative radiation-based approach. The percentage of B10 mouse recipients that engrafted directly correlated with the degree of disparity between donor and recipient and the dose of total body irradiation administered. Although the occurrence of engraftment following conditioning with doses of total body irradiation of > or = 600 cGy was similar for animals receiving bone marrow disparate at MHC or MHC, minor and hematopoietic (Hh-1) loci (67% vs 78%), the level of donor chimerism was significantly less when multiple histocompatibility barriers were present (94.6 +/- 3.8% vs 37.5 +/- 12.5%). Treatment of the recipient with cyclophosphamide 2 days following allogeneic bone marrow transplantation reduced the dose of radiation sufficient for reliable engraftment to only 500 cGy of total body irradiation, regardless of MHC and Hh-1 disparity. Donor chimerism was stable and present in all lineages, with production of lymphoid (T and B cell), NK, and myeloid (erythrocyte, platelet, granulocyte, and macrophage) cells. Mixed chimeras exhibited donor-specific tolerance in vitro, as assessed by mixed lymphocyte culture (MLR) and cytotoxicity (CML) assays, and in vivo to skin and primarily vascularized cardiac allografts. The observation that engraftment and tolerance can be achieved across multiple histocompatibility barriers using nonmyeloablative recipient conditioning may allow allogeneic bone marrow transplantation to be applied to nonmalignant disease states in which lethal conditioning cannot be justified, including the induction of donor-specific tolerance for solid organ transplantation and the treatment of hemoglobinopathies and enzyme deficiency states.

Mixed allogeneic chimerism in the rat. Donor-specific transplantation tolerance without chronic rejection for primarily vascularized cardiac allografts.

Graft loss secondary to chronic rejection remains a major source of morbidity and mortality in solid organ transplantation. Mixed chimerism has been suggested as one potential approach to overcome this limitation. Until now, whether long-term tolerance for primarily vascularized allografts can be achieved with mixed chimerism has not been adequately assessed due to technical limitations in the mouse and the inability to establish a reliable model of mixed chimerism in the rat. We now report that stable multilineage mixed hematopoietic chimerism can be achieved following the transplantation of a mixture of T cell-depleted syngeneic and allogeneic bone marrow cells into myeloablated rat recipients using a number of MHC plus minor antigen-disparate donor and recipient strain combinations (F344+WF-->F344, F344+ACI-->F344, WF+F344-->WF, and WF+ACI-->WF). Ninety-one percent of animals engrafted with a level of lymphoid chimerism ranging between 12% and 93% (73.3 +/- 4.8%). Peripheral blood lymphocyte chimerism remained stable for up to 13 months after reconstitution. Multilineage chimerism for lymphoid (T and B cells) and myeloid (granulocyte and macrophage) lineages was present, which suggests that engraftment of the pluripotent rat stem cell had occurred. There was no clinical or histologic evidence of graft-versus-host disease. Donor-specific skin (mean survival time [MST] > or = 177 days) and primarily vascularized cardiac (MST > or = 213 days) grafts were accepted without evidence for acute or chronic rejection. In contrast, MHC-disparate third-party skin (MST = 14 days) and cardiac grafts (MST = 13 days) were rapidly rejected. The tolerance was systemic, since donor-specific tolerance was present in vitro as assessed by the mixed lymphocyte proliferation assay. These data suggest that mixed chimerism prevents graft loss secondary to chronic rejection in skin as well as primarily vascularized grafts. Furthermore, a rat model for mixed allogeneic chimerism may provide insight into the mechanisms involved in tolerance induction for a variety of allografts (lungs, small bowel, limb, etc.) not readily transplantable in mouse recipients.

Mixed allogeneic chimerism achieved by lethal and nonlethal conditioning approaches induces donor-specific tolerance to simultaneous islet allografts.

We previously reported that donor-specific, but not third party, skin allografts were permanently accepted if mixed allogeneic (B10+BR-->B10) reconstitution and skin graft placement were performed sequentially or simultaneously in lethally conditioned (950 cGy) recipients. The purpose of the present study was to examine whether a similar outcome would occur if islets were placed coincident with the time of bone marrow infusion and to establish the minimum dose of cytoreduction sufficient to achieve chimerism and tolerance for simultaneous islet allografts. B10 (H-2b) mice were rendered diabetic using streptozotocin. After sustained hyperglycemia (> 300 mg/dl), diabetic B10 mice were irradiated (950 cGy) and reconstituted with 5 x 10(6) T cell-depleted (TCD) B10 + 15 x 10(6) TCD B10.BR bone marrow cells. Islet allografts genetically matched or disparate to the bone marrow donor were placed under the renal capsule within 24 hr following infusion of bone marrow cells. All donor-specific B10.BR mouse (H-2k) islet allografts were permanently accepted (n = 8; MST > or = 173 days), while 7 of 9 MHC-disparate third-party BALB/c mouse (H-2d) islet grafts were rejected. The other 2 allografts remained functional over 200 days posttransplantation. We recently established a nonlethal conditioning strategy to achieve multilineage mixed chimerism. We applied this model to examine whether simultaneous islet grafts matched to the donor would be permanently accepted if the donor was incompletely myeloablated. Diabetes was induced in B10 mouse recipients. Animals with hyperglycemia were conditioned with 500 cGy of TBI followed by an infusion of 15 x 10(6) untreated B10.BR bone marrow cells. A simultaneous islet allograft matched or MHC-disparate to the bone marrow donor was performed the same day. Two days following bone marrow transplantation, a single dose of cyclophosphamide (200 mg/kg) was injected via the intraperitoneal route. Islet allografts matched to the bone marrow donor were significantly prolonged (n = 9; MST > or 226 days) and showed no evidence for chronic rejection, while MHC-disparate grafts were rejected (n = 5; MST = 34 days). Animals that received islet grafts but no bone marrow also rejected their grafts with a similar time course. These data suggest that permanent donor-specific tolerance to islet allografts placed coincident with bone marrow transplantation can be achieved after lethal as well as incompletely myeloablative conditioning.

Phenotypic characterization of a novel bone marrow-derived cell that facilitates engraftment of allogeneic bone marrow stem cells.

Bone marrow transplantation is an accepted therapy for hematologic malignancies, aplastic anemia, metabolic disorders, and solid tumors. However, graft-versus-host disease (GVHD) and failure of engraftment have limited the widespread application of this technology to nonmalignant disease states. The use of purified bone marrow stem cells has been suggested as an approach to promote engraftment yet avoid GVHD. Although bone marrow stem cells, purified by cell sorting, engraft and repopulate lethally irradiated genetically identical recipients, they do not engraft in major histocompatibility complex (MHC)-disparate allogeneic recipients. We report for the first time the characterization of a novel cell population of donor bone marrow origin, separate from the hematopoietic stem cell, that facilitates engraftment of purified allogeneic bone marrow stem cells in an MHC-specific fashion without causing GVHD. Although 1,000 purified stem cells (c-kit+/Sca-1+/lineage-) reliably repopulate syngeneic mouse recipients, 10 times that number do not engraft in MHC-disparate allogeneic recipients. The addition of as few as 30,000 facilitating cells (CD8+/CD45R+/TCR-) is sufficient to permit engraftment of purified stem cells in MHC-disparate recipients. The cell surface phenotype of this purified cellular population differs significantly from other characterized lineages of lymphoid or myeloid origin. Based on multiparameter rare-events cell sorting, the facilitating fraction is CD8+, CD3+, CD45R+, Thy 1+, class IIdim/intermediate but alpha beta-TCR- and gamma delta-TCR-. This cellular population comprises approximately 0.4% of the total bone marrow and is separate from the hematopoietic stem cell. The coadministration of purified facilitating cells plus stem cells to optimize engraftment yet avoid GVHD may expand the potential application of bone marrow transplantation to disease states in which the morbidity and mortality associated with conventional BMT cannot be justified.