Xenotransplantation for AIDS.
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Acquired Immunodeficiency Syndrome↗
Biomedical subjects
S T Ildstad
Publications and source records attributed to S T Ildstad.
Mixed allogeneic chimerism induced by a sublethal approach prevents autoimmune diabetes and reverses insulitis in nonobese diabetic (NOD) mice.
Evidence in experimental models suggests that many autoimmune diseases can be prevented by transplantation of bone marrow from disease-resistant donors. For potential clinical application, it would be important to avoid the morbidity and mortality associated with lethal conditioning and achieve mixed chimerism using less than complete recipient ablation. We report here for the first time that stable chimerism achieved in NOD mice using a sublethal radiation-based conditioning approach is sufficient to prevent beta-cell destruction and abrogate insulitis in prediabetic NOD mice. The percentage of NOD mouse recipients (8 wk of age) that engrafted with donor bone marrow correlated with the dose of irradiation and number of bone marrow cells transplanted. Engraftment of B10.BR bone marrow occurred in > or = 94% of animals receiving > or = 750 cGy of total body irradiation before bone marrow transplantation and > or = 30 x 10(6) bone marrow cells, while reproducible engraftment did not occur at radiation doses of less than 700 cGy and cellular doses of less than 30 x 10(6) bone marrow cells. All chimeric animals remained free of diabetes (n = 38) for 10 mo following bone marrow transplantation. Moreover, in all animals examined, no insulitis was present from 12 to 36 wk following reconstitution. In striking contrast, 61% (22 of 36) of NOD recipients that were conditioned but did not receive bone marrow developed acute diabetes by 12 mo. Insulitis was present in all remaining animals. These results suggest that allogeneic chimerism achieved using a sublethal conditioning approach can prevent the onset of diabetes and even reverse preexisting insulitis in NOD mice.
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Current progress in chimerism and donor-specific tolerance.
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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.
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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.
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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.
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Mixed xenogeneic chimerism and tolerance III. A nonlethal approach to induce donor-specific transplantation tolerance to simultaneous islet xenografts.
Previously, we reported that transplantation of rat bone marrow into lethally irradiated (950 rads) mouse recipients results in stable xenogeneic (rat-->mouse) chimerism and confers donor-specific transplantation tolerance for subsequent or simultaneous islet xenografts. For potential clinical application of chimerism to induce tolerance, it would be important to avoid the morbidity and mortality associated with lethal conditioning. Recently, we established a model to achieve durable multilineage xenogeneic chimerism using a nonlethal cytoreductive approach. We report here for the first time that donor-specific rat islet xenografts placed coincident with the bone marrow transplant are permanently accepted by nonlethally conditioned recipients. All recipients conditioned with 700 cGy of total body irradiation and transplanted with 40 x 10(6) F344 rat bone marrow cells repopulated as mixed donor/host chimeras. The chimeras exhibited permanent acceptance of donor islet xenografts, since donor-specific F344 (Rt1A1) rat islet xenografts were significantly prolonged (median survival time > 110 days), while MHC-disparate third-party WF (Rt1Au) rat islets were prolonged but rejected (median survival time = 33.2 days). The islets were functional to maintain normoglycemia and regulated in function to respond to an intraperitoneal glucose challenge. These data suggest that tolerance to donor-specific islet xenografts placed coincident with bone marrow transplantation can be achieved after nonlethal conditioning.
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In vivo dynamic MRI tracking of rat T-cells labeled with superparamagnetic iron-oxide particles.
Dynamic MRI tracking of rat T-cells in vivo is performed in rat testicles after labeling isolated rat T-cells in vitro with superparamagnetic dextran-coated iron-oxide particles, BMS180549. Tissue inflammation induced by the local injection of the calcium ionophore, A23187, is used to attract labeled T-cells. Gradient-echo MR images of rat testicles show a statistically significant decrease (4%) of the signal intensity in areas of injection of A23187 as early as 30 min after intravenous infusion of 2 x 10(8) labeled T-cells. The signal change reaches its maximum (6-7% decrease) at about 60-120 min after cell infusion. T2-mapping also shows a decrease of T2 in the areas with A23187. Image quantitation, which includes a chemical-shift effect, significantly enhances the sensitivity for detection of superparamagnetically labeled T-cells. Localization of labeled T-cells in rat testicles has been verified by fluorescence microscopy studies of T-cells co-labeled with a lipophilic fluorescent carbocyanine dye, 1,1-dioctadecyl-3,3,3',3'-tetramethyl-lindocarbocyanine perchlorate. These results represent the first successful demonstration of dynamic tracking of specific cells in vivo using MRI.
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Effects of FK 506 on chimerism and the induction of donor-specific unresponsiveness following fully allogeneic bone marrow transplantation in mice.
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A minimal conditioning approach to achieve stable multilineage mouse plus rat chimerism.
Transplantation of untreated rat bone marrow into lethally irradiated (950 cGy) mouse recipients results in durable xenogeneic (rat-->mouse) chimerism and confers donor-specific transplantation tolerance for subsequent xenografts. The purpose of the present study was to characterize the minimal dose of total body irradiation (TBI) which would allow engraftment of rat bone marrow in mouse recipients. We report here that durable and stable lymphohaematopoietic cross-species chimerism can be achieved using a less than totally ablative radiation-based conditioning approach. The percentage of B10 mouse recipients which engrafted with rat bone marrow cells correlated with the dose of TBI. Engraftment of rat bone marrow stem cells occurred in all animals receiving 750 cGy prior to bone marrow transplantation, while no engraftment was detected at doses less than 650 cGy. Although most of the recipients were repopulated with mixed mouse and rat multilineage chimerism, some exhibited a predominance of rat cells. Although mixed xenogeneic rat/mouse chimeras prepared by lethal TBI produced only mouse derived RBC (red blood cells), chimeras prepared by sublethal conditioning produced both rat and mouse RBC. Only animals with detectable chimerism exhibited specific functional transplantation tolerance to donor xenoantigens, as assessed in vitro by mixed lymphocyte reaction assay. This model may offer an in vivo approach to study the role of species-specific growth factors in stem cell biology as well as the mechanisms for the induction of tolerance across species barriers.
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Allogeneic chimerism induces donor-specific tolerance to simultaneous islet allografts in nonobese diabetic mice.
BACKGROUND: Nonobese diabetic (NOD) mice develop a systemic autoimmune disease that resembles type I diabetes in human beings. Previous studies in NOD mice have shown that transplantation of bone marrow from normal donors to lethally conditioned recipients prevented the development of diabetes (B10.BR-->NOD, BALB/c-->NOD). The focus of the present study was to examine whether donor-specific transplantation tolerance for islet allografts could be achieved if islet transplantations were performed coincident with the time of bone marrow infusion in diabetic NOD mice. Moreover, the transplanted islets were evaluated for evidence of recurrent autoimmunity. METHODS: Female NOD mice were followed until autoimmune diabetes occurred (urine glucose, to +; blood glucose level, 300 mg/dl or greater). Diabetic NOD mice were irradiated with 950 cGy total body irradiation and received 30 x 10(6) untreated B10.BR bone marrow cells (B10.BR-->NOD). A simultaneous islet allograft was placed under the renal capsule within 24 hours after infusion of the bone marrow cells. Mice were monitored by means of blood glucose levels, and histologic analyses were performed on the transplanted islet. RESULTS: Islet allografts genetically matched to the bone marrow donor were significantly prolonged (n = 5; mean survival time, 206 days or more) and showed no evidence for chronic rejection or recurrent insulitis, whereas major histocompatibility complex-disparate third-party allografts were rejected (n = 3; mean survival time, 37 days) and exhibited lymphocytic infiltration compatible with rejection on histologic evaluation. CONCLUSIONS: These data suggest that permanent donor-specific tolerance to islet allografts placed simultaneously with bone marrow transplantation can be achieved in diabetic NOD mice. Moreover, recurrent autoimmune destruction of the pancreatic tissue is prevented by the bone marrow chimerism.
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Bone marrow-derived dendritic cells pulsed with synthetic tumour peptides elicit protective and therapeutic antitumour immunity.
Dendritic cells, the most potent 'professional' antigen-presenting cells, hold promise for improving the immunotherapy of cancer. In three different well-characterized tumour models, naive mice injected with bone marrow-derived dendritic cells prepulsed with tumour-associated peptides previously characterized as being recognized by class I major histocompatibility complex-restricted cytotoxic T lymphocytes, developed a specific T-lymphocyte response and were protected against a subsequent lethal tumour challenge. Moreover, in the C3 sarcoma and the 3LL lung carcinoma murine models, treatment of animals bearing established macroscopic tumours (up to 1 cm2 in size) with tumour peptide-pulsed dendritic cells resulted in sustained tumour regression and tumour-free status in more than 80% of cases. These results support the clinical use of tumour peptide-pulsed dendritic cells as components in developing effective cancer vaccines and therapies.
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Xenotransplantation.
Transplantation of organs across a species barrier has long been a dream of the transplantation community. Renewed interest in xenotransplantation has emerged due to the short supply of organs available for clinical transplantation. However, transplantation of solid organs across a species barrier has not matched the success of allogeneic combinations, even for closely related species. The rejection response to xenografts is vigorous and not adequately controlled by conventional immunosuppressive agents that control alloreactivity. This may suggest a different mechanism for xenoreactivity, or stronger histocompatibility antigen disparities. This article reviews the current clinical experience with xenografts, mechanisms of xenoreactivity, the induction of tolerance across species disparities, and recent models in which human tissue has been transplanted into other species as an in vivo model of the human immune system.
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Characterization and comparison of the lytic function of NKR-P1+ and NKR-P1-rat natural killer cell clones established from NKR-P1bright/TCR alpha beta-cell lines.
From sorted rat NKR-P1bright/T cell receptor (TCR) alpha beta-cells, we established interleukin (IL)-2-dependent cell lines with the morphology, phenotype and function of natural killer (NK) cells. The cell lines NKbr11.3 and NKbr1.28 had large-granular-lymphocyte morphology, were capable of lysing NK-and lymphokine-activated-killer-susceptible target cells, and had the phenotype NKR-P1bright/CD3-/CD4-/CD5-/CD25-/gp42+/TCR alpha beta-. Both cell lines mediated reverse antibody-dependent cellular cytotoxicity via NKR-P1. NKR-P1-subpopulations, identical in all other aspects of phenotype, spontaneously developed in both cell lines. Cloning of NKbr11.3 and NKbr1.28 by limiting dilution resulted in two NKR-P1+ clones, 11.3(6B) and 1.28(3D), and three NKR-P1- clones, 11.3(8A), 11.3(10B), and 1.28(9F). The NKR-P1- clones were lytic and their target preference resembled that of the parental lines, except that C1498 and P815 appeared to be poor targets for 11.3(8A) and 11.3(10B). These cells represent the first reported rat IL-2-dependent NK cell lines and clones. They will be useful for the study of NK cell function as well as the function and expression of NKR-P1.
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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.
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Mixed xenogeneic chimerism (mouse+rat-->mouse) to induce donor-specific tolerance to sequential or simultaneous islet xenografts.
We previously reported that donor-specific rat islet xenografts were accepted by fully xenogeneic (rat-->mouse) chimeras when the islets were transplanted at least six weeks following reconstitution. The purpose of the present study was to examine whether a similar outcome would occur for mixed xenogeneic (mouse+rat-->mouse) chimeras if the islets were placed coincident with the time of bone marrow infusion. As with fully xenogeneic chimeras (rat-->mouse), synchronous donor-specific F344 rat (Rt1A1) islet xenografts were significantly prolonged (MST > 139 days) in mixed xenogeneic (mouse+rat-->mouse) chimeras, while MHC-disparate third-party WF rat (Rt1Au) grafts were rejected (MST = 21.2 days). The transplanted donor-specific islets were functional to maintain euglycemia and they were regulated in function to respond to a glucose challenge. For potential clinical application, it would be of obvious benefit if the islet xenografts could be placed at the time of bone marrow transplantation. We therefore performed similar studies using donor islets administered simultaneously with bone marrow. Donor-specific islet xenografts were permanently accepted by all mouse recipients (n = 5). When MHC-disparate third-party rat islets were transplanted, only 3 of 8 islet xenografts were rejected; the other 5 remained functional from 77 to 90 days posttransplantation. Although prolonged and functional, the MHC-disparate islets that were accepted exhibited histologic evidence of fibrosis and rejection, while those matched to the donor did not. These data therefore suggest that donor-specific islet xenografts are permanently accepted if placed simultaneously or sequentially following mixed xenogeneic bone marrow reconstitution.
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Characterization and function of the NKR-P1dim/T cell receptor-alpha beta+ subset of rat T cells.
MHC-unrestricted cytotoxicity is mediated primarily by NK cells. However, some subsets of TCR-alpha beta+ and TCR-gamma delta+ T cells also have the capacity to mediate MHC-unrestricted cytotoxicity, particularly after incubation in high concentrations of IL-2. Currently, it is not known what receptors on T cells are responsible for this activity, nor whether such receptors are the same as those on NK cells. We have recently described a type II integral membrane protein, termed NKR-P1, that is expressed at high levels on rat NK cells (NKR-P1bright). NKR-P1 contains a carbohydrate recognition domain characteristic of C-type (Ca(2+)-dependent) animal lectins and is a representative member of a distinct group of this superfamily. By a variety of criteria, NKR-P1 is linked to a signaling pathway that activates NK cell lytic function. Based on its structure and function, NKR-P1 has been implicated as a candidate molecule involved in or contributing to MHC-unrestricted cytotoxicity. We describe herein the expression of NKR-P1 at low levels on a small subset of rat T cells with an NKR-P1dim/TCR-alpha beta+ phenotype and on a small subset of cells with an NKR-P1dim/TCR-alpha beta- phenotype (presumably containing gamma delta+ T cells). Before incubation with IL-2, the NKR-P1dim subsets of cells lack MHC-unrestricted cytolytic capacity and lack the capacity for reverse antibody-dependent cellular cytotoxicity (rADCC) mediated via NKR-P1. However, culture of NKR-P1dim/TCR-alpha beta+ T cells in IL-2 led to the acquisition of both MHC-unrestricted cytotoxicity and the capacity for rADCC via NKR-P1. NK-like cytolytic function was not found among IL-2-activated NKR-P1-/TCR-alpha beta+ T cells. These data suggest that expression of functional NKR-P1 (i.e., ability to signal rADCC) correlates with and potentially contributes to MHC-unrestricted cytotoxicity.
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FK506 inhibits the differentiation of developing thymocytes but not negative selection of T cell receptor V beta 5+ and V beta 11+ T lymphocytes in vivo.
To examine the influence of FK506 on lymphocyte development, we employed a syngeneic bone marrow transplantation model using MHC-disparate B10 (H-2b, I-Ab) and B10.BR (H-2k, I-Ak, I-Ek) mice. B10 mice, which do not express class II I-E, do not delete any known T cell receptor (TCR)-V beta, while B10.BR mice (MHC class II I-Ek, I-Ak) delete V beta 5+ and V beta 11+ TCR. Continuous daily treatment of syngeneically reconstituted B10 mice with FK506 delayed the development of thymocytes from the CD4+CD8+ to CD4+CD8- stage, while no effect was observed at the earlier CD4-CD8- to CD4+CD8+ stage. At the same time, there was a significant reduction in TCRhigh thymocytes compared with untreated, syngeneically reconstituted controls. These results suggest that FK506 treatment interfered with thymic positive selection. We also examined whether FK506 treatment would influence negative selection. Levels of expression of V beta 5+ and V beta 11+ T cells in FK506-treated B10.BR-->B10.BR recipients were similar to those observed in unmanipulated, syngeneically reconstituted B10.BR-->B10.BR controls. This was not due to the inhibition of clonal proliferation by FK506, since 35 days after drug withdrawal complete recovery of the peripheral Thy1.2+ population was observed, while the percentages of V beta 5+ and V beta 11+Thy1.2+ T cells were maintained at values similar to controls. Surprisingly, clonal proliferation stimulated by monoclonal antibody against V beta 5 and V beta 11 TCRs was observed in CsA-treated, syngeneically reconstituted B10.BR mice but not in FK506-treated mice, suggesting that CsA may be more likely to induce autoreactivity. Differences in thymic architecture between FK506- and CsA-treated animals further suggested that the drugs may differ in their effects on T cell development in vivo.
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