Development and analysis of transgenic mice expressing porcine hematopoietic cytokines: a model for achieving durable porcine hematopoietic chimerism across an extensive xenogeneic barrier.

The capacity of mixed hematopoietic chimerism to induce tolerance has not been demonstrated in discordant xenogeneic species combinations because of the difficulty in achieving lasting hematopoietic engraftment. In an effort to create a model of long-lasting disparate xenogeneic hematopoietic chimerism, we have developed transgenic (Tg) mice carrying porcine cytokines. Three lines of Tg mice were generated: one carrying porcine IL-3 and GM-CSF genes only (termed IL/GM) and the remaining two lines carrying in addition, the soluble SCF gene (termed IL/GM/sS) or membrane-bound SCF gene (termed IL/GM/mS). Sera from mice with IL/GM and IL/GM/sS transgenes markedly stimulated the proliferation of swine marrow cells in vitro. However, proliferation of swine marrow cells was not induced in cultures containing IL/GM/mS sera. Consistent with these observations, ELISA assays revealed detectable levels of porcine cytokines in the sera of IL/ GM and IL/GM/sS, but not in sera of IL/GM/mS Tg mice. Marrow stromal cells prepared from all three kinds of Tg mice, but not those from non-Tg littermates, were capable of supporting the growth of porcine hematopoietic cells in vitro. Immunodeficient Tg mice were generated by crossing Tg founders with C.B-17 SCID mice for five generations. All Tg immunodeficient mice showed improved porcine hematopoietic engraftment compared with non-Tg controls. These Tg mice provide a useful model system for studying porcine hematopoietic stem cells, and for evaluating the feasibility of donor-specific tolerance induction by mixed chimerism across highly disparate xenogeneic barriers.

Role of antibody-independent complement activation in rejection of porcine bone marrow cells in mice.

BACKGROUND: Although complement activation has been shown to be important in the rejection of solid organs in some xenogeneic species combinations, its role in the rejection of xenogeneic marrow engraftment is unknown. METHODS: The effect of complement depletion with cobra venom factor on porcine bone marrow cell (BMC) engraftment was examined in 3 Gy-irradiated C.B-17 severe combined immunodeficiency mice receiving 10(8) pig BMC. RESULTS: At 26 days after transplantation, the percentages of swine class I+, myeloid, and CD2+ cells in marrow, spleen, and peripheral blood, and the numbers of porcine myeloid progenitor cells in marrow, were increased in cobra venom factor-treated recipients compared with simultaneous control recipients. Consistent with the in vivo results, preheating serum (56 degrees C for 30 min) reduced the inhibitory effect of severe combined immunodeficiency mouse serum on the proliferation of pig BMC in vitro. CONCLUSION: Murine complement is capable of resisting xenogeneic hematopoietic engraftment through an antibody-independent mechanism.

Donor-derived interferon gamma is required for inhibition of acute graft-versus-host disease by interleukin 12.

We have demonstrated that a single injection of interleukin (IL)-12 on the day of bone marrow transplantation (BMT) inhibits acute graft-versus-host disease (GVHD) in mice. This effect of IL-12 can be diminished by anti-interferon (IFN)-gamma mAb. To determine the mechanism by which IFN-gamma affects IL-12-mediated GVHD protection, we have compared the effect of IL-12 on GVHD in C57BL/6 wild-type (WT) or IFN-gamma gene knockout (GKO) recipients of fully major histocompatibility complex plus minor antigen-mismatched allogeneic BMT from WT or GKO BALB/c mice. Lethal acute GVHD was readily induced in the absence of IFN-gamma. IL-12 inhibited GVHD mortality to a similar extent in WT and GKO recipients of WT allogeneic BMT. However, neither WT nor GKO recipients were protected by IL-12 from GVHD induced by GKO allogeneic BMT. Moreover, the effective inhibition of host-reactive donor T cell activation and expansion that is associated with IL-12-mediated GVHD protection was dependent on the ability of BALB/c donors to produce IFN-gamma. These results demonstrate that (a) acute GVHD can be induced in the absence of IFN-gamma, (b) host IFN-gamma does not play a critical role in IL-12-induced GVHD protection, and (c) the protective effect of IL-12 against GVHD is dependent on the ability of the donor to produce IFN-gamma.

Pig MHC mediates positive selection of mouse CD4+ T cells with a mouse MHC-restricted TCR in pig thymus grafts.

Remarkably normal immune function and specific T cell tolerance to discordant xenogeneic donors can be achieved by grafting fetal pig thymus and liver (FP THY/LIV) tissue to T cell and NK cell-depleted, thymectomized (ATX) mice. To determine whether or not host class II MHC molecules participate in the positive selection of mouse CD4+ T cells in FP THY/LIV grafts, we compared their development in ATX "AND" TCR-transgenic mice with positive selecting or nonselecting host MHC genotypes. Mouse TCR-transgenic CD4 single positive T cells repopulated the periphery significantly and to a similar extent in both T/NK cell-depleted, ATX AND mice with positive-selecting or nonselecting MHC backgrounds after grafting with FP THY/LIV. Therefore, MHC molecules from a widely disparate xenogeneic species can positively select T cells bearing a host class II MHC-restricted TCR without a contribution from the host MHC. These results, in combination with previous studies performed in this model, suggest that the T cell repertoire that is generated by the combination of positive selection on xenogeneic MHC and negative selection on both recipient and xenogeneic porcine MHC is tolerant of both donor and recipient and has sufficient cross-reactivity with host MHC/foreign peptide complexes to confer a high level of immunocompetence. The results have implications for the potential clinical applicability of xenogeneic thymic transplantation and also suggest a predominant role for the TCR recognition of species-conserved MHC residues in positive selection.

B-cell reconstitution and xenoreactive anti-pig natural antibody production in severe combined immunodeficient mice reconstituted with immunocompetent B cells from varying sources.

BACKGROUND: Little is known about the B-cell subsets that produce xenoreactive natural antibodies (NAb). This study was undertaken to investigate the potential role of varying B-cell populations in anti-pig NAb production in mice. METHODS: Severe combined immunodeficient (scid) mice were reconstituted with bone marrow or splenic or peritoneal B cells from immunocompetent mice. B-cell reconstitution and anti-pig NAb were evaluated by flow cytometric analysis. RESULTS: Adult marrow failed to reconstitute normal numbers of CD5+ B1a cells, but fully reconstituted CD5- Mac1- B2 and CD5- Mac1+ B1b cell populations in scid mice. Recipients of peritoneal B cells showed poor reconstitution of B2 cells, and an overshoot of B1 cells in the peritoneal cavity. Although B2 cells predominate in the adult spleen, splenic B cells from immunocompetent mice preferentially reconstituted B cells, including B1 cells, in the peritoneal cavity, but did not reconstitute splenic B2 cells. Therefore, neither adult marrow, splenocytes nor peritoneal cells can fully reconstitute scid mice with all B-cell subpopulations. Nevertheless, serum anti-pig NAb in marrow-reconstituted mice recovered to normal levels by 3 weeks, and were maintained for at least 30 weeks. Serum NAb in scid mice receiving peritoneal B cells reached normal levels by 4-7 weeks after transfer. However, NAb in sera of scid mice receiving splenic B cells took longer (>25 weeks) to reach normal levels. CONCLUSIONS: These results indicate that adult marrow-derived B cells can efficiently produce anti-pig NAb, and that peritoneal B cells have greater NAb-producing ability than splenic B cells or their immediate progeny.

Intrathymic deletion of alloreactive T cells in mixed bone marrow chimeras prepared with a nonmyeloablative conditioning regimen.

BACKGROUND: Mixed hematopoietic chimerism induced with a nonmyeloablative conditioning regimen leads to donor-specific transplantation tolerance. Analyses of specific Vbeta-bearing T-cell families that recognize endogenous superantigens demonstrated that donor-specific tolerance is due mainly to an intrathymic deletional mechanism in these mixed chimeras. However, superantigens are not known to behave as classical transplantation antigens. We therefore used T-cell receptor (TCR) transgenic (Tg) recipients expressing a clonotypic TCR specific for an allogeneic major histocompatibility complex antigen to further assess deletional tolerance. METHODS: 2C TCR Tg mice (H2b), whose Tg TCR recognizes major histocompatibility complex class I Ld, were used as recipients of Ld+ bone marrow cells after conditioning with depleting anti-CD4 and CD8 monoclonal antibodies, 3 Gy whole-body irradiation, and 7 Gy thymic irradiation. Chimerism and deletion of CD8+ 2C recipient T cells was evaluated by flow cytometry and by immunohistochemical staining. Tolerance was tested with in vitro cell-mediated lympholysis assays and in vivo by grafting with donor skin. RESULTS: Intrathymic and peripheral deletion of 2C+ CD8-single-positive T cells was evident in mixed chimeras, and deletion correlated with the presence of donor-type cells with dendritic morphology in the thymus, and with chimerism in lymphohematopoietic tissues. Chimeras showed tolerance to the donor in cell-mediated lympholysis assays and specifically accepted donor skin grafts. CONCLUSIONS: Tolerance to transplantation antigens is achieved through intrathymic deletion of donor-reactive T cells in mixed chimeras prepared with a nonmyeloablative conditioning regimen and allogeneic bone marrow transplantation.

Role of intrathymic rat class II+ cells in maintaining deletional tolerance in xenogeneic rat-->mouse bone marrow chimeras.

BACKGROUND: Mixed xenogeneic bone marrow chimerism and tolerance can be induced in mice conditioned with a nonmyeloablative regimen followed by injection of T cell-depleted rat bone marrow cells. We hypothesized that, despite a gradual decline in rat hematopoiesis observed in these chimeras, as long as rat class II+ antigen-presenting cells remain in their thymi, tolerance will persist as a result of deletion of donor-reactive thymocytes. METHODS: The level of chimerism and of mouse Vbeta5 and Vbeta11 T-cell deletion was followed over time. These results were correlated with the presence of rat class II+ cells in the thymus by immunohistochemistry and the presence of tolerance in long-term chimeras by in vivo and in vitro assays. RESULTS: (1) Proliferation and cytotoxicity assays, as well as skin graft survival, demonstrated the presence of specific tolerance to host and to donor rat, with normal reactivity to third-party rat and mouse stimulators, even as late as 85 weeks after bone marrow transplantation. (2) The absence of mature Vbeta5+ and Vbeta11+ host T cells in the thymus and periphery was always associated with the presence of rat class II+ cells in the thymus, and incomplete deletion of T cells expressing these Vbeta families was observed in thymi in which rat class II+ cells were not detectable. CONCLUSIONS: Donor-specific T-cell tolerance is maintained during the period when donor-type reconstitution declines, and is most likely mediated by intrathymic clonal deletion of T cells that recognize antigens expressed on class II+ rat cells.

Interleukin-12 preserves the graft-versus-leukemia effect of allogeneic CD8 T cells while inhibiting CD4-dependent graft-versus-host disease in mice.

We have recently demonstrated that a single injection of 4,900 IU of interleukin-12 (IL-12) on the day of bone marrow transplantation (BMT) markedly inhibits acute graft-versus-host disease (GVHD) in a fully major histocompatibility complex plus minor antigen-mismatched BMT model (A/J --> B10, H-2(a) --> H-2(b)), in which donor CD4(+) T cells are required for the induction of acute GVHD. We show here that donor CD8-dependent graft-versus-leukemia (GVL) effects against EL4 (H-2(b)) leukemia/lymphoma can be preserved while GVHD is inhibited by IL-12 in this model. In mice in which IL-12 mediated a significant protective effect against GVHD, marked GVL effects of allogeneic T cells against EL4 were observed. GVL effects against EL4 depended on CD8-mediated alloreactivity, protection was not observed in recipients of either syngeneic (B10) or CD8-depleted allogeneic spleen cells. Furthermore, we analyzed IL-12-treated recipients of EL4 and A/J spleen cells which survived for more than 100 days. No EL4 cells were detected in these mice by flow cytometry, tissue culture, adoptive transfer, necropsies, or histologic examination. Both GVL effects and the inhibitory effect of IL-12 on GVHD were diminished by neutralizing anti-interferon-gamma (IFN-gamma) monoclonal antibody. This study demonstrates that IL-12-induced IFN-gamma production plays a role in the protective effect of IL-12 against GVHD. Furthermore, IFN-gamma is involved in the GVL effect against EL4 leukemia, demonstrating that protection from CD4-mediated GVHD and CD8-dependent anti-leukemic activity can be provided by a single cytokine, IFN-gamma. These observations may provide the basis for a new approach to inhibiting GVHD while preserving GVL effects of alloreactivity.

Interleukin-12 prevents severe acute graft-versus-host disease (GVHD) and GVHD-associated immune dysfunction in a fully major histocompatibility complex haplotype-mismatched murine bone marrow transplantation model.

BACKGROUND: We have recently reported that interleukin (IL)-12 prevents acute graft-versus-host disease (GVHD)-induced mortality in a full major histocompatibility complex- plus multiple minor antigen-mismatched A/J-->B10 bone marrow transplantation (BMT) model. Because most patients have access to a haploidentical, one haplotype-mismatched donor, we have now investigated the protective effect of IL-12 against GVHD and GVHD-associated immune dysfunction in a haploidentical CBD2F1 (H2kxd) --> B6D2F1 (H2bxd) strain combination. METHODS: GVHD was induced by injecting CBD2F1 marrow and spleen cells into lethally irradiated B6D2F1 mice. RESULTS: In untreated control mice, GVHD resulted in 87% mortality by day 8 after BMT, with no survivors beyond day 17. Treatment with a single injection of IL-12 on the day of BMT led to 87% long-term survival, with no significant weight loss, diarrhea or GVHD skin changes. The majority of T cells recovering in these mice showed the CD62L+, CD44low, CD45RBhigh naive phenotype. These T cells showed specific tolerance to both host and donor histocompatibility antigens, but normal anti-third party (H2s) alloresponses in vitro. B-cell proliferative responses to lipopolysaccharide were also normal in IL-12-protected mice. Moreover, normal negative selection of thymocytes bearing T cell receptors with Vbeta that recognize endogenous superantigens was observed among CD4+CD8- thymocytes, indicating a lack of GVHD-associated thymic selection abnormalities in IL-12-protected allogeneic BMT recipients. CONCLUSIONS: IL-12 provides permanent protection against an otherwise severe, rapidly lethal GVHD, with no clinical manifestations of chronic GVHD, immunosuppression or autoimmune features, in a full major histocompatibilty complex haplotype-mismatched murine BMT model.

Positive and negative selection of functional mouse CD4 cells by porcine MHC in pig thymus grafts.

Specific tolerance to discordant xenogeneic donors can be achieved by grafting of fetal pig thymic and liver tissue (FP THY/LIV) to T cell and NK cell-depleted, thymectomized (ATX) mice. Mouse CD4+ T cells develop in FP THY/LIV grafts, and demonstrate remarkably normal immune function, including host-restricted responses to keyhole limpet hemocyanin. We have therefore studied the role of host MHC class II in the development of mouse T cells in FP THY/LIV grafts by comparing their development in ATX MHC class II-deficient (IIKO) and wild-type (H-2b) mice. Mouse CD4+ T cells repopulated T/NK cell-depleted, ATX IIKO mice after grafting with FP THY/LIV, indicating that pig MHC can positively select mouse CD4 cells. Expression of TCR, MHC class I, Qa-2, heat-stable Ag, and CD45RB among double-positive and CD4 single-positive (SP) graft thymocytes in wild-type recipients was similar to that in normal mouse thymi, whereas CD4 SP thymocytes in grafts of IIKO mice showed increased Qa-2 and decreased heat-stable Ag expression, suggesting an increased level of maturity. Double-positive cells in grafts of IIKO mice also expressed higher than normal levels of Qa-2. Deletion within the grafts of Vbeta3+, Vbeta5.1/5.2+, and Vbeta11+ but not Vbeta6+, Vbeta7+, or Vbeta8.1/8.2+ mouse CD4 SP thymocytes in ATX IIKO mice demonstrated that swine leukocyte Ag participates in negative selection of the T cell repertoire. Therefore, porcine MHC mediates positive and negative selection of mouse thymocytes, but host class II MHC molecules also regulate thymocyte maturation in xenogeneic thymic grafts.

Immune restoration by fetal pig thymus grafts in T cell-depleted, thymectomized mice.

Donor-specific tolerance can be induced across a discordant xenogeneic barrier in T/NK cell-depleted, thymectomized (ATX) B10 mice by grafting of fetal pig thymic and liver tissue (FP THY/LIV) under the kidney capsule. We have now examined the phenotype and function of murine T cells that develop in FP THY/LIV grafts in these mice. Mouse CD4+ T cells reached normal levels in PBL by 14 wk, and were maintained up to 30 wk. Similar proportions of splenic CD4+ cells expressed the naive phenotype (CD45RBhighMEL-14+CD44low) in FP THY/LIV graft recipients and euthymic control mice. These CD4 cells were functional, demonstrating normal proliferative responses and up-regulation of CD25 and CD69 after activation by mitogens or alloantigens. They proliferated in response to the protein Ag KLH presented by host MHC following in vivo immunization. ATX B10 mice grafted with FP THY/LIV also cleared Pneumocystis carinii infections, whereas simultaneously-treated ATX B10 mice not receiving FP THY were unable to do so. Discordant xenogeneic thymus grafting can therefore restore immune competence. Thus, in addition to tolerance induction, xenogeneic thymic replacement might have a potential role in the reconstitution of immunity in patients afflicted with immunodeficiencies affecting the thymus.

Discordant xenogeneic neonatal thymic transplantation can induce donor-specific tolerance.

The limited supply of human organs for transplantation necessitates the development of methods leading to acceptance of xenografts. To avoid the hazards of the high-dose chronic immunosuppressive pharmacotherapy which would otherwise be required for successful xenografting, it would be desirable to induce permanent tolerance to xenogeneic donors. We have recently demonstrated that xenogeneic donor-specific tolerance can be induced by transplanting fetal pig thymic and hematopoietic tissue into thymectomized, T cell-depleted, and natural killer-cell-depleted mice, or into natural killer cell-depleted nude mice. We have now extended these studies by replacing fetal tissue with neonatal pig thymic and hematopoietic tissue, and by examining the in vivo responses of reconstituted mice to pig skin grafts. Neonatal tissue was studied because it might be more practicable than fetal tissue for the purpose of transplantation to primates. BALB/c nu/nu mice transplanted with neonatal (<24-hr-old) pig thymus and spleen fragments developed circulating mouse CD4+ cells. The pig thymus grafts were necessary for mouse T-cell development, as CD4 recovery did not occur in recipients of neonatal pig splenic tissue alone. The CD4+ cells that developed included Vbeta8.1/2+ T cells in similar proportions as in BALB/c mice, and Vbeta11+ and Vbeta5+ CD4 T cells were deleted almost as completely as in normal BALB/c mice. This deletion was detected among CD4 single-positive graft thymocytes. In 9 of 12 evaluable animals, mixed lymphocyte responses demonstrated tolerance to donor-type pig SLA antigens, with responsiveness to alloantigens and/or third-party pig xenoantigens. Furthermore, grafting of neonatal pig thymus conferred the ability to reject allogeneic mouse skin in 7 of 10 animals. In addition, 7 of 10 animals accepted paternal (donor SLA-matched) skin (median survival time [MST] > 100 days), whereas 4 of 4 animals rejected third-party SLA-mismatched pig skin (MST=40.5 days). We conclude that neonatal pig thymi transplanted to BALB/c nu/nu mice can support the development of mouse CD4+ cells that are functional and specifically tolerant to donor-type pig antigens.

Skin graft tolerance across a discordant xenogeneic barrier.

Specific T-cell tolerance may be essential for successful xenotransplantation in humans. Grafting of thymectomized, T cell-depleted normal mice with xenogeneic fetal pig thymus and liver (FP THY/LIV) tissue results in the recovery of functional CD4 antigen-positive cells. We have tested T-cell tolerance by skin grafting. Donor-matched pig skin survived permanently (> 200 days), whereas allogeneic mouse skin was rapidly rejected. Nontolerant control mice rejected pig skin within 26 days. Both porcine and murine histocompatibility class IIhigh cells were detected in long-term thymus grafts, and T-cell repertoire analyses suggested that tolerance to both donors and recipients developed, at least in part, by intragraft clonal deletion. This study demonstrates the principle that tolerance, measured by the stringent criterion of skin grafting, can be induced across a widely disparate species barrier.

Natural killer cells weakly resist engraftment of allogeneic, long-term, multilineage-repopulating hematopoietic stem cells.

Natural killer (NK) cells effect hybrid resistance, in which parental hematopoietic cell grafts are rejected by F1 recipients. NK cells can also resist engraftment of fully MHC-mismatched allogeneic marrow. However, studies of NK cell-mediated alloresistance have relied on short-term proliferation, colony, or survival assays; therefore, their results may not reflect effects of NK cells on the engraftment of allogeneic pluripotent hematopoietic stem cells (PHSC). We have now addressed the role of NK cells in resisting engraftment of these most primitive hematopoietic cells, which provide long-term repopulation of multiple hematopoietic lineages. We took advantage of a nonmyeloablative conditioning regimen that permits allogeneic marrow engraftment and induction of mixed chimerism in mice to evaluate the effect of host NK cell depletion with mAb PK136 on long-term competitive repopulating ability of allogeneic marrow. Mice were pretreated with depleting anti-CD4 and anti-CD8 mAbs, then received 3 Gy of whole body irradiation and 7 Gy of thymic irradiation prior to allogeneic bone marrow transplantation. Depending on the strain combination used, statistically significant increases in long-term allogeneic repopulation of both myeloid and lymphoid cell lineages were observed in recipients depleted of NK cells before bone marrow transplantation compared with controls. Depletion of host NK cells alone was sufficient to enhance donor PHSC engraftment. However, a statistically significant increase in allogeneic reconstitution in NK cell-depleted chimeras compared with control chimeras was not observed in every experiment, and differences were most readily apparent in a strain combination in which recipient NK cells have been shown to have high resistance to engraftment of donor short-term repopulating cells. Chronic (16 weeks) anti-NK1.1 treatment resulted in higher levels of donor-type repopulation than that in animals receiving only pretransplant NK cell depletion. Our studies demonstrate for the first time that host NK cells resist engraftment of allogeneic long-term repopulating PHSC, and provide a model for studying the elements that determine what is regarded as "self" and "non-self" by newly developing NK cells.

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.