Justin J. Sergio

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.

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.

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.

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.

Evidence for nonimmune mechanisms in the loss of hematopoietic chimerism in rat→mouse mixed xenogeneic chimeras

Abstract: We have recently described a relatively nontoxic, nonmyeloablative conditioning regimen allowing engraftment of T cell‐depleted (TCD) rat bone marrow in mice, leading to a state of mixed xenogeneic chimerism and donor‐specific skin graft tolerance, apparently by a deletional mechanism. The conditioning regimen involves depletion of host T and NK cells with mAbs, followed by administration of 7 Gy thymic irradiation (TI) and 3 Gy whole body irradiation (WBI) prior to transplantation of TCD F344 rat bone marrow cells (BMC). Although the percentage of rat cells gradually declines over time post‐BMT in these animals, they demonstrate prolonged survival of donor‐specific rat skin grafts and an absence of anti‐rat antibody responses long after this decline is underway. We therefore hypothesized that the loss of rat hematopoietic repopulation may not be due to a loss of tolerance at the T cell or B cell level. We have now evaluated the effect of a repeat, late, donor marrow infusion on chimerism and tolerance. Treatment with 3 Gy WBI followed by TCD rat marrow infusion at 22 weeks following the original BMT led to a marked increase in rat cell repopulation of both myeloid and lymphoid lineages and prolonged the period of complete central and peripheral deletion of Vp5+ and Vβ11+ host T cells, which recognize endogenous (presumably mouse) superantigens presented by rat hematopoietic cells. Furthermore, the second marrow infusion did not induce a cytotoxic antibody response to rat marrow. Since these results suggested that the decline in rat chimerism in our model was not associated with a loss of T cell or B cell tolerance to the donor, we evaluated the possibility that a failure of NK cell tolerance led to the decline by comparing chimerism in animals receiving chronically NK cell‐depleting mAb and controls. Chronic NK depletion did not markedly enhance the level of rat repopulation in any lineage. Together, our results are most consistent with the interpretation that the gradual decline in rat repopulation in our chimeras reflects a competitive advantage of host hematopoietic cells over xenogeneic cells rather than immune‐mediated rejection, possibly due to species selectivity of cytokines and adhesion molecule interactions of hematopoietic progenitors and the marrow microenvironment. This host hematopoietic advantage might be counteracted by repeat donor‐specific marrow infusions.

Donor‐specific growth factors promote swine hematopoiesis in severe combined immune deficient mice

Abstract: Induction of a state of mixed hematopoietic chimerism following bone marrow transplantation is associated with donor‐specific tolerance in the concordant xenogeneic rat‐to‐mouse species combination. We are now attempting to induce such tolerance in a discordant species combination, pig to mouse. Our initial studies showed that non‐immune physiologic factors limited the level of swine hematopoietic reconstitution in severe combined immune deficient (SCID) mice. We have now examined the ability of swine‐specific growth factors, interleukin‐3 (IL‐3), granulocyte macrophage‐colony stimulating factor (GM‐CSF), and stem cell factor (SCF) to enhance repopulation by swine bone marrow cells in SCID recipients. The results indicate that swine IL‐3 promotes pig hematopoiesis in SCID mouse recipients. The percentage of swine class I+ cells in bone marrow, spleen, and peripheral blood was markedly increased by a 3‐week treatment course with porcine IL‐3. In longer‐term studies, the effect of IL‐3 was further enhanced by combining it with porcine GM‐CSF. Almost all repopulating porcine cells expressed a swine myeloid marker. Colony‐forming assays showed a correlation of the number of pig‐specific colonies with the number of swine cells detected by flow cytometry in transplanted‐SCID bone marrow recipients. Porcine CD2+ cells which did not express CD4 or CDS coreceptors were also detected in SCID mouse recipients of pig bone marrow, and their numbers were also increased by swine cytokine treatment. Swine IgG, but not B cells were detected in SCID recipients at 3 and 6 weeks following bone marrow transplantation, and declined over time, suggesting that mature B cells engrafted, but that de novo B lymphopoiesis did not occur in these mice. Thus, our study demonstrates that donor‐specific growth factors can help to overcome the physiologic barrier to xenogeneic hematopoiesis in the discordant pig to mouse species combination.

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.

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.

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

Abstract: 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.