Sykes M

Mixed allogeneic chimerism as an approach to transplantation tolerance

Abstract Mixed allogeneic chimerism is currently being investigated as a means to induce donor-specific transplantation tolerance across MHC barriers. Here, Megan Sykes and David Sachs compare the two most extensively studied examples - lethal whole body irradiation followed by reconstitution with mixed allogeneic and syngeneic marrow, and total lymphoid irradiation followed by allogeneic marrow infusion - and assess the likelihood that they will be clinically useful in the future.

The importance of nonimmune factors in reconstitution by discordant xenogeneic hematopoietic cells.

Bone marrow transplantation has been shown to induce donor-specific tolerance in rodent models. This approach could potentially be applied to xenotrans- plantation across discordant species barriers. To evaluate host factors resisting hematopoietic cell engraftment, we have developed two model systems utilizing the combination of swine into severe combined immunodeficient (SCID) mice. SCID mice lack functional B and T lymphocytes, and can therefore be used to evaluate nonimmune factors resisting marrow en-graftment, and for adoptive transfer studies to test the role of immune cells and antibodies. First we transplanted swine bone marrow cells into SCID mice conditioned with whole-body irradiation (4 Gy). For nine weeks following the intravenous administration of 108 swine bone marrow cells, up to 3.8% of peripheral blood leukocytes were of swine origin, as determined by flow cytometry (FCM). These cells were all of the myeloid lineage. Swine IgG was also detectable in the serum for up to 14 weeks. The bone marrow of the reconstituted mice contained low percentages of swine myeloid cells, and swine myeloid progenitors

In vitro and in vivo analysis of bone marrow-derived CD3+, CD4−, CD8−, NK1.1+ cell lines

The development of methods of avoiding graft-versus-host disease (GVHD) while retaining the alloengraftment-promoting and anti-leukemic effects of allogeneic T cells is a major goal of research in bone marrow transplantation (BMT). We have recently obtained evidence suggesting that natural suppressor (NS) cells derived from T cell-depleted (TCD) syngeneic marrow can protect against GVHD while permitting alloengraftment. We have now attempted to enrich and then propagate NS cells in vitro, with the goal of obtaining an enhanced anti-GVHD effect by adoptive transfer in vivo. Two long-term cell lines were generated culturing BMC depleted of Mac1-positive cells and of Mac1-positive plus Thy1-positive cells in high concentrations of IL-2. Both cell lines showed anti-GVHD effects when administered along with a GVHD-producing inoculum, while permitting complete allogeneic reconstitution. A clone derived from Mac1-depleted BMC protected completely against a more chronic pattern of GVHD. These cell lines demonstrated suppressive activity in vitro, cytolytic activity against a broad range of natural killer (NK)-sensitive and NK-resistant targets, and a novel cell surface phenotype, with characteristics of both alpha beta-TcR-bearing T cells and of NK cells. In some respects, these cells resemble LAK cells and differ from fresh NS cells, and from the cloned NS cells derived from spleens of total lymphoid irradiation (TLI)-treated mice and neonatal mice. To our knowledge, this is the first detailed phenotypic analysis of cell lines with in vivo anti-GVHD activity. If applicability can be demonstrated in large animal models, the ability to use bone marrow as a source of such protective cell lines might also have potential utility in clinical BMT.

Enhanced CD4 reconstitution by grafting neonatal porcine tissue in alternative locations is associated with donor-specific tolerance and suppression of preexisting xenoreactive T cells.

BACKGROUNDnDonor-specific xenograft tolerance can be achieved by grafting fetal porcine thymus tissue to thymectomized (ATX) mice treated with natural killer (NK) and T-cell-depleting monoclonal antibodies plus 3 Gy of total body irradiation (TBI). Grafting of neonatal, instead of fetal, thymus, along with neonatal pig spleen, leads to a lower level of mouse CD4 cell reconstitution, with less reliable tolerance induction. For a number of reasons, it would be advantageous to use neonatal rather than fetal pigs as donors. We therefore investigated the possibility that grafting larger amounts of neonatal porcine thymus tissue to different sites could allow improved outcomes to be achieved.nnnMATERIALS AND METHODSnMultiple or single fragments of neonatal porcine thymus tissue were grafted with a splenic fragment to different sites (mediastinum, mesentery, and kidney capsule) of ATX B6 mice treated with T- and NK-cell-depleting antibodies and 3Gy TBI. Mice also received an intraperitoneal injection containing 1 x 10(7) donor splenocytes. Donor-specific skin graft tolerance was evaluated, and CD4 reconstitution and mouse anti-donor xenoantibodies were followed by flow cytometry.nnnRESULTSnPeripheral repopulation of CD4+ cells occurred by 7 weeks after transplantation in mice grafted with four fragments of neonatal porcine tissue in either the mediastinum or the mesentery, but not in mice grafted under both kidney capsules with the same amount of tissue. The level of CD4 reconstitution correlated with skin graft tolerance and an absence of induced anti-donor xenoantibodies. Seventy-five percent of mice with >20% of CD4+ cells among peripheral blood lymphocytes (PBL) by 13 weeks posttransplantation accepted donor porcine skin, while rejecting either non-donor neonatal porcine or mouse BALB/c skin allografts. In contrast, only 29% of grafted mice with <20% CD4+ cells in the peripheral blood at 13 weeks accepted donor porcine skin. Grafted mice with poor reconstitution showed either low or high levels of anti-pig xenoantibodies of the IgM, IgG1, and IgG2a isotypes. Grafted mice with >20% CD4+ cells all had low levels of anti-pig xenoantibodies of these isotypes and displayed mixed lymphocyte reaction (MLR) tolerance to donor pig major histocompatibility complex (MHC), with responsiveness to allogeneic mouse stimulators.nnnCONCLUSIONnGrafting neonatal porcine thymus into either the mediastinum or mesentery provides earlier and more efficient reconstitution of the CD4 compartment than does grafting under the kidney capsule. Good CD4 reconstitution was associated with optimal donor-specific skin graft tolerance and avoidance of the anti-donor xenoantibody responses observed in mice with poor CD4 reconstitution. These results also suggest that there is a suppressive component to the porcine xenograft tolerance induced with this approach.

Natural suppressor cells in spleens of irradiated, bone marrow-reconstituted mice and normal bone marrow: Lack of SCA-1 expression and enrichment by depletion of MAC1-positive cells

We have recently reported the development of natural suppressor (NS) cells in lethally irradiated, bone marrow-reconstituted mice during the early weeks after bone marrow transplantation (BMT). These cells were shown to be derived primarily from the syngeneic marrow component in recipients of mixed allogeneic plus syngeneic (host type) marrow, and it was speculated that they might be responsible for the anti-GVHD effect previously described for T-cell-depleted syngeneic marrow. It was therefore of interest to look for such suppressive activity in normal adult bone marrow, which might serve as an obtainable source of such cells if they were to be isolated and used clinically. Such activity has indeed been found in normal adult bone marrow and its characteristics compared to that in spleens of early BMT recipients. Suppressive cells from both sources were similar in their specificity patterns and radiosensitivity, and were of the null (i.e., non-T, non-B, nonmacrophage) cell phenotype. Suppression from either source can be enriched by removal of Mac1-positive cells, providing a possible approach to obtaining NS-enriched populations for in vitro expansion and adoptive transfer studies. Such depletion of Mac1-positive cells was associated with a threefold enrichment of Thy1-positive cells, of which one half were CD4- and CD8-negative, similar to the reported phenotype of cultured NS cell lines. Even when enriched in this manner, the contribution of Thy1-positive cell populations did not reach statistical significance. A recent report has suggested that NS cells might actually be pluripotent hematopoietic stem cells. In contrast, we report here that depletion of Sca1-positive pluripotent hematopoietic stem cells with monoclonal antibody plus immunomagnetic beads does not remove NS activity.

The induction of specific pig skin graft tolerance by grafting with neonatal pig thymus in thymectomized mice.

BACKGROUNDnXenogeneic donor-specific tolerance can be induced by transplanting fetal pig thymus and liver tissue (FP THY/LIV) to thymectomized (ATX), T/NK cell-depleted mice. By using neonatal pig tissue, we hoped to overcome two obstacles that arise with the use of fetal pig tissue: (1) the inability to keep fetal pigs alive after harvesting their thymic tissue, resulting in unavailability of their skin or other organs for grafting; and (2) the limited fetal thymic tissue yield, making application to large animals and humans more difficult.nnnMETHODSnNeonatal pig thymus tissue (NP THY) was grafted into ATX, T/NK cell-depleted, 3Gy whole body-irradiated, originally immunocompetent B6 mice to evaluate the ability of NP THY to reconstitute mouse CD4+ T cells and to induce xenogeneic tolerance to donor pig skin grafts.nnnRESULTSnRepopulation of mouse CD4+ T cells in the peripheral tissues was observed in T/NK cell-depleted, ATX B6 mice that received NP THY with or without neonatal pig spleen (NP SPL), but not in those receiving NP SPL alone, indicating that pig thymus grafting was necessary and sufficient for mouse T cell recovery. Seven of nine NP THY/SPL-grafted ATX mice and two of six NP THY-grafted ATX mice that reconstituted >5% CD4+ cells in PBL accepted donor pig skin long-term without lymphocyte infiltration, whereas they rejected allogeneic BALB/c skin and third party pig skin grafts as rapidly as euthymic mice.nnnCONCLUSIONSnNP THY can support the development of mouse CD4+ T cells that are functional and specifically tolerant to donor pig antigens in ATX, T/NK cell-depleted, 3 Gy whole body-irradiated, originally immunocompetent B6 mice. Additional grafting of NP SPL with NP THY improves the efficiency of tolerance induction in this model.

Multiple mixed chimeras: Reconstitution of lethally irradiated mice with syngeneic plus allogeneic bone marrow from multiple strains

Reconstitution of lethally irradiated B10 mice with a mixture of 5 x 10(6) B10 plus 15 x 10(6) B10.D2 T-cell-depleted (TCD) bone marrow (BM) cells has previously been shown to produce stable, mixed chimeras which are specifically tolerant to donor skin grafts; the inclusion of TCD syngeneic marrow in the inoculum leads to improved immunocompetence in the resulting chimeras. In order to determine whether this method of transplant tolerance induction could be extended to multiple simultaneous allogeneic donors, we have investigated the engraftment capacity of combinations containing syngeneic and more than one allogeneic source of bone marrow. B10 mice were lethally irradiated and reconstituted with a mixture of (B10 + B10.D2 + B10.BR) or (B10 + B10.RIII + B10.BR) TCD BM. Analysis of each group of animals by flow microfluorometry provided evidence for stable multiple mixed chimerism in the majority of animals. All animals which exhibited such multiple chimerism were also tolerant of skin grafts from both allogeneic donors and promptly rejected fourth party skin grafts. An attempt to produce chimerism with TCD marrow from 5 allogeneic plus syngeneic BM cells was less successful. When animals were given non-TCD allogeneic BM from 2 allogeneic donors along with TCD syngeneic BM, they reconstituted as fully allogeneic chimeras in which one or the other allogeneic donor prevailed. These results indicate that (1) multiple allogeneic donor BM cells can engraft simultaneously in the mixed marrow model, but there may be a limit to the number of marrow strains which can repopulate a single animal; (2) multiple allogeneic engraftment confers transplantation tolerance to multiple donors; and (3) TCD is essential to permit multiple mixed chimerism to develop.

Mixed Bone Marrow Chimeras as an Approach to Transplantation Tolerance

Transplantation tolerance across a complete MHC barrier can be achieved in mice using lethal irradiation followed by reconstitution with a mixture of T-cell-depleted (TCD) syngeneic and allogeneic bone marrow cells.1,2 Such animals repopulate as stable mixed lymphohematopoietic chimeras, and enjoy improved survival compared with recipients of allogeneic TCD marrow alone,3 probably due, in part, to the improved immunocompetence they demonstrate.3 In addition, such animals are free from any clinical sign of graft-versus-host disease (GVHD), and this is also the case even if T-cells are not removed from the allogeneic marrow.4 Experiments in which allogeneic spleen cells were added to the donor inoculum to intentionally produce GVHD indicated that the syngeneic TCD component of the bone marrow inoculum improved the survival of such animals, presumably due to protection from GVHD.4 The data presented in this paper address the possible mechanism of this protective effect. We show that the syngeneic marrow inoculum provides a source of precursor cells which, in recipient spleens, develop the ability to suppress the generation of cytolytic activity by mature T-lymphocytes. We also demonstrate an enhanced protection from GVHD when the syngeneic TCD inoculum is allowed to develop its suppressive activity spontaneously in the recipient before the allogeneic inoculum is given. Such protection could have potential clinical applications.