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Biomedical subjects

M Sykes

Publications and source records attributed to M Sykes.

At least 73 records · Page 4Linked to original sources
PubMed1999

Dose and timing of interleukin (IL)-12 and timing and type of total-body irradiation: effects on graft-vs.-host disease inhibition and toxicity of exogenous IL-12 in murine bone marrow transplant recipients.

Paradoxically, a single injection of recombinant murine interleukin (IL)-12 on the day of bone marrow transplantation (BMT) inhibits graft-vs.-host disease (GVHD) while preserving graft-vs.-leukemia (GVL) effects in lethally irradiated mice receiving fully MHC-mismatched bone marrow and spleen cells. These protective effects are mediated by interferon (IFN)-gamma, whose early secretion is induced by IL-12 treatment. We investigated the relationship of IL-12 dose and timing of administration, as well as timing and type of total-body irradiation (TBI), with the ability of IL-12 to inhibit GVHD or mediate toxicity. The results show that a relatively low dose of IL-12 (as little as 50 U in a single injection) can mediate significant GVHD protection. The timing of IL-12 administration, however, is a critical factor. IL-12 administered 1 hour before BMT was most protective, but protection was still observed when it was administered 1-12 hours after BMT. Delaying IL-12 administration to 36 hours post-BMT completely obviated its protective effect. Administration of a second IL-12 injection 6 days after BMT negated the protective effect of an initial injection at the time of BMT. While IL-12 protection was evident when TBI was administered by 137Cs-irradiator in one or two fractions on day -1 or day 0, the use of an X-irradiator to deliver TBI on day -1 was associated with marked IL-12 toxicity. Whereas the protective effect of IL-12 against GVHD depended on donor-derived IFN-gamma, toxicity depended on the ability of host cells to produce IFN-gamma. Careful studies are warranted to test the effects of IL-12 in the context of BMT with various conditioning regimens in large animal preclinical models before this novel approach to GVHD protection can be applied clinically.

Animals↗
PubMed1999

Lymphohematopoietic graft-vs.-host reactions can be induced without graft-vs.-host disease in murine mixed chimeras established with a cyclophosphamide-based nonmyeloablative conditioning regimen.

Mixed hematopoietic chimerism can be induced in mice receiving allogeneic bone marrow transplantation (BMT) after nonmyeloablative host conditioning with depletion T cells with of anti-T cell monoclonal antibodies (mAbs), low-dose (3 Gy) total-body irradiation (TBI), and local thymic irradiation (7 Gy). These mice are specifically tolerant to donor and host antigens. When nontolerant donor T cells are given to chimeras several months after BMT, full donor-type chimerism develops, but graft-vs.-host disease (GVHD) does not occur. The induction of such lymphohematopoietic GVH reactions without GVHD could provide an approach to separating graft-vs.-leukemia (GVL) from GVHD in patients with hematologic malignancies. To make the nonmyeloablative conditioning regimen described above more cytoreductive for such malignancies, we have now modified it by replacing TBI with cyclophosphamide (CP). Treatment with anti-CD4 and anti-CD8 mAbs on day -5, 200 mg/kg CP on day -1, and 7 Gy thymic irradiation on day 0 was only slightly myelosuppressive and allowed fully major histocompatibility complex (MHC)-mismatched (with or without multiple minor antigen disparities) allogeneic bone marrow to engraft and establish long-term mixed chimerism in 40 to 82% of recipients in three different strain combinations. The administration of nontolerant donor spleen cells at 5 weeks or at 5, 8, and 11 weeks posttransplant was capable of eliminating host hematopoietic cells, leading to full or nearly full donor chimerism in six of six and two of four chimeric animals in two different strain combinations. No clinical evidence of GVHD was observed in any recipients of these donor leukocyte infusions (DLI). These studies demonstrate that induction of mixed chimerism with nonmyeloablative conditioning followed at appropriate times by DLI might allow lymphohematopoietic GVH reactions, and hence GVL effects, to eliminate chronic hematologic malignancies without causing clinically significant GVHD.

Animals↗
PubMed1999

Mixed chimerism induced without lethal conditioning prevents T cell- and anti-Gal alpha 1,3Gal-mediated graft rejection.

Gal alpha 1,3Gal-reactive (Gal-reactive) antibodies are a major impediment to pig-to-human xenotransplantation. We investigated the potential to induce tolerance of anti-Gal-producing cells and prevent rejection of vascularized grafts in the combination of alpha 1,3-galactosyltransferase wild-type (GalT(+/+)) and deficient (GalT(-/-)) mice. Allogeneic (H-2 mismatched) GalT(+/+) bone marrow transplantation (BMT) to GalT(-/-) mice conditioned with a nonmyeloablative regimen, consisting of depleting CD4 and CD8 mAb's and 3 Gy whole-body irradiation and 7 Gy thymic irradiation, led to lasting multilineage H-2(bxd) GalT(+/+) + H-2(d) GalT(-/-) mixed chimerism. Induction of mixed chimerism was associated with a rapid reduction of serum anti-Gal naturally occurring antibody levels. Anti-Gal-producing cells were undetectable by 2 weeks after BMT, suggesting that anti-Gal-producing cells preexisting at the time of BMT are rapidly tolerized. Even after immunization with Gal-bearing xenogeneic cells, mixed chimeras were devoid of anti-Gal-producing cells and permanently accepted donor-type GalT(+/+) heart grafts (>150 days), whereas non-BMT control animals rejected these hearts within 1-7 days. B cells bearing receptors for Gal were completely absent from the spleens of mixed chimeras, suggesting that clonal deletion and/or receptor editing may maintain B-cell tolerance to Gal. These findings demonstrate the principle that induction of mixed hematopoietic chimerism with a potentially relevant nonmyeloablative regimen can simultaneously lead to tolerance among both T cells and Gal-reactive B cells, thus preventing vascularized xenograft rejection.

Animals↗
PubMed1999

The role of interleukin-12 in preserving the graft-versus-leukemia effect of allogeneic CD8 T cells independently of GVHD.

Interleukin (IL)-12 is a potent immunostimulatory cytokine and inducer of Th1 cell activity and of cytotoxic T lymphocyte and natural killer cell function. This cytokine also has anti-tumor activity. Although IL-12 has been shown to be an important pathogenic cytokine in the induction of graft-versus-host disease (GVHD), injection of exogenous IL-12 to murine allogeneic bone marrow transplantation (BMT) recipients paradoxically leads to a significant delay in the onset of GVHD mortality in fully MHC plus multiple minor antigen-mismatched strain combinations, and to complete inhibition of GVHD in a single haplotype-mismatched murine BMT model. IL-12-induced inhibition of GVHD is associated with reduced donor T cell activation and expansion, in part through an interferon (IFN)-gamma-mediated mechanism. Fas-mediated apoptosis of donor T cells also plays a significant role in IL-12-induced GVHD protection. Importantly, IL-12 preserves the graft-versus-leukemia (GVL) effect of allogeneic CD8 T cells against EL4, a host-type leukemia/lymphoma, while inhibiting GVHD. Like the protective effect against GVHD, the GVL effect in IL-12-treated mice is dependent on IFN-gamma. Thus, treatment with IL-12 leads to separation of GVHD-promoting and GVL effects of allogeneic BMT via an IFN-gamma-dependent mechanism.

Animals↗
PubMed1998

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.

Animals↗
PubMed1998

Cross-species interaction of porcine and human integrins with their respective ligands: implications for xenogeneic tolerance induction.

BACKGROUND: Organ transplantation is limited by the number of available donors. One possible solution would be the use of pigs as organ donors. However, current immunosuppressive protocols cannot prevent rejection of these organs. If donor-specific tolerance toward porcine antigens could be induced in recipients, subsequent implantation of porcine organs would be possible without further immunosuppression. Induction of tolerance can be achieved with a bone marrow transplant if donor antigen-presenting cells successfully differentiate in the recipient thymus to induce deletion of donor-reactive host cells. Migration of porcine progenitor cells to the host marrow and thymus and differentiation into tolerance-inducing antigen-presenting cells is likely to require successful interaction of porcine adhesion molecules with human ligands. In this study, we investigated whether very late antigen (VLA)4 and VLA-6 integrins, which play important roles in homing and differentiation of hematopoietic progenitor cells, function across the pig-to-human species barrier. METHODS: Static cell-to-cell and cell-to-extracellular matrix protein adhesion assays were used to examine the cross-species interaction of porcine adhesion molecules with human ligands. RESULTS: Our studies show that porcine cells adhere to various human endothelial cell monolayers and extracellular matrix proteins and demonstrate that porcine VLA-4 and VLA-6 appear to be fully cross-reactive to the human ligands vascular cell adhesion molecule-1 and laminin, respectively. CONCLUSIONS: It is likely that porcine hematopoietic progenitor cells will be able to successfully employ pVLA-4- and pVLA-6-human ligand interactions in a pig-to-human bone marrow transplantation model in order to induce donor-specific tolerance.

Animals↗
PubMed1998

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.

Animals↗
PubMed1998

Function of porcine adhesion molecules in a human marrow microenvironment.

BACKGROUND: One way to circumvent the need for chronic immunosuppression in solid organ xenografting may be to induce donor-specific tolerance using bone marrow transplantation. If this approach is to succeed in the pig-to-human species combination, pig marrow must be capable of maturing into relevant tolerance-inducing cells and replenishing itself in host human marrow. One possible barrier is adhesion molecule incompatibility. We have studied the compatibility across the pig-human species barrier of two well-characterized ligands known to be important in hematopoiesis, CD44 and very late antigen (VLA)-4. METHODS: In vitro long-term bone marrow cultures were studied in which the effects of blocking antibodies were assessed by measuring cell numbers and colony-forming units. RESULTS: The blocking of CD44 had a comparable inhibitory effect on the hematopoiesis of human and pig marrow, even if the latter was maintained on a human stromal layer. Both cellular proliferation and colony-forming activity were inhibited by anti-CD44 monoclonal antibody. By contrast, a significant difference was observed in VLA-4 usage by hematopoietic cells of the two species. Blocking VLA-4 markedly inhibited human hematopoietic cellular proliferation but had no effect on pig hematopoiesis, on either porcine or human stroma. CONCLUSIONS: The data suggest that the incompatibility of either CD44 or VLA-4 is unlikely to limit the efficiency of porcine hematopoiesis in a human marrow environment. However, the difference in VLA-4 utilization between these species raises the possibility that other interactions may be important for effective porcine hematopoiesis and that their failure to function between species may contribute to the poor function of porcine hematopoietic cells in primate marrow microenvironments.

Animals↗
PubMed1998

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.

Animals↗
PubMed1998

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.

Animals↗
PubMed1998

Extrathymic T cell deletion and allogeneic stem cell engraftment induced with costimulatory blockade is followed by central T cell tolerance.

A reliable, nontoxic method of inducing transplantation tolerance is needed to overcome the problems of chronic organ graft rejection and immunosuppression-related toxicity. Treatment of mice with single injections of an anti-CD40 ligand antibody and CTLA4Ig, a low dose (3 Gy) of whole body irradiation, plus fully major histocompatibility complex-mismatched allogeneic bone marrow transplantation (BMT) reliably induced high levels (>40%) of stable (>8 mo) multilineage donor hematopoiesis. Chimeric mice permanently accepted donor skin grafts (>100 d), and rapidly rejected third party grafts. Progressive deletion of donor-reactive host T cells occurred among peripheral CD4(+) lymphocytes, beginning as early as 1 wk after bone marrow transplantation. Early deletion of peripheral donor-reactive host CD4 cells also occurred in thymectomized, similarly treated marrow recipients, demonstrating a role for peripheral clonal deletion of donor-reactive T cells after allogeneic BMT in the presence of costimulatory blockade. Central intrathymic deletion of newly developing T cells ensued after donor stem cell engraftment had occurred. Thus, we have shown that high levels of chimerism and systemic T cell tolerance can be reliably achieved without myeloablation or T cell depletion of the host. Chronic immunosuppression and rejection are avoided with this powerful, nontoxic approach to inducing tolerance.

Abatacept↗
PubMed1998

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.

Animals↗
PubMed1998

Interleukin-12 inhibits graft-versus-host disease through an Fas-mediated mechanism associated with alterations in donor T-cell activation and expansion.

We have recently made the paradoxical observation that a single injection of recombinant murine interleukin-12 (IL-12) on the day of bone marrow transplantation (BMT) inhibits graft-versus-host disease (GVHD) in lethally irradiated mice receiving fully major histocompatability complex (MHC)-mismatched bone marrow and spleen cells. We have now examined the mechanism of this effect of IL-12 on acute GVHD. By day 4 post-BMT, IL-12-treated mice showed marked reductions in splenic donor CD4(+) and CD8(+) T cells compared with GVHD controls. Expression of the early activation markers IL-2R alpha chain (CD25) and CD69 on splenic donor CD4(+) cells was considerably higher at early time points (36 and 72 hours post-BMT) in IL-12-treated mice compared with GVHD controls. However, the later, GVHD-associated increase in CD25 and very late antigen-4 (VLA-4) expression on donor T cells was greatly depressed in IL-12-protected mice compared with GVHD controls. The marked GVHD-associated expansion of host-reactive T helper cells by day 4 was also completely inhibited in the IL-12-treated group. Expression of Fas was increased on donor CD4 cells of IL-12-treated mice compared with those of controls on days 3 through 7 post-BMT. Furthermore, the ability of IL-12 to protect against GVHD was at least partially dependent on the ability of donor cells to express functional Fas molecules. We conclude that IL-12 treatment at the time of BMT markedly perturbs the activation of alloreactive donor CD4(+) T cells that play a critical role in the pathogenesis of acute GVHD. We hypothesize that these perturbations culminate in Fas-dependent apoptosis of donor T cells, thus impeding their expansion and their GVHD-promoting activity.

Animals↗
PubMed1998

Tolerization of anti-Galalpha1-3Gal natural antibody-forming B cells by induction of mixed chimerism.

Xenotransplantation could overcome the severe shortage of allogeneic organs, a major factor limiting organ transplantation. Unfortunately, transplantation of organs from pigs, the most suitable potential donor species, results in hyperacute rejection in primate recipients, due to the presence of anti-Galalpha1-3Gal (Gal) natural antibodies (NAbs) in their sera. We evaluated the ability to tolerize anti-Gal NAb-producing B cells in alpha1,3-galactosyltransferase knockout (GalT KO) mice using bone marrow transplantation (BMT) from GalT+/+ wild-type (WT) mice. Lasting mixed chimerism was achieved in KO mice by cotransplantation of GalT KO and WT marrow after lethal irradiation. The levels of anti-Gal NAb in sera of mixed chimeras were reduced markedly 2 wk after BMT, and became undetectable at later time points. Immunization with Gal+/+ xenogeneic cells failed to stimulate anti-Gal antibody production in mixed chimeras, whereas the production of non-Gal-specific antixenoantigen antibodies was stimulated. An absence of anti-Gal-producing B cells was demonstrated by enzyme-linked immunospot assays in mixed KO + WT --> KO chimeras. Thus, mixed chimerism efficiently induces anti-Gal-specific B cell tolerance in addition to T cell tolerance, providing a single approach to overcoming both the humoral and the cellular immune barriers to discordant xenotransplantation.

Animals↗
PubMed1998

Natural killer cells and their role in graft rejection.

Natural killer cells can weakly resist engraftment of allogeneic bone marrow transplants in mice. Functional studies suggest that natural killer cell tolerance can be induced by bone marrow transplantation. Human natural killer cell inhibitory receptor repertoires differ between individuals, depending on their MHC genotype. This supports the concept that the human natural killer cell repertoire, like that of mice, adapts to the MHC molecules presented in its environment. Natural killer cells play a greater role in rejecting xenogeneic than allogeneic bone marrow and have been implicated in the rejection of xenogeneic solid organ transplants. Natural killer cell inhibitory receptors may have a lower likelihood of cross-reacting with xenogeneic than with allogeneic MHC class I molecules; important glycosylation differences between species may affect the propensity of natural killer cells to kill xenogeneic targets.

Adaptation, Physiological↗