Bone Marrow Rejection Research Articles

The abrogation of allosensitization following the induction of mixed allogeneic chimerism.

The association of preformed anti-donor Abs with the hyperacute rejection of bone marrow and solid organ allografts and the persistence of the anti-donor immune response secondary to immunologic memory make allosensitization an absolute contraindication to transplantation. Mixed allogeneic (A + B-->A) bone marrow chimerism has been demonstrated to confer donor-specific tolerance in nonsensitized recipients, but has not been evaluated in the setting of allosensitization. The current study documents that despite significant anti-donor sensitization, mixed allogeneic engraftment is possible and provides a marked advantage over fully allogeneic (B-->A) models. Moreover, the acceptance of donor skin grafts and loss of circulating anti-donor Abs suggest that allosensitization can be abrogated with the induction of stable mixed allogeneic chimerism.

MHC class Ia molecules alone control NK-mediated bone marrow graft rejection

Mice with functionally deleted genes encoding MHC class I heavy (H-2Kb, H-2Db) and light (β2-microglobulin) chains were used in bone marrow cell transfer experiments to study the role of class Ia and Ib molecules in NK cell function. Absence of H-2Kb and absence of H-2Db on bone marrow cells resulted in complete and in almost complete NK-mediated rejection, respectively. Absence of either H-2 class Ib (at least when expressed in H-2 class Ia-deficient mice) or cell surface class Ia free heavy chains did not result in bone marrow rejection. Thus, in C57BL / 6 adult mice, the inactivation of NK cells required for bone marrow cell engraftment relies entirely upon-H-2 class Ia molecules. These results imply the existence of an inhibitory receptor which recognizes either directly or indirectly H-2Db molecules and further suggest that in C57BL / 6 mice the NK cells which do not express a H-2Kb specific inhibitory receptor necessarily express an H-2Db-specific one.

MHC class Ia molecules alone control NK-mediated bone marrow graft rejection.

Mice with functionally deleted genes encoding MHC class I heavy (H-2K(b), H-2D(b)) and light (beta2-microglobulin) chains were used in bone marrow cell transfer experiments to study the role of class Ia and Ib molecules in NK cell function. Absence of H-2K(b) and absence of H-2D(b) on bone marrow cells resulted in complete and in almost complete NK-mediated rejection, respectively. Absence of either H-2 class Ib (at least when expressed in H-2 class Ia-deficient mice) or cell surface class Ia free heavy chains did not result in bone marrow rejection. Thus, in C57BL/6 adult mice, the inactivation of NK cells required for bone marrow cell engraftment relies entirely upon-H-2 class Ia molecules. These results imply the existence of an inhibitory receptor which recognizes either directly or indirectly H-2D(b) molecules and further suggest that in C57BL/6 mice the NK cells which do not express a H-2K(b) specific inhibitory receptor necessarily express an H-2D(b)-specific one.

Ly-49 G2+ NK cells are responsible for mediating the rejection of H-2b bone marrow allografts in mice.

NK cells can mediate the specific rejection of bone marrow but not solid tissue allografts in lethally irradiated mice. NK cells are also responsible for the phenomenon of "hybrid resistance' in which F1 hybrid H-2 heterozygous mice can reject parental H-2 homozygous bone marrow grafts. Ly-49C and Ly-49 G2 are markers identified on subsets of NK cells. While Ly-49C+ NK cells have been demonstrated to mediate the specific rejection of H-2d bone marrow allografts, the role of the Ly-49 G2+ NK subset is unclear because depletion of this subset in vivo did not affect splenic NK activity against tumor targets. Through bone marrow transplantation typing studies, we demonstrate that Ly-49 G2+ NK cells complement Ly-49C+ NK cells in that they specifically mediate the rejection of H-2b bone marrow allografts in lethally irradiated mice. In support of this, depletion of the Ly-49C+ NK subset in vivo also enhanced the ability of the mice to reject H-2b bone marrow cells suggesting that the depletion was augmenting the ability of the Ly-49 G2+ NK cells to reject the marrow allografts. Depletion of Ly-49 G2+ NK cells in F1 hybrid mice abrogated their ability to reject parental H-2b but not H-2d bone marrow grafts. Therefore, Ly-49 G2 denotes a subset of NK cells that appears to play a critical role in the recognition of H-2b bone marrow cells in allogeneic and F1 hybrid mice.

PREVENTION OF BONE MARROW AND CARDIAC GRAFT REJECTION IN AN H-2 HAPLOTYPE DISPARATE MOUSE COMBINATION BY AN ANTI-LFA-1 ANTIBODY

The aim of the present study was to compare the preventive effect of a nondepleting monoclonal antibody specific for the LFA-1 alpha chain (CD11a) on the rejection of bone marrow, vascularized cardiac, and nonvascularized skin grafts in the same haplotype-disparate mouse strain combination. A 7-day treatment with a total dose of 0.5 mg of anti-LFA-1 antibody (H-129) had no effect on the rejection of BDF1(H-2b/d) skin grafts by CDF1 (H-2k/d)-treated mice. In contrast, the same treatment regimen significantly prolonged the survival of BDF1 cardiac allografts in CDF1 mice: 7 out of 10 mice had a functional graft after 70 days, whereas all control mice had rejected their graft by 11 days. Nevertheless, cardiac allografts were ultimately rejected. In contrast, infusion of anti-LFA-1 antibody was able to promote definitive engraftment of T-depleted BDF1 marrow in 9 gray-irradiated CDF1 recipients: in surviving mice, engraftment increased from 10% in controls to 78% in antibody-treated recipients. In mice that tolerated their cardiac graft for more than 70 days, there was a slight delay in the rejection of donor skin graft but no in vitro evidence of tolerance. In contrast, mice with successful marrow engraftment did not reject donor skin graft and failed to mount proliferative and cytotoxic responses against donor alloantigens, whatever the percentage of engrafted donor leukocytes. These results indicate that a nondepleting anti-LFA-1 antibody can efficiently protect against rejection of MHC-incompatible heart and bone marrow. The protective effect of anti-LFA-1 antibody was associated with the induction of T cell tolerance toward donor antigens after bone marrow transplantation.

Prevention of Graft-Versus-Host Disease and Bone Marrow Rejection: Kinetics of Induction of Tolerance by UVB Modulation of Accessory Cells and T Cells in the Bone Marrow Inoculum

AbstractUVB irradiation (700 J/m2) of bone marrow cells (UVB-BMC) before transplantation into lethally gamma-irradiated (10.5 Gy) allogeneic rats prevents graft-versus-host disease (GVHD) and induces a stable complete lymphohematopoietic chimerism. To better understand the underlying mechanism of the development of stable chimerism and induction of tolerance to donor organs in this model, we examined if the addition of T cells or dendritic cells (DC), as antigen presenting cells (APC), would restore the immunogenicity of UVB-BMC in in vitro mixed lymphocyte reaction (MLR) and induce in vivo bone marrow (BM) graft rejection. Whereas gamma-irradiated, unfractionated BMC induce allogeneic T cells to proliferate, UVB irradiation of BMC abolishes the stimulatory capacity of such cells in a primary MLR. Addition of purified T cells, CD4+ T cells, CD8+ T cells or B cells, respectively, failed to restore the capacity of UVB-BMC to stimulate allogeneic T- cell proliferation. In contrast, the addition of only a small number of splenic accessory cells or purified DC, which by themselves were relatively ineffective in stimulating T-cell proliferation, restored the accessory function and the allostimulatory capacity of UVB-BMC. To define the molecular defect induced by UVB irradiation, cytokines were added as costimulatory factors to primary MLRs and the results showed that the addition of interleukin (IL)-2 or IL-6 but not IL-1 or interferon gamma (IFN-gamma) restored the stimulatory capacity of UVB BMC. This finding suggests that UVB may alter the production, and/or utilization of IL-2 and IL-6 either at the membrane or cytoplasmic level. Parallel in vivo studies showed that addition of DC to UVB BM inoculum resulted in failure of BM engraftment, whereas addition of T cells led to development of fatal GVHD, thus suggesting that UVB modulation of accessory cells reduces graft immunogenicity and prevents BMT rejection, while modulation of T cells prevents GVHD. Our data provide evidence that UVB modulation of APC and mature T cells contained within BMC is potentially useful in preventing GVHD without endangering successful engraftment and may serve as a model for induction of adult chimerism and tolerance without the development of GVHD.

Prevention of graft-versus-host disease and bone marrow rejection: kinetics of induction of tolerance by UVB modulation of accessory cells and T cells in the bone marrow inoculum

UVB irradiation (700 J/m2) of bone marrow cells (UVB-BMC) before transplantation into lethally gamma-irradiated (10.5 Gy) allogeneic rats prevents graft-versus-host disease (GVHD) and induces a stable complete lymphohematopoietic chimerism. To better understand the underlying mechanism of the development of stable chimerism and induction of tolerance to donor organs in this model, we examined if the addition of T cells or dendritic cells (DC), as antigen presenting cells (APC), would restore the immunogenicity of UVB-BMC in in vitro mixed lymphocyte reaction (MLR) and induce in vivo bone marrow (BM) graft rejection. Whereas gamma-irradiated, unfractionated BMC induce allogeneic T cells to proliferate, UVB irradiation of BMC abolishes the stimulatory capacity of such cells in a primary MLR. Addition of purified T cells, CD4+ T cells, CD8+ T cells or B cells, respectively, failed to restore the capacity of UVB-BMC to stimulate allogeneic T- cell proliferation. In contrast, the addition of only a small number of splenic accessory cells or purified DC, which by themselves were relatively ineffective in stimulating T-cell proliferation, restored the accessory function and the allostimulatory capacity of UVB-BMC. To define the molecular defect induced by UVB irradiation, cytokines were added as costimulatory factors to primary MLRs and the results showed that the addition of interleukin (IL)-2 or IL-6 but not IL-1 or interferon gamma (IFN-gamma) restored the stimulatory capacity of UVB BMC. This finding suggests that UVB may alter the production, and/or utilization of IL-2 and IL-6 either at the membrane or cytoplasmic level. Parallel in vivo studies showed that addition of DC to UVB BM inoculum resulted in failure of BM engraftment, whereas addition of T cells led to development of fatal GVHD, thus suggesting that UVB modulation of accessory cells reduces graft immunogenicity and prevents BMT rejection, while modulation of T cells prevents GVHD. Our data provide evidence that UVB modulation of APC and mature T cells contained within BMC is potentially useful in preventing GVHD without endangering successful engraftment and may serve as a model for induction of adult chimerism and tolerance without the development of GVHD.

Rejection of class I MHC-deficient haemopoietic cells by irradiated MHC-matched mice

Irradiated MHC-heterozygous mice often reject bone marrow cells transplanted from one of the homozygous parental strains, a phenomenon ('hybrid resistance') that appears to violate the laws of transplantation. Rejection of parental and allogeneic marrow cells also differs from conventional T cell-mediated rejection mechanisms as it is effected by NK1.1+ cells. To account for the unusual specificity of bone marrow rejection, it has been proposed that NK1.1+ cells destroy marrow cells that fail to express the full complement of self MHC class I (MHC-I) molecules. We show here that NK1.1+ cells in normal mice reject haemopoietic transplants from mice that are deficient for normal cell-surface MHC-I expression because of a targeted mutation in the beta 2-microglobulin gene. These findings demonstrate that deficient expression of MHC-I molecules renders marrow cells susceptible to rejection.

Natural killer cells activated with interleukin 2 in vitro can be adoptively transferred and mediate hematopoietic histocompatibility‐1 antigen‐specific bone marrow rejection in vivo

An adoptive transfer model was developed for examining natural killer (NK) cell-mediated bone marrow cell (BMC) graft rejection. Homogeneous populations of NK cells were obtained by culturing spleen cells from mice with severe combined immune deficiency in the presence of recombinant interleukin 2 for 7 days. These cells maintained a phenotypic (CD3-CD4-CD8-NK-2.1+ and asialo GM1+) and lytic pattern consistent with that of activated NK cells. The cells were infused into lethally irradiated recipients whose own NK cells were depleted by anti-NK-1.1 antibody treatment and could no longer reject marrow allografts. The transferred NK cells restored the ability of the hosts to reject BMC in a hematopoietic histocompatibility-1 (Hh-1) antigen-specific manner. Immunogenetic studies demonstrated that the rejection was a function of the donor, not host, NK cells. These studies demonstrate that NK cells can be cultured in vitro with recombinant interleukin 2 and are capable of mediating Hh-1-specific BMC rejection in vivo in the absence of other immune cell types.

Induction of bone marrow allograft rejection and hybrid resistance in nonresponder recipients by antibody: is there evidence for a dual receptor interaction in acute marrow graft rejection?

Acute marrow graft rejection in allogeneic or semiallogeneic donor-recipient mouse combinations has been suggested to be caused by natural killer (NK) cells. The unique in vitro specificity of NK cells for tumor cells, however, does not explain the specific rejection of bone marrow grafts by NK cells. Recent experiments have implicated antibody in marrow graft recipients as the specificity-inducing component that guides NK cells in an antibody-dependent cytotoxic (ADCC) reaction to attack the marrow graft. On the basis of this hypothesis, one would postulate that nonresponder marrow graft recipients can be converted into responders by injection with antibody of appropriate specificity. Results presented in this report show that this is indeed possible. Specific monoclonal or polyclonal antibody of IgG isotype induces marrow graft rejection in nonresponder recipients. This can be demonstrated in allogeneic as well as in semi-allogeneic (hybrid resistance) donor-recipient strain combinations. Antibody-induced marrow graft rejection is independent of complement and dependent on the presence of NK cells. Surprisingly, graft rejection induced by antibody is quite efficient in allogeneic and semiallogeneic marrow donor-recipient combinations, whereas it is generally poor in syngeneic combinations. This result is not understood if NK cells lyse bone marrow cells solely in an ADCC-type reaction. Because NK cells can lyse targets in an antibody-dependent as well as independent reaction, it is proposed that the binding of NK cells to targets via their receptors plays an additional role in the rejection of bone marrow in vivo. Preliminary evidence for this possibility is that NK cells in the apparent absence of antibody may have a detectable suppressive effect on the growth of marrow grafts in F1 hybrid mice transplanted with parental marrow grafts.

Immunohistological skin alterations in allogeneic bone marrow transplantation.

Skin biopsies of 26 patients with leukemia and seven patients with aplastic anemia were investigated before and at different stages after allogeneic bone marrow transplantation (BMT) to establish the immunological criteria which distinguish skin alterations during normal reconstitution from dermal lesions mediated by graft-versus-host disease (GvHD). Of the 33 patients studied 27 presented with clinically diagnosed acute and/or chronic GvHD, one patient died of bone marrow rejection. Immunohistological analysis of the respective skin biopsies with selected monoclonal antibodies against human leukocyte antigens (HLA) and differentiation antigens of the lympho-hematopoietic cells revealed low dermal mononuclear cell counts with phenotypically normal constituents in five cases with uncomplicated reconstitution post-grafting. In contrast, increased dermal cellular infiltrates predominantly consisting of Lyt 3+, OKT 8+ T-lymphocytes, as well as of a large number of Ia-like (immune response associated = HLA-D) determinant + monocytes/macrophages were observed in all patients with active acute/chronic GvH reactivity. As sign of activation simultaneous expression of HLA-D region products was also found on a subset of the invading OKT8+ T-lymphocytes. Progression of GvHD was associated with additional surface staining of keratinocytes for Ia-like determinants. Loss of Ia-like determinant+, OKT6+ dentritic epithelial cells in all leukemic patients, as well as in patients with aplastic anemia with or without GvHD suggested damage of Langerhans cells due to the previous radiotherapy and/or specific immunological destruction. In patients with fatal outcome of GvHD prolonged reduction of these dentritic epithelial cells seemed to be indicative of impaired immune reconstitution or bone marrow dysfunction. Thus immunopathological features of skin GvHR may enable early recognition and prognostic evaluation of this disease possibly allowing more effective therapy.

Experience with incompatible maternal donors for bone marrow transplantation.

Marrow transplantation in aplastic anemia and leukemia has generally been limited to siblings who have been histocompatible at both the serological (A and B) and lymphocyte determined (D or MLC) loci of the HLA system. We studied three male patients, two with aplastic anemia and one with acute myelogenous leukemia, who received transplants from their histoincompatible mothers. MLC studies between donors and recipients showed varying degrees of stimulation. Definite engraftment occurred in one patient and transient engraftment in another. Engraftment in the third patient could not be evaluated. In the patient with sustained engraftment, there was clinical evidence of severe graft versus host disease (GVHD) however, this was not substantiated by histologic findings. This preliminary study suggests that MLC incompatibility may be more of an indicator of the risk of GVHD than of bone marrow rejection. If more effective control of GVHD can be accomplished, marrow transplantation between MLC-reactive individuals may become feasible.