Mixed chimerism and tolerance can be successfully induced in rodents through allogeneic bone marrow transplantation (BMT) with costimulation blockade (CB), but varying success rates have been ...reported with distinct models and protocols. We therefore investigated the impact of minor antigen disparities on the induction of mixed chimerism and tolerance. C57BL/6 (H2b) mice received nonmyeloablative total body irradiation (3 Gy), costimulation blockade (anti-CD40L mAb and CTLA4Ig), and 2×107 bone marrow cells (BMC) from either of three donor strains: Balb/c (H2d) (MHC plus multiple minor histocompatibility antigen (mHAg) mismatched), B10.D2 (H2d) or B10.A (H2a) (both MHC mismatched, but mHAg matched). Macrochimerism was followed over time by flow cytometry and tolerance was tested by skin grafting. 20 of 21 recipients of B10.D2 BMC but only 13 of 18 of Balb/c BMC and 13 of 20 of B10.A BMC developed stable long-term multilineage chimerism (p<0.05 for each donor strain versus B10.D2). Significantly superior donor skin graft survival was observed in successfully established long-term chimeras after mHAg matched BMT compared to mHAg mismatched BMT (p<0.05). Both minor and major antigen disparities pose a substantial barrier for the induction of chimerism while the maintenance of tolerance after nonmyeloablative BMT and costimulation blockade is negatively influenced by minor antigen disparities.
The only way to overcome the need for life-long immunosuppression in a transplant recipient is to induce tolerance. Deletional tolerance can be reliably achieved with the induction of mixed chimerism ...through transplantation of donor bone marrow (BM). Despite the development of increasingly milder BM transplantation (BMT) animal models, BM engraftment in humans still requires considerably toxic conditioning and puts patients at risk for the development of GVHD. However, in a proof-of-concept trial, mixed chimerism and tolerance have been successfully induced in highly selected patients suffering from both end-stage renal disease and multiple myeloma. Meanwhile, there has been notable progress in developing advanced experimental BMT regimens, in particular through the use of costimulation blockers. Costimulation blockade in rodent models allowed the design of BMT protocols entirely devoid of irradiation. Costimulation blockers have also succeeded in more complex protocols in non-human primates. They are under clinical evaluation in renal transplantation as immunosuppressive therapy. Costimulation blockade may lead the way for the development of milder BMT protocols and broader application of mixed chimerism in organ transplantation.
Peripheral and central clonal deletion are important tolerance mechanisms in models using bone marrow transplantation (BMT) with costimulation blockade (CB). However, since tolerance can be found ...before peripheral deletion is complete and since elimination of recipient CD4+ cells at the time of BMT prevents tolerance induction, we investigated the potential roles of regulation and anergy in such a murine model. We found that transient elimination of CD25+ cells or neutralization of IL2 immediately after BMT and CB prevented the induction of skin graft tolerance. Cotransfer into SCID mice of CD4+ cells taken from chimeras early after BMT, together with naïve recipient‐type CD4+ cells significantly prolonged donor skin graft survival. In contrast, cotransfer of CD4+ cells harvested from chimeras late after BMT did not prolong donor skin graft survival. Besides, depletion of CD25+ cells in established chimeras several months post‐BMT did not break tolerance. In vivo administration of recombinant IL2 inhibited chimerism and tolerance neither early nor late post‐BMT, arguing against a decisive role for classical anergy. Thus, CD4 cell‐mediated regulation contributes significantly to tolerance induction early after BMT, but appears to have no critical role in the maintenance of tolerance.
Induction of mixed chimerism (i.e., coexistence of donor and recipient hematopoietic cells) through transplantation of allogeneic donor bone marrow under appropriate host conditioning, is one of the ...most reliable strategies to induce transplantation tolerance. Robust tolerance is evident in mixed chimeras as they permanently accept donor skin grafts while promptly rejecting third party grafts. Although historically, myeloablative and T-cell depleting regimens have been described, milder protocols involving costimulation blockade have recently been developed. The prototypical murine protocol described in this chapter, involves the use of CTLA4Ig and a monoclonal antibody-specific for CD154 (CD40L) for costimulation blockade, 3 Gy of nonmyeloablative total body irradiation and a conventional number of 20 x 10(6) fully allogeneic bone marrow cells. Flow cytometry is used to determine levels of multilineage hematopoietic chimerism and deletion of donor-reactive CD4+ T cells. Tolerance is assessed in vivo by grafting of donor and third party skin.
Previously, we have constructed recombinant derivatives of the major birch pollen allergen, Bet v 1, with a more than 100-fold reduced ability to induce IgE-mediated allergic reactions. These ...derivatives differed from each other because the two recombinant Bet v 1 fragments represented unfolded molecules whereas the recombinant trimer resembled most of the structural fold of the Bet v 1 allergen. In this study, we analyzed the Ab (IgE, IgG subclass, IgA, IgM) response to Bet v 1, recombinant and synthetic Bet v 1-derived peptides in birch pollen allergic patients who had been vaccinated with the derivatives or adjuvant alone. Furthermore, we studied the induction of IgE-mediated skin responses in these patients using Bet v 1 and Bet v 1 fragments. Both types of vaccines induced a comparable IgG1 and IgG4 response against new sequential epitopes which overlap with the conformational IgE epitopes of Bet v 1. This response was 4- to 5-fold higher than that induced by immunotherapy with birch pollen extract. Trimer more than fragments induced also IgE responses against new epitopes and a transient increase in skin sensitivity to the fragments at the beginning of therapy. However, skin reactions to Bet v 1 tended to decrease one year after treatment in both actively treated groups. We demonstrate that vaccination with folded and unfolded recombinant allergen derivatives induces IgG Abs against new epitopes. These data may be important for the development of therapeutic as well as prophylactic vaccines based on recombinant allergens.
Life-long immunosuppressive drug therapy is required after organ transplantation to prevent immunological rejection. Outcome after transplantation remains limited by the severe side effects and ...incomplete efficacy associated with currently available immunosuppressants. The co-stimulation blockers anti-CD40L and CTLA4Ig are members of a new, promising class of biologicals which are currently under development as immunosuppressants. Selected co-stimulation blockers have the potential to eventually replace current therapies to become the new standard of immunosuppression after organ transplantation.
Claudine Bruck – GlaxoSmithKline, King of Prussia, USA
Michel Goldman – University of Brussels, Brussels, Belgium
Bone marrow transplantation (BMT) under costimulation blockade induces mixed chimerism and tolerance in rodent models. Recent data, predominantly from in vitro studies, suggest that in addition to ...blocking the CD28 costimulation pathway CTLA4Ig also acts through upregulating the tryptophan-catabolizing enzyme indoleamine-2,3-dioxygenase (IDO). Here we demonstrate that even though CTLA4Ig is critically required for the induction of chimerism and tolerance in a murine model of nonmyeloablative BMT, IDO activity is not. No significant differences were detectable in the kynurenine to tryptophan ratios (indicative of IDO activity) in sera of BMT recipients treated with CTLA4Ig (tolerant group) versus BMT recipients treated without CTLA4Ig (nontolerant group) versus naïve controls. In vivo inhibition of IDO immediately after BMT with CTLA4Ig or several months thereafter did not block achievement of chimerism and tolerance. Thus, IDO does not play a critical role in the induction or maintenance of chimerism and tolerance in a CTLA4Ig-based BMT model.
Organ transplant recipients require life-long treatment with immunosuppressive drugs. Currently available immunosuppression is associated with substantial morbidity and mortality, and is ineffective ...in inhibiting chronic rejection and graft loss. Therefore, a permanent state of donor-specific tolerance remains a primary goal for transplantation research. The induction of mixed hematopoietic chimerism is an attractive concept in this regard. Hematopoietic chimerism modulates the immunologic repertoire by extending the mechanisms of self-tolerance to donor-specific allotolerance. Despite recent progress in developing nontoxic bone marrow transplantation protocols for rodents, translation to large animals has remained difficult. Here, we outline the concept of tolerance via mixed chimerism, and review recent progress and remaining challenges in bringing this approach to the clinical setting.
Allogeneic bone marrow transplantation (BMT) under costimulation blockade allows induction of mixed chimerism and tolerance without global T‐cell depletion (TCD). The mildest such protocols without ...recipient cytoreduction, however, require clinically impracticable bone marrow (BM) doses. The successful use of mobilized peripheral blood stem cells (PBSC) instead of BM in such regimens would provide a substantial advance, allowing transplantation of higher doses of hematopoietic donor cells. We thus transplanted fully allogeneic murine granulocyte colony‐stimulating factor (G‐CSF) mobilized PBSC under costimulation blockade (anti‐CD40L and CTLA4Ig). Unexpectedly, PBSC did not engraft, even when very high cell doses together with nonmyeloablative total body irradiation (TBI) were used. We show that, paradoxically, T cells contained in the donor PBSC triggered rejection of the transplanted donor cells. Rejection of donor BM was also triggered by the cotransplantation of unmanipulated donor T cells isolated from naïve (nonmobilized) donors. Donor‐specific transfusion and transient immunosuppression prevented PBSC‐triggered rejection and mixed chimerism and tolerance were achieved, but graft‐versus‐host disease (GVHD) occurred. The combination of in vivo TCD with costimulation blockade prevented rejection and GVHD. Thus, if allogeneic PBSC are transplanted instead of BM, costimulation blockade alone does not induce chimerism and tolerance without unacceptable GVHD‐toxicity, and the addition of TCD is required for success.
In a murine mixed chimerism model, T cells contained in high numbers in the donor peripheral blood stem cells triggered rejection of the transplanted donor cells.
Summary
Distinct lymphocyte populations have been identified that either promote or impede the establishment of chimerism and tolerance through allogeneic bone marrow transplantation (BMT). Natural ...killer T (NKT) cells have pleiotropic regulatory properties capable of either augmenting or downmodulating various immune responses. We investigated in this study whether NKT cells affect outcome in mixed chimerism models employing fully mismatched nonmyeloablative BMT with costimulation blockade (CB). The absence of NKT cells had no detectable effect on chimerism or skin graft tolerance after conditioning with 3Gy total body irradiation (TBI), and a limited positive effect with 1Gy TBI. Stimulation of NKT cells with alpha‐galactosylceramide (alpha‐gal) at the time of BMT prevented chimerism and tolerance. Activation of recipient (as opposed to donor) NKT cells was necessary and sufficient for the alpha‐gal effect. The detrimental effect of NKT activation was also observed in the absence of T cells after conditioning with in vivo T‐cell depletion (TCD). NKT cells triggered rejection of BM via NK cells as chimerism and tolerance were not abrogated when NKT cells were stimulated in the absence of both NK cells and T cells. Thus, activation of NKT cells at the time of BMT overcomes the effects of CB, inhibiting the establishment of chimerism and tolerance.
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