Allograft rejection is associated with development of functional IgE specific for donor MHC antigens.

BACKGROUND: Donor-specific antibodies of the IgG isotype are measured routinely for diagnostic purposes in renal transplant recipients and are associated with antibody-mediated rejection and long-term graft loss. OBJECTIVE: This study aimed to investigate whether MHC-specific antibodies of the IgE isotype are induced during allograft rejection. METHODS: Anti-MHC/HLA IgE levels were measured in sera of mice grafted with skin or heart transplants from various donor strains and in sera of kidney transplant patients with high levels of HLA IgG. Mediator release was triggered in vitro by stimulating basophils that were coated with murine or human IgE-positive serum, respectively, with specific recombinant MHC/HLA antigens. Kidney tissue samples obtained from organ donors were analyzed by using flow cytometry for cells expressing the high-affinity receptor for IgE (FcεRI). RESULTS: Donor MHC class I- and MHC class II-specific IgE was found on acute rejection of skin and heart grafts in several murine strain combinations, as well as during chronic antibody-mediated heart graft rejection. Anti-HLA IgE, including donor HLA class I and II specificities, was identified in a group of sensitized transplant recipients. Murine and human anti-MHC/HLA IgE triggered mediator release in coated basophils on stimulation with specific MHC/HLA antigens. HLA-specific IgE was not linked to atopy, and allergen-specific IgE present in allergic patients did not cross-react with HLA antigens. FcεRI+ cells were found in the human renal cortex and medulla and provide targets for HLA-specific IgE. CONCLUSION: These results demonstrate that MHC/HLA-specific IgE develops during an alloresponse and is functional in mediating effector mechanisms.

The site of allergen expression in hematopoietic cells determines the degree and quality of tolerance induced through molecular chimerism.

The transplantation of allergens (e.g. Phl p 5 or Bet v 1) expressed on BM cells as membrane-anchored full-length proteins leads to permanent tolerance at the T-cell, B-cell, and effector-cell levels. Since the exposure of complete allergens bears the risk of inducing anaphylaxis, we investigated here whether expression of Phl p 5 in the cytoplasm (rather than on the cell surface) is sufficient for tolerance induction. Transplantation of BALB/c BM retrovirally transduced to express Phl p 5 in the cytoplasm led to stable and durable molecular chimerism in syngeneic recipients (∼20% chimerism at 6 months). Chimeras showed allergen-specific T-cell hyporesponsiveness. Further, Phl p 5-specific TH 1-dependent humoral responses were tolerized in several chimeras. Surprisingly, Phl p 5-specific IgE and IgG1 levels were significantly reduced but still detectable in sera of chimeric mice, indicating incomplete B-cell tolerance. No Phl p 5-specific sIgM developed in cytoplasmic chimeras, which is in marked contrast to mice transplanted with BM expressing membrane-anchored Phl p 5. Thus, the expression site of the allergen substantially influences the degree and quality of tolerance achieved with molecular chimerism in IgE-mediated allergy.

Advanced Therapy Medicinal Products for Rare Diseases: State of Play of Incentives Supporting Development in Europe.

In 2008, the European Union introduced the Advanced Medicines Regulation aiming to improve regulation of advanced therapy medicinal products (ATMPs). We applied the ATMPs classification definitions in this Regulation to understand the link of this emerging group of medicinal products and the use of the Orphan Regulation. A total of 185 products that can be classified as ATMPs based on this Regulation have been submitted for orphan designation. Prior to its introduction in 2008, 4.5% of the products submitted for orphan designation met these criteria. This percentage went up to 15% after 2008. We analyzed several parameters associated with active ATMP ODDs focusing on sponsor type and EU-Member State origin, therapeutic area targeted, and ATMP classification [i.e., somatic cell therapy medicinal product, tissue-engineered product (TEP), or gene therapy medicinal product (GTMP)] and the use of regulatory services linked to incentives such as the use of protocol assistance (PA) and other Committees [Committee for Advanced Therapies (CAT) and the Pediatric Committee]. The aim here was to gain insight on the use of different services. The UK submits the largest number of ATMPs for ODD representing ~30% of the total to date. Few submissions have been received from central and Eastern European Member States as well as some of the larger Member States such as Germany (3.6%). ATMPs ODDs were primarily GTMPs (48.7%) and SCTMPs (43.3%). TEPs only represented 8% of all submissions for this medicinal class. This is different from non-ODDs ATMPs where GTMPs make only 20% of ATMPs. A total of 11.7% of ATMP ODDs had received formal CAT classification. A total of 29.8% of all orphan drug (OD) ATMPs requested PA. A total of 71.8% did not have an agreed pediatric investigation plan (PIP). Four products (Glybera one PA; Zalmoxis two; Holoclar one; Strimvelis three) have received a marketing authorization (MAA) and a 10-year market exclusivity. Strimvelis also completed their PIP, which was compliant and received the additional 2-year extension to their 10-year market exclusivity. One OD ATMP (Cerepro) received a negative opinion for MAA. The use of services linked to incentives offered by different legislations for ATMP ODDs is low, indicating a need for increasing awareness.

Anti-Interleukin-6 Promotes Allogeneic Bone Marrow Engraftment and Prolonged Graft Survival in an Irradiation-Free Murine Transplant Model.

Transfer of recipient regulatory T cells (Tregs) induces mixed chimerism and tolerance in an irradiation-free bone marrow (BM) transplantation (BMT) model involving short-course co-stimulation blockade and mTOR inhibition. Boosting endogenous Tregs pharmacologically in vivo would be an attractive alternative avoiding the current limitations of performing adoptive cell therapy in the routine clinical setting. Interleukin-6 (IL-6) potently inhibits Treg differentiation and its blockade was shown to increase Treg numbers in vivo. Therefore, we investigated whether IL-6 blockade can replace adoptive Treg transfer in irradiation-free allogeneic BMT. Treatment with anti-IL-6 instead of Treg transfer led to multi-lineage chimerism (persisting for ~12 weeks) in recipients of fully mismatched BM and significantly prolonged donor skin (MST 58 days) and heart (MST > 100 days) graft survival. Endogenous Foxp3+ Tregs expanded in anti-IL-6-treated BMT recipients, while dendritic cell (DC) activation and memory CD8+ T cell development were inhibited. Adding anti-IL-17 to anti-IL-6 treatment increased Treg frequencies, but did not further prolong donor skin graft survival significantly. These results demonstrate that IL-6 blockade promotes BM engraftment and donor graft survival in non-irradiated recipients and might provide an alternative to Treg cell therapy in the clinical setting.

Cell Therapy for Prophylactic Tolerance in Immunoglobulin E-mediated Allergy.

BACKGROUND: Therapeutic strategies for the prophylaxis of IgE-mediated allergy remain an unmet medical need. Cell therapy is an emerging approach with high potential for preventing and treating immunological diseases. We aimed to develop a cell-based therapy inducing permanent allergen-specific immunological tolerance for preventing IgE-mediated allergy. METHODS: Wild-type mice were treated with allergen-expressing bone marrow cells under a short course of tolerogenic immunosuppression (mTOR inhibition and costimulation blockade). Bone marrow was retrieved from a novel transgenic mouse ubiquitously expressing the major grass pollen allergen Phl p 5 as a membrane-anchored protein (BALB/c-Tg[Phlp5-GFP], here mPhl p 5). After transplantation recipients were IgE-sensitized at multiple time points with Phl p 5 and control allergen. RESULTS: Mice treated with mPhl p 5 bone marrow did not develop Phl p 5-specific IgE (or other isotypes) despite repeated administration of the allergen, while mounting and maintaining a strong humoral response towards the control allergen. Notably, Phl p 5-specific T cell responses and allergic airway inflammation were also completely prevented. Interestingly allergen-specific B cell tolerance was maintained independent of Treg functions indicating deletional tolerance as underlying mechanism. CONCLUSION: This proof-of-concept study demonstrates that allergen-specific immunological tolerance preventing occurrence of allergy can be established through a cell-based therapy employing allergen-expressing leukocytes.

Incomplete clonal deletion as prerequisite for tissue-specific minor antigen tolerization.

Central clonal deletion has been considered the critical factor responsible for the robust state of tolerance achieved by chimerism-based experimental protocols, but split-tolerance models and the clinical experience are calling this assumption into question. Although clone-size reduction through deletion has been shown to be universally required for achieving allotolerance, it remains undetermined whether it is sufficient by itself. Therapeutic Treg treatment induces chimerism and tolerance in a stringent murine BM transplantation model devoid of myelosuppressive recipient treatment. In contrast to irradiation chimeras, chronic rejection (CR) of skin and heart allografts in Treg chimeras was permanently prevented, even in the absence of complete clonal deletion of donor MHC-reactive T cells. We show that minor histocompatibility antigen mismatches account for CR in irradiation chimeras without global T cell depletion. Furthermore, we show that Treg therapy-induced tolerance prevents CR in a linked suppression-like fashion, which is maintained by active regulatory mechanisms involving recruitment of thymus-derived Tregs to the graft. These data suggest that highly efficient intrathymic and peripheral deletion of donor-reactive T cells for specificities expressed on hematopoietic cells preclude the expansion of donor-specific Tregs and, hence, do not allow for spreading of tolerance to minor specificities that are not expressed by donor BM.

Strategies to overcome the ABO barrier in kidney transplantation.

Kidney transplantation across the ABO blood group barrier was long considered a contraindication for transplantation, but in an effort to increase donor pools, specific regimens for ABO-incompatible (ABOi) transplantation have been developed. These regimens are now widely used as an integral part of the available treatment options. Various desensitization protocols, commonly based on transient depletion of preformed anti-A and/or anti-B antibodies and modulation of B-cell immunity, enable excellent transplant outcomes, even in the long-term. Nevertheless, the molecular mechanisms behind transplant acceptance facilitated by a short course of anti-humoral treatment are still incompletely understood. With the evolution of efficient clinical programmes, tailoring of recipient preconditioning based on individual donor-recipient blood type combinations and the levels of pretransplant anti-A/B antibodies has become possible. In the context of low antibody titres and/or donor A2 phenotype, immunomodulation and/or apheresis might be dispensable. A concern still exists, however, that ABOi kidney transplantation is associated with an increased risk of surgical and infectious complications, partly owing to the effects of extracorporeal treatment and intensified immunosuppression. Nevertheless, a continuous improvement in desensitization strategies, with the aim of minimizing the immunosuppressive burden, might pave the way to clinical outcomes that are comparable to those achieved in ABO-compatible transplantation.

Polyclonal Recipient nTregs Are Superior to Donor or Third-Party Tregs in the Induction of Transplantation Tolerance.

Induction of donor-specific tolerance is still considered as the "Holy Grail" in transplantation medicine. The mixed chimerism approach is virtually the only tolerance approach that was successfully translated into the clinical setting. We have previously reported successful induction of chimerism and tolerance using cell therapy with recipient T regulatory cells (Tregs) to avoid cytotoxic recipient treatment. Treg therapy is limited by the availability of cells as large-scale expansion is time-consuming and associated with the risk of contamination with effector cells. Using a costimulation-blockade based bone marrow (BM) transplantation (BMT) model with Treg therapy instead of cytoreductive recipient treatment we aimed to determine the most potent Treg population for clinical translation. Here we show that CD4(+)CD25(+) in vitro activated nTregs are superior to TGFß induced iTregs in promoting the induction of chimerism and tolerance. Therapy with nTregs (but not iTregs) led to multilineage chimerism and donor-specific tolerance in mice receiving as few as 0.5 × 10(6) cells. Moreover, we show that only recipient Tregs, but not donor or third-party Tregs, had a beneficial effect on BM engraftment at the tested doses. Thus, recipient-type nTregs significantly improve chimerism and tolerance and might be the most potent Treg population for translation into the clinical setting.

T-regulatory cell treatment prevents chronic rejection of heart allografts in a murine mixed chimerism model.

BACKGROUND: The mixed chimerism approach induces donor-specific tolerance in both pre-clinical models and clinical pilot trials. However, chronic rejection of heart allografts and acute rejection of skin allografts were observed in some chimeric animals despite persistent hematopoietic chimerism and tolerance toward donor antigens in vitro. We tested whether additional cell therapy with regulatory T cells (Tregs) is able to induce full immunologic tolerance and prevent chronic rejection. METHODS: We recently developed a murine "Treg bone marrow (BM) transplantation (BMT) protocol" that is devoid of cytoreductive recipient pre-treatment. The protocol consists of a moderate dose of fully mismatched allogeneic donor BM under costimulation blockade, together with polyclonal recipient Tregs and rapamycin. Control groups received BMT under non-myeloablative irradiation and costimulation blockade without Treg therapy. Multilineage chimerism was followed by flow cytometry, and tolerance was assessed by donor-specific skin and heart allografts. RESULTS: Durable multilineage chimerism and long-term donor skin and heart allograft survival were successfully achieved with both protocols. Notably, histologic examination of heart allografts at the end of follow-up revealed that chronic rejection is prevented only in chimeras induced with the Treg protocol. CONCLUSIONS: In a mouse model of mixed chimerism, additional Treg treatment at the time of BMT prevents chronic rejection of heart allografts. As the Treg-chimerism protocol also obviates the need for cytoreductive recipient treatment it improves both efficacy and safety over previous non-myeloablative mixed chimerism regimens. These results may significantly impact the development of protocols for tolerance induction in cardiac transplantation.