The mixed chimerism approach achieves donor-specific tolerance in organ transplantation, but clinical use is inhibited by the toxicities of current bone marrow (BM) transplantation (BMT) protocols. Blocking the CD40:CD154 pathway with anti-CD154 monoclonal antibodies (mAbs) is exceptionally potent in inducing mixed chimerism, but these mAbs are clinically not available. Defining the roles of donor and recipient CD40 in a murine allogeneic BMT model, we show that CD4 or CD8 activation through an intact direct or CD4 T cell activation through the indirect pathway is sufficient to trigger BM rejection despite CTLA4Ig treatment. In the absence of CD4 T cells, CD8 T cell activation via the direct pathway, in contrast, leads to a state of split tolerance. Interruption of the CD40 signals in both the direct and indirect pathway of allorecognition or lack of recipient CD154 is required for the induction of chimerism and tolerance. We developed a novel BMT protocol that induces mixed chimerism and donor-specific tolerance to fully mismatched cardiac allografts relying on CD28 costimulation blockade and mTOR inhibition without targeting the CD40 pathway. Notably, MHC-mismatched/minor antigen-matched skin grafts survive indefinitely whereas fully mismatched grafts are rejected, suggesting that non-MHC antigens cause graft rejection and split tolerance.

Introduction: Preformed donor-specific antibodies (DSA) detected by single antigen bead technology are associated with positive crossmatch (XM) results, antibody-mediated rejection (AMR) and inferior allograft survival. IgG elimination by peri-Tx immunoadsorption (IA) may enable a rapid reduction of preformed alloantibodies and XM conversion immediately before transplantation without prolonged desensitization on the waiting list. Methods: This study included 110 DSA-positive recipients of a deceased donor renal allograft (re-transplant: 71%; ≥ third transplant: 25%), who had been subjected to peri-Tx IA (transplantation between June 2009 and September 2013; follow-up until January 2014). The protocol consisted of (i) a single pre-Tx IA session (protein A/GAM peptide columns), serial post-Tx IA, (ii) induction therapy (ATG or IL-2 receptor antibody) and (iii) tacrolimus-based immunosuppression. Patients with a positive complement-dependent cytotoxicity crossmatch (CDCXM) proceeded to transplantation if XM converted negative upon 6L plasma treatment. Results: Twenty-eight (26%) of 110 DSA+ recipients had a positive CDCXM that rendered negative by pre-Tx IA (DSA+/XM+). Except re-transplantation (93 vs. 63%, p=0.003) there was no significant difference between DSA+/XM+ versus DSA+/XM- recipients with respect to baseline data. Patient and death-censored transplant survival were 93% and 87% at 1 year and 92% and 78% at 3 years respectively without any significant differences between DSA+/XM+ and DSA+/XM- recipients (p=0.39; p=0.37). Causes of graft loss during 4 to 54 months of follow-up were acute or chronic rejection (n=6), surgical complications (vascular complications in 7patients, most of them re-Tx recipients) or other non-immunological causes (n=7). Rates of clinical C4d-positive AMR were 25% versus 13% (p=0.15), C4d-negative AMR 21% versus 18% (p=0.7) and T-cell-mediated rejection 11% versus 15% (p=0.6) for XM+ vs XM- patients respectively. Conclusions: The protocol of peri-Tx enables a favourable deceased donor allograft survival in sensitized DSA-positive recipients even after pre-operative conversion of a positive CDCXM.

Specific immunotherapy is the only curative treatment currently available for IgE-mediated allergy and preventive strategies are lacking altogether. We have recently reported that molecular chimerism induces durable tolerance in experimental models of allergy, thus potentially providing a new approach for the treatment and prevention of allergic diseases. Molecular chimerism is a gene-therapy approach for tolerance induction toward defined disease-causing antigens. In proof-of-concept studies, we introduced a clinically relevant grass pollen allergen into hematopoietic stem cells and transplanted those modified cells into preconditioned syngeneic mice. Long-lasting and robust tolerance toward the allergen was achieved. In our most recent studies published in Clinical and Experimental Allergy we demonstrated that milder, non-myeloablative conditioning is sufficient to induce tolerance. Our results revealed that, in contrast to other rodent models of chimerism, persistent microchimerism suffices to induce lasting tolerance at the T cell, B cell and effector cell levels in IgE-mediated allergy. This article addendum provides a summary of the recent paper and its implications.

Summary Background Development of antigen‐specific preventive strategies is a challenging goal in IgE ‐mediated allergy. We have recently shown in proof‐of‐concept experiments that allergy can be successfully prevented by induction of durable tolerance via molecular chimerism. Transplantation of syngeneic hematopoietic stem cells genetically modified to express the clinically relevant grass pollen allergen P hl p 5 into myeloablated recipients led to high levels of chimerism (i.e. macrochimerism) and completely abrogated P hl p 5‐specific immunity despite repeated immunizations with P hl p 5. Objective It was unclear, however, whether microchimerism (drastically lower levels of chimerism) would be sufficient as well which would allow development of minimally toxic tolerance protocols. Methods Bone marrow cells were transduced with recombinant viruses integrating P hl p 5 to be expressed in a membrane‐anchored fashion. The syngeneic modified cells were transplanted into non‐myeloablated recipients that were subsequently immunized repeatedly with P hl p 5 and B et v 1 (control). Molecular chimerism was monitored using flow cytometry and PCR . T cell, B ‐cell and effector‐cell tolerance were assessed by allergen‐specific proliferation assays, isotype levels in sera and RBL assays. Results Here we demonstrate that transplantation of P hl p 5‐expressing bone marrow cells into recipients having received non‐myeloablative irradiation resulted in chimerism persisting for the length of follow‐up. Chimerism levels, however, declined from transient macrochimerism levels to persistent levels of microchimerism (followed for 11 months). Notably, these chimerism levels were sufficient to induce B ‐cell tolerance as no P hl p 5‐specific IgE and other high affinity isotypes were detectable in sera of chimeric mice. Furthermore, T ‐cell and effector‐cell tolerance were achieved. Conclusions and Clinical Relevance Low levels of persistent molecular chimerism are sufficient to induce long‐term tolerance in IgE ‐mediated allergy. These results suggest that it will be possible to develop minimally toxic conditioning regimens sufficient for low level engraftment of genetically modified bone marrow.

Schwaiger, E; Christoph, K; Baranyi, U; Pilat, N; Korom, S; Matheeussen, V; De Meester, I; Mühlbacher, F; Wekerle, T Author Information

Klaus, C; Pilat, N; Schwaiger, E; Gattringer, M; Muehlbacher, F; Wekerle, T Author Information

Pilat, N1; Baranyi, U1; Klaus, C1; Jaeckel, E2; Mpofu, N2; Muehlbacher, F1; Wekerle, T1 Author Information