Benedikt Mahr

Anti-inflammatory properties of the PI3K pathway are mediated by IL-10/DUSP regulation

Resolution of inflammation is an important hallmark in the course of infectious diseases. Dysregulated inflammatory responses may have detrimental consequences for the affected organism. Therefore, tight regulation of inflammation is indispensable. Among numerous modulatory signaling pathways, the PI3K/PTEN signaling pathway has been proposed recently to be involved in the regulation of innate immune reactions. Here, we attempted to elucidate molecular mechanisms that contribute to the modulatory properties of the PI3K signaling pathway in inflammation. PTEN‐deficient macrophages, which harbor constitutively active PI3Ks, were analyzed in response to gram‐negative bacteria and PAMPs such as LPS. PTEN‐deficient cells showed reduced inflammatory cytokine production, which was accompanied by reduced MAPK signaling activation in early‐ as well as late‐phase activation. Simultaneously, we found increased levels of the MKP DUSP1, as well as the anti‐inflammatory cytokine IL‐10. Our data suggest that differential DUSP1 regulation coupled with enhanced IL‐10 production contributes to the anti‐inflammatory properties of the PI3K pathway.

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.

Deletional and regulatory mechanisms coalesce to drive transplantation tolerance through mixed chimerism.

Establishing donor‐specific immunological tolerance could improve long‐term outcome by obviating the need for immunosuppressive drug therapy, which is currently required to control alloreactivity after organ transplantation. Mixed chimerism is defined as the engraftment of donor hematopoietic stem cells in the recipient, leading to viable coexistence of both donor and recipient leukocytes. In numerous experimental models, cotransplantation of donor bone marrow (BM) into preconditioned (e.g., through irradiation or cytotoxic drugs) recipients leads to transplantation tolerance through (mixed) chimerism. Mixed chimerism offers immunological advantages for clinical translation; pilot trials have established proof of concept by deliberately inducing tolerance in humans. Widespread clinical application is prevented, however, by the harsh preconditioning currently necessary for permitting BM engraftment. Recently, the immunological mechanisms inducing and maintaining tolerance in experimental mixed chimerism have been defined, revealing a more prominent role for regulation than historically assumed. The evidence from murine models suggests that both deletional and regulatory mechanisms are critical in promoting complete tolerance, encompassing also the minor histocompatibility antigens. Here, we review the current understanding of tolerance through mixed chimerism and provide an outlook on how to realize widespread clinical translation based on mechanistic insights gained from chimerism protocols, including cell therapy with polyclonal regulatory T cells.

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.

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 TGFβ 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 × 106 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.

Donor CD4 T Cells Trigger Costimulation Blockade-Resistant Donor Bone Marrow Rejection Through Bystander Activation Requiring IL-6

Bone marrow (BM) transplantation under costimulation blockade induces chimerism and tolerance. Cotransplantation of donor T cells (contained in substantial numbers in mobilized peripheral blood stem cells and donor lymphocyte infusions) together with donor BM paradoxically triggers rejection of donor BM through undefined mechanisms. Here, nonmyeloablatively irradiated C57BL/6 recipients simultaneously received donor BM (BALB/c) and donor T cells under costimulation blockade (anti‐CD154 and CTLA4Ig). Donor CD4, but not CD8 cells, triggered natural killer‐independent donor BM rejection which was associated with increased production of IL‐6, interferon gamma (IFN‐γ) and IL‐17A. BM rejection was prevented through neutralization of IL‐6, but not of IFN‐γ or IL‐17A. IL‐6 counteracted the antiproliferative effect of anti‐CD154 in vitro. Rapamycin and anti‐lymphocyte function‐associated antigen 1 negated this effect of IL‐6 in vitro and prevented BM rejection in vivo. Simultaneous cotransplantation of (BALB/cxB6)F1, recipient or irradiated donor CD4 cells, or late transfer of donor CD4 cells did not lead to BM rejection, whereas cotransplantation of third party CD4 cells did. Transferred donor CD4 cells became activated, rapidly underwent apoptosis and triggered activation and proliferation of recipient T cells. Collectively, these results provide evidence that donor T cells recognizing the recipient as allogeneic lead to the release of IL‐6, which abolishes the effect of anti‐CD154, triggering donor BM rejection through bystander activation.

IL-2/α-IL-2 Complex Treatment Cannot Be Substituted for the Adoptive Transfer of Regulatory T cells to Promote Bone Marrow Engraftment.

Cell therapy with recipient Tregs achieves engraftment of allogeneic bone marrow (BM) without the need for cytoreductive conditioning (i.e., without irradiation or cytotoxic drugs). Thereby mixed chimerism and transplantation tolerance are established in recipients conditioned solely with costimulation blockade and rapamycin. However, clinical translation would be substantially facilitated if Treg-stimulating pharmaceutical agents could be used instead of individualized cell therapy. Recently, it was shown that interleukin-2 (IL-2) complexed with a monoclonal antibody (mAb) (clone JES6-1A12) against IL-2 (IL-2 complexes) potently expands and activates Tregs in vivo. Therefore, we investigated whether IL-2 complexes can replace Treg therapy in a costimulation blockade-based and irradiation-free BM transplantation (BMT) model. Unexpectedly, the administration of IL-2 complexes at the time of BMT (instead of Tregs) failed to induce BM engraftment in non-irradiated recipients (0/6 with IL-2 complexes vs. 3/4 with Tregs, p<0.05). Adding IL-2 complexes to an otherwise effective regimen involving recipient irradiation (1Gy) but no Treg transfer indeed actively triggered donor BM rejection at higher doses (0/8 with IL-2 complexes vs. 9/11 without, p<0.01) and had no detectable effect at two lower doses (3/5 vs. 9/11, p>0.05). CD8 T cells and NK cells of IL-2 complex-treated naïve mice showed an enhanced proliferative response towards donor antigens in vitro despite the marked expansion of Tregs. However, IL-2 complexes also expanded conventional CD4 T cells, CD8 T cells, NK cells, NKT cells and notably even B cells, albeit to a lesser extent. Notably, IL-2 complex expanded Tregs featured less potent suppressive functions than in vitro activated Tregs in terms of T cell suppression in vitro and BM engraftment in vivo. In conclusion, these data suggest that IL-2 complexes are less effective than recipient Tregs in promoting BM engraftment and in contrast actually trigger BM rejection, as their effect is not sufficiently restricted to Tregs but rather extends to several other lymphocyte populations.

The Immunosuppressive Effect of CTLA4 Immunoglobulin Is Dependent on Regulatory T Cells at Low But Not High Doses

B7.1/2‐targeted costimulation blockade (CTLA4 immunoglobulin [CTLA4‐Ig]) is available for immunosuppression after kidney transplantation, but its potentially detrimental impact on regulatory T cells (Tregs) is of concern. We investigated the effects of CTLA4‐Ig monotherapy in a fully mismatched heart transplant model (BALB/c onto C57BL/6). CTLA4‐Ig was injected chronically (on days 0, 4, 14, and 28 and every 4 weeks thereafter) in dosing regimens paralleling clinical use, shown per mouse: low dose (LD), 0.25 mg (≈10 mg/kg body weight); high dose (HD), 1.25 mg (≈50 mg/kg body weight); and very high dose (VHD), 6.25 mg (≈250 mg/kg body weight). Chronic CTLA4‐Ig therapy showed dose‐dependent efficacy, with the LD regimen prolonging graft survival and with the HD and VHD regimens leading to >95% long‐term graft survival and preserved histology. CTLA4‐Igs effect was immunosuppressive rather than tolerogenic because treatment cessation after ≈3 mo led to rejection. FoxP3‐positive Tregs were reduced in naïve mice to a similar degree, independent of the CTLA4‐Ig dose, but recovered to normal values in heart recipients under chronic CTLA4‐Ig therapy. Treg depletion (anti‐CD25) resulted in an impaired outcome under LD therapy but had no detectable effect under HD therapy. Consequently, the immunosuppressive effect of partially effective LD CTLA4‐Ig therapy is impaired when Tregs are removed, whereas CTLA4‐Ig monotherapy at higher doses effectively maintains graft survival independent of Tregs.

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.

Regulatory T Cells Promote Natural Killer Cell Education in Mixed Chimeras

Therapeutic administration of regulatory T cells (Tregs) leads to engraftment of conventional doses of allogeneic bone marrow (BM) in nonirradiated recipient mice conditioned with costimulation blockade and mammalian target of rapamycin inhibition. The mode of action responsible for this Treg effect is poorly understood but may encompass the control of costimulation blockade–resistant natural killer (NK) cells. We show that transient NK cell depletion at the time of BM transplantation led to BM engraftment and persistent chimerism without Treg transfer but failed to induce skin graft tolerance. In contrast, the permanent absence of anti–donor NK reactivity in mice grafted with F1 BM was associated with both chimerism and tolerance comparable to Treg therapy, implying that NK cell tolerization is a critical mechanism of Treg therapy. Indeed, NK cells of Treg‐treated BM recipients reshaped their receptor repertoire in the presence of donor MHC in a manner suggesting attenuated donor reactivity. These results indicate that adoptively transferred Tregs prevent BM rejection, at least in part, by suppressing NK cells and promote tolerance by regulating the appearance of NK cells expressing activating receptors to donor class I MHC.