Chronic CD40L blockade is required for long-term cardiac allograft survival with a clinically relevant CTLA4-Ig dosing regimen.

Introduction: In de-novo kidney transplantation, the CTLA4-Ig fusion protein belatacept is associated with improved graft function but also an increased risk of acute rejection compared to calcineurin inhibitor therapy. The combination with a second costimulation blocker could potentially improve outcome while avoiding calcineurin inhibitor toxicity. The aim of this study was to define the conditions under which the combination of CTLA4-Ig and CD40L blockade leads to rejection-free permanent graft survival in a stringent murine heart transplantation model. Methods: Naïve wild-type or CD40L (CD154) knock-out mice received a fully mismatched BALB/c cardiac allograft. Selected induction and maintenance protocols for CTLA4-Ig and blocking αCD40L monoclonal antibodies (mAB) were investigated. Graft survival, rejection severity and donor-specific antibody (DSA) formation were assessed during a 100-day follow-up period. Results and Discussion: Administering αCD40L mAb as monotherapy at the time of transplantation significantly prolonged heart allograft survival but did not further improve the outcome when given in addition to chronic CTLA4-Ig therapy (which prolongs graft survival to a median of 22 days). Likewise, chronic αCD40L mAb therapy (0.5mg) combined with perioperative CTLA4-Ig led to rejection in a proportion of mice and extensive histological damage, despite abrogating DSA formation. Only the permanent interruption of CD40-CD40L signaling by using CD40L-/- recipient mice or by chronic αCD40L administration synergized with chronic CTLA4-Ig to achieve long-term allograft survival with preserved histological graft integrity in all recipients without DSA formation. The combination of α-CD40L and CTLA4-Ig works most effectively when both therapeutics are administered chronically.

In vivo Treg expansion under costimulation blockade targets early rejection and improves long-term outcome.

CTLA4Ig has been shown to improve kidney allograft function, but an increased frequency of early rejection episodes poses a major obstacle for more widespread clinical use. The deleterious effect of CTLA4Ig on Treg numbers provides a possible explanation for graft injury. Therefore, we aimed at improving CTLA4Ig's efficacy by therapeutically increasing the number of Tregs. Murine cardiac allograft transplantation (BALB/c  to B6) was performed under CTLA4Ig therapy modeled after the clinically approved dosing regimen and Tregs were transferred early or late after transplant. Neither early nor late Treg transfer prolonged allograft survival. Transferred Tregs were traceable in various lymphoid compartments but only modestly increased overall Treg numbers. Next, we augmented Treg numbers in vivo by means of IL2 complexes. A short course of IL2/anti-IL2-complexes administered before transplantation reversed the CTLA4Ig-mediated decline in Tregs. Of note, the addition of IL2/anti-IL2-complexes to CTLA4Ig therapy substantially prolonged heart allograft survival and significantly improved graft histology on day 100. The depletion of Tregs abrogated this effect and resulted in a significantly diminished allograft survival. The increase in Treg numbers upon IL2 treatment was associated with a decreased expression of B7 on dendritic cells. These results demonstrate that therapy with IL2 complexes improves the efficacy of CTLA4Ig by counterbalancing its unfavorable effect on Tregs.

Distinct roles for major and minor antigen barriers in chimerism-based tolerance under irradiation-free conditions.

Eliminating cytoreductive conditioning from chimerism-based tolerance protocols would facilitate clinical translation. Here we investigated the impact of major histocompatibility complex (MHC) and minor histocompatibility antigen (MiHA) barriers on mechanisms of tolerance and rejection in this setting. Transient depletion of natural killer (NK) cells at the time of bone marrow (BM) transplantation (BMT) (20 × 106 BALB/c BM cells → C57BL/6 recipients under costimulation blockade [CB] and rapamycin) prevented BM rejection. Despite persistent levels of mixed chimerism, BMT recipients gradually rejected skin grafts from the same donor strain. Extending NK cell depletion did not improve skin graft survival. However, F1 (C57BL/6×BALB/c) donors, which do not elicit NK cell-mediated rejection, induced durable chimerism and tolerance. In contrast, if F1 donors with BALB/c background only were used (BALB/c×BALB.B), no tolerance was observed. In the absence of MiHA disparities (B10.D2 donors, MHC-mismatch only), temporal NK cell depletion established stable chimerism and tolerance. Conversely, MHC identical BM (BALB.B donors, MiHA mismatch only) readily engrafted without NK cell depletion but no skin graft tolerance ensued. Therefore, we conclude that under CB and rapamycin, MHC disparities provoke NK cell-mediated BM rejection in nonirradiated recipients whereas MiHA disparities do not prevent BM engraftment but impede skin graft tolerance in established mixed chimeras.

Treg-mediated prolonged survival of skin allografts without immunosuppression.

Injection of Interleukin-2 (IL-2) complexed with a particular anti-IL-2 monoclonal antibody (mab) JES6-1 has been shown to selectively expand CD4+Foxp3+ T regulatory T cells (Tregs) in vivo. Although the potency of this approach with regard to transplantation has already been proven in an islet transplantation model, skin graft survival could not be prolonged. Since the latter is relevant to human allograft survival, we sought to improve the efficiency of IL-2 complex (cplx) treatment for skin allograft survival in a stringent murine skin graft model. Here, we show that combining low doses of IL-2 cplxs with rapamycin and blockade of the inflammatory cytokine IL-6 leads to long-term (>75 d) survival of major histocompatibility complex-different skin allografts without the need for immunosuppression. Allograft survival was critically dependent on CD25+FoxP3+ Tregs and was not accompanied by impaired responsiveness toward donor alloantigens in vitro after IL-2 cplx treatment was stopped. Furthermore, second donor-type skin grafts were rejected and provoked rejection of the primary graft, suggesting that operational tolerance is not systemic but restricted to the graft. These findings plus the lack of donor-specific antibody formation imply that prolonged graft survival was largely a reflection of immunological ignorance. The results may represent a potentially clinically translatable strategy for the development of protocols for tolerance induction.

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.

Hybrid resistance to parental bone marrow grafts in nonlethally irradiated mice.

Resistance to parental bone marrow (BM) grafts in F1 hybrid recipients is due to natural killer (NK) cell-mediated rejection triggered through "missing self" recognition. "Hybrid resistance" has usually been investigated in lethally irradiated F1 recipients in conjunction with pharmacological activation of NK cells. Here, we investigated BM-directed NK-cell alloreactivity in settings of reduced conditioning. Nonlethally irradiated (1-3 Gy) or nonirradiated F1 (C57BL6 × BALB/c) recipient mice received titrated doses (5-20 x 106 ) of unseparated parental BALB/c BM without pharmacological NK cell activation. BM successfully engrafted in all mice and multilineage donor chimerism persisted long-term (24 weeks), even in the absence of irradiation. Chimerism was associated with the rearrangement of the NK-cell receptor repertoire suggestive of reduced reactivity to BALB/c. Chimerism levels were lower after transplantation with parental BALB/c than with syngeneic F1 BM, indicating partial NK-mediated rejection of parental BM. Activation of NK cells with polyinosinic-polycytidylic acid sodium salt poly(I:C), reduced parental chimerism in nonirradiated BM recipients but did not prevent hematopoietic stem cell engraftment. In contrast, equal numbers of parental lymph node cells were completely rejected. Hence, hybrid resistance leads to incomplete rejection of parental BM under reduced conditioning settings.

CTLA4Ig Improves Murine iTreg Induction via TGFß and Suppressor Function In Vitro.

Blockade of the CD28:CD80/86 costimulatory pathway has been shown to be potent in blocking T cell activation in vitro and in vivo. The costimulation blocker CTLA4Ig has been approved for the treatment of autoimmune diseases and transplant rejection. The therapeutic application of regulatory T cells (Tregs) has recently gained much attention for its potential of improving allograft survival. However, neither costimulation blockade with CTLA4Ig nor Treg therapy induces robust tolerance on its own. Combining CTLA4Ig with Treg therapy would be an attractive approach for minimizing immunosuppression or for possibly achieving tolerance. However, since the CD28 pathway is more complex than initially thought, the question arose whether blocking CD80/86 would inadvertently impact immunological tolerance by interfering with Treg generation and function. We therefore wanted to investigate the compatibility of CTLA4Ig with regulatory T cells by evaluating direct effects of CTLA4Ig on murine Treg generation and function in vitro. For generation of polyclonal-induced Tregs, we utilized an APC-free in vitro system and added titrated doses of CTLA4Ig at different time points. Phenotypical characterization by flow cytometry and functional characterization in suppressor assays did not reveal negative effects by CTLA4Ig. The costimulation blocker CTLA4Ig does not impair but rather improves murine iTreg generation and suppressor function in vitro.

Belatacept/CTLA4Ig: an update and critical appraisal of preclinical and clinical results.

INTRODUCTION: The B7/CD28/CTLA4 signaling cascade is the most thoroughly studied costimulatory pathway and blockade with CTLA4Ig (abatacept) or its derivative belatacept has emerged as a valuable option for pharmacologic immune modulation. Several clinical studies have ultimately led to the approval of belatacept for immunosuppression in kidney transplant recipients. Areas covered: This review will discuss the immunological background of costimulation blockade and recent preclinical data and clinical results of CTLA4Ig/belatacept. Expert commentary: The development of belatacept is a major advance in clinical transplantation. However, in spite of promising results in preclinical and clinical trials, clinical use remains limited at present, in part due to increased rates of acute rejection. Recent efforts showing encouraging progress in refining such protocols might be a step toward harnessing the full potential of costimulation blockade-based immunosuppression.

Blockade of adhesion molecule lymphocyte function-associated antigen-1 improves long-term heart allograft survival in mixed chimeras.

BACKGROUND: The mixed chimerism approach for intentional induction of donor-specific tolerance was shown to be successful in various models from mice to humans. For transplant patients, the approach would obviate the need for long-term immunosuppression and associated side effects; moreover, it would preclude the risk of late graft loss due to chronic rejection. Widespread clinical application is hindered by toxicities related to recipient pre-conditioning. Herein we aimed to investigate a clinically relevant protocol for tolerance induction to cardiac allografts, sparing CD40 blockade or T-cell depletion. METHODS: B6 mice were conditioned with non-myeloablative total body irradiation, fully mismatched BALB/c bone marrow cells, and short-term therapy, based on either anti- lymphocyte function-associated antigen-1 (anti-LFA-1) or anti-CD40L. Multilineage chimerism was followed by flow-cytometric analysis, tolerance was assessed with skin and heart allografts from fully or major histocompatibility complex-mismatched donors. Mechanisms of tolerance were investigated by analysis of donor-specific antibodies (DSAs), mixed lymphocyte reaction (MLR) assays, and deletion of donor-reactive T cells. RESULTS: We found that the combination of cytotoxic T-lymphocyte antigen 4 immunoglobulin (CTLA4Ig) and rapamycin with LFA-1 blockade enhanced bone marrow engraftment and led to more efficient T-cell engraftment and subsequent tolerization. Although fully mismatched skin grafts were chronically rejected, primarily vascularized heart allografts survived indefinitely and without signs of chronic rejection, independent of minor antigen mismatches. CONCLUSIONS: We have demonstarted a robust protocol for the induction of tolerance for cardiac allografts in the absence of CD40 blockade. Our findings demonstrate the potential of a clinically relevant minimal conditioning protocol designed to induce lifelong immunologic tolerance toward cardiac allografts.

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.

Transplantation Tolerance through Hematopoietic Chimerism: Progress and Challenges for Clinical Translation.

The perception that transplantation of hematopoietic stem cells can confer tolerance to any tissue or organ from the same donor is widely accepted but it has not yet become a treatment option in clinical routine. The reasons for this are multifaceted but can generally be classified into safety and efficacy concerns that also became evident from the results of the first clinical pilot trials. In comparison to standard immunosuppressive therapies, the infection risk associated with the cytotoxic pre-conditioning necessary to allow allogeneic bone marrow engraftment and the risk of developing graft-vs.-host disease (GVHD) constitute the most prohibitive hurdles. However, several approaches have recently been developed at the experimental level to reduce or even overcome the necessity for cytoreductive conditioning, such as costimulation blockade, pro-apoptotic drugs, or Treg therapy. But even in the absence of any hazardous pretreatment, the recipients are exposed to the risk of developing GVHD as long as non-tolerant donor T cells are present. Total lymphoid irradiation and enriching the stem cell graft with facilitating cells emerged as potential strategies to reduce this peril. On the other hand, the long-lasting survival of kidney allografts, seen with transient chimerism in some clinical series, questions the need for durable chimerism for robust tolerance. From a safety point of view, loss of chimerism would indeed be favorable as it eliminates the risk of GVHD, but also complicates the assessment of tolerance. Therefore, other biomarkers are warranted to monitor tolerance and to identify those patients who can safely be weaned off immunosuppression. In addition to these safety concerns, the limited efficacy of the current pilot trials with approximately 40-60% patients becoming tolerant remains an important issue that needs to be resolved. Overall, the road ahead to clinical routine may still be rocky but the first successful long-term patients and progress in pre-clinical research provide encouraging evidence that deliberately inducing tolerance through hematopoietic chimerism might eventually make it from dream to reality.

Minor Antigen Disparities Impede Induction of Long Lasting Chimerism and Tolerance through Bone Marrow Transplantation with Costimulation Blockade.

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. .

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.

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.

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.

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.

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.

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.

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.