Lukas Unger

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-LFA-1 or rapamycin overcome costimulation blockade-resistant rejection in sensitized bone marrow recipients.

While costimulation blockade‐based mixed chimerism protocols work well for inducing tolerance in rodents, translation to preclinical large animal/nonhuman primate models has been less successful. One recognized cause for these difficulties is the high frequency of alloreactive memory T cells (Tmem) found in the (pre)clinical setting as opposed to laboratory mice. In the present study, we therefore developed a murine bone marrow transplantation (BMT) model employing recipients harboring polyclonal donor‐reactive Tmem without concomitant humoral sensitization. This model was then used to identify strategies to overcome this additional immune barrier. We found that B6 recipients that were enriched with 3 × 107 T cells isolated from B6 mice that had been previously grafted with Balb/c skin, rejected Balb/c BM despite costimulation blockade with anti‐CD40L and CTLA4Ig (while recipients not enriched developed chimerism). Adjunctive short‐term treatment of sensitized BMT recipients with rapamycin or anti‐LFA‐1 mAb was demonstrated to be effective in controlling Tmem in this model, leading to long‐term mixed chimerism and donor‐specific tolerance. Thus, rapamycin and anti‐LFA‐1 mAb are effective in overcoming the potent barrier that donor‐reactive Tmem pose to the induction of mixed chimerism and tolerance despite costimulation blockade.

Non-professional marathon running: RAGE axis and ST2 family changes in relation to open-window effect, inflammation and renal function

Conflicting data exist on the relevance of marathon (M) and half marathon (HM) running for health. The number of non-professional athletes finishing M and HM events is steadily growing. In order to investigate molecular changes occurring in amateur athletes, we enrolled 70 non-professional runners finishing a single M (34) or HM (36) event at baseline, the finish line and during recovery, and 30 controls. The measurement of the Receptor for Advanced Glycation Endproducts, Interleukin 1 receptor antagonist, ST2 and cytokeratin 18 was combined with molecules measured during clinical routine. Results were analyzed in the light of blood cell analysis, lactate measurements, correction for changes in plasma volume and body composition assessments. There were intrinsic differences in body mass index, abdominal body fat percentage and training time between M and HM runners. C-reactive protein changes in M and HM runners. While soluble RAGE, AGEs and ST2 increased immediately after the race in HM runners, HMGB1 increased in HM and M after the race and declined to baseline after a recovery period. We give insights into the regulation of various molecules involved in physical stress reactions and their possible implications for the cardiovascular system or renal function.

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.

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.

Implication for Bone Marrow Derived Stem Cells in Hepatocyte Regeneration after Orthotopic Liver Transplantation

The liver has the outstanding ability to regenerate itself and restore parenchymal tissue after injury. The most common cell source in liver growth/regeneration is replication of preexisting hepatocytes although liver progenitor cells have been postulated to participate in liver regeneration in cases of massive injury. Bone marrow derived hematopoietic stem cells (BM-HSC) have the formal capacity to act as a source for hepatic regeneration under special circumstances; however, the impact of this process in liver tissue maintenance and regeneration remains controversial. Whether BM-HSC are involved in liver regeneration or not would be of particular interest as the cells have been suggested to be an alternative donor source for the treatment of liver failure. Data from murine models of liver disease show that BM-HSC can repopulate liver tissue and restore liver function; however, data obtained from human liver transplantation show only little evidence for liver regeneration by this mechanism. The cell source for liver regeneration seems to depend on the nature of regeneration process and the extent of injury; however, the precise mechanisms still need to be resolved. Current data suggest, that in human orthotopic liver transplantation, liver regeneration by BM-HSC is a rather rare event and therefore not of clinical relevance.

Management of portal hypertension before and after liver transplantation

Orthotopic liver transplantation (OLT) represents a curative treatment option for end‐stage liver disease (ESLD). Although epidemiology of ESLD has recently changed due to the rising prevalence of nonalcoholic fatty liver disease and the decreased burden of hepatitis C virus infections due to highly effective antiviral regimens, the management of portal hypertension (PHT) remains a clinical challenge in the pre‐ and post‐OLT setting. The measurement of the hepatic venous pressure gradient represents the most reliable but invasive tool for assessment of the severity of PHT. Although novel liver ultrasound and magnetic resonance–based elastography methods have been developed, their value to screen for liver fibrosis and PHT in transplanted patients remains to be established. Nonselective beta‐blockers represent the cornerstone of medical treatment of PHT, but more studies on their effects on clinical endpoints after OLT are needed. Statins are widely used to treat hyperlipidemia, which is a common condition after OLT. Although a growing body of evidence suggests that statins decrease portal pressure and PHT‐related complications in ESLD, studies on potential benefits of statins after OLT are lacking. Finally, transjugular intrahepatic portosystemic shunts (TIPS) are effective in decreasing PHT and seem to decrease mortality on the OLT waiting list. Moreover, TIPS does not have an impact on liver function nor complicate the transplant surgical procedures. TIPS may also be used after OLT, but the evidence is limited. In conclusion, whereas the management of PHT in patients with ESLD is based on strong evidence, further data on the value of noninvasive monitoring tools as well as on medical and invasive treatment options in the post‐OLT setting are needed to improve management strategies in patients with recurrent PHT after liver transplantation. Liver Transplantation 24 112–121 2018 AASLD.

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.