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Background: Induction of donor-specific tolerance is a promising approach to achieve long-term graft patency in transplantation with little to no maintenance immunosuppression. Changes to the recipient's T cell receptor (TCR) repertoire are understood to play a pivotal role in the establishment of a robust state of tolerance in chimerism-based transplantation protocols., Methods: We investigated changes to the TCR repertoires of patients participating in an ongoing prospective, controlled, phase I/IIa trial designed to evaluate the safety and efficacy of combination cell therapy in living donor kidney transplantation. Using high-throughput sequencing, we characterized the repertoires of six kidney recipients who also received bone marrow from the same donor (CKBMT), together with an infusion of polyclonal autologous Treg cells instead of myelosuppression., Findings: Patients undergoing combination cell therapy exhibited partial clonal deletion of donor-reactive CD4 + T cells at one, three, and six months post-transplant, compared to control patients receiving the same immunosuppression regimen but no cell therapy (p = 0.024). The clonality, R20 and turnover rates of the CD4 + and CD8 + TCR repertoires were comparable in both groups, showing our protocol caused no excessive repertoire shift or loss of diversity. Treg clonality was lower in the case group than in control (p = 0.033), suggesting combination cell therapy helps to preserve Treg diversity., Interpretation: Overall, our data indicate that combining Treg cell therapy with CKBMT dampens the alloimmune response to transplanted kidneys in humans in the absence of myelosuppression., Funding: This study was funded by the Vienna Science and Technology Fund (WWTF)., Competing Interests: Declaration of interests The authors have declared that no conflict of interest exists., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

The proportion of patients with type I allergy in the world population has been increasing and with it the number of people suffering from allergic symptoms. Recently we showed that prophylactic cell therapy employing allergen-expressing bone marrow (BM) cells or splenic B cells induced allergen-specific tolerance in naïve mice. Here we investigated if cell therapy can modulate an established secondary allergen-specific immune response in pre-immunized mice. We sensitized mice against the grass pollen allergen Phl p 5 and an unrelated control allergen, Bet v 1, from birch pollen before the transfer of Phl p 5-expressing BM cells. Mice were conditioned with several combinations of low-dose irradiation, costimulation blockade, rapamycin and T cell-depleting anti-thymocyte globulin (ATG). Levels of allergen-specific IgE and IgG1 in serum after cell transfer were measured via ELISA and alterations in cellular responses were measured via an in vitro proliferation assay and transplantation of Phl p 5 + skin grafts. None of the tested treatment protocols impacted Phl p 5-specific antibody levels. Transient low-level chimerism of Phl p 5 + leukocytes as well as a markedly prolonged skin graft survival were observed in mice conditioned with high numbers of Phl p 5 + BMC or no sensitization events between the day of cell therapy and skin grafting. The data presented herein demonstrate that a pre-existing secondary allergen-specific immune response poses a substantial hurdle opposing tolerization through cell therapy and underscore the importance of prophylactic approaches for the prevention of IgE-mediated allergy.

The efficacy of costimulation blockade with CTLA4-Ig (belatacept) in transplantation is limited due to T cell-mediated rejection, which also persists after induction with anti-thymocyte globulin (ATG). Here, we investigate why ATG fails to prevent costimulation blockade-resistant rejection and how this barrier can be overcome. ATG did not prevent graft rejection in a murine heart transplant model of CTLA4-Ig therapy and induced a pro-inflammatory cytokine environment. While ATG improved the balance between regulatory T cells (Treg) and effector T cells in the spleen, it had no such effect within cardiac allografts. Neutralizing IL-6 alleviated graft inflammation, increased intragraft Treg frequencies, and enhanced intragraft IL-10 and Th2-cytokine expression. IL-6 blockade together with ATG allowed CTLA4-Ig therapy to achieve long-term, rejection-free heart allograft survival. This beneficial effect was abolished upon Treg depletion. Combining ATG with IL-6 blockade prevents costimulation blockade-resistant rejection, thereby eliminating a major impediment to clinical use of costimulation blockers in transplantation., (© 2024. The Author(s).)

Donor-specific tolerance remains a goal in transplantation because it could improve graft survival and reduce morbidity. Cotransplantation of donor hematopoietic cells to achieve chimerism is a promising approach for tolerance induction, which was successfully tested in clinical trials. However, current protocols are associated with side effects related to the myelosuppressive recipient conditioning, which makes it difficult to introduce them as standard therapy. More recently, adoptive cell therapy with polyclonal or donor-specific regulatory T cells (Treg) proved safe and feasible in several transplant trials, but it is unclear whether it can induce tolerance on its own. The combination of both approaches-Treg therapy and hematopoietic cell transplantation-leads to chimerism and tolerance without myelosuppressive treatment in murine models. Treg therapy promotes engraftment of allogeneic hematopoietic cells, reducing conditioning requirements and enhancing regulatory mechanisms maintaining tolerance. This review discusses possible modes of action of transferred Treg in experimental chimerism models and describes translational efforts investigating the potent synergy of Treg and chimerism., Competing Interests: T.W. received speaker’s honoraria from eGenesis and Mallinckrodt/Therakos and is a DSMB member for Quell Therapeutics. The other author declares no conflicts of interest., (Copyright © 2023 Wolters Kluwer Health, Inc. All rights reserved.)

Up to a third of the world's population suffers from allergies, yet the effectiveness of available preventative measures remains, at large, poor. Consequently, the development of successful prophylactic strategies for the induction of tolerance against allergens is crucial. In proof-of-concept studies, our laboratory has previously shown that the transfer of autologous hematopoietic stem cells (HSC) or autologous B cells expressing a major grass pollen allergen, Phl p 5, induces robust tolerance in mice. However, eventual clinical translation would require safe allergen expression without the need for retroviral transduction. Therefore, we aimed to chemically couple Phl p 5 to the surface of leukocytes and tested their ability to induce tolerance. Phl p 5 was coupled by two separate techniques, either by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) or by linkage via a lipophilic anchor, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-poly(ethylene glycol)-maleimide (DSPE-PEG-Mal). The effectiveness was assessed in fresh and cultured Phl p 5-coupled cells by flow cytometry, image cytometry, and immunofluorescence microscopy. Chemical coupling of Phl p 5 using EDC was robust but was followed by rapid apoptosis. DSPE-PEG-Mal-mediated linkage was also strong, but antigen levels declined due to antigen internalization. Cells coupled with Phl p 5 by either method were transferred into autologous mice. While administration of EDC-coupled splenocytes together with short course immunosuppression initially reduced Phl p 5-specific antibody levels to a moderate degree, both methods did not induce sustained tolerance towards Phl p 5 upon several subcutaneous immunizations with the allergen. Overall, our results demonstrate the successful chemical linkage of an allergen to leukocytes using two separate techniques, eliminating the risks of genetic modifications. More durable surface expression still needs to be achieved for use in prophylactic cell therapy protocols.

Hematopoietic chimerism remains the most promising strategy to bring transplantation tolerance into clinical routine. The concept of chimerism-based tolerance aims to extend the recipient's mechanisms of self-tolerance (ie, clonal deletion, anergy, and regulation) to include the tolerization of donor antigens that are introduced through the cotransplantation of donor hematopoietic cells. For this to be successful, donor hematopoietic cells need to engraft in the recipient at least temporarily. Three pioneering clinical trials inducing chimerism-based tolerance in kidney transplantation have been published to date. Within this review, we discuss the mechanisms of tolerance that are associated with the specific therapeutic protocols of each trial. Recent data highlight the importance of regulation as a mechanism that maintains tolerance. Insufficient regulatory mechanisms are also a likely explanation for situations of tolerance failure despite persisting donor chimerism. After decades of preclinical development of chimerism protocols, mechanistic data from clinical trials have recently become increasingly important. Better understanding of the required mechanisms for tolerance to be induced in humans will be a key to design more reliable and less invasive chimerism protocols in the future., Competing Interests: T.W. received speaker’s honoraria from eGenesis and Mallinckrodt/Therakos and is a Data and Safety Monitoring Board member for Quell Therapeutics. The other authors declare no conflicts of interest., (Copyright © 2023 Wolters Kluwer Health, Inc. All rights reserved.)

Introduction: Prophylactic strategies to prevent the development of allergies by establishing tolerance remain an unmet medical need. We previously reported that the transfer of autologous hematopoietic stem cells (HSC) expressing the major timothy grass pollen allergen, Phl p 5, on their cell surface induced allergen-specific tolerance in mice. In this study, we investigated the ability of allergen-expressing immune cells (dendritic cells, CD4 + T cells, CD8 + T cells, and CD19 + B cells) to induce allergen-specific tolerance in naive mice and identified CD19 + B cells as promising candidates for allergen-specific cell therapy., Methods: For this purpose, CD19 + B cells were isolated from Phl p 5-transgenic BALB/c mice and transferred to naive BALB/c mice, pre-treated with a short course of rapamycin and an anti-CD40L antibody. Subsequently, the mice were subcutaneously sensitized three times at 4-week intervals to Phl p 5 and Bet v 1 as an unrelated control allergen. Allergen-expressing cells were followed in the blood to monitor molecular chimerism, and sera were analyzed for Phl p 5- and Bet v 1-specific IgE and IgG 1 levels by RBL assay and ELISA, respectively. In vivo allergen-induced lung inflammation was measured by whole-body plethysmography, and mast cell degranulation was determined by skin testing., Results: The transfer of purified Phl p 5-expressing CD19 + B cells to naive BALB/c mice induced B cell chimerism for up to three months and prevented the development of Phl p 5-specific IgE and IgG 1 antibody responses for a follow-up period of 26 weeks. Since Bet v 1 but not Phl p 5-specific antibodies were detected, the induction of tolerance was specific for Phl p 5. Whole-body plethysmography revealed preserved lung function in CD19 + B cell-treated mice in contrast to sensitized mice, and there was no Phl p 5-induced mast cell degranulation in treated mice., Discussion: Thus, we demonstrated that the transfer of Phl p 5-expressing CD19 + B cells induces allergen-specific tolerance in a mouse model of grass pollen allergy. This approach could be further translated into a prophylactic regimen for the prevention of IgE-mediated allergy in humans., Competing Interests: RV received research grants from Worg Pharmaceuticals, Hangzhou, China; HVD Biotech, Vienna, Austria; and Viravaxx, Vienna, Austria. He serves as a consultant for Viravaxx and Worg. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2023 Prickler, Baranyi, Mengrelis, Weijler, Kainz, Kratzer, Steiner, Mucha, Rudoph, Pilat, Bohle, Strobl, Pickl, Valenta, Linhart and Wekerle.)

Introduction: Antibody mediated rejection (ABMR) is a major factor limiting outcome after organ transplantation. Anti-HLA donor-specific antibodies (DSA) of the IgG isotype are mainly responsible for ABMR. Recently DSA of the IgE isotype were demonstrated in murine models as well as in a small cohort of sensitized transplant recipients. In the present study, we aimed to determine the frequency of pre-existing and de novo anti-HLA IgE antibodies in a cohort of 105 solid organ transplant recipients., Methods: We prospectively measured anti-HLA IgE antibodies in a cohort of kidney (n=60), liver, heart and lung (n=15 each) transplant recipients before and within one-year after transplantation, employing a single-antigen bead assay for HLA class I and class II antigens. Functional activity of anti-HLA IgE antibodies was assessed by an in vitro mediator release assay. Antibodies of the IgG1-4 subclasses and Th1 and Th2 cytokines were measured in anti-HLA IgE positive patients., Results: Pre-existing anti-HLA IgE antibodies were detected in 10% of renal recipients (including 3.3% IgE-DSA) and in 4.4% of non-renal solid organ transplant recipients (heart, liver and lung cohort). Anti-HLA IgE occurred only in patients that were positive for anti-HLA IgG, and most IgE positive patients had had a previous transplant. Only a small fraction of patients developed de novo anti-HLA IgE antibodies (1.7% of kidney recipients and 4.4% of non-renal recipients), whereas no de novo IgE-DSA was detected. IgG subclass antibodies showed a distinct pattern in patients who were positive for anti-HLA IgE. Moreover, patients with anti-HLA IgE showed elevated Th2 and also Th1 cytokine levels. Serum from IgE positive recipients led to degranulation of basophils in vitro , demonstrating functionality of anti-HLA IgE., Discussion: These data demonstrate that anti-HLA IgE antibodies occur at low frequency in kidney, liver, heart and lung transplant recipients. Anti-HLA IgE development is associated with sensitization at the IgG level, in particular through previous transplants and distinct IgG subclasses. Taken together, HLA specific IgE sensitization is a new phenomenon in solid organ transplant recipients whose potential relevance for allograft injury requires further investigation., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Mucha, Cho, Weijler, Muckenhuber, Hofmann, Wahrmann, Heinzel, Linhart, Gattinger, Valenta, Berlakovich, Zuckermann, Jaksch, Oberbauer and Wekerle.)

Reducing the recipient's T cell repertoire is considered to increase the efficacy of regulatory T cell (Treg) therapy. This necessitates timing the administration of antithymocyte globulin (ATG) early enough before adoptive cell therapy (ACT) so that residual serum ATG does not deplete the transferred Tregs. The optimum time point in this regard has not been defined. Herein, we report the effects of residual serum ATG on the viability of an in vitro expanded Treg cell product used in a clinical trial of ACT in kidney transplant recipients (NCT03867617). Patients received ATG monotherapy (either 6 or 3 mg/kg body weight) without concomitant immunosuppression 2 to 3 weeks before transplantation and Treg transfer. An anti-ATG immunoglobulin G (IgG) immune response was elicited in all patients within 14 days. In turn, the elimination of total and Treg-specific ATG was accelerated substantially over control patients receiving the same dose of ATG with concomitant immunosuppression. However, ATG serum concentrations of <1 μg/mL, which had previously been reported as subtherapeutic threshold, triggered apoptosis of Tregs in vitro. Therefore, ATG levels need to decline to lower levels than those previously thought for efficacious Treg transfer. In 5 of 6 patients, such low levels of serum ATG considered safe for Treg transfer were reached within 2 weeks after ATG administration., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

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., Competing Interests: TW received speaker’s honoraria from eGenesis and Mallinckrodt/Therakos and is a DSMB member for Quell Therapeutics. BM is currently employed by Astra Zeneca. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Unger, Muckenhuber, Mahr, Schwarz, Pilat, Granofszky, Regele and Wekerle.)

Regulatory T cells (Tregs) play a critical role in maintaining self-tolerance and in containing allo-immune responses in the context of transplantation. Recent advances yielded the approval of the first pharmaceutical costimulation blockers (abatacept and belatacept), with more of them in the pipeline. These costimulation blockers inhibit effector cells with high clinical efficacy to control disease activity, but might inadvertently also affect Tregs. Treg homeostasis is controlled by a complex network of costimulatory and coinhibitory signals, including CD28, the main target of abatacept/belatacept, and CTLA4, PD-1 and ICOS. This review shall give an overview on what effects the therapeutic manipulation of costimulation has on Treg function in transplantation., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Muckenhuber, Wekerle and Schwarz.)

Background: Tacrolimus is the calcineurin inhibitor of choice for preventing acute rejection episodes in kidney transplant patients. However, tacrolimus has a narrow therapeutic range that requires regular monitoring of blood concentrations to minimize toxicity. A new once-daily tacrolimus formulation, LCP-tacrolimus (LCPT), has been developed, which uses MeltDose™ drug-delivery technology to control drug release and enhance overall bioavailability. Our study compared dosing of LCPT with current standard-of-care tacrolimus [immediate-release tacrolimus (IR-Tac) or prolonged-release tacrolimus (PR-Tac)] during the 6 months following de novo kidney transplantation. Comparisons of graft function, clinical outcomes, safety, and tolerability for LCPT versus IR-Tac/PR-Tac were also performed. Methods: Standard immunological risk patients with end-stage renal disease who had received a de novo kidney transplant were randomized (1:1) to LCPT (N = 200) or IR-Tac/PR-Tac (N = 201). Results: Least squares (LS) mean tacrolimus total daily dose from Week 3 to Month 6 was significantly lower for LCPT than for IR-Tac/PR-Tac. Although LS mean tacrolimus trough levels were significantly higher for LCPT than IR-Tac/PR-Tac, tacrolimus trough levels remained within the standard reference range for most patients. There were no differences between the groups in treatment failure measures or safety profile. Conclusion: LCPT can achieve similar clinical outcomes to other tacrolimus formulations, with a lower daily dose. Clinical Trial Registration: https://clinicaltrials.gov/, identifier NCT02432833., Competing Interests: GP, DS, SG, CP, and GN are all employees of Chiesi Farmaceutici S.p.A. KB has received honoraria and/or research funding from Alexion, Astellas, Bristol-Myers Squibb, Chiesi, Fresenius, Hansa, Hexal, Merck, Novartis, Otsuka, Pfizer, Roche, Sandoz, Siemens, and Veloxis Pharmaceuticals. UM received participation fees from Chiesi for scientific advisory boards. LR has been an advisor for Hansa and Biotest; has received speaker fees from Astellas, Novartis, Chiesi and Bristol-Myers Squibb; and has received grants from Chugai, Terumo and HemaT. GP, who is now an employee of Chiesi Farmaceutici S.p.A., worked at Dipartimento di Medicina e Chirurgia, UO Nefrologia, Azienda Ospedaliero-Universitaria di Parma, Parma, Italy at the time of the study and received consulting fees from Chiesi for work as a medical research physician for the present study. OW has received research grants for clinical studies, speaker's fees, honoraria, and travel expenses from Amgen, Astellas, Bristol-Myers Squibb, Chiesi, Hexal, Janssen-Cilag, MSD, Novartis, Pfizer, Roche, and Sanofi. DS received consulting fees from Chiesi for work as a clinical study manager for the present study. NK has received speaker fees and participated in advisory boards for Abbvie, Amgen, Astellas, Chiesi, Fresnius Medical Care, Gilead, Merck Sharp and Dohme, Neovii, Novartis, Roche, Sanofi, and Shire. MB has received funds for travel from Astellas and Chiesi. JP has received consulting fees from Chiesi. DK has received honoraria from Chiesi and Astellas. TW has received honoraria from Chiesi and Therakos/Mallinckrodt, and research funding from Astellas, Chiesi, Neovii, Novartis, Sandoz, Sanofi, and Teva. AG-D has received travel grants, speaker fees, and/or advisory board honoraria from Alexion, Chiesi, MSD and Novartis, and has participated in clinical trials sponsored by Alexion, Astellas, Chiesi and Novartis. LW had received research funding from Chiesi. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The authors declare that this study (ClinicalTrials.gov, NCT02432833) was funded by Chiesi Farmaceutici SpA. The funder was involved in study design, collection, analysis and interpretation of data. All co-authors have contributed substantially to the analyses and interpretation of the data, and have provided important intellectual input and approval of the final version of the manuscript. Fishawack Inc. has received funding by Chiesi Farmaceutici SpA to provide medical writing support., (Copyright © 2022 Budde, Rostaing, Maggiore, Piotti, Surace, Geraci, Procaccianti, Nicolini, Witzke, Kamar, Albano, Büchler, Pascual, Gutiérrez-Dalmau, Kuypers, Wekerle, Głyda, Carmellini, Tisone, Midtvedt, Wennberg and Grinyó.)

Background: Pre-sensitized kidney transplant recipients have a higher risk for rejection following kidney transplantation and therefore receive lymphodepletional induction therapy with anti-human T-lymphocyte globulin (ATLG) whereas non-sensitized patients are induced in many centers with basiliximab. The time course of lymphocyte reconstitution with regard to the overall and donor-reactive T-cell receptor (TCR) specificity remains elusive., Methods/design: Five kidney transplant recipients receiving a 1.5-mg/kg ATLG induction therapy over 7 days and five patients with 2 × 20 mg basiliximab induction therapy were longitudinally monitored. Peripheral mononuclear cells were sampled pre-transplant and within 1, 3, and 12 months after transplantation, and their overall and donor-reactive TCRs were determined by next-generation sequencing of the TCR beta CDR3 region. Overall TCR repertoire diversity, turnover, and donor specificity were assessed at all timepoints., Results: We observed an increase in the donor-reactive TCR repertoire after transplantation in patients, independent of lymphocyte counts or induction therapy. Donor-reactive CD4 T-cell frequency in the ATLG group increased from 1.14% + -0.63 to 2.03% + -1.09 and from 0.93% + -0.63 to 1.82% + -1.17 in the basiliximab group in the first month. Diversity measurements of the entire T-cell repertoire and repertoire turnover showed no statistical difference between the two induction therapies. The difference in mean clonality between groups was 0.03 and 0.07 pre-transplant in the CD4 and CD8 fractions, respectively, and was not different over time (CD4: F(1.45, 11.6) = 0.64 p = 0.496; CD8: F(3, 24) = 0.60 p = 0.620). The mean difference in R20, a metric for immune dominance, between groups was -0.006 in CD4 and 0.001 in CD8 T-cells and not statistically different between the groups and subsequent timepoints (CD4: F(3, 24) = 0.85 p = 0.479; CD8: F(1.19, 9.52) = 0.79 p = 0.418)., Conclusion: Reduced-dose ATLG induction therapy led to an initial lymphodepletion followed by an increase in the percentage of donor-reactive T-cells after transplantation similar to basiliximab induction therapy. Furthermore, reduced-dose ATLG did not change the overall TCR repertoire in terms of a narrowed or skewed TCR repertoire after immune reconstitution, comparable to non-depletional induction therapy., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Aschauer, Jelencsics, Hu, Gregorich, Reindl-Schwaighofer, Wenda, Wekerle, Heinzel and Oberbauer.)

The importance and exact role of graft-resident leucocytes (also referred to as passenger leucocytes) in transplantation is controversial as these cells have been reported to either initiate or retard graft rejection. T cell activation to allografts is mediated via recognition of intact or processed donor MHC molecules on antigen-presenting cells (APC) as well as through interaction with donor-derived extracellular vesicles. Reduction of graft-resident leucocytes before transplantation is a well-known approach for prolonging organ survival without interfering with the recipient's immune system. As previously shown by our group, injecting mice with IL-2/anti-IL-2 complexes (IL-2cplx) to augment expansion of CD4 T regulatory cells (Tregs) induces tolerance towards islet allografts, and also to skin allografts when IL-2cplx treatment is supplemented with rapamycin and a short-term treatment of anti-IL-6. In this study, we investigated the mechanisms by which graft-resident leucocytes impact graft survival by studying the combined effects of IL-2cplx-mediated Treg expansion and passenger leucocyte depletion. For the latter, effective depletion of APC and T cells within the graft was induced by prior total body irradiation (TBI) of the graft donor. Surprisingly, substantial depletion of donor-derived leucocytes by TBI did not prolong graft survival in naïve mice, although it did result in augmented recipient leucocyte graft infiltration, presumably through irradiation-induced nonspecific inflammation. Notably, treatment with the IL-2cplx protocol prevented early inflammation of irradiated grafts, which correlated with an influx of Tregs into the grafts. This finding suggested there might be a synergistic effect of Treg expansion and graft-resident leucocyte depletion. In support of this idea, significant prolongation of skin graft survival was achieved if we combined graft-resident leucocyte depletion with the IL-2cplx protocol; this finding correlated along with a progressive shift in the composition of T cells subsets in the grafts towards a more tolerogenic environment. Donor-specific humoral responses remained unchanged, indicating minor importance of graft-resident leucocytes in anti-donor antibody development. These results demonstrate the importance of donor-derived leucocytes as well as Tregs in allograft survival, which might give rise to new clinical approaches., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Steiner, Weijler, Wekerle, Sprent and Pilat.)

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., (© 2021 The Authors. American Journal of Transplantation published by Wiley Periodicals LLC on behalf of The American Society of Transplantation and the American Society of Transplant Surgeons.)

Background: Antigen recognition of allo-peptides and HLA molecules leads to the activation of donor-reactive T-cells following transplantation, potentially causing T-cell-mediated rejection (TCMR). Sequencing of the T-cell receptor (TCR) repertoire can be used to track the donor-reactive repertoire in blood and tissue of patients after kidney transplantation., Methods/design: In this prospective cohort study, 117 non-sensitized kidney transplant recipients with anti-CD25 induction were included. Peripheral mononuclear cells (PBMCs) were sampled pre-transplant and at the time of protocol or indication biopsies together with graft tissue. Next-generation sequencing (NGS) of the CDR3 region of the TCRbeta chain was performed after donor stimulation in mixed lymphocyte reactions to define the donor-reactive TCR repertoire. Blood and tissue of six patients experiencing a TCMR and six patients without rejection on protocol biopsies were interrogated for these TCRs. To elucidate common features of T-cell clonotypes, a network analysis of the TCR repertoires was performed., Results: After transplantation, the frequency of circulating donor-reactive CD4 T-cells increased significantly from 0.86 ± 0.40% to 2.06 ± 0.40% of all CD4 cells (p < 0.001, mean dif.: -1.197, CI: -1.802, -0.593). The number of circulating donor-reactive CD4 clonotypes increased from 0.72 ± 0.33% to 1.89 ± 0.33% (p < 0.001, mean dif.: -1.168, CI: -1.724, -0.612). No difference in the percentage of donor-reactive T-cells in the circulation at transplant biopsy was found between subjects experiencing a TCMR and the control group [p = 0.64 (CD4 + ), p = 0.52 (CD8 + )]. Graft-infiltrating T-cells showed an up to six-fold increase of donor-reactive T-cell clonotypes compared to the blood at the same time (3.7 vs. 0.6% and 2.4 vs. 1.5%), but the infiltrating TCR repertoire was not reflected by the composition of the circulating TCR repertoire despite some overlap. Network analysis showed a distinct segregation of the donor-reactive repertoire with higher modularity than the overall TCR repertoire in the blood. These findings indicate an unchoreographed process of diverse T-cell clones directed against numerous non-self antigens found in the allograft., Conclusion: Donor-reactive T-cells are enriched in the kidney allograft during a TCMR episode, and dominant tissue clones are also found in the blood., Trial Registration: Clinicaltrials.gov: NCT: 03422224 (https://clinicaltrials.gov/ct2/show/NCT03422224)., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Aschauer, Jelencsics, Hu, Heinzel, Gregorich, Vetter, Schaller, Winkler, Weinberger, Pimenov, Gualdoni, Eder, Kainz, Troescher, Regele, Reindl-Schwaighofer, Wekerle, Huppa, Sykes and Oberbauer.)