MHC Mismatch Research Articles

CAR Treg synergy with anti-CD154 mediates infectious tolerance to dictate heart transplant outcomes.

Successful allograft specific tolerance induction would eliminate the need for daily immunosuppression and improve post-transplant quality of life. Adoptive cell therapy with regulatory T cells expressing donor-specific Chimeric Antigen Receptors (CAR-Tregs) is a promising strategy, but as monotherapy, cannot prolong the survival with allografts with multiple MHC mismatches. Using an HLA-A2-transgenic haplo-mismatched heart transplantation model in immunocompetent C57Bl/6 recipients, we show that HLA-A2-specific (A2) CAR Tregs was able to synergize with low dose of anti-CD154 to enhance graft survival. Using haplo-mismatched grafts expressing the 2W-OVA transgene and tetramer-based tracking of 2W- and OVA-specific T cells, we showed that in mice with accepted grafts, A2.CAR Tregs inhibited endogenous non-A2 donor- specific T cell, B cell and antibody responses, and promoted a significant increase in endogenous FoxP3 + Tregs with indirect donor-specificity. By contrast, in mice where A2.CAR Tregs failed to prolong graft survival, FoxP3 neg A2.CAR T cells preferentially accumulated in rejecting allografts and endogenous donor-specific responses were not controlled. This study therefore provides the first evidence for synergy between A2.CAR Tregs and CD154 blockade to promote infectious tolerance in immunocompetent recipients of haplo-mismatched heart grafts and defines features of A2.CAR Tregs when they fail to reshape host immunity towards allograft tolerance.

Comparison of Autologous and Allogeneic Adipose-Derived Stem Cells in Kidney Transplantation: Immunological Considerations and Therapeutic Efficacy.

Regenerative medicine shows significant potential in treating kidney diseases through the application of various types of stem and progenitor cells, including mesenchymal stem cells (MSCs), renal stem/progenitor cells, embryonic stem cells (ESCs), and induced pluripotent stem cells (iPSCs). Stem cells possess the unique ability to repair injured organs and improve impaired functions, making them a key element in the research of therapies for kidney tissue repair and organ regeneration. In kidney transplantation, reperfusion injury can cause tissue destruction, leading to an initially low glomerular filtration rate and long-term impact on function by creating irreversible interstitial fibrosis. MSCs have proven useful in repairing early tissue injury in animal models of kidney, lung, heart, and intestine transplantation. The use of stem cell therapies in solid organ transplantation raises the question of whether autologous or allogeneic cells should be preferred. Adipose-derived stem cells (ASCs), characterized by the lack of HLA Class II molecules and low expression of HLA Class I and co-stimulatory signals, are considered immune-privileged. However, the actual risk of graft rejection associated with allogeneic ASCs remains unclear. It has been demonstrated that donor-derived ASCs can promote the development of Treg cells in vitro, and some degree of tolerance induction has been observed in vivo. Nevertheless, a study comparing the efficacy of autologous and allogeneic ASCs in a rat model with a total MHC mismatch for kidney transplantation showed that donor-derived administration of ASCs did not improve the grafts' survival and was associated with increased mortality through an immunologically mediated mechanism. Given the lack of data, autologous ASCs appear to be a safer option in this research context. The aim of this review was to examine the differences between autologous and allogeneic ASCs in the context of their application in kidney transplantation therapies, considering potential immune reactions and therapeutic efficacy. Some have argued that ASCs harvested from end-stage renal disease (ESRD) patients may have lower regenerative potential due to the toxic effects of uremia, potentially limiting their use in transplantation settings. However, evidence suggests that the beneficial properties of ASCs are not affected by uremia or dialysis. Indeed, some investigators have demonstrated that ASCs harvested from chronic kidney disease (CKD) patients exhibit normal characteristics and function, maintaining consistent proliferative capacity and genetic stability over time, even after prolonged exposure to uremic serum Furthermore, no differences were observed in the response of ASCs to immune activation or their inhibitory effect on the proliferation of alloantigen-activated peripheral blood mononuclear cells between patients with normal or impaired renal function. This review presents the current achievements in stem cell research aimed at treating kidney diseases, highlighting significant progress and ongoing efforts in the development of stem cell-based therapies. Despite the encouraging results, further research is needed to overcome the current limitations and fully realize the potential of these innovative treatments. Advances in this field are crucial for developing effective therapies that can address the complex challenges associated with kidney damage and failure.

Immunomodulation by juglone alleviates acute graft-versus-host disease without compromising the graft-versus-leukaemia activity in mice.

Acute graft-versus-host disease (GVHD) remains a major barrier to successful transplantation outcomes. Recent studies have shown that pharmacotherapy for GVHD should target both the innate and adaptive inflammatory immune responses. Juglone, a redox-active phytochemical found in walnuts, has shown potent anti-inflammatory effects in models of colitis and inflammatory bowel disease. However, its effects on T-cell-mediated immune responses remain largely unknown. Considering the overlapping mediators of inflammation in GVHD and the aforementioned conditions, we investigated the use of juglone as a prophylactic agent for GVHD. Immunomodulatory activity and mechanism of action of juglone were studied using murine splenic leukocytes in vitro. The GVHD prophylactic efficacy of orally administered juglone was evaluated using a murine model of allogeneic haematopoietic stem cell transplantation based on an MHC mismatch. Juglone exhibited immunomodulatory activity by (i) inhibiting the activation of dendritic cells and CD4+ T-cells, (ii) inhibiting cytokine secretion and lymphocyte proliferation, and (iii) inducing exhaustion of CD4+ T-cells, as shown by increased expression of CTLA-4 (CD152) and Fas (CD95). Oral administration of juglone significantly reduced mortality and morbidity associated with GVHD while maintaining graft-versus-leukaemia activity. This was accompanied by a decrease in the number of naïve CD4+ cells, and an increase in the number of CD4+ and CD8+ central memory T-cells. Juglone is a potent immunomodulator for GVHD prophylaxis. Our study is the first to provide a dosage framework for the oral administration of juglone that can be used for clinical development.

Comparison of NK alloreactivity prediction models based on KIR-MHC interactions in haematopoietic stem cell transplantation.

The biological processes underlying NK cell alloreactivity in haematopoietic stem cell transplantation (HSCT) remain unclear. Many different models to predict NK alloreactivity through KIR and MHC genotyping exist, raising ambiguities in its utility and application for clinicians. We assessed 27 predictive models, broadly divided into six categories of alloreactivity prediction: ligand-ligand, receptor-ligand, educational, KIR haplotype-based, KIR matching and KIR allelic polymorphism. The models were applied to 78 NGS-typed donor/recipient pairs undergoing allogeneic HSCT in genoidentical (n=43) or haploidentical (n=35) matchings. Correlations between different predictive models differed widely, suggesting that the choice of the model in predicting NK alloreactivity matters. For example, two broadly used models, educational and receptor-ligand, led to opposing predictions especially in the genoidentical cohort. Correlations also depended on the matching fashion, suggesting that this parameter should also be taken into account in the choice of the scoring strategy. The number of centromeric B-motifs was the only model strongly correlated with the incidence of acute graft-versus-host disease in our set of patients in both the genoidentical and the haploidentical cohorts, suggesting that KIR-based alloreactivity, not MHC mismatches, are responsible for it. To our best knowledge, this paper is the first to experimentally compare NK alloreactivity prediction models within a cohort of genoidentical and haploidentical donor-recipient pairs. This study helps to resolve current discrepancies in KIR-based alloreactivity predictions and highlights the need for deeper consideration of the models used in clinical studies as well as in medical practice.

Elimination of 4T1 Mammary Tumor Cells by BALB/cBy UBC-GFP Transgenics following Stable Inheritance of the H-2b MHC Allele.

The human ubiquitin C promoter (UBC)-driven GFP-transgenic mouse (UBC-GFP) transgene integration site was mapped recently to chromosome 17, linked closely to the MHC locus. In this study, we demonstrate a functional consequence of this insertion site in the backcrossed UBC-GFP BALB/c congenic strain [CByJ.B6-Tg(UBC-GFP) 30Scha/J]: rejection of transplanted "syngeneic" 4T1 mammary tumor cells. Rejection of BALB/c-derived 4T1 cells is in all likelihood a consequence of MHC mismatch due to stable inheritance of C57BL/6-derived H-2b (rather than prototypical H-2d) by the BALB/c UBC-GFP strain. These data are a valuable resource to researchers who have previously employed the UBC-GFP congenic strain for attempted syngeneic MHC-matched and allogenic MHC-mismatched studies, as their data likely require reinterpretation. Further, this study reemphasizes the impact of mapping transgene integration sites of commonly used mouse strains as a way of increasing scientific rigor and reproducibility.

APRIL/BLyS deficient rats prevent donor specific antibody (DSA) production and cell proliferation in rodent kidney transplant model.

APRIL (A proliferation inducing ligand) and BLyS (B Lymphocyte Stimulator) are two critical survival factors for B lymphocytes and plasma cells, the main source of alloantibody. We sought to characterize the specific effects of these cytokines in a kidney transplant model of antibody mediated rejection (AMR). We engineered APRIL-/- and BLyS-/- Lewis rats using CRISPR/Cas9. APRIL-/- and BLyS-/- rats were sensitized with Brown Norway (BN) blood (complete MHC mismatch). Twenty-one days following sensitization, animals were harvested and collected tissues were analyzed using flow cytometry, ELISPOT, and immunohistochemistry. Flow cross match and a 3 day mixed lymphocyte reaction (MLR) was performed to assess donor specific antibody (DSA) production and T-cell proliferation, respectively. Sensitized dual knock out Lewis rats (APRIL-/-/BLyS-/-) underwent kidney transplantation and were sacrificed on day 7 post-transplant. Sensitized BLyS-/- had significant decreases in DSA and cell proliferation compared to WT and APRIL-/- (p<0.02). Additionally, BLyS-/- rats had a significant reduction in IgG secreting cells in splenic marginal zone B lymphocytes, and in cell proliferation when challenged with alloantigen compared to WT and APRIL-/-. Transplanted APRIL-/-/BLyS-/- rodents had significantly less DSA and antibody secreting cells compared to WT (p<0.05); however, this did not translate into a significant difference in AMR seen between groups. In summary, our studies suggest that APRIL and BLyS play a greater role in DSA generation rather than AMR, highlighting the role of cellular pathways that regulate AMR.

Tolerance of a Vascularized Composite Allograft Achieved in MHC Class-I-mismatch Swine via Mixed Chimerism.

BackgroundVascularized composite allografts (VCAs) allow reconstruction of devastating injuries and amputations, yet require lifelong immunosuppression that is associated with significant morbidity. Induction of immune tolerance of VCAs would permit widespread use of these procedures. VCAs are acquired from deceased donors most likely to be fully-MHC-mismatched (in contrast to living-related renal transplant donor-recipient pairs matched at one MHC haplotype). After achieving VCA tolerance in a swine model equivalent to clinical living-related renal transplants (single-haplotype MHC mismatches: e.g., “mother-daughter”/haploidentical), we tested our protocol in MHC class I, class II, and fully-MHC-mismatched pairs. Although class II mismatched swine demonstrated similar results as the haploidentical scenario (stable mixed chimerism and tolerance), our protocol failed to prevent rejection of class I and full mismatch VCAs. Here, we describe a new adapted conditioning protocol that successfully achieved tolerance across MHC class-I-mismatch barriers in swine.MethodsSwine were treated with non-myeloablative total body and thymic irradiation two days prior to infusion of bone marrow cells from an MHC class I-mismatched donor. They also received a short-term treatment with CTLA4-Ig (Belatacept®) and anti-IL6R mAb (Tociluzimab®) and were transplanted with an osteomyocutaneous VCA from the same donor.ResultsStable mixed chimerism and tolerance of MHC class-I-mismatched VCAs was achieved in 3 recipients. Allograft tolerance was associated with a sustained lack of anti-donor T cell response and a concomitant expansion of double negative CD4-CD8- T cells producing IL-10.ConclusionsThis study demonstrates the first successful mixed chimerism-induced VCA tolerance in a large animal model across a MHC class-I-mismatch. Future studies aimed at fully-mismatched donor-recipient pairs are under investigation with this protocol.

Tissue Engineering Scaffolds for Minimally-Invasive Immunosurveillance of Acute Cellular Graft Rejection

<h3>Purpose</h3> As there is no assay to predict the risk of acute cellular allograft rejection (ACAR), clinicians rely on protocol graft biopsy and aggressive, <i>one-size-fits-all</i> immunosuppression. Immunosuppression protects grafts from ACAR but increases systemic toxicities. Endomyocardial biopsy (EMB) histology suffers from variability and lags behind molecular biomarkers. <b>A minimally invasive surveillance method is urgently needed to quantify early risk of rejection for minimizing graft biopsy and personalizing treatment.</b> <h3>Methods</h3> Biomaterial scaffolds accumulate immune cells producing biomarkers of ACAR as an engineered immunological niche. We implanted subcutaneous poly-caprolactone scaffolds in murine heart or skin transplants to determine if they could identify ACAR. Rag2KO mice received full MHC mismatch allografts or syngeneic controls, and ACAR was initiated as a tunable event by C57BL/6 T cell transfer. Scaffolds were explanted and analyzed via histology and differential gene expression by RNA sequencing. <h3>Results</h3> The scaffolds provided a novel measure of graft health. We algorithmically identified <b>11 differentially-expressed genes as biomarkers</b> that distinguish mice with rejecting grafts from those with healthy grafts. <i>Importantly, the scoring system detects oncoming ACAR prior to clinical signs of rejection.</i> Also, histology of these niches identified changes associated with ACAR progression. <h3>Conclusion</h3> Gene expression in the cell-capture scaffold identified a biomarker signature of early ACAR, without invasive biopsy. <b>Scaffold cell capture technology provides a leading indicator of ACAR by monitoring tissue-specific immune responses associated with allograft rejection.</b> This implantable scaffold enables minimally invasive histological evaluation and molecular calculation of the early risk of rejection to reduce the frequency of routine EMB and personalize immune suppression that ultimately could prolong transplant life while minimizing risk.

Infusion of iPSC-Derived CD34 + Hematopoietic Progenitors Following Myeloablative HSC Transplantation and Assessment of Their Immunogenicity in MHC-Defined Nonhuman Primate Model

Bone marrow suppression and associated neutropenia that renders the patient more susceptible to infection is a major complication and limiting factor for current chemotherapy. Strategies aimed at neutrophil reconstitution using transfusions or accelerating neutrophil recovery with G-CSF show a limited effect in reducing the rate of infection. To overcome these limitations, administration of ex vivo expanded somatic hematopoietic progenitors (HPs) has been explored as a way to facilitate a more rapid myeloid recovery and improve overall survival following myeloablation. Recent advances in reprogramming and induced pluripotent stem cell (iPSC) technologies have created alternative platforms for off-the-shelf supply of immunologically compatible HPs, including cellular products derived from MHC homozygous superdonors which can increase the degree of MHC matching and may provide a maximum utility for stem cell banking. In these studies, we developed Mauritian cynomolgus macaque (MCM) model to evaluate the utility and safety of CD34 +CD45 + hematopoietic progenitors derived from iPSCs (iHPs) generated from MHC homozygous animals in the treatment of cytopenia following myeloablative stem cell transplantation. MCM iPSCs were generated from MHC homozygous animals and used to generate iHPs in coculture with OP9. Three groups of MCMs underwent myeloablative total body irradiation (TBI) followed by transplantation of cryopreserved autologous CD34 + HSPCs. Four days after autologous HSPC transplantation, animals received transfusion of 30x10 6/kg cryopreserved iCD34 + cells tagged with eGFP or tdTomato from homozygous MHC-matched iPSCs (group 2) or homozygous MHC mismatched iPSCs (group 3). We have demonstrated that infusion of iHPs is safe and well tolerated. No teratoma or tumor formation was observed in animals one year after infusion of iHPs. Although we detected few iPSC derived hematopoietic cells in the blood within 1-week post-infusion, we did not see any significant differences in blood counts or other peripheral blood parameters between all animal groups. Intriguingly we found footprints of iPSC-derived cells in the colon, lymph node, skin and brain specimens collected 25 days after iHP infusion from one animal. This suggests a possibility of migration and retention within these tissues of iHP and iHP-derived myeloid cells. To assess immunogenicity, MHC homozygous iHPs were infused into immunocompetent MCMs across different MHC barriers. 10x10 6/kg of cryopreserved iCD34+ from homozygous iPSCs were transfused every week for 3 weeks, into animals that were divided into 4 groups namely autologous (group I), MHC matched homozygous (group II), MHC matched heterozygous (group III) and MHC mismatch (group IV). Overall, these studies revealed low immunogenicity of MHC homozygous iHPs in MHC matched homozygous and heterozygous animals, while weak immune response was detected following iHP infusion in some MHC mismatched animals. DisclosuresSlukvin: Cynata Therapeutics: Consultancy, Current equity holder in publicly-traded company.

Sensitization and Desensitization in Vascularized Composite Allotransplantation.

Vascularized composite allotransplantation (VCA) is a field under research and has emerged as an alternative option for the repair of severe disfiguring defects that result from severe tissue loss in a selected group of patients. Lifelong immunosuppressive therapy, immunosuppression associated complications, and the effects of the host immune response in the graft are major concerns in this type of quality-of-life transplant. The initial management of extensive soft tissue injury can lead to the development of anti-HLA antibodies through injury-related factors, transfusion and cadaveric grafting. The role of antibody-mediated rejection, donor-specific antibody (DSA) formation and graft rejection in the context of VCA still remain poorly understood. The most common antigenic target of preexisting alloantibodies are MHC mismatches, though recognition of ABO incompatible antigens, minor histocompatibility complexes and endothelial cells has also been shown to contribute to rejection. Mechanistically, alloantibody-mediated tissue damage occurs primarily through complement fixation as well as through antibody-dependent cellular toxicity. If DSA exist, activation of complement and coagulation cascades can result in vascular thrombosis and infarction and thus rejection and graft loss. Both preexisting DSA but especially de-novo DSA are currently considered as main contributors to late allograft injury and graft failure. Desensitization protocols are currently being developed for VCA, mainly including removal of alloantibodies whereas treatment of established antibody-mediated rejection is achieved through high dose intravenous immunoglobulins. The long-term efficacy of such therapies in sensitized VCA recipients is currently unknown. The current evidence base for sensitizing events and outcomes in reconstructive transplantation is limited. However, current data show that VCA transplantation has been performed in the setting of HLA-sensitization.

A Novel Role for AMP-Activated Protein Kinase (AMPK) in Controlling T Cell Sensitivity to IL-6

Allogeneic hematopoietic stem cell transplantation (alloHSCT) is a curative treatment for high-risk leukemia and many non-malignant hematologic disorders. However, the use of alloHSCT as a therapy remains limited by acute graft-versus-host disease (GVHD), where activated donor T cells attack and destroy host tissues in the skin, gastrointestinal tract, and liver. We have previously shown that alloreactive T cells increase phosphorylation of the intracellular energy sensor AMP-activated protein kinase (AMPK) as early as day 3 post-transplant (levels 8-fold higher in alloreactive T cells compared to naive or syngeneic controls (p=0.0003)) and donor T cells lacking AMPK (AMPK KO) cause significantly less GVHD in both major- and minor-histocompatibility mismatch models of GVHD. At the same time, AMPK KO T cells equivalently clear leukemia cells and extend recipient survival in leukemia models with high tumor burden. Transplantation of cells lacking AMPK lowers the number of donor T cells recovered on day 7 post-transplant (3.15 +/- 0.49x106 versus1.87 +/- 0.53x106, p=0.0006, wildtype (wt) versus AMPKKO respectively) but interestingly, increases both the percentage and total number of regulatory T cells (Treg) (0.85 +/- 0.32x104 vs. 1.69 +/- 0.34x104, wt vs. KO, p=0.004).To understand these findings mechanistically, we sought to study the role of AMPK in donor Treg expansion and stabilization post-alloHSCT. Canonically, interleukin-2 (IL-2) signaling, through phosphorylation of Stat5, promotes Treg generation and stability, while inflammatory signals (e.g. IL-6 signaling through Stat3) are known to decrease Treg stability post-transplant. Studies testing IL-2 sensitivity in KO cells, or the degree of Stat5 phosphorylation post-transplant, were unrevealing. However, AMPK KO Tregs significantly decrease their sensitivity to IL-6, with a 50% reduction in phosphorylated-Stat3 levels following intermediate levels of cytokine stimulation (Figure 1A). To demonstrate the functional consequences of this difference in cytokine sensitivity, we exposed in vitro generated Treg to increasing concentrations of IL-6. As expected, the percentage of wt FoxP3+ cells decreased sharply with increasing IL-6 concentrations (decreasing from 50% to 14%), while AMPK KO Tregs were minimally affected over this dose range (47% versus 42%; Figure 1B, p<0.02).To determine if increased Treg stability was the predominant reason for improvements in GVHD seen with AMPK KO cells, CD25+ cells were removed from the donor inoculum of AMPK KO or wt mice by magnetic selection (to eliminate >90% of Treg), and then either CD25-replete or CD25-depleted sets of cells were transplanted into B6xDBA2 F1 recipients in a major MHC mismatch model of GVHD. Despite a decrease in AMPK KO Treg numbers on day 7 to levels commiserate with wt donors (Figure 2A), elimination of AMPK KO Treg had only a modest effect on GVHD severity (Figure 2B), implicating a substantial role for AMPK in the non-Treg compartment.To explore the role of AMPK in non-Tregs further, we investigated the sensitivity of unmanipulated wt and AMPK KO T cells to IL-6 stimulation. Similar to FoxP3+ cells, both naïve CD4 and CD8 T cells lacking AMPK experienced lower levels of Stat3 phosphorylation in response to graded concentrations of IL-6 (Figure 3A). Furthermore, if IL-6 was added to a 4-day culture with anti-CD3 stimulation, the number of wt T cells recovered after 96 hours increased >2.2-fold. In contrast, AMPK KO cells experienced minimal changes in T cell number over a wide range of IL-6 concentrations (Figure 3B).In conclusion, we define in these studies a novel role for AMPK in controlling donor T cell sensitivity to IL-6, both at the level of Stat3 phosphorylation, and in terms of functional consequences. Not only does this striking result elegantly explain our previous observation of increased Treg percentages, and decreased donor T cell numbers, following transplantation of AMPK KO cells, it continues to make the case that inhibition of AMPK represents an attractive clinical target for the amelioration of GVHD. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Obesity induces gut microbiota alterations and augments acute graft-versus-host disease after allogeneic stem cell transplantation.

The efficacy of allogeneic hematopoietic stem cell transplantation (allo-HSCT) is limited by acute and chronic graft-versus-host disease (GVHD). The impact of obesity on allo-HSCT outcomes is poorly understood. Here, we report that obesity had a negative and selective impact on acute gut GVHD after allo-HSCT in mice with diet-induced obesity (DIO). These animals exhibited increased gut permeability, endotoxin translocation across the gut, and radiation-induced gastrointestinal damage after allo-HSCT. After allo-HSCT, both male and female DIO mouse recipients showed increased proinflammatory cytokine production and expression of the GVHD marker ST2 (IL-33R) and MHC class II molecules; they also exhibited decreased survival associated with acute severe gut GVHD. This rapid-onset, obesity-associated gut GVHD depended on donor CD4+ T cells and occurred even with a minor MHC mismatch between donor and recipient animals. Retrospective analysis of clinical cohorts receiving allo-HSCT transplants from unrelated donors revealed that recipients with a high body mass index (BMI, >30) had reduced survival and higher serum ST2 concentrations compared with nonobese transplant recipients. Assessment of both DIO mice and allo-HSCT recipients with a high BMI revealed reduced gut microbiota diversity and decreased Clostridiaceae abundance. Prophylactic antibiotic treatment protected DIO mouse recipients from endotoxin translocation across the gut and increased inflammatory cytokine production, as well as gut pathology and mortality, but did not protect against later development of chronic skin GVHD. These results suggest that obesity-induced alterations of the gut microbiota may affect GVHD after allo-HSCT in DIO mice, which could be ameliorated by prophylactic antibiotic treatment.

Tissue-Derived IL-33 Is a Critical Local Signal That Targets Th1 Cells in the Small Intestine to Sustain Graft Versus Host Disease in the Absence of IL-12

Background: Graft vs. host disease (GVHD) remains a major complication of allogeneic stem cell transplantation (alloSCT). To create space for donor stem cells and prevent their rejection, alloSCT protocols rely on conditioning regimens involving chemotherapy, frequently combined with radiation. Interleukin (IL)-33 is a nuclear protein that is found in the secondary lymphoid organs and barrier tissues. IL-33 is augmented in recipient barrier tissues by conditioning. We have established that IL-33 is a clinically-relevant signal. While we have further elucidated that IL-33 is acting directly on donor T cells to augment GVHD, neither the critical place nor timing of IL-33 stimulation of donor T cells during the alloimmune response mediating GVHD have been defined. Methods: We used both IL-33 knockout mice as recipients and ST2 (IL33 receptor) knockout mice as T cell donors in MHC mismatch alloSCT models to define the spatial and temporal importance of IL-33 in GVHD tissue damage. More specifically, to establish the role of IL-33 in T cell effector function in the small intestine during GVHD, irradiated Il33-/- and Il33+/+ C57BL/6 (B6) recipients were given BALB/c bone marrow (BM) and T cells. Additionally, irradiated BALB/c mice were given St2+/+ B6 BM and Cd4 Cre x St2fl/fl or St2-/- and St2+/+ T cells. To determine the role of IL-33 on T cell persistence in GVHD, we used an MHCII-disparate model. Irradiated Il33-/- and Il33+/+ bm12 recipients were reconstituted with B6 BM, with or without CD90.1+ B6 T cells. Results: When donor T cell responses were characterized by flow cytometry at days 7, 14, and 28 post alloSCT, the bulk of these data suggest that IL-33 sustains CD4+ T helper Type 1 (Th1) cells in the barrier tissues at late points following GVHD initiation. Specifically, B6 Il33-/- recipients had fewer BALB/c CD4+Tbet+ cells in the small intestine lamina propria (SI LP; WT 15.9 ± 1.31, KO 9.53 ± 1.32; p=0.03; n=3/group), but not in the spleen (WT 54.8 ± 7.37, KO 57.68 ± 4.90; p=0.75; n=4/group) at day 7. Additionally, we found that when we used therapeutic doses of anti-IL-12p40 to neutralize IL-12 there was no impact on the CD4+Tbet+ cells in the SI of B6 il33-/- recipients (KO control IgG 7.29 ± 0.63, KO anti-IL-12p40 6.61 ± 0.75; p=0.52; n=4/group) suggesting that the decrease in the frequency of CD4+Tbet+ T cells in Il33-/- in the SI is independent of IL-12. In the CD4-dependent B6 to bm12 GVHD model, we find that IL-33-deficient recipients have reduced overall CD4+ donor T cells in the SI LP at both day 14 and day 28 (d14: WT 16.15 ± 2.55, KO 0.35 ± 0.07; p=0.003; n=3/group; d28: WT 33.07 ± 7.17, KO 6.27 ± 1.59; p=0.022; n=3/group). Consistent with our B6 recipients of BALB/c T cells above, this decrease was not observed in the spleen or lymph nodes. Reduced clusters of CD3+ cells were evident in immunohistochemistry of bm12 Il33-/- relative to bm12 Il33+/+ at day 28 post-alloSCT. In corroboration with our IL-33-deficient recipient results, B6 CD4+ T cells lacking the ST2 receptor when co-transferred with ST2 competent T cells were not sustained in the SI LP at day 7 (ST2 WT 18.55 ± 0.77, ST2 KO 6.35 ± 1.42; p=0.0003; n=4/group). ST2 deficient CD4+ donor cells were of equal frequency to the ST2 competent donor cells in the spleen of BALB/c recipients (ST2 WT 54.58 ± 1.914, ST2 KO 50 ± 2.53; p=0.19; n=4/group). Conclusions: Our data reveal that IL-33 is not required for donor Th1 responses in the spleen and LN, but IL-33 is critical for Th1 cells to persist in barrier tissues like the small intestine after alloSCT in the absence of IL-12 stimulation. These data suggest that targeting IL-33 signaling may be an effective therapy to diminish GVHD in target tissues. Disclosures Blazar: Fate Therapeutics Inc.: Research Funding; Childrens' Cancer Research Fund: Research Funding; BlueRock Therapeutics: Research Funding; BlueRock Therapeuetic: Consultancy; Magenta Therapeutics: Consultancy; KidsFirst Fund: Research Funding; Tmunity: Other: Co-founder. Shlomchik:Bluesphere Bio: Consultancy, Other: shareholder.

Plasmacytoid Dendritic Cell-driven Induction of Treg Is Strain Specific and Correlates With Spontaneous Acceptance of Kidney Allografts.

DBA/2J kidney allografts, but not heart allografts, are spontaneously accepted indefinitely in C57BL/6 (B6) mice, through regulatory tolerance mechanism dependent on Foxp3 cells. In contrast, B6 kidneys are rejected within a week in DBA/2J recipients. We hypothesized that the tolerogenic difference of the kidneys might be due to differences in number or function of plasmacytoid dendritic cells (pDCs), because these cells are potent inducers of Foxp3 cells. pDCs from murine bone marrow, native kidneys, and spontaneously accepted kidney allografts were analyzed using flow cytometry and immunohistochemical staining. Naive T cells were cocultured with pDCs in specific strain combinations and analyzed for FoxP3 induction and functionality. MEK/ERK and NFκB inhibitors were used to assess the regulatory T-cell induction pathways. pDCs and T-cell cultures were adoptively transferred before heterotopic heart transplantation to assess allograft survival. DBA/2J pDCs were more potent in inducing Foxp3 in B6 T cells than the reverse combination, correlating with survival of the kidney allografts. Foxp3 induction by pDCs in vitro was dependent on pDC viability, immaturity, and class II MHC mismatch and blocked by MEK/ERK and NFκB inhibition. pDC-induced Foxp3 T cells suppressed proliferation of B6 T cells in vitro, and adoptive transfer into B6 recipients 2 weeks before heterotopic DBA/2J heart transplantation resulted in prolonged allograft survival. These data suggest that pDC-induced regulatory T cells are dependent on downstream signaling effects and on strain-dependent, MHC class II disparity with naive T cells, which may explain organ- and strain-specific differences in spontaneous tolerance.

Obesity-Induced Microbiome Alterations Result in Severe Gastrointestinal Graft-Versus-Host Disease Following Allogeneic Hematopoietic Stem Cell Transplantation

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) remains a viable treatment option for many cancers but its clinical utility is limited due to the occurrence of graft-versus-host disease (GVHD). Understanding the impact of obesity on immune function has become increasingly important in the setting of the current obesity pandemic. We report here that obesity has a negative and selective impact on acute gut GVHD. Diet-induced obese (DIO) mice exhibited increased gut permeability, endotoxin translocation and radiation-induced gastrointestinal damage. After allo-HSCT, DIO recipients across strains and sex had markedly increased pro-inflammatory cytokines (IL-6, TNF), GVHD biomarker ST2, MHC class II expression and exhibited rapid mortality associated with severe acute gut pathology. This obesity-associated lethal acute gut GVHD was dependent on donor CD4 T cells and occurred even in minor MHC mismatch strain combination in which only a delayed skin chronic GVHD resulted in lean recipients. Pro-inflammatory cytokine blockade targeting both IL-6 and TNF ameliorated obesity-associated acute gut GVHD while maintaining graft-versus-tumor (GVT) effects. Microbiome assessment of DIO mice revealed markedly reduced microbiome diversity and decreased Clostridiaceae abundance. Additionally, DIO mice had a significant increase of GVHD-associated Akkermansia muciniphila before and after allo-HSCT compared to the controls. Extended antibiotic treatment of DIO mice protected from the endotoxin translocation, cytokine storm as well as gut GVHD pathology but did not protect later development of chronic skin GVHD. These results demonstrate that obesity alters the microbiome and imparts differential effects on GVHD following allo-HSCT with decreased survival and this inferior outcome can be pre-empted by combined pro-inflammatory cytokine blockade or antibiotic pretreatment. Disclosures Pai: Roche-Genentech: Employment. Holtan:CSL Behring: Consultancy; Incyte: Consultancy; Bristol-Myers Squibb: Consultancy; Janssen: Consultancy. Ferrara:ViraCor: Consultancy; Incyte: Consultancy; Kamada: Consultancy; Mallinckrodt: Consultancy; Enlivex: Consultancy; Xenikos: Consultancy; CSL Behring: Consultancy; National Institutes of Health: Research Funding. Levine:Novartis: Honoraria; Kamada: Research Funding; bluebird bio: Consultancy; National Cancer Institute: Research Funding; Incyte: Consultancy, Research Funding; Ironwood: Honoraria; Biogen: Other: non-financial support; Viracor: Patents &amp; Royalties: biomarker patent. Abedi:Abbie: Speakers Bureau; Gilead: Speakers Bureau; Celgene: Speakers Bureau; BMS: Speakers Bureau; Takeda: Speakers Bureau. Blazar:Kamon Pharmaceuticals, Inc: Membership on an entity's Board of Directors or advisory committees; Five Prime Therapeutics Inc: Co-Founder, Membership on an entity's Board of Directors or advisory committees; Regeneron Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Magenta Therapeutics and BlueRock Therapeuetics: Membership on an entity's Board of Directors or advisory committees; Fate Therapeutics, Inc.: Research Funding; RXi Pharmaceuticals: Research Funding; Alpine Immune Sciences, Inc.: Research Funding; Abbvie Inc: Research Funding; Leukemia and Lymphoma Society: Research Funding; Childrens' Cancer Research Fund: Research Funding; KidsFirst Fund: Research Funding; Tmunity: Other: Co-Founder; BlueRock Therapeutics: Membership on an entity's Board of Directors or advisory committees.

Autoantibody production significantly decreased with APRIL/BLyS blockade in murine chronic rejection kidney transplant model.

Chronic antibody mediated rejection (cAMR) remains a significant barrier to achieving long-term graft survival in kidney transplantation, which results from alloantibody production from B lymphocytes and plasma cells. APRIL (A proliferation-inducing ligand) and BLyS (B lymphocyte stimulator) are critical survival factors for B lymphocytes and plasma cells. Here we describe the results of APRIL/BLyS blockade in a murine cAMR kidney transplant model. c57/B6 mice underwent kidney transplantation with Bm12 kidneys (minor MHC mismatch), a well-described model for chronic rejection where animals cannot make donor specific antibody but rather make antinuclear antibody (ANA). Following transplantation, animals received TACI-Ig (to block APRIL and BLyS) or no treatment. Animals were continued on treatment until harvest 4 weeks following transplant. Serum was analyzed for circulating anti-nuclear autoantibodies using HEp-2 indirect immunofluorescence. Spleen and transplanted kidneys were analyzed via H&E. ANA production was significantly decreased in APRIL/BLyS blockade treated animals (p<0.0001). No significant difference in autoantibody production was found between syngeneic transplant control (B6 to B6) and APRIL/BLyS blockade treated animals (p = 0.90). Additionally, disruption of splenic germinal center architecture was noted in the APRIL/BLyS blockade treated animals. Despite the significant decrease in autoantibody production and germinal center disruption, no significant difference in lymphocyte infiltration was noted in the transplanted kidney. APRIL/BLyS blockade resulted in a significant decrease of autoantibody production and disrupted splenic germinal center formation in a chronic kidney transplant model, however in this model no difference in kidney transplant pathology was seen, which may have to do with the absence of any T cell centric immunosuppression. Regardless, these findings suggest that APRIL/BLyS blockade may play a role in decreasing antibody formation long-term in kidney transplantation. Future investigations will use APRIL/BLyS blockade in conjunction with T lymphocyte depleting agents to determine its efficacy in chronic rejection.

ST2 and ROR-γ-t Roles in Intestinal Regulatory T Cells

Allogeneic hematopoietic cell transplantation (allo-HCT) is an essential therapeutic modality for patients with hematological malignancies and benign blood disorders. Unfortunately, acute graft-versus-host disease (aGVHD) remains the major complication of allo-HCT and is associated with high mortality. Soluble STimulation-2 (sST2) is increased during aGVHD while regulatory T cells (Tregs) that express membrane-bound ST2 prevent GVHD through unknown mechanisms. Herein, we studied nonlymphoid tissue ST2+ Tregs in murine models of allo-HCT. Transplantation of Foxp3- T cells and Tregs sorted from different Foxp3 reporter mice indicated that ST2+Tregs isolated from aGVHD mice were of donor origin, thymus-derived, and predominantly intestine localized (data not shown). To further understand the mechanism, we performed a transcriptome analysis of sorted Foxp3GFP Tregs from aGVHD mice receiving wild type (WT) CD25-depleted total T cells with either WT or ST2-/- Foxp3GFP Tregs in both major (B6, H-2bàBALB/c, H-2d, Mhc) and minor (B6, H-2bàC3H.SW, H-2b, miH) mismatched aGVHD models. ST2-/- Foxp3GFP Tregs showed decreased expression of key molecules needed for Treg function such as Foxp3, Il2ra, Icos, Il27, Ctla4, and Ahr. Strikingly, transcription of RAR-related-orphan-receptor-c (Rorc), essential for proinflammatory IL-17+ T cells development, was the most upregulated transcript in ST2-/- Foxp3GFP Treg isolated from the gut in both models (Figure 1A). The transcriptome finding led us to investigate the frequency and function of ST2+Tregs in the gut of naive mice deficient for Rorc. Isolation and staining of intestinal T cells showed a significant increase of ST2+Treg frequency in the gut of Rorc-/- mice compared to WT mice: 55% vs. 12%, respectively (Figure 1B). Rorc-/- T cells transplantation enhanced the day 10 post-HCT frequency of intestinal ST2+Tregs (Figure 1C, left) that co-expressed TIGIT and Helios expression (data not shown), and decreased aGVHD score and mortality (Figure 1C, right). Previous studies have shown that IL-33 signaling in Tregs elicits their amphiregulin expression and increases their ability to suppress effector T cells (Teffs). IL-23, a key proinflammatory cytokine in the maintenance and amplification of Th17/Tc17, can restrain Treg responses by inhibiting IL-33 responsiveness. Using ex vivo polyclonal Tregs from donor B6 cultured with IL-2 (control Tregs) vs IL-2+IL-33 (TregIL-33) vs IL-2+IL-33+IL-23+IL-17, we found that adding IL-33 doubled the frequencies of ST2+ Tregs compared to control Tregs, and adding IL-23+IL-17 reversed this activity (Figure 2A). Markers of Tregs activation (KLRG1) (Figure 2B) and function (Helios, LAG3) (data not shown) were augmented in TregIL-33 compared to other Treg conditions. TregIL-33 also expressed more amphiregulin than Tregs cultured in the two other conditions (Figure 2C). Next, we evaluated their function in vitro and in vivo. TregIL-33 displayed better immunosuppression than other Tregs on CD4+ T cells at a physiological Tregs:Teffs ratio of 1:12; differences were not seen at higher ratios or on suppression of CD8+ T cells (Figure 3A). Then we examined the in vivo capacity of donor TregIL-33 to protect from aGVHD intestinal damage by co-adoptive transfer of Tregs cultured in the three aforementioned conditions with CD25-depleted WT T cells in Mhc and miH mismatch aGVHD models. We used a physiological ratio of 1 donor Treg per 20 donor Teffs (5% of total T cells). Mice receiving TregIL-33 showed significantly less severe aGVHD and better survival compared to those cultured either without IL-33 or with IL-33 and IL23/IL17 in both Mhc (Figure 3B) and miH (data not shown) mismatched aGVHD models. Analysis of intestinal T cells of these mice showed decreased total lymphocytes infiltration in the gut at day 10 after HCT, and decreased intestinal Tbet and RORγt expressing Teffs in both Mhc (Figure 3C) and miH (not shown) mismatched aGVHD models. Altogether, we conclude that within GVHD target organs, ST2+Tregs are predominantly intestine localized, and inversely correlated with RORgt in steady state and inflammatory conditions. Triggering the ST2/IL-33 pathway in Tregs prevents severe aGVHD, possibly through a dual mechanism: upregulation of amphiregulin and immunosuppression on effector T cells. Thus, TregIL-33 is a potential cellular therapy avenue for preventing or treating aGVHD. DisclosuresBlazar:Kadmon Corporation, LLC: Consultancy, Research Funding. Paczesny:Viracor IBT Laboratories: Patents & Royalties.

Obesity predisposes to rapid gastrointestinal graft-versus-host disease lethality after allogeneic hematopoietic stem cell transplantation in mice

Abstract Obesity is a form of chronic inflammation, termed “meta-inflammation”, resulting in the development of many chronic diseases such as diabetes and cancer. In this study, we assessed the impact of obesity on recipient graft-versus-host disease (GVHD) outcome post allogeneic HSCT using mouse models. In a model of sclerodermatous chronic GVHD, lean or diet-induced obese (DIO) BALB/c mice received 800 cGy total body irradiation (TBI), bone marrow cells, and splenocytes from H2-identical but minor MHC-mismatched B10.D2 mice. To mimic major MHC mismatch transplant which results in acute GVHD impacting gut, skin and liver, lean or DIO C57BL/6 mice received 1050 cGy TBI, bone marrow cells, and different doses of purified T cells from BALB/c mice. Strikingly, after the minor mismatch HSCT, all DIO mice succumbed to gut pathology while all lean mice survived and developed sclerodermatous GVHD at week 3 post transplant. In both models, pathological assessment indicated marked gut pathology in DIO mice correlating with lethality. Lean mice in the major MHC mismatch strain combination had significantly increased survival and less severe gut pathology compared to the DIO mice. The DIO mice had higher expression of pro-inflammatory cytokines in the gut, liver, visceral fat, and serum post-HSCT. Flow cytometric analysis demonstrated increased numbers of donor CD8+ T cells in the gut of DIO mice compared to lean mice. In an initial evaluation, untreated DIO mice displayed increased gut permeability as well as having limited diversity in their microbiome which would contribute to severe GVHD post-HSCT. Taken together, these results indicate that obesity exacerbates GVHD after allogeneic HSCT primarily affecting the gastrointestinal tract.

Reprogramming MHC specificity by CRISPR-Cas9-assisted cassette exchange

The development of programmable nucleases has enabled the application of new genome engineering strategies for cellular immunotherapy. While targeted nucleases have mostly been used to knock-out or knock-in genes in immune cells, the scarless exchange of entire immunogenomic alleles would be of great interest. In particular, reprogramming the polymorphic MHC locus could enable the creation of matched donors for allogeneic cellular transplantation. Here we show a proof-of-concept for reprogramming MHC-specificity by performing CRISPR-Cas9-assisted cassette exchange. Using murine antigen presenting cell lines (RAW264.7 macrophages), we demonstrate that the generation of Cas9-induced double-stranded breaks flanking the native MHC-I H2-Kd locus led to exchange of an orthogonal H2-Kb allele. MHC surface expression allowed for easy selection of reprogrammed cells by flow cytometry, thus obviating the need for additional selection markers. MHC-reprogrammed cells were fully functional as they could present H2-Kd-restricted peptide and activate cognate T cells. Finally, we investigated the role of various donor template formats on exchange efficiency, discovering that templates that underwent in situ linearization resulted in the highest MHC-reprogramming efficiency. These findings highlight a potential new approach for the correcting of MHC mismatches in cellular transplantation.

Abstract 44: Tolerance Induction with Post Transplantation High-Dose Cyclophosphamide relies on Recipient-derived Immune Regulation and Donor Bone Marrow in a Murine Model of Vascularized Composite Allotransplantation

PURPOSE: Developing novel treatment concepts to minimize/avoid immunosuppression by induction of immune tolerance represents the prime task in the field of transplantation. In the clinic to date, this was achieved in highly selected patients in clinical trials of kidney transplantation combined with concomitant stem cell or bone marrow transplantation. Vascularized composite allotransplantation (VCA) is evolving as viable treatment modality for patients suffering from functional and esthetic defects to craniofacial structures, extremities and urogenital tissues. In certain VCAs transplantation of vascularized bone marrow may be inherently part of the allograft designs (i.e. hands and faces) and thereby representing a unique opportunity in combining bone marrow and VCA. METHODS: Murine skin, heart, and hind limb (VCA) transplants were performed across a full MHC mismatch barrier. Recipients treatment comprised non-myeloablative TBI and T-cell depletion and a single dose of post-transplant cyclophosphamide (PTCy). Donor BM and splenocytes (DBM) were injected at the time of transplantation. Post-transplant multi-lineage and, Foxp3 chimerism as well as Vβ-TCR staining was performed. Donor-specific unresponsiveness was tested by MLR and by 2° skin and solid organ transplantation (SOT). Mechanistic studies were undertaken using transgenic mice (DEREG and Foxp3-DTR) to investigate the role of regulatory T cells (Tregs). VCA was performed in animals thymectomized prior to receiving PTCy and the transplant to highlight the role of the thymus in induction and maintenance of tolerance in this murine model. RESULTS: Untreated animals rejected skin grafts, SOT and VCA acutely within 14 ± 1 days, 9 ± 2 days, and 8 ± 1 days, respectively. The treatment regimen extended skin and SOT graft survival (32 ± 8; 65 ± 4, respectively). Additional DBM augmentation lead to allograft survival of >150 days in skin and SOT. However, indefinite graft survival of >150 days was observed in all animals receiving the induction regimen and a VCA ± DBM. In groups receiving a VCA ± DBM, donor chimerism was detected at 22.51% ± 5.96% and 30.17% ± 8.72%, respectively. Prior or post transplantation depletion of recipient-derived Tregs in VCA recipients did not lead to abrogation of tolerance. In recipients of skin graft + DBM, chimerism was not achieved after recipient-derived Treg depletion prior to transplantation (POD-2) and consequently the allografts were rejected (MST = 23.5 days, N=4). In-vitro, Vβ-T cell receptor staining indicates clonal deletion as an additional central tolerance mechanism. However, transplantation into a thymectomized host did not abrogate long-term allograft survival (N=6). All long-term survivors showed donor-specific T cell unresponsiveness in-vitro (MLR) while demonstrated proliferation against 3rd party stimulators. In-vivo, tolerant animals accepted donor-matched secondary skin, while 3rd party FVB/N skin was acutely rejected. Donor-matched hearts were accepted long-term. CONCLUSION: Taken together, robust tolerance and immunosuppression-free long-term allograft survival can be achieved with PTCy in stringent fully MHC mismatched murine models of skin, heart, and vascularized composite allotransplantation. Stable multi lineage chimerism and recipient derived regulatory T cells play a critical role in maintaining tolerance in this model, in particular in the peritransplant time period.