Donor Antigen-presenting Cells Research Articles

Depletion of donor dendritic cells ameliorates immunogenicity of both skin and hind limb transplants.

Acute cellular rejection remains a significant obstacle affecting successful outcomes of organ transplantation including vascularized composite tissue allografts (VCA). Donor antigen presenting cells (APCs), particularly dendritic cells (DCs), orchestrate early alloimmune responses by activating recipient effector T cells. Employing a targeted approach, we investigated the impact of donor-derived conventional DCs (cDCs) and APCs on the immunogenicity of skin and skin-containing VCA grafts, using mouse models of skin and hind limb transplantation. By post-transplantation day 6, skin grafts demonstrated severe rejections, characterized by predominance of recipient CD4 T cells. In contrast, hind limb grafts showed moderate rejection, primarily infiltrated by CD8 T cells. Notably, the skin component exhibited heightened immunogenicity when compared to the entire VCA, evidenced by increased frequencies of pan (CD11b-CD11c+), mature (CD11b-CD11c+MHCII+) and active (CD11b-CD11c+CD40+) DCs and cDC2 subset (CD11b+CD11c+ MHCII+) in the lymphoid tissues and the blood of skin transplant recipients. While donor depletion of cDC and APC reduced frequencies, maturation and activation of DCs in all analyzed tissues of skin transplant recipients, reduction in DC activities was only observed in the spleen of hind limb recipients. Donor cDC and APC depletion did not impact all lymphocyte compartments but significantly affected CD8 T cells and activated CD4 T in lymph nodes of skin recipients. Moreover, both donor APC and cDC depletion attenuated the Th17 immune response, evident by significantly reduced Th17 (CD4+IL-17+) cells in the spleen of skin recipients and reduced levels of IL-17E and lymphotoxin-α in the serum samples of both skin and hind limb recipients. In conclusion, our findings underscore the highly immunogenic nature of skin component in VCA. The depletion of donor APCs and cDCs mitigates the immunogenicity of skin grafts while exerting minimal impact on VCA.

Pretransplant, Th17 dominant alloreactivity in highly sensitized kidney transplant candidates

IntroductionSensitization to donor human leukocyte antigen (HLA) molecules prior to transplantation is a significant risk factor for delayed access to transplantation and to long-term outcomes. Memory T cells and their cytokines play a pivotal role in shaping immune responses, thereby increasing the risk of allograft rejection among highly sensitized patients. This study aims to elucidate the precise contribution of different CD4+ memory T cell subsets to alloreactivity in highly sensitized (HS) kidney transplant recipients.Methods and resultsStimulation of peripheral blood mononuclear cells (PBMC) with various polyclonal stimulating agents to assess non-specific immune responses revealed that HS patients exhibit elevated immune reactivity even before kidney transplantation, compared to non-sensitized (NS) patients. HS patients' PBMC displayed higher frequencies of CD4+ T cells expressing IFNγ, IL4, IL6, IL17A, and TNFα and secreted relatively higher levels of IL17A and IL21 upon stimulation with PMA/ionomycin. Additionally, PBMC from HS patients stimulated with T cell stimulating agent phytohemagglutinin (PHA) exhibited elevated expression levels of IFNγ, IL4 and, IL21. On the other hand, stimulation with a combination of resiquimod (R848) and IL2 for the activation of memory B cells demonstrated higher expression of IL17A, TNFα and IL21, as determined by quantitative real-time PCR. A mixed leukocyte reaction (MLR) assay, employing third-party donor antigen presenting cells (APCs), was implemented to evaluate the direct alloreactive response. HS patients demonstrated notably higher frequencies of CD4+ T cells expressing IL4, IL6 and IL17A. Interestingly, APCs expressing recall HLA antigens triggered a stronger Th17 response compared to APCs lacking recall HLA antigens in sensitized patients. Furthermore, donor APCs induced higher activation of effector memory T cells in HS patients as compared to NS patients.ConclusionThese results provide an assessment of pretransplant alloreactive T cell subsets in highly sensitized patients and emphasize the significance of Th17 cells in alloimmune responses. These findings hold promise for the development of treatment strategies tailored to sensitized kidney transplant recipients, with potential clinical implications.

Polyfunctional donor-reactive T cells are associated with acute T-cell mediated rejection of the kidney transplant.

Acute T-cell mediated rejection (aTCMR) still remains a clinical problem after kidney transplantation despite significant improvements in immunosuppressive regimens. Polyfunctional T-cells, i.e. T-cells producing multiple pro-inflammatory cytokines, are believed to be the most relevant T-cells in an immune response. The aim of this study was to determine whether polyfunctional donor-reactive T-cells are associated with aTCMR. In a case-control study, 49 kidney transplant recipients with a biopsy-proven aTCMR in the first year after transplantation were included, as well as 51 controls without aTCMR. Circulating donor-reactive T-cells were identified by the expression of CD137 after short-term co-culture with donor antigen-presenting cells. Polyfunctional donor-reactive T-cells were further characterized by dissection into different T-cell subsets encompassing the spectrum of naïve to terminally-differentiated effector T-cells. Prior to kidney transplantation, proportions of donor-reactive CD4+ (0.03% versus 0.02%; P<0.01) and CD8+ (0.18% versus 0.10%; P<0.01) CD137++ T-cells were significantly higher in recipients with a biopsy-proven aTCMR versus non-rejectors. Polyfunctionality was higher (P=0.03) in this subset of CD137-expressing T-cells. These cells were predominantly of the EM/EMRA-phenotype, with polyfunctional donor-reactive CD137++CD4+ T-cells predominantly co-expressing CD28 whereas approximately half of the polyfunctional CD137++CD8+ T-cells co-expressed CD28. In addition, at the time of aTCMR, polyfunctional donor-reactive CD137++ CD4+, but not CD8+, T-cells, were specifically decreased by 75% compared to before transplantation in recipients with as well as those without an aTCMR. Prior to transplantation, the proportion of polyfunctional donor-reactive CD137++ T-cells is associated with the occurrence of a biopsy-proven aTCMR within the first year after transplantation.

Anesthetic concerns in recipients of hematopoietic stem cell transplant.

Dear Editor, Since the advent of bone marrow transplant in 1957, our understanding of the biology behind Hematopoietic Stem Cell Transplant (HSCT) has improved[1] and almost 50,000 HSCT take place globally in a year.[2] Although anesthetic concerns of patients undergoing solid organ transplantation have been addressed, there is a dearth of literature relating to HSCT, especially after the recent advancements like targeted therapy.[3,4] HSCT entails the regeneration of different blood cell lineages from donor stem cells and embarks on the interplay between recipient T lymphocytes, natural killer (NK) cells and donor antigen presenting cells (APC) and lymphocytes. Allogenic transplant brings forth graft vs. tumor effect (GVT) which helps to prevent recurrences but is shadowed by the graft vs. host disease (GVHD), necessitating chronic immunosuppression. Post HSCT patients are at increased risk of opportunistic infections, especially before engraftment and subtle signs of infections may be missed in the preoperative period as well as there may be delayed wound healing after surgery. While exaggerated increase in host immunity may enhance GVHD, overt diminution by immunosuppressant may lead to secondary infections and reactivation of cytomegalovirus. Therefore, it is prudent to delay elective surgeries before engraftment and while the patient is on immune-modulators.[5] Moreover, anesthesiologist should be aware of long-term adverse effects of chemotherapeutic drugs including potential difficult airway due to gum hypertrophy (cyclosporine) and lymph node enlargement as part of post-transplant lymphoproliferative disorder (PTLD). Acute GVHD may involve different organ systems, leading to potentially dreaded complications like acute lung injury, veno-occlusive disease of liver, thrombotic micro angiopathy (TMA) which should be evaluated during the pre-anesthetic check-up.[5] Surgical stress leads to a temporary state of immunosuppression via various mechanisms like raised catecholamines, angiogenic factors, matrix metalloproteinases (MMPs) and attenuated NK cell activity and cell mediated immunity (CMI). Volatile agents, especially isoflurane may promote tumor growth and spread by reducing NK cell activity, T cell depletion, increased angiogenesis by up-regulation of hypoxia inducible factor (HIF-1 alpha), whereas N2O causes bone marrow suppression. However, sevoflurane was found to stabilize the anti-cancer gene P38. Similar to inhaled agents, ketamine and thiopentone induces T cell apoptosis and NK cell underactivity. Propofol appears to be beneficial by reducing angiogenesis and maintaining T cell activity. Despite report showing impairment of macrophage-monocyte function, midazolam appears to be safer. Action of muscle relaxants may get prolonged due to immunomodulators like cyclosporine, as seen in renal transplant recipients.[6] Although anecdotal reports showed that IV opioids reduced NK cell, T cell and TLR (Toll like Receptor) activity on macrophage, it might enhance tumor lysis by stabilizing P53 and P38 and induce tumor death via NF-kB (nuclear factor kappa beta) and Fas-FasL pathway. Thus, immunomodulating effects of opioids are dose and concentration dependent and judicial perioperative analgesia by opioids seems logical.[7] Whenever feasible, use of regional anesthesia must be emphasized as local anesthetics increase local NK cell activity. Propofol along with paravertebral block have been found to be associated with reducing tumor burden in ER-negative breast cancer. Specific concerns of immune-modulators are provided in Table 1 and perioperative concerns are summarized in Table 2.Table 1: Side effects of commonly used agents and their concernsTable 2: Perioperative concerns in a nutshellInduction by propofol/etomidate, followed by maintenance with total intravenous anesthesia (TIVA) with propofol, intermediate acting muscle relaxants with minimal organ dependent metabolism (e.g., atrcurium, cisatracurium) and use of regional anesthesia may be considered as preferred techniques of choice. With the growing number of HSCTs, the authors feel the need for renewed interest and good quality research in this area. Financial support and sponsorship Nil. Conflicts of interest There are no conflicts of interest.

Interaction of immune cells and lymphatic vessels in corneal transplantation

AbstractImmune‐mediated graft rejections remain the most common cause for graft failure after organ and tissue transplantation. There is a great unmet medical need for pharmacologic strategies to promote graft survival without unduly compromising the health of the recipient.Corneal allograft rejection is histologically characterized by massive infiltration of CD4+ T‐cells, macrophages and the invasion of blood and lymphatic vessels.We could show that lymphatic vessels determine the high‐risk status of a corneal graft recipient. The healthy cornea is devoid of blood and lymphatic vessels. In contrast, in the high risk situation graft rejection is highly correlated with the degree of corneal vascularisation and presence of numerous pro‐inflammatory immune cells.CD11c+ CD11b+ myeloid dendritic cells (DCs) are present throughout the anterior stroma – even in the healthy cornea. Inflammatory stimuli determine the maturation stage of DCs and their immunogenic properties. It is known that an increasing number of MHC class II‐bearing, antigen specific DCs in cervical draining lymph nodes arise only a few hours after transplantation. This implies that DCs at different maturation stages might be the main initiators of this devastating tissue destruction.The origin of donor antigen presenting cells can be either from the donor tissue or the host. Thereby allo‐antigens from the grafted tissues travel via lymphatic vessels towards the draining lymph nodes of the recipient. Here they are recognized by host T cells either by the direct pathway, which involves the recognition of intact donor MHC molecules on donor‐derived APCs, or by the indirect pathway, which involves the recognition of donor major or minor MHC‐derived peptides, processed and presented by recipient APCs to host T cells. Nowadays it is widely accepted that resident MHC class II APC populations are present in the cornea. These cells are capable of expressing MHC class II antigen and accessory molecules (CD40, CD80) after transplantation. We and others could demonstrate that the immune reaction is triggered by the above described “direct allo‐sensitisation” where APCs travel from the donor tissue via corneal pathological lymphatic vessels into the local draining lymph nodes of the recipient and present the foreign antigen.Recent studies could show that lymphatic vessels not just display an exit route for leukocytes but are in close interaction with these cells and regulate the entry and trafficking of antigen presenting cells into and via lymphatic vessels.This presentation will summarize the current knowledge about the interaction of immune cells and lymphatic vessels in corneal transplantation.

Indirectly Recognizable Epitopes Derived from Recipient Mismatched HLA-B Are Associated with Adverse Prognosis in Single-Unit Cord Blood Transplantation

Backgrounds Despite cord blood is an important alternative stem cell source for the patients without HLA matched donors, engraftment failure is the most significant obstacle in cord blood transplantation (CBT). Donor-specific anti-HLA antibody and hemophagocytic syndrome (HPS) after severe pre-engraftment immune reaction are the major causes of engraftment failure. However, the allo-immunity associated with graft rejection has not been fully understood. In HLA-mismatched transplantation, mismatched HLA is an important target for allo-immunity but the immunogenicity is different depending on the mismatched HLA pair of donor and recipient. Therefore, we hypothesized that by evaluating the antigenicity of mismatched HLA, we could improve donor selection criteria for CBT. Methods: We performed a retrospective analysis of clinical data on 4,100 single-unit CBT in Japan between 2001 and 2017 deposited in Transplant Registry Unified Management Program (TRUMP). Using the PIRCHE algorithm (PIRCHE AG, Berlin), the number of mismatched HLA-derived epitopes to be presented by HLA was predicted. PIRCHE was quantified separately for the mismatched recipient loci (HLA-A, -B, -C, -DRB1) and the presenting donor-HLA (ClassI or ClassII, matched or mismatched HLA). Specifically, we evaluated binding affinities of peptides that could be made from recipient mismatched HLA to donor HLA using netMHCpan and netMHCIIpan. Epitopes with IC50 < 500 nM for Class I and < 1000 nM for Class II were defined as PIRCHE-I and PIRCHE-II, respectively. Results: The death-censored median observation period was 2.7 years (0.5-17.1), median age at transplantation was 53 years (16-75), and 96.4% of patients were infused with HLA-mismatched cord blood. The present cohort includes patients with AML (49.6%), ALL (14.0%), Lymphoma (12.9%), MDS (10.8%), CML (2.2%), and other diseases (10.5%). Full-intensity conditioning was performed in 50.4% of patients. All patients received calcineurin inhibitors (CNI) for graft-versus-host disease (GvHD) prophylaxis, 1410 (34.5%) of them received MMF and 2110 (51.5%) received MTX in combination with CNI. The number of PIRCHEs derived from each HLA-loci showed zero-inflated distributions. Patients with PIRCHE-I derived from recipient mismatched HLA-B (rB-PIRCHE-I) had a higher non-relapse mortality (NRM, Hazard ratio [HR] = 1.19, p = 0.004) and lower progression free survival (PFS, HR = 1.12, p = 0.002). They tended to have higher risk for engraftment failure and infection was leading cause of death. rB-PIRCHE-I was not associated with incidence of acute and chronic GvHD. Interestingly, rB-PIRCHE-I on matched HLA (rB-PIRCHE-I on match) was significantly associated with prognosis (PFS, HR = 1.11, p = 0.005), but those presented on mismatched HLA were not (PFS, HR = 1.07, p = 0.1). The numbers of infused nucleated cells and CD34+ cells in the graft were higher in the patients with rB-PIRCHE-I on match, but there was no difference in other patient backgrounds with or without rB-PIRCHE-I on match. In patients transplanted with HLA-B mismatched cord blood, rB-PIRCHE-I on match was prognostic. In patients who received MMF, rB-PIRCHE-I on match was associated with engraftment failure (Figure 1) and poor prognosis (NRM, HR = 1.48, p < 0.001; PFS, HR = 1.26, p < 0.001), whereas there was no prognostic impact in the patients with MTX. In order to elucidate the mechanism by which rB-PIRCHE-I increases engraftment failure in patients with MMF, we performed a detailed analysis of 100 patients who underwent CBT with MMF in the cohort of Kyushu University Hospital. As a result, we found that the frequency of HPS was significantly higher in patients with rB-PIRCHE-I on match (HR = 5.75, p = 0.001) Discussion: In the early period after CBT, donor antigen-presenting cells (APCs) are not reconstituted and donor T-cell is restricted to donor HLA. Therefore, we speculate that recipient APCs are inducing severe allo-immune reaction by presenting rB-PIRCHE-I on matched HLAs which are recognizable to donor T-cells. Furthermore, NK cells in the graft have been reported to prevent GvHD via suppression of recipient APCs, and MMF has the ability to suppress NK cells; it is possible that MMF enhanced the allo-immune response induced by recipient APCs. (Figure 2) Conclusion: HLA-mismatched cord blood without rB-PIRCHE-I is more desirable as a donor; when selecting a donor with rB-PIRCHE-I, it may be better to use MTX instead of MMF. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Donor extracellular vesicle trafficking via the pleural space represents a novel pathway for allorecognition after lung transplantation

Restoration of lymphatic drainage across the bronchial anastomosis after lung transplantation requires several weeks. As donor antigen and antigen presenting cell trafficking via lymphatics into graft-draining lymph nodes is an important component of the alloresponse, alternative pathways must exist that account for rapid rejection after pulmonary transplantation. Here, we describe a novel allorecognition pathway mediated through donor extracellular vesicle (EV) trafficking to mediastinal lymph nodes via the pleural space. Pleural fluid collected early after lung transplantation in rats and humans contains donor-specific EVs. In a fully MHC mismatched rat model of lung transplantation, we demonstrate EVs carrying donor antigen preferentially accumulate in mediastinal lymph nodes and colocalize with MHC II expressing cells within 4h of engraftment. Injection of allogeneic EVs into pleural space of syngeneic lung transplant recipients confirmed their selective trafficking to mediastinal lymph nodes and resulted in activation of T cells in mediastinal, but not peripheral lymph nodes. Thus, we have uncovered an alternative pathway of donor antigen trafficking where pulmonary EVs released into the pleural space traffic to locoregional lymph nodes via pleural lymphatics. This pathway obviates the need for restoration of lymphatics across the bronchial anastomosis for trafficking of donor antigen to draining lymph nodes.

184-OR: Antigen-Presenting Cells Can Generate Hybrid Peptides Identified in Type 1 Diabetes

Type 1 Diabetes (T1D) is an autoimmune disease characterized by selective loss of pancreatic β-cells. Aberrant reactivity to insulin and insulin precursor peptides has long been considered important in the pathogenesis of T1D. Recently, T cells recognizing hybrid insulin peptides (HIPs) have been described, first in a rodent model and later in human T1D. A HIP is a CD4 T-cell epitope that is formed by the post-translational fusion of two peptide fragments with at least one of the peptide fragments derived from insulin or a precursor. HIP formation can occur intra- or intermolecularly. The role of these HIPs in the pathogenesis of T1D remains unclear. Others have proposed that HIPs are formed in β-cells during crinophagy of secretory granules. As ultrastructural studies have demonstrated that β-cells can transfer secretory vesicles to local antigen presenting cells (APCs) , we hypothesized that APCs could similarly generate HIPs via protease mediated transpeptidation. To test this, we manufactured peptide-containing nanocarriers mimicking β-cell secretory vesicles to transfer selected peptides to APCs in both mouse and human in vitro systems utilizing donor APCs and CD4 TCR transgenic T cells or a CD4 transductant reporter cells respectively. Here, we demonstrate the subsets of APCs which are capable of intramolecular and intermolecular HIP formation. We further demonstrate that this formation can be blocked by proteosome inhibition. Our study identifies a novel mechanism for the generation of HIPs recognized in T1D. Disclosure S.A.Khokhar: None. M.Liu: None. J.L.Gaglia: Advisory Panel; Dompé, Regeneron Pharmaceuticals Inc., Consultant; Vertex Pharmaceuticals Incorporated, Research Support; Avotres Inc., Dompé, Janssen Research &amp; Development, LLC, Provention Bio, Inc., Stock/Shareholder; Vertex Pharmaceuticals Incorporated. Funding National Institutes of Health 5P30DK036836JDRF 5-ECR-2016-186-A-N

Targeting fatty acid β-oxidation impairs monocyte differentiation and prolongs heart allograft survival.

Monocytes play an important role in the regulation of alloimmune responses after heart transplantation (HTx). Recent studies have highlighted the importance of immunometabolism in the differentiation and function of myeloid cells. While the importance of glucose metabolism in monocyte differentiation and function has been reported, a role for fatty acid β-oxidation (FAO) has not been explored. Heterotopic HTx was performed using hearts from BALB/c donor mice implanted into C57BL/6 recipient mice and treated with etomoxir (eto), an irreversible inhibitor of carnitine palmitoyltransferase 1 (Cpt1), a rate-limiting step of FAO, or vehicle control. FAO inhibition prolonged HTx survival, reduced early T cell infiltration/activation, and reduced DC and macrophage infiltration to heart allografts of eto-treated recipients. ELISPOT demonstrated that splenocytes from eto-treated HTx recipients were less reactive to activated donor antigen-presenting cells. FAO inhibition reduced monocyte-to-DC and monocyte-to-macrophage differentiation in vitro and in vivo. FAO inhibition did not alter the survival of heart allografts when transplanted into Ccr2-deficient recipients, suggesting that the effects of FAO inhibition were dependent on monocyte mobilization. Finally, we confirmed the importance of FAO on monocyte differentiation in vivo using conditional deletion of Cpt1a. Our findings demonstrate that targeting FAO attenuates alloimmunity after HTx, in part through impairing monocyte differentiation.

Prediction of Graft-Versus-Host Disease in Recipients of Single Mismatched Unrelated Hematopoietic Cell Transplantation Donor Using a Highly Multiplexed Proteomic Assay, MHC-Pepseq

Graft-versus-host disease (GVHD) remains a major cause of treatment failure after allogeneic hematopoietic cell transplantation (alloHCT). In HLA-mismatched donor setting, indirect presentation of allogeneic peptides from recipient's mismatched HLA class I or II proteins by donor or recipient antigen presenting cells can be an immunogenic driver of GVHD. However, the potential diversity of such antigens is large, and predicting them in a systematic manner has proven challenging. Using a novel, highly-multiplexed peptide-MHC binding assay (MHC-PepSeq) we sought to 1) identify allogeneic peptides derived from mismatched HLA protein that can be efficiently presented by HLA-DR, and 2) explore the possibility that the frequency of these HLA-DRB1 binding allopeptides may be predictive of clinical GVHD in HLA-DPB1 mismatched donor/recipient pairs.Using publicly-available population allele frequency data (allelefrequencies.net), we identified a set of class I and II sequences that cover >95% of alleles at each of 9 human HLA-loci (-A, -B, -C, -DRA1,-DRB1, -DQA1, -DQB1, -DPA1, -DPB1) in 3 major US populations (European Caucasian, African American, Mexican Chicano). When represented in the form of densely overlapping tiled 15-mer peptides, 7,744 unique 15mers were identified. We encoded these peptides into DNA oligonucleotides and used the PepSeq parallel synthesis protocol to generate a library of the corresponding DNA-barcoded peptides. The library was incubated with recombinantly-expressed full-length HLA proteins, washed, eluted, amplified, and sequenced to identify the various HLA-derived peptides that bind to the assayed HLA proteins (Figure 1). In the current study, DPB1-derived allopeptides in the setting of HLA-A, B, C, DRB1, and DQB1 (10/10) matched unrelated (MUD) HCT donors with a mismatch in DPB1 were investigated. The peptide library was assayed for binding to the DRB1*07:01 protein, selected since it was the common allele in this cohort.We identified 327 patients who were transplanted at our center and met these criteria. For each case, we used comprehensive in silico tiling to identify HLA-DPA and DPB-derived peptides present in the recipient but absent in the donor. This set was intersected with the peptides identified as binders to HLA-DRB1*07:01 in the 7,744-plex MHC-PepSeq assay, to arrive at a donor-recipient pair-specific set of ‘allopeptides’ Overall, we identified such allopeptide at the median of 0 (range: 0-8) across the 327 cases.Next, we examined an association between the number of allopeptides and acute GVHD in the cohort of 94 patients with positive HLA-DRB1*07:01. Median age at alloHCT was 60 years (range: 19-78), 53% males, 1.% bone marrow graft and only 7% female to male donors. Ablative (TBI) conditioning was delivered to 34%) pts. 83% received Tacrolimus/Sirolimus-based, and 9% received post-transplant cyclophosphamide-based GVHD prophylaxis. Patient/HCT characteristics are summarized in Table 1.In this cohort, 18% had no DPB1 mismatch, 54% had a single and 28% had double mismatches, with 21% pts carrying non-permissive DPB1 mismatches. Allopeptide score was 0 in 75% of pts. Non-permissive mismatch 9 (39%) vs. 11 (16%) were more likely to have allopeptide score ≥1 and similarly double mismatches 11 (48%) vs. 15 (21%) were more likely to have allopeptide score of ≥1. Among pts with ≥1 allopeptide score 14 (61%) had DPB1 matched or permissive mismatch.The cumulative incidence of grade 2-4 acute GVHD was 40.8% (range: 29-52) in pts with no allopeptides from DPB1 compared with 56% (range: 34-74) in those with ≥1 allopeptides (p=0.259) (Figure 2). The cumulative incidence of grade 3-4 acute GVHD and chronic GVHD were similar between allopeptide 0 vs. ≥1.Together, we show that the “MHC-PepSeq” assay can identify novel candidate HLA-derived allopeptides in 10/10 MUD HCTs. The number of such peptides are relatively low - with a majority having no allopeptide. In an exploratory analysis in a selected cohort of patients with HLA-DRB1*0701 in the setting of 10/10 MUD HCT, the number of allopeptides in our assay may be predictive of GVHD. The expanded analyses on other HLA-DRB1 restriction elements are underway. [Display omitted] DisclosuresAl Malki: CareDx: Consultancy; Hansa Biopharma: Consultancy; Neximmune: Consultancy; Jazz Pharmaceuticals, Inc.: Consultancy; Rigel Pharma: Consultancy. Ali: BMS: Speakers Bureau; Incyte: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; CTI BioPharma: Membership on an entity's Board of Directors or advisory committees. Forman: Mustang Bio: Consultancy, Current holder of individual stocks in a privately-held company; Lixte Biotechnology: Consultancy, Current holder of individual stocks in a privately-held company; Allogene: Consultancy.

Recipient Hematopoietic, but Not Non-Hematopoietic, Antigen-Presenting Cells Are Indispensable for CD4+ T-Cell-Mediated Xenogeneic Graft-Versus-Host Disease

BackgroundAs a major drawback of conventional GVHD models using inbred mouse strains, they are not suitable for assessing the safety and effectiveness of current human-specific therapies. To overcome this limitation, xenogeneic GVHD models using highly immunodeficient mice are currently being developed and are widely used to study human diseases. However, the underlying mechanisms of human immune responses against host are not fully understood. We have previously reported that human CD4+ T-cells and, to a lesser extent, CD8+ T-cells play a critical role in the induction and development of xenogeneic GVHD. (Kawasaki et al. Blood Abstract 2016 #701). Donor antigen-presenting cells (APCs) were not necessarily required for T-cell activation. In this context, the present study focused on the immunobiology of CD4+ T-cell-mediated GVHD, and defined the individual role of host hematopoietic, and non-hematopoietic, APCs in inducing human immune responses in NOD/Shi-scid-IL2rg null (NOG) mice.MethodsA xenogeneic GVHD model was established by intravenously injection of human CD4+ T-cells into NOG mice. Bone marrow (BM) chimeras were generated as summarized in Figure 1A. Briefly, BM cells from wild-type (MHC+/+) or MHC−/− NOG mice, respectively, were transplanted into other mice, in which only non-hematopoietic cells including target organs, or only hematopoietic cells including APCs, respectively, were deficient in MHC molecules. After human CD4+ T-cells were injected into these BM chimeras, survival and clinical GVHD scores were monitored.ResultsControl mice (MHC+/+ → MHC+/+) uniformly developed progressive GVHD, and they all died within 12 weeks following the injection of human CD4+ T-cells. The former chimeras (MHC+/+ → MHC−/−) showed similar GVHD severity and mortality, whereas the latter chimeras (MHC−/− → MHC+/+) showed significantly less body weight loss and lower clinical GVHD scores than the control (Figure 1B, C), which resulted in longer survival (Figure 1D). These results indicate that host hematopoietic, but not non-hematopoietic, APCs play a critical role in the development of xenogeneic GVHD.To verify these results, we then analyzed T-cell subsets in the lungs and spleen of NOG mice one week after the injection of purified human naïve (CD45RA+CCR7+) CD4+ T-cells. Flow cytometric analysis showed a markedly increased percentage of central memory (CD45RA−CCR7+) and effector memory (CD45RA−CCR7−) CD4+ T-cells in the lungs and spleen of MHC+/+ mice. Notably, the memory differentiation was also observed in MHC−/− mice. However, the proportion of terminally differentiated effector memory (CD45RA+CCR7−) CD4+ T-cells was significantly increased in MHC−/− mice, suggesting that a certain number of effector T-cells failed to develop a memory phenotype due to a lack of antigen-specific stimulation. Splenectomized NOG mice also showed rapid memory differentiation of CD4+ T-cells in the lungs during early GVHD. These findings suggest that neither MHC molecule expression on host tissues nor host hematopoietic APCs are required for the memory differentiation of CD4+ T-cells in lungs during early GVHD in xenogeneic chimeras.Focusing on memory T-cells during early xenogeneic GVHD, we revealed that most memory CD4+ T-cells in lungs were less activated and showed a slowly proliferative phenotype (CD69low and CFSElow) compared to those in spleen. Intracellular IFN-γ production was also decreased in these cells. These findings suggest that most memory T-cells in lungs have less potential to cause sustained inflammation, and are therefore less associated with the pathogenesis of GVHD. Increased susceptibility of T-cells to peripheral apoptosis appears to be one of the characteristic properties of spontaneous proliferation. Consistent with this, we revealed that the memory CD4+ T-cells in lungs showed a significantly decreased expression of Bcl-2 compared to those in the spleen, indicating that these cells spontaneously acquire the memory phenotype, but not in response to non-hematopoietic APCs.ConclusionsOur data clearly indicate that the immunobiology of xenogeneic GVHD is highly dependent on human CD4+ T-cells and mouse hematopoietic, but not non-hematopoietic, APCs. We believe that our findings provide a better understanding of the immunobiology of humanized mice and support the development of novel options for the prevention and treatment for GVHD. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Natural Killer Cells are Essential for Allograft Vasculopathy in a Model of CD4+ T Cell-Mediated Chronic Rejection

Purpose In transplantation, recipient CD4+ T cells have two potential means of MHC class II-restricted donor antigen detection: (1) the direct recognition of allogeneic MHC class II expressed by donor antigen-presenting cells (APCs) and (2) the indirect recognition of donor antigens processed and presented by recipient APCs. As donor-derived APCs are gradually depleted over time, the indirect pathway emerges as the dominant mode of allorecognition in the long-term. A clear role of direct CD4+ T cells as effector cells has been shown in acute rejection, however, the role of indirect CD4+ T cells in chronic rejection is less clear. The function of NK cells in solid organ transplantation is multifaceted, but they have been implicated in several different animal models of chronic rejection. Our study sought to test the hypothesis that NK cells are the key effectors in sustaining an anti-donor response in a model of indirect CD4+ T cell mediated chronic allograft vasculopathy (CAV). Methods We utilized a mouse model of cardiac transplantation with MHC class II-deficient B6.C2D (H-2b) hearts engrafted into immune-deficient BALB/c.rag-/- (H-2d) recipients. Recipients had no functional adaptive immunity but an intact innate immune system. After transplantation, the recipients received purified polyclonal BALB/c CD4+ T cells with or without concurrent administration of an NK cell depleting antibody (anti-Asialo GM1). Allografts and recipient spleens were subsequently assessed after 30 days. Results All allografts demonstrated cardiac contraction, as detected by abdominal palpation, for 30 days both with and without CD4+ T cell reconstitution. Only 1/9 (11%) of allografts from un-reconstituted recipients demonstrated evidence of CAV, while 5/7 (72%) of allografts from recipients that received adoptively transferred CD4+ T cells showed disease (p Conclusion In the context of immunodeficient hosts, CD4+ T cells are sufficient to mediate chronic allograft vasculopathy, independent of recipient CD8+ T cell and B cell responses. Recipient NK cells are necessary for the development of this form of chronic rejection.

Recent developments in prophylaxis and treatment of graft-versus-host disease

Acute graft-versus-host disease (GVHD) is a systemic immune reaction in which mature T cells in the donor inoculum are abnormally activated by the host and donor antigen-presenting cells. These activated cells then attack normal host tissue after allogeneic hematopoietic stem cell transplantation (HSCT). Clinical GVHD develops when the damage exceeds the threshold for tissue tolerance. Furthermore, functional damage of hematopoietic and non-hematopoietic organs due to this acute immune response triggers the subsequent development of chronic GVHD. The endpoint of HSCT is shifting from just surviving the acute phase to having an enriched life until later years; therefore, seamless and detailed immune management is required from the acute to chronic phase after HSCT. Acute GVHD prophylaxis consisted of calcineurin inhibitors and short-term methotrexate for a long time. However, various novel immune-modulating strategies have been developed against the background of recent diversification in donor sources and changes in treatment goals. In this review, we discuss recent clinical developments in basic and clinical research regarding acute or chronic GVHD.

Alternative mechanisms that mediate graft-versus-host disease in allogeneic hematopoietic cell transplants.

Allogeneic hematopoietic cell transplantation (alloHCT) benefits increasing numbers of patients with otherwise lethal diseases. Graft-versus-host disease (GVHD), however, remains one of the most potentially life-threatening complications due to its own comorbidities and the side effects of its treatment. In this issue of the JCI, two groups have turned dogma on its head by providing evidence for alternative mechanisms of acute GVHD (aGVHD) in humans. The principle of donor T cell reactivity elicited by host antigen-presenting cells (APCs) expressing MHC-encoded major HLA disparities or expressing minor histocompatibility antigen (miHA) differences presented by identical HLA molecules remains intact. These reports, however, demonstrate that GVHD can additionally result from peripheral host T cells resident in skin and gut being stimulated against donor APCs in the form of monocyte-derived macrophages. Moreover, these donor monocyte-derived macrophages can themselves mediate cytopathic effects against resident host T cells in skin explants and against a keratinocyte-derived cell line.

B lymphocytes contribute to indirect pathway T cell sensitization via acquisition of extracellular vesicles.

B cells have been implicated in transplant rejection via antibody-mediated mechanisms and more recently by presenting donor antigens to T cells. We have shown in patients with chronic antibody-mediated rejection that B cells control the indirect T cell alloresponses. To understand more about the role of B cells as antigen-presenting cells for CD4+ T cell with indirect allospecificity, B cells were depleted in C57BL/6 mice, using an anti-CD20 antibody, prior to receiving MHC class I-mismatched (Kd ) skin. The absence of B cells at the time of transplantation prolonged skin graft survival. To study the mechanisms behind this observation, T cells with indirect allospecificity were transferred in mice receiving a Kd skin transplant. T cell proliferation was markedly inhibited in the absence of recipient B cells, suggesting that B cells contribute to indirect pathway sensitization. Furthermore, we have shown that a possible way in which B cells present alloantigens is via acquisition of MHC-peptide complexes. Finally, we demonstrate that the addition of B cell depletion to the transfer of regulatory T cells (Tregs) with indirect alloresponse further prolonged skin graft survival. This study supports an important role for B cells in indirect T cell priming and further emphasizes the advantage of combination therapies in prolonging transplant survival.

Moving Toward Transplant Tolerance: Is Targeting Donor Antigen-presenting Cells the Key?

Moving Toward Transplant Tolerance: Is Targeting Donor Antigen-presenting Cells the Key?

Targeting Intestinal Vitamin D Receptor Signaling to Mitigate Graft-Versus-Host Disease

Graft-versus-host disease (GVHD) remains the major cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation (HSCT). Impaired mucosal epithelial barrier contributes significantly to amplifying systemic inflammatory responses characteristic of GVHD. Emerging evidence demonstrates that protecting or restoring intestinal barrier function represents a feasible approach to mitigate GVHD.There is growing interest in how vitamin D and the vitamin D receptor (VDR) signaling regulate immune responses in intestinal inflammatory diseases. The role of VDR signaling in regulating alloimmunity is unknown. We therefore tested the novel hypothesis that VDR signaling plays an important role in maintaining intestinal epithelial barrier function during GVHD. In initial studies, we examined whether intestinal VDR signaling is altered in recipient mice undergoing either syngeneic or allogeneic bone marrow transplantation (BMT). We found a progressive decline in VDR gene expression over time in colon tissues of allogeneic, but not syngeneic, BMT recipients compared with that of naïve animals. Notably, the expression of CYP27B1, the key enzyme participating in the biosynthesis of the active form of vitamin D, 1α,25-dihydroxyvitamin D3 (1,25VitD3), was also significantly decreased in the intestines of allogeneic BMT recipients compared to that of naïve animals. Caco-2 is an epithelial cell line that is widely used as a model for the intestinal barrier. We showed that proinflammatory cytokines TNF-α and IL-6 reduced Caco-2 VDR protein levels in a dose and time dependent manner by western blot analysis. To assess monolayer barrier function, an in vitro permeability assay was performed using FITC-dextran. We found 1,25VitD3 significantly attenuated the increase in epithelial monolayer permeability induced by TNF-α and IL-6. Tight junctions are important components of the intestinal barrier. We also found proinflammatory cytokines reduced gene expression of tight junctions such as occludin in Caco-2 cells. Remarkably, 1,25VitD3 treatment significantly restored occludin expression.We then examined how vitamin D/VDR pathway regulates the development of GVHD. 1,25VitD3 supplementation after BMT significantly increased the survival of recipient mice in a well-established C57Bl/6→Balb/c GVHD model (p=0.0045). 1,25VitD3 treatment did not appear to modify donor T cell alloreactivity. Specifically, donor T cell activation, proliferation, and polarization were not affected by 1,25VitD3 treatment. Notably, infusion of donor T cells from VDR-WT (VDR+/+) or VDR-deficient (VDR-/-) mice caused similar GVHD-associated mortality in Balb/c recipient mice (p=0.98), further suggesting that the beneficial effects of 1,25VitD3 treatment is not due to its effects on donor T cells. 1,25VitD3 also did not appear to affect host and donor antigen presenting cells to modulate GVHD severity. Specifically, dendritic cells from VDR+/+ or VDR-/- mice showed equal ability to stimulate the proliferation of alloreactive T cells in mixed lymphocyte reactions. There was also no difference in post-transplant survival when recipient mice were reconstituted with donor T-cell-depleted bone marrow cells from either VDR+/+ or VDR-/- mice (recipients were transplanted with the same donor T cells from VDR-WT animals; p=0.58). Instead, 1,25VitD3 treatment significantly increased tight junction protein expression in the gut, suggesting its effect on intestinal epithelial cells. Finally, transgenic mice selectively overexpress VDR in the intestinal epithelium (villin promoter-driven FLAG-tagged human VDR gene expression) were used as BMT recipients in gain-of-function studies. We found a significantly decreased GVHD-associated mortality in these mice compared to that of control littermates (p=0.0002), providing direct evidence that intestinal epithelial VDR signaling protects against GVHD. Our results provide new evidence supporting the notion that impaired mucosal barrier function is a major pathogenic factor in the development of GVHD. Intestinal VDR signaling plays an important role in maintaining intestinal barrier integrity and enhancing this pathway protects against GVHD. Thus, our study provides pre-clinical data supporting the use of vitamin D or its analogs as a simple and inexpensive therapeutic modality with minimal side effects for mitigating GVHD. DisclosuresNo relevant conflicts of interest to declare.

Receptor tyrosine kinase MerTK suppresses an allogenic type I IFN response to promote transplant tolerance.

Recipient infusion of donor apoptotic cells is an emerging strategy for inducing robust transplantation tolerance. Daily clearance of billions of self-apoptotic cells relies on homeostatic engagement of phagocytic receptors, in particular, receptors of the tyrosine kinase family TAM (Tyro3, Axl, and MerTK), to maintain self-tolerance. However, an outstanding question is if allogeneic apoptotic cells trigger the same receptor system for inducing allogeneic tolerance. Here, we employed allogeneic apoptotic splenocytes and discovered that the efferocytic receptor MerTK on recipient phagocytes is a critical mediator for transplantation tolerance induced by this strategy. Our findings indicate that the tolerogenic properties of allogeneic apoptotic splenocytes require MerTK transmission of intracellular signaling to suppress the production of inflammatory cytokine interferon α (IFN-α). We further demonstrate that MerTK is crucial for subsequent expansion of myeloid-derived suppressor cells and for promoting their immunomodulatory function, including maintaining graft-infiltrating CD4+ CD25+ Foxp3+ regulatory T cells. Consequently, recipient MerTK deficiency resulted in failure of tolerance by donor apoptotic cells, and this failure could be effectively rescued by IFN-α receptor blockade. These findings underscore the importance of the efferocytic receptor MerTK in mediating transplantation tolerance by donor apoptotic cells and implicate MerTK agonism as a promising target for promoting transplantation tolerance.

Human CD8+CD28- T Suppressor Cells Expanded by IL-15 In Vitro Suppress in an Allospecific and Programmed Cell Death Protein 1-Dependent Manner.

CD8+CD28− T suppressor cells (Ts) have been recently documented to play an important role in alloimmunity. Therefore, understanding and optimizing the conditions under which these cells are generated and/or expanded would greatly facilitate further research and potential clinical use. In this study, we describe rapid expansion of human allospecific CD8+CD28− Ts cells through coculture of CD8+ T cells with human leukocyte antigen-mismatched donor antigen-presenting cells plus IL-15 in a relative short period of time in vitro. Interestingly, IL-15 promotes the expansion of CD8+CD28− Ts cells through several parallel mechanisms. The expanded CD8+CD28− Ts cells upregulate expression of CD132, CD25, and programmed cell death protein 1 (PD-1), but downregulate expression of CD122, GZM-B, and perforin, while exhibiting no cytotoxicity. Most importantly, the expanded CD8+CD28− Ts cells vigorously inhibit CD4+ T cells proliferation in a contact-dependent and donor-specific manner both in vitro and in vivo. Interestingly, the co-inhibitory molecules PD-1 and programmed death-ligand 1 play an obligatory role in the mechanisms of CD8+CD28− Ts cells suppression. Taken together, our study report novel methodology for IL-15-induced expansion of human CD8+CD28− Ts cells and possible mechanisms. These findings may facilitate understanding of transplant rejection and promote clinical application of CD8+CD28− Ts cell-based strategies for inducing and monitoring transplant tolerance in the future.

Equal Expansion of Endogenous Transplant-Specific Regulatory T Cell and Recruitment Into the Allograft During Rejection and Tolerance.

Despite numerous advances in the definition of a role for regulatory T cells (Tregs) in facilitating experimental transplantation tolerance, and ongoing clinical trials for Treg-based therapies, critical issues related to the optimum dosage, antigen-specificity, and Treg-friendly adjunct immunosuppressants remain incompletely resolved. In this study, we used a tractable approach of MHC tetramers and flow cytometry to define the fate of conventional (Tconvs) and Tregs CD4+ T cells that recognize donor 2W antigens presented by I-Ab on donor and recipient antigen-presenting cells (APCs) in a mouse cardiac allograft transplant model. Our study shows that these endogenous, donor-reactive Tregs comparably accumulate in the spleens of recipients undergoing acute rejection or exhibiting costimulation blockade-induced tolerance. Importantly, this expansion was not detected when analyzing bulk splenic Tregs. Systemically, the distinguishing feature between tolerance and rejection was the inhibition of donor-reactive conventional T cell (Tconv) expansion in tolerance, translating into increased percentages of splenic FoxP3+ Tregs within the 2W:I-Ab CD4+ T cell subset compared to rejection (~35 vs. <5% in tolerance vs. rejection). We further observed that continuous administration of rapamycin, cyclosporine A, or CTLA4-Ig did not facilitate donor-specific Treg expansion, while all three drugs inhibited Tconv expansion. Finally, donor-specific Tregs accumulated comparably in rejecting tolerant allografts, whereas tolerant grafts harbored <10% of the donor-specific Tconv numbers observed in rejecting allografts. Thus, ~80% of 2W:I-Ab CD4+ T cells in tolerant allografts expressed FoxP3+ compared to ≤10% in rejecting allografts. A similar, albeit lesser, enrichment was observed with bulk graft-infiltrating CD4+ cells, where ~30% were FoxP3+ in tolerant allografts, compared to ≤10% in rejecting allografts. Finally, we assessed that the phenotype of 2W:I-Ab Tregs and observed that the percentages of cells expressing neuropilin-1 and CD73 were significantly higher in tolerance compared to rejection, suggesting that these Tregs may be functionally distinct. Collectively, the analysis of donor-reactive, but not of bulk, Tconvs and Tregs reveal a systemic signature of tolerance that is stable and congruent with the signature within tolerant allografts. Our data also underscore the importance of limiting Tconv expansion for high donor-specific Tregs:Tconv ratios to be successfully attained in transplantation tolerance.