Solid Organ Transplant Rejection Research Articles

Isohemagglutinin titering performed on an automated solid-phase and hemagglutinin-based analyzer is comparable to results obtained by manual gel testing.

Isohemagglutinins (anti-A and anti-B) mediate hemolytic transfusion reactions, antibody-mediated rejection of solid-organ transplants, and delayed engraftment after stem cell transplant. However, quantification of isohemagglutinins is often labor intensive and operator dependent, limiting availability and interfacility comparisons. We evaluated an automated, solid-phase and agglutination-based antibody titer platform versus manual gel testing. Plasma samples were obtained from 54 randomly selected patients. Titers were determined by our laboratory's standard assay (manual dilution followed by manual gel testing) and were compared to results obtained on a fully automated blood bank analyzer (Galileo NEO, Immucor). The analyzer determined immunoglobulin G (IgG) antibodies using solid-phase and immunoglobulin M (IgM) antibodies by direct hemagglutination. Isohemagglutinin titers obtained by manual gel versus the automated assay generally (>80%) agreed within one doubling dilution, and always (100%) agreed within two dilutions. Among O samples, the gel titer and the highest titer obtained with the automated assay (either IgG or IgM) were similar in paired, nonparametric analysis (p = 0.06 for anti-A; p = 0.13 for anti-B). Gel titers from group A and group B patients were slightly higher than the highest titer obtained using the automated assay (p = 0.04 for group A; p = 0.009 for group B), although these differences were within the accepted error of measurement. Manual and automated methodologies yielded similar isohemagglutinin titers. Separate quantification of IgM and IgG isohemagglutinins via automated titration may yield additional insight into hemolysis, graft survival after ABO-incompatible transplantation, and red blood cell engraftment after ABO-incompatible stem cell transplant.

Impact of the microbiota on solid organ transplant rejection.

The microbiota in mammalian hosts can affect maturation and function of the immune system and has been associated with health and disease. We will review new findings on how this dynamic environmental factor impacts alloimmunity and therapy in transplant hosts. The microbiota changes after transplantation and immunosuppressive therapy. New data indicate that different microbial community structures have distinct impact on graft outcome, from promoting, to inhibiting or being neutral to transplant survival. In addition, we will address reciprocal interactions between the microbiota and immunosuppressive drugs, as well as the suitability of the microbiota as a predictive biomarker and its utility as adjunct therapy in transplantation. Advances in microbiome sequencing and wider availability of gnotobiotic facilities are enabling mechanistic investigations into the commensal communities and pathways that modulate allograft outcome, responsiveness to immunosuppression and side effects of drugs. A better understanding of the functions of the microbiota may help mitigate drug toxicity, predict drug dosage and dampen alloimmunity in transplant patients.

Human CD8+ Tregs expressing a MHC-specific CAR display enhanced suppression of human skin rejection and GVHD in NSG mice

AbstractPolyclonal CD8+CD45RClow/− Tregs are potent regulatory cells able to control solid organ transplantation rejection and even induce tolerance. However, donor major histocompatibility complex (MHC)–specific Tregs are more potent than polyclonal Tregs in suppressing T-cell responses and preventing acute as well as chronic rejection in rodent models. The difficulty of identifying disease-relevant antigens able to stimulate Tregs has reduced the possibility of obtaining antigen-specific Tregs. To bypass this requirement and gain the advantage of antigen specificity, and thus improve the therapeutic potential of CD8+ Tregs, we stably introduced a chimeric antigen receptor (CAR) derived from a HLA-A*02 antigen-specific antibody (A2-CAR) in human CD8+ Tregs and developed a clinically compatible protocol of transduction and expansion. We demonstrated that A2-CAR CD8+ Tregs were not phenotypically altered by the process, were specifically activated, and did not exhibit cytotoxic activity toward HLA-A*02+ kidney endothelial cells (ECs). We showed that A2-CAR CD8+ Tregs were more potent suppressors of immune responses induced by HLA-A*02 mismatch than control-CAR CD8+ Tregs, both in vitro and in vivo, in models of human skin graft rejection and graft-versus-host disease (GVHD) in NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice. We showed that integrity of human skin graft was preserved with A2-CAR CD8+ Tregs at least 100 days in vivo after administration, and that interaction between the A2-CAR CD8+ Tregs and HLA-A*02+ kidney ECs resulted in a fine-tuned and protolerogenic activation of the ECs without cytotoxicity. Together, our results demonstrated the relevance of the CAR engineering approach to develop antigen-specific CAR-CD8+ Tregs for clinical trials in transplantation, and potentially in other diseases.

Missing self triggers NK cell-mediated chronic vascular rejection of solid organ transplants

Current doctrine is that microvascular inflammation (MVI) triggered by a transplant -recipient antibody response against alloantigens (antibody-mediated rejection) is the main cause of graft failure. Here, we show that histological lesions are not mediated by antibodies in approximately half the participants in a cohort of 129 renal recipients with MVI on graft biopsy. Genetic analysis of these patients shows a higher prevalence of mismatches between donor HLA I and recipient inhibitory killer cell immunoglobulin-like receptors (KIRs). Human in vitro models and transplantation of β2-microglobulin-deficient hearts into wild-type mice demonstrates that the inability of graft endothelial cells to provide HLA I-mediated inhibitory signals to recipient circulating NK cells triggers their activation, which in turn promotes endothelial damage. Missing self-induced NK cell activation is mTORC1-dependent and the mTOR inhibitor rapamycin can prevent the development of this type of chronic vascular rejection.

Solid Organ Transplant Rejection Associated with the Use of the Immunomodulatory Drugs (IMIDs)

Introduction: IMIDs are a cornerstone of multiple myeloma treatment and have a recently expanded role in the treatment of selected lymphomas. However, the safety of chemotherapeutic regimens containing IMIDs in patients with a history of solid organ transplantation (SOT) is uncertain. Recently, several published case reports have described SOT rejection (SOTr) among patients taking IMIDs. Methods: To investigate a potential association between IMID exposure and SOTr, we evaluated postmarketing adverse event reports submitted to the FDA Adverse Event Reporting System (FAERS) and published in the literature. We descriptively characterized the clinical outcomes and severity of SOTr and utilized the World Health Organization-Uppsala Monitoring (WHO-UMC) scale to assess drug causality. Results: We identified 22 cases of SOTr associated with the use of lenalidomide (n=16), pomalidomide (n=3), or thalidomide (n=3) from FAERS and the literature. After IMID initiation, the median time to onset of SOTr was 39 days (range 3-70 days) with 89% of cases occurring by 60 days. Notably, SOTr occurred within 2 weeks of lenalidomide and pomalidomide administration in 5 cases, including 2 patients with a distant history of SOT (lenalidomide, n=1; pomalidomide, n=1). The most commonly affected organs were the kidney (n=13) and heart (n=10), with 3 of these cases involving rejection of both organs. In 12 cases, rejection was confirmed with biopsy findings of acute T-cell mediated rejection. Notably, 6 of these 12 cases provided baseline biopsy findings prior to IMID initiation that indicated stable organ function and showed no signs of rejection. All cases were complicated by serious outcomes including 7 deaths described as related to graft rejection. Other serious clinical manifestations included surgical excision of the original transplanted organ (n=2), requirement of a second transplant (n=3), and dialysis (n=4), including 2 cases that required long-term renal replacement therapy. We determined the causality as probable (no confounding factor identified) in 6 cases and possible (confounding factor identified) in the remaining 16 cases. The most frequently reported confounding factor was concomitant change of the baseline immunosuppressive regimen with the initiation of the IMID (n=7). Notably, 5 of the 7 cases with fatal outcome had immunosuppression reduced at IMID therapy initiation, including 2 cases with a distant history of SOT and stable graft function. Conclusion: To our knowledge, this is the largest case series describing SOTr with the IMIDs. These cases are characterized by serious events including death and reduced long-term survival of the graft. Although this analysis is limited by the quality of spontaneous adverse event reporting, the strong temporal relationship and the histologic findings of T-cell mediated rejection provide reasonable evidence of a contributory role of IMIDs to SOTr. This risk is plausible because IMIDs induce T-cell proliferation, enhance interleukin-2 and interferon-gamma production, and inhibit regulatory T-cell function. Increased surveillance for early signs of acute rejection is warranted for SOT recipients receiving IMID therapy. The US prescribing information is currently being revised to reflect the risk of SOTr across all IMIDs. Disclosures No relevant conflicts of interest to declare.

In vitro selection of tacrolimus binding aptamer by systematic evolution of ligands by exponential enrichment method for the development of a fluorescent aptasensor for sensitive detection of tacrolimus

Tacrolimus (TAC) is an immunosuppressant for preventing solid-organ transplant rejection. Because of its narrow therapeutic window, analytical methods which can detect TAC in serum samples with high accuracy and reliability are required. In this study, specific aptamers (Apt122 and Apt125) for TAC were isolated via systematic evolution of ligands by exponential enrichment method using magnetic beads to immobilize the target. After determination of binding constants of aptamers by flow cytometry analysis, Apt122 was selected and labeled with ATTO 647 N as a fluorophore to develop a fluorescent sensing platform for detection of TAC using graphene oxide (GO) as a fluorescence quencher. The designed aptasensor could detect TAC in phosphate buffer saline (10 mM PBS) and serum samples with detection limits as low as 1.4 and 2.5 nM, respectively.

Iron deficiency anemia associated with extracorporeal photopheresis: A retrospective analysis.

Extracorporeal photopheresis (ECP) is associated with few adverse effects. We have anecdotally noted patients treated with long-term ECP develop iron deficiency anemia (IDA). We performed a retrospective chart review of adult patients who received ECP for any indication at Mayo Clinic Rochester and Mayo Clinic Arizona. The primary objective was to describe the cumulative incidence of IDA at 1 year of ECP therapy. A total of 123 patients were eligible for analysis. Graft-vs-host disease was the most common indication for ECP (n = 76, 61.8%). At 1 year of ECP therapy, the cumulative incidence of IDA was 24.1% (95% CI, 14.2%-32.9%). At 5 years, the cumulative incidence of IDA was 68.3% (95% CI, 38%-83.8%). Risk factors for the development of IDA included: cumulative number of ECP sessions (HR 1.34, 95% CI, 1.05-1.73 per 10 additional sessions, P = .022), an indication for ECP of solid organ transplant rejection (compared to cutaneous T-cell lymphoma, HR 5.46, 95% CI, 2.06-14.49, P < .001), and proton pump inhibitor use at baseline (HR 2.15, 95% CI, 1.1-4.21, P = .03). Iron supplementation was initiated in 29 of 37 evaluable patients who developed IDA, with a cumulative incidence of supplementation in 77.2% patients within 3 months of recognition of IDA (95% CI, 55.8%-88.3%). Hemoglobin normalized in 50.1% of patients started on iron supplementation for IDA within 7 months (95% CI, 25.2%-66.7%). Iron deficiency anemia is common in patients receiving long-term ECP and occurs throughout ECP therapy. IDA resolved with iron supplementation in half of patients.

Composite Graft Pretreatment With Hydrogen Sulfide Delays the Onset of Acute Rejection.

Vascularized composite allotransplantation can reconstruct devastating tissue loss by replacing like-with-like tissues, most commonly in the form of hand or face transplantation. Unresolved technical and ethical challenges have meant that such transplants remain experimental treatments. The most significant barrier to expansion of this field is the requirement for systemic immunosuppression, its toxicity and effect on longevity.Hydrogen sulfide (H2S) has been shown experimentally to ameliorate the ischemia reperfusion injury associated with composite tissue autotransplantation, which has been linked to acute rejection in solid organ transplantation. In this protocol, a large-animal model was used to evaluate the effect of H2S on acute rejection after composite tissue allotransplantation. A musculocutaneous flap model in SLA-mismatched swine was used to evaluate acute rejection of allotransplants in 2 groups: control animals (n = 8) and a treatment group in which the allografts were pretreated with hydrogen sulfide (n = 8). Neither group was treated with systemic immunosuppression. Acute rejection was graded clinically and histopathologically by an independent, blinded pathologist. Data were analyzed by t tests with correction for multiple comparisons by the Holm-Šídák method. Clinically, H2S-treated tissue composites showed a delay in the onset of rejection that was statistically significant from postoperative day 6. Histopathologically, this difference between groups was also apparent, although evidence of a difference in groups disappeared beyond day 10. Targeted hydrogen sulfide treatment of vascularized composite allografts immediately before transplantation can delay acute rejection. This may, in turn, reduce or obviate the requirement for systemic immunosuppression.

Laboratory monitoring requirements during mycophenolate mofetil therapy for dermatologic conditions: A single-institution retrospective chart review

To the Editor: The Food and Drug Administration recommends routine laboratory monitoring for patients receiving mycophenolate mofetil because of evidence that mycophenolate mofetil causes neutropenia when used to prevent solid organ transplant rejection.1 Without supporting studies, similar recommendations have been applied to the use of mycophenolate for dermatologic conditions. On the basis of our observations that mycophenolate does not cause neutropenia, hepatic injury, or renal injury when used to treat dermatologic conditions, we performed a retrospective chart review to evaluate the utility of laboratory monitoring of individuals receiving mycophenolate for skin disease.

Simultaneous Detection of Beta and Gamma Human Herpesviruses by Multiplex qPCR Reveals Simple Infection and Coinfection Episodes Increasing Risk for Graft Rejection in Solid Organ Transplantation.

Herpesviruses are common components of the human microbiome that become clinically relevant when a competent immunosurveillance is compromised, such as in transplantation. Members of the beta and gamma subfamilies are associated with a wide diversity of pathologies, including end-organ disease and cancer. In this study, we developed a multiplex qPCR technique with high specificity, sensitivity, efficiency and predictability that allowed the simultaneous detection and quantification of beta and gamma human herpesviruses. The technique was tested in a cohort of 34 kidney- or liver-transplanted pediatric patients followed up for up to 12 months post-transplant. Viral load was determined in 495 leukocyte-plasma paired samples collected bi-weekly or monthly. Human herpesvirus (HHV) 7 was the herpesvirus most frequently found in positive samples (39%), followed by Epstein-Barr virus (EBV) (20%). Also, EBV and HHV7 were present in the majority of coinfection episodes (62%). The share of positive samples exclusively detected either in leukocytes or plasma was 85%, suggesting that these herpesviruses tended to take a latent or lytic path in an exclusive manner. Infection by human cytomegalovirus (HCMV) and HHV6, as well as coinfection by EBV/HHV7 and EBV/HHV6/HHV7, were associated with graft rejection (RR = 40.33 (p = 0.0013), 5.60 (p = 0.03), 5.60 (p = 0.03) and 17.64 (p = 0.0003), respectively). The routine monitoring of beta and gamma herpesviruses should be mandatory in transplant centers to implement preventive strategies.

Comparison of Outcomes between the Cellex and Uvar-Xts Closed-System Extracorporeal Photopheresis (ECP) Devices When Used for Graft-Versus-Host Disease; A Single Center Experience

Abstract BACKGROUND: Graft versus host disease (GVHD) is a significant contributor to non-relapse mortality after allogeneic stem cell transplant (SCT). Steroids are first line therapy and extracorporeal photopheresis (ECP) is used as second line therapy for steroid refractory or intolerant patients. ECP has immunomodulatory effects rather than immunosuppressive effect which decreases the risk of infection, and may decreases the risk of disease relapse. There are two ECP instruments approved in the US for the treatment of Cutaneous T Cell Lymphoma (CTLC), however both have been used "off-label" to prevent or treat solid organ transplant rejection, acute and chronic GVHD, and other autoimmune diseases. Until recently the UVAR-XTS instrument has been the most commonly used closed system in the US until the CELLEX was approved in 2009. The CELLEX and the UVAR-XTS procedures are similar in that buffy coat is collected, exposed to methoxsalen (UVADEX®) ex vivo and then UVA light before returning the treated cells back to the patient in a closed looped system. The instruments differ in the collection of the buffy coat; collection is continuous by the CELLEX, and it is intermittent by UVAR-XTS. In addition CELLEX has been shown to be more efficient in collecting mononuclear cells when compared to the UVAR -XTS. Little clinical data is available comparing the clinical efficacy of the two instruments in the setting of GVHD. We designed a single institution retrospective study to compare the efficacy and safety of the two instruments. The primary outcomes analyzed were &gt;/=50% reduction of steroid dose between the initial and final dose during the study period, and the number and type of adverse events that occurred during the ECP procedures. METHODS: Study Population: We reviewed charts of allogeneic stem cell transplant patients who received ECP for the treatment of acute or chronic GVHD between 1/2009 and 12/2015. The patients were divided into two groups based on the type of ECP instrument that was used. One group was exclusively treated with UVAR-XTS while the second group with CELLEX. Patients who received treatments with both instruments were excluded. The frequency of steroid dose reduction by &gt;/=50%, and toxicity was compared between the two instruments while adjusting for age, gender, GVHD severity, number of organs involved by GVHD, type of allogeneic stem cell transplant, conditioning regimen, number of other immunosuppressants, type of anticoagulants (ACDA vs heparin), type of access line, and baseline blood counts. Statistical Analysis: The baseline characteristics of the patients in the two groups were compared using Chi square, Fischer's exact test for categorical variables and one-way analysis of variance (ANOVA) for continuous variables. Chi square analysis was used to determine the difference in frequency of &gt;/=50% steroid dose reduction and adverse events between the two groups. Logistic multivariate regression was used to evaluate the potential interactions of all significant covariates. A p value of less than 0.05 was considered to be statistically significant. Statistical analyses were performed using STATA14 software (StataCorp, College Station, TX). RESULTS: We identified 242 allogeneic SCT recipients who received ECP for acute or chronic GVHD in the study period, 146 of whom met the selection criteria. 69 patients had all procedure performed with UVAR-XTS and 77 patients had all procedures performed with CELLEX. There was no significant difference in age, gender, percent of acute GVHD, anticoagulant used, type of transplant, number of organs involved, number of immunosuppressants, vascular access and baseline blood counts between the two treatment cohorts as shown in table 1. Although year of transplant and conditioning regimen were different between the two cohorts, neither of these covariates influenced the impact of instrument on the primary outcome. In multivariate analysis, the patients who underwent ECP with CELLEX were 3 times more likely to have &gt;/=50% steroid dose reduction (p =0.01). The total number of adverse events was similar between the instruments (p = 0.73). (Table 2) CONCLUSION: More than twice as many patients with GVHD treated with the CELLEX had a steroid dose reduction by &gt;/=50% reflecting clinical improvement when compared to the patients treated with the UVAR-XTS. Similar safety profile was observed between the two instruments based on a similar number of adverse events. Disclosures No relevant conflicts of interest to declare.

Sirolimus Augments Hematopoietic Stem and Progenitor Cell Recovery from Hematopoietic Stress

Sirolimus is an approved drug to prevent solid organ transplant rejection and to treat lymphangioleiomyomatosis. Sirolimus also has been employed in a broad range of clinical circumstances, including induction of tolerance in nonmyeloablative HLA-mismatched bone marrow (BM) transplantation and treatment of graft-versus-host disease following allogeneic stem cell transplantation. Sirolimus suppresses mammalian target of rapamycin (mTOR), a serine-threonine protein kinase that responds to multiple signals to regulate cellular metabolism, survival, and growth. We recently reported the application of sirolimus on murine models of immune-mediated BM failure (Feng X, Haematologica, 2017;102:1691-1703) in which sirolimus alleviated pancytopenia and attenuated BM destruction by depleting active cytotoxic T cells and expanding regulatory T cells. Controls for these experiments suggested that sirolimus might also act directly to preserve hematopoietic stem and progenitor cells (HSPCs) in non-immune BM damage and hematopoietic cell injury. In the current study, we systematically tested the effects of sirolimus on HSPC regeneration following irradiation and exposure to cytotoxic drugs. B6 mice subjected to 5 Gys total body irradiation (TBI) showed large declines in HSPC numbers, which were abrogated by sirolimus at 2 mg/Kg/day by intraperitoneal (i.p.) injection for 13 days: we observed significant increases (2-4-fold) in the proportions and absolute numbers of HSPCs relative to TBI controls when animals were evaluated at 14 days (Fig. 1A). Sirolimus did not alter peripheral blood cell counts or BM cellularity. Functionally, BM cells from sirolimus-treated TBI mice contained 2-3-fold more colonies than did marrow of TBI controls. In a competitive repopulation stem cell assay, recipients of sirolimus-treated TBI donor BM cells had enhanced donor engraftment, as measured in recipient animals' peripheral blood and BM. There was also an increased recovery of donor HSPCs in recipient BM, relative to recipients of TBI-only donor BM cells, evaluated at 7 months after donor cell infusion. In other experiments, sirolimus at the same dose following busulfan (10 mg/kg i.p. three times weekly for 10 injections; Fig. 1B) or 5-fluorouracil (150 mg/Kg on days 0, 15, and 29 respectively; Fig. 1C) induced similar improved HSPC regeneration. Sirolimus-mediated HSPC regeneration was associated with c-Kitup-regulation in BM cells. In a PCR-based transcriptome assay of hematopoiesis genes performed on sorted Lin-CD150+ BM cells, sirolimus-treated TBI mice showed a 90% increase in c-Kit mRNA relative to TBI controls. Expression of other stem cell and transcription factor genes, including Cd34, Gata2,Angpt1, and Etv6, was also increased, while expression of T cell differentiation and Notch signaling genes, such as Cd2, Cd4, Cd8a, Tnfsf11, Jag1a, and Il-1a, was decreased in sirolimus-treated TBI mice relative to TBI controls. In parallel experiments, sirolimus had no effect on HSPCs in steady-state B6 mice that were not subjected to hematopoietic injury. Sirolimus stimulated HSPC expansion in mice carrying the Wv mutation in the c-Kit locus (Fig. 1D). To test the potential utility of sirolimus in humans, we pre-irradiated human BM CD34+ cells with 5 Gys to induce injury, then transplanted the radiated cells into immunodeficient NSG mice, followed by sirolimus treatment (2 mg/Kg/day i.p. for 4 injections). Sirolimus significantly increased engraftment of human CD45+ cells in recipient blood and BM at 5 weeks relative to the mice without sirolimus treatment (Fig. 1E). In summary, sirolimus augments c-Kit expression and HSPC recovery in response to hematopoietic stress. This novel functional property detected in murine models and xenotransplantation of human cells suggests potential clinical applicability of sirolimus to ameliorate hematopoietic cell injury and accelerate HSPC regeneration after BM hematopoietic injury. [Display omitted] DisclosuresYoung:CRADA with Novartis: Research Funding; GlaxoSmithKline: Research Funding; National Institute of Health: Research Funding.

An optimized protocol to quantify signaling in human transitional B cells by phospho flow cytometry

Background and purposePhospho flow cytometry is a powerful technique to analyze signaling in rare cell populations. This technique, however, requires harsh conditions for cell fixation and permeabilization, which can denature surface antigens or antibody-conjugated fluorochromes. These are among several technical limitations which have been a barrier to quantify signaling in unique B cell subsets. One such immature subset, transitional B cells (TrBs), may play a role in suppressing solid organ transplant rejection, graft-versus-host disease, autoimmunity, and even the immune response to malignancy. Here we sought to optimize a protocol for quantification of signaling in human TrBs compared with mature B cell subsets. ResultsTrBs were defined by surface marker expression as CD19+CD24hiCD38hi. Key parameters optimized included antibody clone selection, sequence of surface epitope labeling in relation to paraformaldehyde-based fixation and methanol-based permeabilization, photomultiplier tube (PMT) voltages, and compensation. Special attention was paid to labeling of CD38 with regard to these parameters, and an optimized protocol enabled reliable identification of TrBs, naïve (CD24+CD38+), early memory (CD24hiCD38−), and late memory (CD24−CD38−) B cells. Phospho flow cytometry enabled simultaneous quantification of phosphorylation among at least three different signaling molecules within the same sample. Among normal donors, transitional B cells exhibited diminished mitogen activated protein kinase/extracellular signal-regulated kinase and Akt phospho signaling upon nonspecific stimulation with phorbol 12-myristate 13-acetateand ionomycin stimulation. ConclusionsWe optimized an effective protocol to quantify B cell subset signaling upon stimulation. Such a protocol may ultimately serve as the basis for assessing dysfunctional B cell signaling in disease, predict clinical outcomes, and monitor response to B cell-directed therapies.

Effective suppression of donor specific antibody production by Cathepsin S inhibitors in a mouse transplantation model

Donor-specific antibodies (DSA) are a major risk factor for antibody-mediated rejection (ABMR) in solid organ transplantation, and ABMR remains a medical challenge. Therefore, effective anti-ABMR therapies are needed to improve overall graft survival. Cathepsin S (Cat S) is an essential protease for antigen peptide loading onto lysosomal/endosomal major histocompatibility complex (MHC) class II molecules to promote antigen presentation. Cat S deficiency produces immuno-deficient phenotypes including a suppressed humoral immune response, and Cat S inhibition reportedly prevents autoimmunity. However, little is known about the effects of Cat S inhibitors on organ transplantation, especially ABMR. Here, we report the pharmacological profile of novel Cat S inhibitors, AS2761325 and AS2863995, and explore their preventive potential on DSA production and acute rejection in a mouse cardiac transplantation model. Cat S inhibitors potently inhibited upregulation of antigen peptide loading MHC class II expression on the surface of splenic B cells and suppressed ovalbumin-induced T cell-dependent antibody production in mice. In a mouse cardiac transplantation model, oral administration of AS2761325 monotherapy inhibited DSA production without affecting graft survival. When combined with a suboptimal dose of tacrolimus, AS2761325 significantly prolonged graft survival. The more potent Cat S inhibitor AS2863995 also prolonged graft survival and almost completely suppressed DSA production. These results suggest that Cat S inhibitors may be promising ABMR prophylaxis drug candidates. Combination therapy comprising a Cat S inhibitor and calcineurin inhibitors may be a more effective immunosuppressive maintenance therapy for controlling both cell-mediated and antibody-mediated rejection.

OR35 A novel mixed lymphocyte culture method to evaluate donor HLA specific immunity in different HLA type mismatched donors and recipients

Aim A novel flow cytometry based mixed lymphocyte culture assay (Flow Cellular Crossmatch: Flow-CXM) was developed for detecting antigen or donor specific T cell immunity. The preliminary study demonstrated that the donor specific T cell response in recipient was correlated with HLA mismatch (MM) numbers between donors & recipients. The aim of this study is to further evaluate the significance of Flow-CXM in different HLA type MM donors and recipients. Methods CXM (Fig. 1): 0.1 × 106 irradiated donor PBMCs were incubated with 0.1 × 106 recipient’s PBMCs and Th1 (IFNγ & IL-2) or Th2 (IL-4, 5, 10) cytokine capture beads for 16 h. The cells were lysed and the captured cytokines were detected by corresponding PE-labeled 2nd antibodies. The beads with different fluorescence were gated and PE stained beads (Flowspot %) were counted to represent the relative number of donor specific responding T cells. HLA Typing High-resolution HLA typing of all donor and recipient samples were performed by NGS or SSO. Results A total 109 paired bone marrow donor and recipient samples were tested by Flow-CXM. Compared to the HLA identical group, IL-2 but not IFNγ response significantly increased in HLA-class I (CI) (P Conclusions The newly developed Flow-CXM is able to detect earlier T cell activation of donor HLA specific immunity in recipient within 24 h. The multiple T cell activation markers (Th1/Th2 cytokines) can be measured in a single reaction. The methodology of Flow-CXM is simple, sensitive, and HLA specific. It hold a potential to be used in clinical transplantation for predicting and monitoring GVHD/GVL in BMT and cellular rejection in solid organ transplantation. Download : Download high-res image (404KB) Download : Download full-size image

P100 Complete single mica gene sequencing-based typing using next generation sequecers

Aim The MICA gene located in the HLA gene cluster region close to HLA-B is highly polymorphic. It is associated with solid organ transplant rejection and bone marrow transplant GVHD. To test a novel Next Generation Sequencing (NGS) based MICA high resolution typing method, we used both Sanger Based Typing (SBT) and NGS in a parallel double blind study. Methods Genomic DNA samples from 54 healthy Han people in southern China were genotyped by both SBT and NGS methods. Well established MICA SBT was performed using an amplicon covering the MICA gene from exon 2 to exon 5 (2.2kb) and MICA-Seq specific software. NGS was performed with a PCR product that includes the entire length of the MICA gene from mid 5’UTR to the end of the 3’UTR (12.6 Kb). Library preparation was performed with the standard library preparation method and reagents from Omixon’s V2 Holotype HLA kits and sequenced on an Illumina MiSeq using paired-end 2x250 bp V2 chemistry. MICA typing was assigned using Omixon Twin software and GenDx’s NGSEngine. Results 2.04 million reads with an average quality score ⩾Q30 were generated. 93.3% of reads produced were high quality and used for MICA consensus assembly with an average coverage depth of 300 across the exonic regions. 26 MICA genotypes consisting of 17 unique alleles among 54 samples were identified, and were 100% concordant between NGS and SBT. MICA NGS was able to resolve all ambiguities obtained by MICA SBT. Alleles initially typed as ambiguous MICA*008:01/04 by SBT were determined to be MICA*008:01:01, *008:01:02 and *008:04. MICA*008:04 was the most frequent at 19.4% (21/108 alleles) by NGS. In addition, 4 samples typed ambiguously as MICA*002:01/MICA*008 or MICA*023/MICA*035, and 3 samples typed as MICA*002:01/MICA*010 or MICA*022/MICA*025 by SBT were resolved by NGS. Furthermore a new *008:01 allele was found with 5 differences in intron 1 and 1 difference in intron 5. This new allele was represented three times in the population of 108 allele calls. Additionally we were able the complete the sequencing of exon 1 for *009:01 and *033 and exon 6 for *007:01 and *033 alleles. Conclusions A NGS based MICA typing method has been established and is capable of resolving ambiguities observed with SBT, identifies new alleles and allows the completion of IMGT reported incomplete allele sequences.

Heparan sulfate is a plasma biomarker of acute cellular allograft rejection.

Despite advances in management of immunosuppression, graft rejection remains a significant clinical problem in solid organ transplantation. Non-invasive biomarkers of graft rejection can facilitate earlier diagnosis and treatment of acute rejection. The purpose of this study was to investigate the potential role of heparan sulfate as a novel biomarker for acute cellular rejection. Heparan sulfate is released from the extracellular matrix during T-cell infiltration of graft tissue via the action of the enzyme heparanase. In a murine heart transplant model, serum heparan sulfate is significantly elevated during rejection of cardiac allografts. Moreover, expression of the enzyme heparanase is significantly increased in activated T-cells. In human studies, plasma heparan sulfate is significantly elevated in kidney transplant recipients with biopsy-proven acute cellular rejection compared to healthy controls, recipients with stable graft function, and recipients without acute cellular rejection on biopsy. Taken together, these findings support further investigation of heparan sulfate as a novel biomarker of acute cellular rejection in solid organ transplantation.

Visualizing the Rapid and Dynamic Elimination of Allogeneic T Cells in Secondary Lymphoid Organs.

Allogeneic organ transplants are rejected by the recipient immune system within several days or weeks. However, the rejection process of allogeneic T (allo-T) cells is poorly understood. In this study, using fluorescence-based monitoring and two-photon live imaging in mouse adoptive transfer system, we visualized the fate of allo-T cells in the in vivo environment and showed rapid elimination in secondary lymphoid organs (SLOs). Although i.v. transferred allo-T cells efficiently entered host SLOs, including lymph nodes and the spleen, ∼70% of the cells had disappeared within 24 h. At early time points, allo-T cells robustly migrated in the T cell area, whereas after 8 h, the numbers of arrested cells and cell fragments were dramatically elevated. Apoptotic breakdown of allo-T cells released a large amount of cell debris, which was efficiently phagocytosed and cleared by CD8+ dendritic cells. Rapid elimination of allo-T cells was also observed in nu/nu recipients. Depletion of NK cells abrogated allo-T cell reduction only in a specific combination of donor and recipient genetic backgrounds. In addition, F1 hybrid transfer experiments showed that allo-T cell killing was independent of the missing-self signature typically recognized by NK cells. These suggest the presence of a unique and previously uncharacterized modality of allorecognition by the host immune system. Taken together, our findings reveal an extremely efficient and dynamic process of allogeneic lymphocyte elimination in SLOs, which could not be recapitulated in vitro and is distinct from the rejection of solid organ and bone marrow transplants.

SYK Inhibition Induces Apoptosis in Germinal Center-Like B Cells by Modulating the Antiapoptotic Protein Myeloid Cell Leukemia-1, Affecting B-Cell Activation and Antibody Production.

B cells play a major role in the antibody-mediated rejection (AMR) of solid organ transplants, a major public health concern. The germinal center (GC) is involved in the generation of donor-specific antibody-producing plasma cells and memory B cells, which are often poorly controlled by current treatments. Myeloid cell leukemia-1 (Mcl-1), an antiapoptotic member of the B-cell lymphoma-2 family, is essential for maintenance of the GC reaction and B-cell differentiation. During chronic AMR (cAMR), tertiary lymphoid structures resembling GCs appear in the rejected organ, suggesting local lymphoid neogenesis. We report the infiltration of the kidneys with B cells expressing Mcl-1 in patients with cAMR. We modulated GC viability by impairing B-cell receptor signaling, by spleen tyrosine kinase (SYK) inhibition. SYK inhibition lowers viability and Mcl-1 protein levels in Burkitt’s lymphoma cell lines. This downregulation of Mcl-1 is coordinated at the transcriptional level, possibly by signal transducer and activator of transcription 3 (STAT3), as shown by (1) the impaired translocation of STAT3 to the nucleus following SYK inhibition, and (2) the lower levels of Mcl-1 transcription upon STAT3 inhibition. Mcl-1 overproduction prevented cells from entering apoptosis following SYK inhibition. In vitro studies with primary tonsillar B cells confirmed that SYK inhibition impaired cell survival and decreased Mcl-1 protein levels. It also impaired B-cell activation and immunoglobulin G secretion by tonsillar B cells. These findings suggest that the SYK–Mcl-1 pathway could be targeted, to improve graft survival by manipulating the humoral immune response.

The Role of the Endothelium during Antibody-Mediated Rejection: From Victim to Accomplice.

Antibody-mediated rejection (AMR) of solid organ transplants is characterized by the activation and injury of the allograft endothelium. Histological and transcriptomic studies have associated microvascular inflammation and endothelial lesions with the severity of rejection and poor graft outcomes. The allograft endothelium forms the physical barrier between the donor organ and the recipient; this position directly exposes the endothelium to alloimmune responses. However, endothelial cells are not just victims and can actively participate in the pathogenesis of rejection. In healthy tissues, the endothelium plays a major role in vascular and immune homeostasis. Organ transplantation, however, subjects the endothelium to an environment of inflammation, alloreactive lymphocytes, donor-specific antibodies, and potentially complement activation. As a result, endothelial cells become activated and have modified interactions with the cellular effectors of allograft damage: lymphocytes, natural killer, and myeloid cells. Activated endothelial cells participate in leukocyte adhesion and recruitment, lymphocyte activation and differentiation, as well as the secretion of cytokines and chemokines. Ultimately, highly activated endothelial cells promote pro-inflammatory alloresponses and become accomplices to AMR.