Donor-specific Abs Research Articles

Tregs integrate native and CAR-mediated costimulatory signals for control of allograft rejection.

Tregs expressing chimeric antigen receptors (CAR-Tregs) are a promising tool to promote transplant tolerance. The relationship between CAR structure and Treg function was studied in xenogeneic, immunodeficient mice, revealing advantages of CD28-encoding CARs. However, these models could underrepresent interactions between CAR-Tregs, antigen-presenting cells (APCs), and donor-specific Abs. We generated Tregs expressing HLA-A2-specific CARs with different costimulatory domains and compared their function in vitro and in vivo using an immunocompetent model of transplantation. In vitro, the CD28-encoding CAR had superior antigen-specific suppression, proliferation, and cytokine production. In contrast, in vivo, Tregs expressing CARs encoding CD28, ICOS, programmed cell death 1, and GITR, but not 4-1BB or OX40, all extended skin allograft survival. To reconcile in vitro and in vivo data, we analyzed effects of a CAR encoding CD3ζ but no costimulatory domain. These data revealed that exogenous costimulation from APCs can compensate for the lack of a CAR-encoded CD28 domain. Thus, Tregs expressing a CAR with or without CD28 are functionally equivalent in vivo, mediating similar extension of skin allograft survival and controlling the generation of anti-HLA-A2 alloantibodies. This study reveals a dimension of CAR-Treg biology and has important implications for the design of CARs for clinical use in Tregs.

Lipophilic Statins Inhibit YAP Nuclear Localization, Coactivator Activity, and Migration in Response to Ligation of HLA Class I Molecules in Endothelial Cells: Role of YAP Multisite Phosphorylation.

Solid-organ transplant recipients exhibiting HLA donor-specific Abs are at risk for graft loss due to chronic Ab-mediated rejection. HLA Abs bind HLA molecules expressed on the surface of endothelial cells (ECs) and induce intracellular signaling pathways, including the activation of the transcriptional coactivator yes-associated protein (YAP). In this study, we examined the impact of lipid-lowering drugs of the statin family on YAP localization, multisite phosphorylation, and transcriptional activity in human ECs. Exposure of sparse cultures of ECs to cerivastatin or simvastatin induced striking relocalization of YAP from the nucleus to the cytoplasm and inhibited the expression of the YAP/TEA domain DNA-binding transcription factor-regulated genes connective tissue growth factor and cysteine-rich angiogenic inducer 61. In dense cultures of ECs, statins prevented YAP nuclear import and expression of connective tissue growth factor and cysteine-rich angiogenic inducer 61 stimulated by the mAb W6/32 that binds HLA class I. Exposure of ECs to either cerivastatin or simvastatin completely blocked the migration of ECs stimulated by ligation of HLA class I. Exogenously supplied mevalonic acid or geranylgeraniol reversed the inhibitory effects of statins on YAP localization either in low-density ECs or high-density ECs challenged with W6/32. Mechanistically, cerivastatin increased the phosphorylation of YAP at Ser127, blunted the assembly of actin stress fiber, and inhibited YAP phosphorylation at Tyr357 in ECs. Using mutant YAP, we substantiated that YAP phosphorylation at Tyr357 is critical for YAP activation. Collectively, our results indicate that statins restrain YAP activity in EC models, thus providing a plausible mechanism underlying their beneficial effects in solid-organ transplant recipients.

Detection of Antibodies Against Human Leukocyte Antigen Class II in the Sera of Patients Receiving Intravenous Immunoglobulin.

Anti-HLA Ab titers were determined in 4 types of IVIG preparations, fresh frozen plasma, and the sera of 11 patients with hematological diseases before and after IVIG administration. Although anti-HLA Abs were not detected in any of the fresh frozen plasma products, various anti-HLA class I and II Abs were detected in all 4 IVIG preparations. Six out of 11 patients who had received IVIG showed a low titer of anti-HLA class II Abs, which were not detected before IVIG administration. Conversely, no anti-HLA class I Abs were detected in any of the 11 patients. Furthermore, all 4 (100%) patients who were positive for anti-HLA class II Abs initially and were assessable became negative for anti-HLA Abs after the discontinuation of IVIG treatment (median, d 79; range, d 22-192). IVIG preparations consist of high-titer anti-HLA class I and II Abs, but the latter can be transiently detected in the sera of patients who had received IVIG. When these patients are screened for the presence of donor-specific Abs, some may be incorrectly deemed positive for HLA class II Abs. Thus, caution is necessary when only donor-specific Abs specific to class II HLAs are detected in patients.

Correlation analysis between virtual and Complement-Dependent-Cytotoxicity crossmatch in a monocenter retrospective series of 118 allografted patients

Purpose of the StudyThe detection of patients' anti-HLA antibodies before allogeneic hematopoietic stem cell transplantation (HSCT) may affect post-transplant outcome, due to a potential detrimental impact on engraftment or toxicity-related issues. Crossmatch (XM) techniques provide support to physicians during the pre-transplant phase but the role of Complement-Dependent Cytotoxicity XM (CDC-XM) is not well-defined when performed routinely and in parallel with the virtual XM. Patients and MethodsWe report here our experience with both virtual and CDC-XM tests on n = 118 patients undergoing search for a donor other than HLA-identical sibling from July 2013 to June 2018 at our Institution. When anti-HLA antibodies (Abs) were present, they were classified as donor-specific Abs (DSA) or non-DSA. ResultsOn the n = 118 patients, n = 35 (29.7 %) had a positive virtual XM test (of which one of more DSA were found in n = 8; 6.8 %) and n = 5 had a positive CDC-XM test. These latter, positive for HLA class II only, were interpreted as false-positive results due to prior administration of anti-CD20 to the patients, all affected by lymphoma; none of them had a positive virtual XM for anti-HLA Abs of class II. Importantly, all these patients successfully engrafted, further supporting the lack of significant impact of CDC-XM positive results in this series. ConclusionsAccording to our data on more than a hundred patients, routinely performed CDC-XM does not seem to add significant information with respect to virtual XM. We cannot exclude the usefulness of CDC-XM in specific situations, although a positive CDC-XM result was an unfrequent event.

Post-Transplant Outcome of the Highly Sensitized Patient Awaiting Heart Transplant Treated with Desensitization

<h3>Purpose</h3> Approximately 30% of patients (pts) awaiting heart transplantation (HT) develop anti-HLA antibodies (Abs) due to sensitizing events. Highly sensitized pts have peak Panel Reactive Antibody (PRA) levels≥80%. We assessed the efficacy of desensitization therapy (DTx) on these pts awaiting HT. <h3>Methods</h3> Between 2010-19, we assessed 78 pts awaiting HT with peak PRA≥80% (mean peak PRA 91.6%, range 80-100%). These pts received DTx with intravenous immunoglobulin (IVIG) alone (n=18), rituximab-based therapy (n=5), plasma exchange (PE) alone (n=15), bortezomib-based therapy (BTZ)(n=30), and finally combination therapy with rituximab/BTZ (n=-10). Peak PRAs (mean fluorescent intensity, MFI) were obtained before and after treatment. All pts received ATG induction and IVIG with eculizumab given to pts crossing high level donor-specific Ab (DSA). 3-year outcomes including survival, freedom from cardiac allograft vasculopathy (CAV, stenosis ≥30%), freedom from non-fatal major adverse cardiac events (NF-MACE: myocardial infarction, new onset heart failure, percutaneous coronary intervention, cardioverter defibrillator/pacemaker implant, stroke), and 1-year freedom from rejection [acute cellular rejection (ACR), Ab-mediated rejection (AMR)] were obtained. A group of non-sensitized pts without pre-HT anti-HLA Abs was used as control. <h3>Results</h3> With each DTx, peak PRA was reduced (table). Post-HT, there was lower freedom from 1-year AMR in the highly sensitized pt groups compared to the control group. 3-year survival, freedom from CAV and NF-MACE were similar among all groups. 26 highly sensitized pts received eculizumab perioperatively for crossing high level DSA. <h3>Conclusion</h3> Although there is variability in how highly sensitized pts respond, pre-HT DTx appears beneficial and enables pts to undergo successful HT. Further studies and longer follow-up are needed to identify the optimal therapy for each highly sensitized individual.

HLA Antibody Titer and C1Q Reactivity Reflect Response to Desensitization and Facilitate Donor Selection

Purpose Recent reports indicate that >50% of pediatric heart transplant (HT) candidates are HLA-sensitized at time of HT. Clinicians have a difficult choice: wait for an ideal organ offer without HLA antibody (Ab) or take the first acceptable offer. Desensitization can be useful to facilitate HT by decreasing or abrogating HLA Ab. We have evolved to use desensitization to evaluate which HLA Ab may be safely crossed and which to avoid. To illustrate our approach, we present: a non-responder, a partial-responder, and a total-responder. Methods HLA Ab analysis was done by Luminex® single antigen bead assay (SAB), with serial dilutions and C1q-fixing assay. Ab ≥2000 MFI by IgG-SAB and ≥500 MFI by C1q-SAB were used to determine the calculated PRA (cPRA). One desensitization cycle consisted of rituximab (x1), bortezomib (BTZ) (x4), and plasmapheresis (x5). Cycles are repeated leaving 2 weeks between last and first doses of each cycle. Results Non-responder (Fig 1A): Bw4/Aw4 sensitized patient did not respond to desensitization. Ab reassessment showed rebound, and after the 3rdcycle the titer increased and Ab was C1q+. The patient received HT with a Bw6/Bw6 donor without Aw4 antigens; no donor specific Ab (DSA) is detected 1 year post-HT. Partial-responder (Fig 1B): selective response for 1 HLA Ab group. Both anti-DR53 and DQ7/DQ8/DQ9 Abs were C1q+. However, DQ7/DQ8/DQ9 Ab had lower baseline titer. After 2 cycles, DR53 Ab did not change while DQ7/DQ8/DQ9 Ab was reduced in titer and became C1q-. HT proceeded with a DQ7+ donor and DSA remains negative 2.5 years post-HT. Total responder (Fig 1C): Ab to all but self HLA. A complete response was seen; by the end of 3rdcycle, most Ab was reduced to low titer and C1q-. HT was done crossing 2 C1q- Abs. No DSA detected 118 days post-HT. Conclusion Multi-dimensional testing of HLA Ab titer and C1q binding and serial monitoring pre- and post-desensitization helps to determine if more treatment is needed and allows for safe HT risk assessment when crossing HLA Abs.

De Novo Development of Antibodies to Cardiac Myosin and Vimentin in Pediatric Heart Transplant Recipients: A Single Center Pilot Study

Purpose Pre and post heart transplant (Tx) HLA antibody (ab) surveillance has become routine. Recent studies show a relationship between donor specific HLA abs (DSA), rejection and coronary vasculopathy (CAV). Little is known about the frequency of non HLA abs to cardiac self antigens (myosin, vimentin) in pediatric heart tx recipients. Aim of this pilot study is to characterize the frequency of non HLA abs in a cohort of pediatric heart tx pts and begin to explore their association with clinical events. Methods Residual sera from 30 post tx HLA ab studies from 12 pts were available. Abs to Vimentin (Anti-V) and cardiac myosin (Anti-M) were determined using an ELISA developed in our lab. Positive (pos) values were based upon a standard curve with known concentrations of myosin or vimentin. Values > 300 ng/ml for Vimentin and > 140 ng/ml for myosin were deemed pos which is 2 standard dev from negative sera. Additionally, pre tx samples were available from 10/12 pts for ab analysis. Clinical events: rejection, cav and death were recorded along with HLA ab results from each sample. Results HLA ab sensitization was noted in pre tx sera from 5/12 (PRA 3-64%) children whereas only 1/10 had sensitization to non HLA abs prior to tx. Of the 30 post tx samples, 27 were pos for Anti-V and 28 for Anti-M; while only 7 were pos for DSA. Of those with DSA, 5 had MFI 5K. Clinically, 4 pts had no rejection, 3 had pAMR1i+ only and 5 had CR ≥ 2R and/or pAMR 2 or 3. Three pts developed CAV, two of whom died. Anti-V and Anti-M abs had a moderate correlation (R=0.649) with each other and there was a trend toward higher levels of Anti-V over time in pts with CAV and death. Table 1 includes summary data on all 12 pts; Anti-V and Anti-M ab levels are shown in tertiles. Conclusion De novo Anti-V and Anti-M abs are common post pediatric heart tx and not related to DSA. These abs may be implicated as contributing factors to detrimental clinical events post tx. The significance and prevalence of these abs post heart tx warrant further study.

Early Acute Microvascular Kidney Transplant Rejection in the Absence of Anti-HLA Antibodies Is Associated with Preformed IgG Antibodies against Diverse Glomerular Endothelial Cell Antigens.

Although anti-HLA antibodies (Abs) cause most antibody-mediated rejections of renal allografts, non-anti-HLA Abs have also been postulated to contribute. A better understanding of such Abs in rejection is needed. We conducted a nationwide study to identify kidney transplant recipients without anti-HLA donor-specific Abs who experienced acute graft dysfunction within 3 months after transplantation and showed evidence of microvascular injury, called acute microvascular rejection (AMVR). We developed a crossmatch assay to assess serum reactivity to human microvascular endothelial cells, and used a combination of transcriptomic and proteomic approaches to identify non-HLA Abs. We identified a highly selected cohort of 38 patients with early acute AMVR. Biopsy specimens revealed intense microvascular inflammation and the presence of vasculitis (in 60.5%), interstitial hemorrhages (31.6%), or thrombotic microangiopathy (15.8%). Serum samples collected at the time of transplant showed that previously proposed anti-endothelial cell Abs-angiotensin type 1 receptor (AT1R), endothelin-1 type A and natural polyreactive Abs-did not increase significantly among patients with AMVR compared with a control group of stable kidney transplant recipients. However, 26% of the tested AMVR samples were positive for AT1R Abs when a threshold of 10 IU/ml was used. The crossmatch assay identified a common IgG response that was specifically directed against constitutively expressed antigens of microvascular glomerular cells in patients with AMVR. Transcriptomic and proteomic analyses identified new targets of non-HLA Abs, with little redundancy among individuals. Our findings indicate that preformed IgG Abs targeting non-HLA antigens expressed on glomerular endothelial cells are associated with early AMVR, and that in vitro cell-based assays are needed to improve risk assessments before transplant.

Pre-Existing Maternal Antibodies Cause Rapid Prenatal Rejection of Allotransplants in the Mouse Model of In Utero Hematopoietic Cell Transplantation.

In utero hematopoietic cell transplantation (IUHCT) is a nonmyeloablative nonimmunosuppressive alternative to postnatal hematopoietic stem cell transplantation for the treatment of congenital hemoglobinopathies. Anti-HLA donor-specific Abs (DSA) are associated with a high incidence of graft rejection following postnatal hematopoietic stem cell transplantation. We determine if DSA present in the mother can similarly cause graft rejection in the fetus following IUHCT. Ten million C57BL/6 (B6, H2kb) bone marrow cells were transplanted in utero into gestational day 14 BALB/c (H2kd) fetuses. The pregnant BALB/c dams carrying these fetuses either had been previously sensitized to B6 Ag or were injected on gestational days 13-15 with serum from B6-sensitized BALB/c females. Maternal-fetal Ab transmission, Ab opsonization of donor cells, chimerism, and frequency of macrochimeric engraftment (chimerism >1%) were assessed by flow cytometry. Maternal IgG was transmitted to the fetus and rapidly opsonized donor cells following IUHCT. Donor cell rejection was observed as early as 4 h after IUHCT in B6-sensitized dams and 24 h after IUHCT in dams injected with B6-sensitized serum. Efficient opsonization was strongly correlated with decreased chimerism. No IUHCT recipients born to B6-sensitized dams or dams injected with B6-sensitized serum demonstrated macrochimeric engraftment at birth compared with 100% of IUHCT recipients born to naive dams or dams injected with naive serum (p < 0.001). In summary, maternal donor-specific IgG causes rapid, complete graft rejection in the fetus following IUHCT. When a third-party donor must be used for clinical IUHCT, the maternal serum should be screened for DSA to optimize the chance for successful engraftment.

P157 Virtual crossmatch can be used successfully as pre-transplant crossmatch for most deceased donor transplants at a high volume center

Aim We developed a reliable virtual crossmatch (VXM) method for use as pre-transplant XM for deceased donor (DD) kidney transplantation (Ktx). Here we report the outcomes of 248 Ktx performed based on VXM. Methods Our VXM method includes 3 integral processes: accurate identification of HLA antibodies (Ab) using right tools and principles, assessment of correlation between the strength of donor specific Ab (DSA) and T and B cell bindings by flow XM (FXM), and listing of clinically relevant unacceptable antigens that will not impact DD offers or graft survival. From the start of the new KAS through Dec. 2016, 434 DD-Ktx were performed at UCSF, of which 375 (86%) were performed based on VXM (350 were VXM−ve with DSA+/− and 25 were VXM+ve with DP/DQα DSA+). 59/434 (14%) of the Ktxs were performed based on pre-tx FXM (54 were FXM−ve with DSA+/− and 5 were FXM+ve with DP-DSA+). Six month protocol biopsy (Bx) and cause Bx were evaluated for C4d rejection. Results All 350 pre-tx VXM−ve assessments were negative by retrospective FXM. There were no hyper acute rejections in these 350 Ktx. Biopsies of 273 negative VXM-based Ktx revealed no significant difference in the rate of antibody-mediated rejection (ABMR) or in acute cellular rejection (ACR) between 3 distinct CPRA groups (Table). Females, re-tx, well-matched tx, and de nova DSA producers were more frequent in both high CPRA groups compared to 0–79% CPRA group. Pre-tx DSA was more common in 99–100% CPRA group compared to other groups. Conclusions Our VXM protocol is reliable, has excellent graft survival, and works even for those with 100% CPRA. Accurate VXM method improves organ allocation for broadly sensitized patients, increases the efficiency of regional/national sharing, and avoids reallocation into unintended recipients. Download : Download high-res image (137KB) Download : Download full-size image

P120 Preventing false positive crossmatches due to rituximab by using anti rituximab antibody

Aim Rituximab (Rtx) is a humanized monoclonal antibody (Ab) to CD20 receptor expressed on B cells. Sera from patients who have received Rtx will have positive B-cell crossmatches (XMs) challenging the detection of clinically relevant Abs. There is a commercially available Ab advertised as blocking the binding of Rtx to CD20. We wanted to test if the addition of this anti-Rituximab (anti-Rtx) Ab to our XMs will prevent false positive (FP) results, allowing the detection of potential donor specific Abs (DSA). Methods Negative control (NCS) and patient sera were spiked or not with Rit. Samples were incubated or not with anti-Rtx Ab (MB2A4, GeneTex) for 20 min. at known concentrations. These mixtures were used for XMs (flow cytometric (FC) and complement dependent cytotoxic (CDC)). Our FCXM uses a 256 channel scale. B-cell FCXM MCS above 9 is weak positive and above 20 is positive. Results Addition of anti-Rtx blocked Rtx binding to B-cells: NCS spiked with Rtx causes positive B-cell XMs, which were negative in the presence of anti-Rtx (Table 1.A). Anti-Rtx prevented FP B-cell XMs in two samples from patients known to have received Rtx (Table 1.B). Anti-Rtx blocks Rtx in a dose-dependent manner. Anti-Rtx allowed the detection of DSA: A serum with anti-DQ2 Abs (4000 MFI) was spiked with Rtx and incubated with anti-Rtx before crossmatching with cells expressing DQ2 antigen or not. The addition of anti-Rtx converted the XM with DQ2-negative cells to negative, but the XM with DQ2-positive cells was weakly positive, as was the XM performed with untreated serum (Table 2). Conclusions Treatment of the sera of patients under Rtx immunotherapy with anti-Rtx before FC and CDC XMs prevents FP results and allow the detection of clinical relevant Abs. Download : Download high-res image (183KB) Download : Download full-size image Download : Download high-res image (97KB) Download : Download full-size image

Virtual Crossmatch Permits Kidney Sharing; Avoiding Transplant into Unintended Recipients

Aim The US Kidney Allocation System successfully exposes highly sensitized patients (pts) with a cPRA of 99 or 100% to deceased donor offers through regional and national sharing. Frequently a positive physical cross match (PXM) occurs while a kidney is being transported, requiring subsequent reallocation. It was estimated that 19.2% of kidneys transported between regions are eventually transplanted (tx) into an unintended recipient(UR), most commonly due to a positive crossmatch (AJT 2017, 17:2139). Using a virtual cross match (VXM) strategy, we aimed to tx highly sensitized pts with kidneys from other regions, without reallocating to an UR. Methods We developed a VXM strategy to avoid a positive PXM. Our VXM method includes 3 integral processes: accurate identification of HLA antibodies (Ab) using right tools and scientific principles on quarterly samples, assessment of correlation between the strength of donor specific HLA Ab (DSA) and the level of T/B cell bindings by flow XM (PXM), and listing of clinically relevant unacceptable antigens that will not impact donor offers or graft survival. When a kidney is offered a VXM is performed, and likelihood of positive PXM is estimated by the HLA lab director. If there is concern due to recent transfusions or infections treated with antibiotics a PXM is done when the pt arrives for tx, but is only required in a minority of cases. Records were reviewed of all kidneys shipped from another region since Dec, 2014 to identify kidneys not used in the intended recipient from Dec, 2014 to Sept, 2017. Results 252 kidneys were shipped to our center from another region based on the negative VXM (Fig 1). Of these imported kidneys 210 (83.3%) were transplanted without a pre-tx PXM, and the remaining 42(16.7%) required a PXM on admission, most commonly necessitated by a recent blood transfusion. 109 pts with 100% cPRA had kidneys imported, and 93 (85.3%) were transplanted after VXM alone. 7 kidneys went to UR, but only 3 (1.2%) were due to a pos PXM or new DSA from recent blood transfusion. The other 4 kidneys went to UR because the intended recipient had a new medical problem upon admission. Mean creatinine at 1-year was 1.30 for pts at highest immunologic risk(cPRA 100%), and 1.31(p=NS) for all other DDRT recipients. VXM alone was used in 114(86.3%) of females, and 92(79%) of re-transplant pts. Consistent with the neg VXM, retrospective PXM performed by flow cytometry for all recipients were negative (Fig 2). Conclusions Sharing is possible, and late reallocations can be almost entirely avoided with a strategy that relies heavily on VXM, without limiting access for highly sensitized pts on the waitlist. A centralized VXM within the allocation system would reduce transplantation into unintended recipients significantly. More frequent antibody screening, especially following blood transfusions and infections would further reduce the need for a pre-tx PXM.

Pre-transplant AT1R antibodies correlate with early allograft rejection

Studies investigating the potential pathogenic effects of non-HLA antibodies (Ab) have identified Ab against the angiotensin II type 1 receptor (AT1R-Ab) as a risk factor for rejection and kidney graft loss. This study sought to validate the risk of AT1R-Ab for acute rejection and to explore the role of other non-HLA Abs in this capacity.Pre- and post-transplant sera from a cohort of 101 patients (n=453 samples total) were tested for AT1R-Ab and other non-HLA Ab using a commercially available ELISA kit and the Luminex platform, respectively.Patients positive for pre-transplant AT1R-Ab were more likely to develop de novo donor-specific Ab (dnDSA) compared to patients that were negative for AT1R-Ab (28% vs 10%, p=0.027). Pre-transplant positivity for AT1R-Ab was associated with TCMR in the first year post-transplant (p=0.034), but did not predict graft loss independent of dnDSA (p=0.063). AT1R-Ab positivity was significantly associated with positivity for Ab against the endothelin A type 1 receptor (ETAR-Ab) inclusive of all study time points (p=0.0021).Given the high prevalence of AT1R-Ab pre-transplant (20%) and its association with dnDSA and early TCMR, a prospective study to determine if more intense immunosuppression and/or AT1R blockade has an impact on outcomes in these patients is warranted.

P004 The challenge of virtual crossmatching in the presence of weak donor specific antibodies

Aim Our laboratory uses a normalized 2500 MFI cut off in the Luminex single-antigen bead assay to define unacceptable antigens. Antibodies (Abs) between 1000 and 2500 MFI are considered weak positive, but are not used to strictly rule out potential donors. The challenge is to accurately predict flow crossmatch (FXM) results in the presence of weak donor-specific Abs (DSA). Method We identified 62 FXM performed in the presence of weak DSA. For simplicity we considered only the B-cell FXM results. We analyzed the data based on the DSA MFI values or DSA epitope profile (DSA against shared epitopes (SE), DSA against a single antigen (Ag), and multiple weak DSA (M)). Results 39/62 (64%) were positive and 22/62 (36%) were negative. All (10/10) FXM with multiple weak DSA were positive. The majority (13/17) of FXM in the presence of weak DSA against a single antigen (Ag) were negative (76%). On the other hand, most (25/34) FXM with weak DSA that shares epitopes with other antigens (SE) were positive (74%). Table 1 summarizes these results. When we analyzed the FXM results based on DSA MFI (excluding multiple DSA cases), we observed that DSA with MFI of 1,000-2,500 resulted in a positive FXM in 50% of cases (Table 2). Unexpectedly, we got more positive FXM (14) than negative (7) when the DSA was under 1,000 MFI. Further analysis revealed that 13/14 of these positive FXM were caused by Abs with reactivity to SE and in particular Abs against HLA-B and -DRB1. Conclusion MFI values of weak DSA are not a reliable predictor of FXM results. When performing a virtual XM in the presence of weak DSA, the Ab profile is a better predictor for FXM reactivity (PPV for DSA with SE is 73.5, independent of the MFI value). Abs that target broadly-shared epitopes frequently cause positive FXM. Download high-res image (105KB) Download full-size image Download high-res image (104KB) Download full-size image

Pretransplant human leukocyte antigen antibodies detected by single-antigen bead assay are a risk factor for long-term kidney graft loss even in the absence of donor-specific antibodies.

Clinical relevance of ELISA- and single-antigen bead assay (SAB)-detected pretransplant HLA antibodies (SAB-HLA-Ab) for kidney graft survival was evaluated retrospectively in 197 patients transplanted between 2002 and 2009 at the University Clinic Frankfurt. Having adjusted for retransplantation and delayed graft function, a significantly increased risk for death-censored graft loss was found in patients with pretransplant SAB-HLA-Ab [HR: 4.46; 95% confidence interval (CI): 1.47-13.48; P = 0.008]. The risk for increased graft loss was also significant in patients with pretransplant SAB-HLA-Ab but without SAB-detected donor-specific Ab (SAB-DSA) (HR: 4.91; 95% CI of 1.43-16.991; P = 0.012). ELISA was not sufficient to identify pretransplant immunized patients with an increased risk for graft loss. In immunized patients, graft loss was predominantly present in patients who received transplants with a mismatch on the HLA-DR locus. In conclusion, even if our study is limited due to small sample size, the results show an increased risk for long-term graft loss in patients with pretransplant SAB-HLA, even in the absence of DSA. SAB-HLA-Ab-positive patients, being negative in ELISA or CDC assay, might profit from a well-HLA-DR-matched graft and intensified immunosuppression.

(768) - Proliferation Signal Inhibitors Prevent Donor-Specific Antibody Production in Sensitized Patients after Heart Transplantation

Everolimus and sirolimus are proliferation signal inhibitors (PSI) reported to decrease incidence and progression of transplant coronary artery disease, incidence of cardiac rejection, and development of de novo circulating antibodies (Abs) in the 1st year after heart transplant (Htx). Donor-specific Abs (DSA) appear to be a main factor associated with poor outcome in sensitized patients post-Htx. It is not established whether PSIs affect development of DSA in sensitized patients after Htx. Between 2010 and 2014, we evaluated 78 Htx patients (pts) with circulating Abs after Htx. Several were switched from mycophenolate mofetil (MMF) to PSI including everolimus (n=19) or sirolimus (n=11) in combination with a calcineurin inhibitor (CNI). Pts with pre-existing DSA were excluded. Pts were divided into 2 groups: maintenance of MMF use ≥1-year (Group A), and PSI use ≥ 1-year (Group B). Maintenance MMF group was matched to the switch-to-PSI group for time from Htx. Assessment of circulating Abs, including development of de novo DSA at 12 months after initiation of PSI was performed. In addition, 1-year subsequent survival and freedom from subsequent DSA development, cellular rejection, antibody-mediated rejection (AMR) were recorded. Reduction of PRA over 1 year was recorded for both groups. The PSI group compared to the MMF group had significantly greater freedom from de novo DSA development over the course of 1 year (see table). There was no significant difference between study groups in 1-year subsequent survival and 1-year subsequent freedom from cellular rejection and AMR. There was no significant difference with treatment of PSIs for subsequent 12-month Class I Abs levels %PRA (38 ± 37 vs. 33 ± 34, P=0.58) or Class II Abs levels %PRA (19 ± 29 vs. 20 ± 28, p=0.81). The use of PSIs in sensitized Htx pts appears to lower de novo development of subsequent DSA compared to maintenance MMF. Further study is warranted with a larger population.Tabled 1EndpointsGroup A Mycophenolate ≥ 1-Year (N=48)Group B PSI ≥ 1-Year (N=30)P-Value1-Year Subsequent Survival after PSI or MMF Initiation100.0%100.0%1.0001-Year Subsequent Freedom from De Novo Antibody Development after PSI or MMF Initiation86.9%100.0%0.0421-Year Subsequent Freedom from Any Treated Rejection93.8%96.7%0.5721-Year Subsequent Freedom from Acute Cellular Rejection97.9%96.7%0.7431-Year Subsequent Freedom from Antibody-Mediated Rejection97.9%93.2%0.3061-Year Subsequent Freedom from Biopsy Negative Rejection97.9%100.0%0.429 Open table in a new tab

Cutting Edge: CTLA-4Ig Inhibits Memory B Cell Responses and Promotes Allograft Survival in Sensitized Recipients.

Sensitized recipients with pretransplant donor-specific Abs are at higher risk for Ab-mediated rejection than nonsensitized recipients, yet little is known about the properties of memory B cells that are central to the recall alloantibody responses. Using cell enrichment and MHC class I tetramers, C57BL/6 mice sensitized with BALB/c splenocytes were shown to harbor H-2K(d)-specific IgG(+) memory B cells with a post-germinal center phenotype (CD73(+)CD273(+)CD38(hi)CD138(-)GL7(-)). These memory B cells adoptively transferred into naive mice without memory T cells recapitulated class-switched recall alloantibody responses. During recall, memory H-2K(d)-specific B cells preferentially differentiated into Ab-secreting cells, whereas in the primary response, H-2K(d)-specific B cells differentiated into germinal center cells. Finally, our studies revealed that, despite fundamental differences in alloreactive B cell fates in sensitized versus naive recipients, CTLA-4Ig was unexpectedly effective at constraining B cell responses and heart allograft rejection in sensitized recipients.

The complement interference phenomenon as a cause of underestimation of high titer anti-HLA antibodies in a renal transplant recipient

Single antigen bead assay is the most sensitive laboratory tool for the anti-HLA antibody (Ab) identification in renal transplant recipients. However, this assay was shown to be prone to the prozone effect (PrE), giving false negative results in sera with high titer anti-HLA Ab. The ethylenediaminetetraacetic acid-EDTA’s Ca++ chelating effect on the C1 complement was described to eliminate the PrE allowing the anti-HLA Ab identification. A mean fluorescence intensity (MFI) ⩾ 2 fold increase in neat versus EDTA treated sera was considered relevant to PrE. We report a case of chronic Ab mediated rejection in a renal transplant recipient with anti-HLA-DQA1∗05:05,DQB1∗03:01 donor specific Ab (DSA), revealed after treatment of the sera with EDTA. Female patient, 36 years old, diagnosed with juvenile nephronophthisis, received a first graft from a living related donor in 1991. During the first year post transplantation the patient experienced two biopsy proven reversible rejection episodes and until 2010 she had no other evidence of graft malfunction. De novo DSA were detected in 2010, as shown on the table. In 2015, due to inconsistency between the sudden elevation of serum creatinine levels and absence of DSA, the patient’s sera were further analyzed after EDTA treatment. DSA with high MFI were detected post-EDTA treatment. The presence of DSA was confirmed with positive B-Flow crossmatch with the donor. Also, a kidney biopsy on 03/26/2015 showed chronic antibody mediated rejection with C4d deposition. Taking into account that the follow up post-transplantation is based on HLA Ab detection and identification, these results highlight the importance of using patients’ sera with EDTA treatment to abolish the PrE before the performance of single antigen bead assay, in order to detect the presence of HLA antibodies. Download : Download full-size image

Family history of graft versus host

Graft Versus Host Disease (GVHD) occurs in 20–35% of bone marrow transplants. It occurs in less than 6% of solid organ transplants, though it has been reported in liver and small intestine. GVHD occurs when donor cells, transplanted into the recipient, see the recipient cells as foreign and attack. We report two cases of GVHD in sisters who received deceased donor kidney pancreas transplants. MeML, African American (AA), 29 year old, and MiM, AA, 27 year old. Antibody (Ab) analysis on MeML, immediately prior to transplant, indicated the presence of Abs to HLA B7 and B81, not Donor Specific Abs (DSA). No HLA class II Ab was detected. The final crossmatch (XM) was negative T cell by FWA, AHG, DTT FWA and DTT AHG. However, the B cell XM was positive by all methods. Flow XMs had a T cell MCS of 26 and B cell MCS of 36. The positive cutoffs for T Cell = 40 and B Cell = 80. One month post transplant there were no HLA class I Abs but class II Abs were detected to DQB1∗06:09, 06:03 and DQ5, none DSA. MiM had no Abs to HLA class I or II except on 5/24/2014 and 11/11/2013. Antibodies to HLA B81 was identified in 2013, not DSA, and DPB1∗04:01 in 2014, the donor DP was not available. The final XM was negative for HLA class I and class II by all methods. The flow XM was also negative for both T and B cells. GVHD diagnosis was based on Short Tandem Repeat Analysis by the Molecular Diagnostic Laboratory. The HLA laboratory identified the possibility of Chimerism in MiM while performing confirmatory typing as she was being worked up for Living Related Bone Marrow Transplant with her father. MeML recovered from her GVHD and continues to do well. MiM succumbed to her GVHD, 74 days post transplant. These two cases clearly demonstrate the importance of analyzing for GVHD following kidney pancreas transplantation. Download full-size image Download full-size image

Potential hyperacute renal rejection due to non-hla antibody in a pediatric patient

Non-HLA antigen targets have been associated with hyperacute, acute, and chronic kidney allograft rejection. Although there is controversy regarding the predictive role of non-HLA antibodies (Abs) in transplant (Txp) outcome - there are cases that indicate causality between the presence of such Abs and graft loss. A 17 month old boy with end-stage kidney disease, secondary to kidney dysplasia, received a living donor Txp from his uncle in April 2014. The presence of HLA abs was tested in 5 sera samples prior to Txp with a PRA of 7% and 0% for class I and II respectively (flow PRA). Single antigen testing confirmed the absence of donor specific HLA Abs (DSA). Initial and final flow cross matches were negative, as expected. During the Txp surgery, it was noted that the kidney became firm, urine output was below expectation and the child became increasingly pressor-dependent. Subsequently, the patient had an episode of ventricular tachycardia and an exploratory laparotomy demonstrated that the Txp kidney became tense and enlarged, with thrombosis of the renal vein. The kidney was nephrectomized within 48 hours and the patient’s status improved significantly. To investigate a potential role for HLA DSA, we tested a 2 days post-Txp serum sample, confirming the absence of HLA-DSA. We also repeated Abs testing on the pre-Txp serum sample ruling out HLA-DSA as the cause of rejection. To assess potential causes for this apparently hyperacute Ab mediated rejection, pre-Txp (pre-14 days) and post-Txp (post-24 days) samples were sent out for AT1R Abs screening and donor specific endothelial cell crossmatch (XM-One). Retrospective testing of a pre- and post-Txp sera for AT1R antibody was out of range for pre-Txp sample and the post-Txp sample was positive ( > 30 Units/ml). The XM-One assay using endothelial precursors isolated from the donor as targets was strongly positive using a pre-Txp serum but negative using post-Txp serum. Approximately two month’s post-Txp, the patient developed HLA Abs, on top of the AT1R antibodies. In conclusion, this patient still requires a kidney Txp and the question is how to proceed – shall we consider only obtaining an organ from a living donor? This would allow for the necessary time to run XM-One and potentially inhibit AT1R pathway with an AT 1 receptor antagonist. What to do if no living donor becomes available?