Search

Donor human leukocyte antigen (HLA)-specific antibodies (DSA) and non-HLA antibodies can cause allograft injury, possibly leading to chronic lung allograft dysfunction (CLAD) after lung transplantation. It remains unclear whether these antibodies are produced locally in the graft or derived solely from circulation. We hypothesized that DSA and non-HLA antibodies are produced in CLAD lungs. Lung tissue was prospectively collected from 15 CLAD patients undergoing retransplantation or autopsy. 0.3 g of fresh lung tissue was cultured for 4 days without or with lipopolysaccharide or CD40L: lung culture supernatant (LCS) was sampled. Protein eluate was obtained from 0.3 g of frozen lung tissue. The mean fluorescence intensity (MFI) of DSA and non-HLA antibodies was measured by Luminex and antigen microarray, respectively. LCS from all 4 patients who had serum DSA at lung isolation were positive for DSA, with higher levels measured after CD40L stimulation (CD40L+LCS). Of these, only 2 had detectable DSA in lung eluate. MFI of non-HLA antibodies from CD40L+LCS correlated with those from lung eluate but not with those from sera. Flow cytometry showed higher frequencies of activated lung B cells in patients whose CD40L+LCS was positive for DSA (n = 4) or high non-HLA antibodies (n = 6) compared to those with low local antibodies (n = 5). Immunofluorescence staining showed CLAD lung lymphoid aggregates with local antibodies contained larger numbers of IgG+ plasma cells and greater IL-21 expression. We show that DSA and non-HLA antibodies can be produced within activated B cell-rich lung allografts. [ABSTRACT FROM AUTHOR]

Lung transplantation. like other transplants, carries a risk of graft rejection due to genetic differences between the donor and the recipient. In this paper, we focus on antibody-mediated rejection, which can cause acute and more importantly chronic graft dysfunction and subsequently shortened allograft survival. We present the case of a 46-year-old patient who, two months after lung transplantation (LTx), developed AMR manifested by the deterioration of graft function and de novo production of donor-specific antibodies (DSA): DQ3 (DQ7, DQ8. DQ9). As the patient was after left single LTx and heavily oxygen dependent a transbronchial biopsy was deemed to be high risk and it was decided to determine the clinical significance of the detected antibodies by their ability to bind complement. The test confirmed that the detected DSAs have the ability cause cytotoxicity of the transplanted organ. After treatment with methotrexate, intravenous immunoglobulin G (IVIg) and alemtuzumab, the patient's condition improved and a complete decrease in DSA was obtained. However, after a year, the production of antibodies increased sharply. Treatment with IVIg, cyclophosphamide and plasmapheresis slightly improved the patient's condition, reducing the MFI DSA values by half, but leaving them at high levels. Based on this clinical case, we discuss problems with making a diagnosis, choosing the right AMR treatment and monitoring the patient's condition during treatment. We also indicate a poor prognosis in the case of the production of DSA antibodies at tlie DQ locus. [ABSTRACT FROM AUTHOR]

Background: We have performed a retrospective analysis of anti-HLA class I MHC and class II MHC antibodies measured using a single antigen bead (SAB) assay and a panel reactive antibody (PRA) assay. Material and methods: A group of 256 patients with end-stage renal disease (ESRD) was tested for anti-HLA antibodies in the tissue typing laboratory between 2017 and 2020. In the cohort, the serum samples of patients waiting for transplantation were tested. Both the PRA and SAB tests of these patients were analyzed using the Luminex (Immucor) method. The threshold of positivity was accepted as median fluorescence intensities (MFI) ≥1000 for PRA screening and MFI ≥750 for SAB screening. Results: Overall, antibodies to HLA antigens were detected in 202 (78.9%) out of 256 patients in the PRA study. Antibodies against both class I/II antigens were detected only in 15.6% of these patients, whereas antibodies against only against class I HLA in 31.3% and only against class II HLA in 32.0%. By comparison, the SAB study found that 66.8% of patients were positive for HLA antigens. Furthermore, donor-specific antibodies (DSA) were detected in 52.0% of PRA-positive patients and 52.6% of SAB-positive patients. It was shown that 168 patients (83.2%) out of 202 PRA-positive patients were found to be SAB-positive. In addition, 51 patients negative in the SAB assay (94.4%) were also negative in the PRA assay. Statistical analysis established a significant correlation between the PRA and SAB positivity (p > 0.001). It was also shown that MFI ≥3000 PRA positivity for class I HLA antigens (p = 0.049) and MFI ≥5000 PRA positivity for class II antigens (p < 0.001) correlated with the SAB positivity in patients. Conclusion: Our results showed the importance of both PRA and SAB assays to define the status of sensitization in patients. [ABSTRACT FROM AUTHOR]

Allosensitization represents a major barrier to heart transplantation. We previously reported favorable 1-year outcomes of complement inhibition at transplant in highly sensitized recipients. We now report a longer follow-up. In this single-arm trial (NCT02013037), 20 patients with panel reactive antibodies ≥70% and preformed donor-specific antibodies received eculizumab during the first 2 months post-transplant. The primary end-point was antibody-mediated rejection ≥ pAMR2 and/or left ventricular dysfunction. The median follow-up was 4.8 years. Beyond the first year post-transplant, there were no episodes of pAMR2 or greater and no Left Ventricular (LV) dysfunction. There were 3 deaths, 1 episode of pAMR1, and 1 patient with minimal de novo cardiac allograft vasculopathy. Compared to a matched control group, we observed a nonstatistically significant benefit of eculizumab with a lower incidence of the primary end-point or death (primary end-point: hazard ratio = 0.50, 95% confidence interval = 0.15-1.67, and p = 0.26; mortality: hazard ratio = 0.51, 95% confidence interval = 0.13-2.07, and p = 0.35). Our results support the utility of complement inhibition for high-immunological-risk recipients. ClinincalTrials.gov, NCT02013037. https://clinicaltrials.gov/ct2/show/NCT02013037?term=eculizumab&cond=heart+transplantation&draw=2&rank=1 [ABSTRACT FROM AUTHOR]

Tocilizumab (TCZ), an IL-6 inhibitor, has shown promise in the treatment of donor-specific antibodies (DSA) and chronic antibody-mediated rejection (AMR) in renal transplant recipients. However, its use in lung transplantation has not been described. This retrospective case–control study compared AMR treatments containing TCZ in 9 bilateral lung transplant recipients to 18 patients treated for AMR without TCZ. Treatment with TCZ resulted in more clearance of DSA, lower recurrence of DSA, lower incidence of new DSA, and lower rates of graft failure when compared to those treated for AMR without TCZ. The incidence of infusion reactions, elevation in transaminases, and infections were similar between the 2 groups. These data support a role for TCZ in pulmonary AMR and establish preliminary evidence to design a randomized controlled trial of IL-6 inhibition for the management of AMR. [ABSTRACT FROM AUTHOR]

Pretransplant immunological assessment of a transplant donor has evolved significantly over the last few decades with the advent of testing platforms with enhanced sensitivity and varying formats. The single antigen bead assay (SAB) assay, a virtual crossmatch (vXM) is used extensively and considered the gold standard for defining donorspecific antibodies (DSA) in many parts of the World. A country like India, is however challenged by the lack of adequate representation of locally frequent HLA alleles and hence in our institution, we continue to perform a physical crossmatch (pXM) on the Complement Dependent Cytotoxicity and flow cytometry platforms alongside the SAB. We report here a case report where the discrepancy between platforms of testing have raised certain pertinent questions in our interpretation of the vXM. [ABSTRACT FROM AUTHOR]

Background: The main objective of this study was to determine the results of the cell-based assay (CDC-XM and FC-XM), and correlate with the results of solid phase assay (L-SAB). Methods. In this retrospective study, 350 prospective renal transplant recipients were tested for the presence of HLA antibodies by CDC-XM, FC-XM and L-SAB screening with their corresponding donor. Results: T-cell-FC-XM showed a sensitivity of 71.43% and a specificity of 91.50% for detecting class I L-SAB (+), while B-cell-FCXM showed a sensitivity of 94.94% and a specificity of 61.99% for detecting class II L-SAB (t). On the other hand, T-CDC-XM showed a sensitivity of 32.14% and a specificity of 98.64% for detecting class I L-SAB (-4-), while B-CDC-XM showed a sensitivity of 44.30% and a specificity of 94.83% for detecting class H L-SAB (-). In this study, the results indicated that DSA class I MFI value of 2845 and above significantly (p 50.001) correlated with T-cell-FC-XM positivity, while MFI value of 4585 and above (p 50.001) showed strong predictive accuracy of a positive T-cell-CDC-XM. However, DSA class II MFI cut-off of 1988 and above significantly (p 50.001) correlated with B-cell-FC-XM positivity, while MFI value of 5986 and above (p %0.001) showed strong predictive accuracy of a positive B-cell-CDC-XM. Conclusions: Our study showed that CDC-XM has poor sensitivity, while FC-XM has poor specificity to detect DSA. L-SAB has good correlation with T-cell-FC-XM (p < 0.0001) but not with B-cell-FC-XM (P = 0.31). DSA strength >2845 and > 1988 significantly correlated with T-cell-FC-XM positivity and B-cell-FC-XM positivity, respectively. While, a MFI value of >4585 and > 5986 significantly correlated with T-tell-CDC-XM posilivity and Bcell- CDC-XM positivity, respectively. These MFI cut-off values could serve as a surrogate marker for CDC-XMl and FC-XM tests and may help in resolving the limitations of cell-based techniques. Iii conclusion, we found that LSAB is more sensitive and specific than CDC-XM and FC-XM and therefore may be used as a test of choice. [ABSTRACT FROM AUTHOR]

Background Patients awaiting solid organ transplantation may develop anti-HLA antibodies after sensitization events such as transfusions, pregnancies, or previous transplantations. However, the effects of a particular sensitization event on HLA alloimmunization have not been well studied in parallel using cell-based assays and solid-phase assays. In this study, we evaluated and compare how different sensitization events affect the HLA antibody screening (HLA-Ab) and donor specific antibody (DSA) status in solid renal organ transplantation patients. Methods HLA antibody (HLA-Ab) screening tests like complement-dependent cytotoxicity crossmatch (CDC-XM), flow cytometry crossmatch (FC-XM) and Luminex panel-reactive antibody (L-PRA) were performed in all 1066 patients (635 males and 431 females). If any of these tests turned out to be positive, a Luminex single antigen bead (L-SAB) assay was performed for DSA identification. Test positive rates and antibody strengths were analyzed according to the different sensitization events and gender. Results In this study, HLA-Ab screening tests positive rates (L-PRA, FC-XM and CDC-XM) were significantly higher in patients with previous transplantation (73.91%, 100% and 56.52% p < 0.001), previous pregnancy (57.46%, 70.14% and 18.85% p < 0.001) or blood transfusion (27.33%, 35.55% and 7.33% p < 0.001) compared with patients without a sensitizing event (6.17%, 13.58% and 1.09). In this study, re-transplantation group showed significantly stronger antibody strength (DSA) than non sensitized group (class I and II MFI 11418.04, 17,837.78 vs class I and II MFI 2659, 3329; P < 0.001) and those with single sensitization events of transfusion (class I and II MFI 11418.04, 17,837.78 vs class I and II MFI 5790.26, 6004.16; P < 0.001) or pregnancy (class I & II MFI 11418, 17,837 vs class I and II MFI 8631.71, 7253.29; P < 0.001). Conclusions Pregnancy and blood transfused had high allo-immunization rate for class I HLA antigens. While re-transplantation patients had high allo-immunization rate for both the HLA classes (HLA- class I and HLA- class II). Re-transplantation group showed significantly stronger antibody strength, followed by pregnancy and then transfusion. [ABSTRACT FROM AUTHOR]

Background: Donor-specific HLA antibodies are important risk factors in antibody-mediated rejection and graft loss after renal transplantation and are associated with higher rejection rates and lower graft survival. Most de novo donor specific antibodies (dnDSA) after renal transplantation are directed toward donor HLA-DQ antigens. An HLA-DQ antigen is a heterodimer consisting of an alpha and beta chain. Traditionally, HLA-DQAl typing has not been part of pretransplant evaluation. Therefore, DQ alpha proteins are not usually considered in the interpretation of HLA-DQ antibody reactions. Methods: The renal transplant recipient had a 0% panel reactive antibody pretransplant. Two years after transplantation, he developed symptoms of abdominal distension and bilateral lower extremity edema. Histopathological findings on renal puncture biopsy showed a combination of T-cell-mediated acute rejection type ILA and antibody-mediated rejection with a trend toward chronicity in the transplanted kidney. DSAs were investigated by HLA-I (HLA-AR) and HLA-Il (HLA-DRBI/DQAl/DQB 1) single antigen bead (SAB) assay. HLA typing was performed to explain the antibody reactivity patterns by PCR-SSO and Sequencing-based typing (SBT). HLA-Matchmaker analysis was performed to identify eplets that explain antibody reactivity patterns, Results: HLA-II SAB analysis of the patient's serum at the time of rejection showed positive reactions with all DQB1*03:03-carrying beads with high mean fluorescence intensity (MFI). However, DQB1 *03:03 was not a dnDSA antigen. High-resolution HLA typing revealed that HLA-DQAl *05:01 and DQAl *03:02 were mismatched donor antigens. HLA Matchmaker analysis demonstrated reactivity toward 13OR and 116 V eplet on DQAl and DQB1. Conclusions: Antibodies specific to DQα chains after renal transplantation were highlighted. [ABSTRACT FROM AUTHOR]

B cells contribute to chronic transplant rejection by producing donor-specific antibodies and promoting T cell response, but how these processes are regulated at the transcriptional level remains unclear. Herein, we investigate the role of transcription factor interferon regulatory factor 4 (IRF4) in controlling B cell response during chronic transplant rejection. We generated the Irf4 gfp reporter mice to determine IRF4 expression in B cell lineage. We then used mice with B cell-specific IRF4 deletion to define the role of IRF4 in B cell response after NP-KLH immunization or allogeneic heart transplantation. In particular, graft survival and histology, as well as B and T cell responses, were evaluated after transplantation. IRF4 is dynamically expressed at different stages of B cell development and is absent in germinal center (GC) B cells. However, IRF4 ablation in the B cell lineage primarily eliminates GC B cells in both naïve and NP-KLH immunized mice. In the transplantation setting, IRF4 functions intrinsically in B cells and governs allogeneic B cell responses at multiple levels, including GC B cell generation, plasma cell differentiation, donor-specific antibody production, and support of T cell response. B cell-specific IRF4 deletion combined with transient CTLA4-Ig treatment abrogates acute and chronic cardiac allograft rejection in naïve recipient mice but not in donor skin-sensitized recipients. B cells require IRF4 to mediate chronic transplant rejection. IRF4 ablation in B cells abrogates allogeneic B cell responses and may also inhibit the ability of B cells to prime allogenic T cells. Targeting IRF4 in B cells represents a potential therapeutic strategy for eliminating chronic transplant rejection. [ABSTRACT FROM AUTHOR]

Donor exchange programs were designed to allocate organs for highly sensitized (HS) patients. The allocation algorithm differs slightly among countries and includes different strategies to improve access to transplants in HS patients. However, many HS patients with a calculated panel reactive of antibodies (cPRA) of 100 % remain on the waiting list for a long time. Some allocation algorithms assume immunological risk, including Imlifidase treatment, to increase the chance of transplantation in very HS patients. Here, we describe our unicenter experience of low-risk delisting strategy in 15 HS patients included in the Spanish donor exchange program without donor offers. After delisting, 7 out of 15 HS patients reduced the cPRA below 99.95 % and impacted the reduction of time on the waiting list (p = 0.01), where 5 out of 7 achieved transplantation. Within those HS that remained above 99.95 %, 1 out of 8 was transplanted. All the HS were transplanted with delisted DSA, and only one with DSA level rebounded early after transplantation. All HS transplanted after delisting maintain graft function. The transplant immunology laboratories are challenged to search intermediate risk assessment methods for delisting high HS patients. [ABSTRACT FROM AUTHOR]

Humoral immunity emerges as a risk factor for graft failure after visceral transplantation (VTx) and development of donor-specific anti-HLA antibodies (DSAs) has been linked with poor outcomes. In most cases, a simultaneous liver transplant can be safely performed in sensitized patients with DSA and appears protective against lymphocytotoxic antibodies. We investigated the incidence of acute (AR) and chronic rejection (CR) in 32 VTx without any B cell-depleting pre-treatment (6 isolated intestinal transplants (IT) and 26 liver-containing, multivisceral transplants (MVT) and assessed the presence of donor-specific antibodies (DSA) pre- and post-transplantation. Twenty-one patients (65 %) developed AR, 15 (57 %) of the MVT and 6 (100 %) of the IT (p = 0.05). CR occurred in 4 IT (60 %, p < 0.001). At one month, de novo DSA were present in 71 % of VTx (66 % MVT vs 100 % IT, p = 0.09). At the last available follow-up, 69 % of the MVT and 50 % of the IT patients were DSA-free. D e novo DSA seemed more persistent (7/19, 37 %) than pre-Tx DSA (1/6, 17 %; p = n.s.), de novo DSA were more frequently specific for HLA class II than class I, 16/19 (84 %) vs. 7/19 (37 %; p = 0.003), and HLA-DQ was their most frequent target HLA. DQ mismatches appeared to be a risk factor for developing de novo DSA. In conclusion, liver-containing visceral allografts have superior short- and long-term outcomes compared with liver-free allografts. De novo DSA develop early and frequently after VTx performed without B cell-depleting induction therapy, but the exact role of DSA in the pathogenesis of rejection remains unclear. [ABSTRACT FROM AUTHOR]

Antibody-mediated rejection (AMR) is an important cause of lung allograft loss in some patients. Challenges with current diagnostic criteria limit timely detection. Ultrastructural studies of endothelia allow the early detection of AMR in kidney allografts. This study aimed to define the ultrastructural changes of the endothelium in lung allografts in the setting of AMR and determine its specificity for AMR. Ultrastuctural studies were performed on lung allograft biopsies of 12 patients using glutaraldehyde-fixed or paraffin-embedded material. AMR had been classified according to the International Society of Heart and Lung Transplant 2016 consensus report criteria. Endothelial changes (swelling [ES], vacuolization [EV], surface irregularity, detachment, neutrophil margination [NM]) and basement membrane changes were graded semi quantitatively using electron microscopy (EM). Grades were compared between AMR, acute cellular rejection, and non-transplant controls. Significant differences were found between AMR and acute cellular reaction biopsies, particularly in ES (p = 0.006), EV (p = 0.023) and NM (p = 0.038). Using a combined score of all categories of assessment, the total EM score was significantly higher in AMR (p = 0.007) and provided excellent sensitivity and specificity with a receiver operator characteristic curve of 1.0. C4d did not correlate with EM changes associated with AMR. The use of paraffin-embedded material samples did not significantly affect the analysis compared with glutaraldehyde-fixed tissue, although ES was reduced in the former. Endothelial structural analysis using EM can facilitate improved diagnostic accuracy of AMR and needs to be validated in larger cohorts, but it also allows retrospective studies to be performed. [ABSTRACT FROM AUTHOR]

Chronic-active antibody-mediated rejection (CAABMR) is associated with poor kidney graft survival and has no clear effective treatment. Forty-one cases of CAABMR were detected in indication graft biopsies and evaluated according to current Banff classification. We investigated the impact of concurrent donor-specific antibodies (DSA) and their characteristics, together with non-adherence regarding immunosuppression on CAABMR histopathological phenotypes and prognosis. Twenty-four (59%) patients had detectable DSA at biopsy, with 15 of them being considered non-adherent. Graft function at biopsy was similar in DSA (+) and (−) patients. DSA (+) patients had significantly higher tubulointerstitial inflammation (i and ti) and acute humoral (g+ptc+v+C4d) composite score than DSA (−). DSA (+)/non-adherent cases presented additionally with increased microvascular inflammation (ptc and v), besides having a distinctively lower ah score. C1q DSA strength was higher ( P  = .046) in non-adherent patients and correlated closely with C4d score ( P  = .002). Lower graft function and ah score, higher proteinuria and ci + ct score, and, separately per each model, DSA (+) (HR = 2.446, P  = .034), DSA (+)/non-adherent (HR = 3.657, P  = .005) and DSA (+)/C1q (+) (HR = 4.831, P  = .003) status were independent predictors of graft failure. CAABMR with concomitant DSA pose a higher risk of graft failure. Adherence should be evaluated, and histopathological phenotyping and DSA characterization may add critical information. [ABSTRACT FROM AUTHOR]

Preformed HLA donor-specific antibodies (DSA) only detected with Luminex have been associated with increased risk of antibody-mediated rejection (ABMR) and graft failure after kidney transplantation (KT). Their evolution after KT may modify this risk. We analyzed postransplant evolution of preformed DSA identified retrospectively and their impact on outcomes of 370 KT performed 2006–2014. Antibodies were monitored prospectively at 1-3-5 years after KT and if any dysfunction. Early acute ABMR was more frequent among patients with preformed DSA class-I or I + II than isolated class-II (29.4% vs 4.5%, p = 0.02). One year post-KT, 20 of 34 patients with functioning KT had persistent DSA. Preformed DSA class-II persisted more frequently than class-I/I + II (66.7% vs 33.3%; p = 0.031). The only risk factor independently associated with persistence was pretransplant MFI. Patients with de novo DSA had the highest risk of ABMR (HR 22.2 [CI 6.1–81.2]). Although recipients with persisting preformed DSA had significantly increased ABMR risk (HR 14.7 [CI 6.5–33.0]), those with cleared preformed DSA also had a higher risk than those without DSA (HR 7.01 [CI 2.2–21.8]). Preformed DSA are a very important risk factor for ABMR and graft loss. Patients who clear preformed DSA still show an increased risk of ABMR and graft loss after KT. [ABSTRACT FROM AUTHOR]

In this retrospective study, our aim was to find the effect of leucodepleted (LD) blood transfusions on the formation of anti-HLA-antibodies when compared to non-leucodepleted (non-LD) transfusions using Luminex-based method. In this study, Luminex single antigen bead assay (L-SAB) and HLA typing were performed on 310 patients. Test positivity rates (as MFI - Mean florescence intensity) were analyzed according to the different sensitization events and gender. Of the 310 patients included in the study, 58.06% (180) patients were male and 41.93% (130) were female. The average age of the patients was 42.86 (±12.37) years. In this study, test positivity rates were significantly lower in the patients who received LD RBC units than in those who received non-LD RBC units (28.43% = 29 of 102 Vs 55.22% = 74 of 134, p < 0.05). In our study, transfusion combined with a history of pregnancy had higher number of significant HLA antibodies compared to cases where transfusion was the only sensitization event (81.81% = 18/22 Vs 39.71% = 85/214, p < 0.05). In addition, anti-HLA-antibodies-MFI were significantly (p < 0.01) higher in non-LD patients compared to LD patients. Patients who received LD RBC units had a significantly lower rate of transfusion-associated alloimmunization compared to those who received non-LD RBC units. Multiparous women had a high risk for transfusion-related alloimmunization compared to both nulliparous women and male patient. Furthermore, class I-anti-HLA-antibodies (HLA-B and HLA-A + B) were significantly associated with pregnancy sensitization and/or blood transfusion as a single sensitization. [ABSTRACT FROM AUTHOR]

Introduction Pretransplant donor-specific HLA alloantibodies detected with the Single Antigen Bead (SAB) assay reflect an increased risk for acute antibody-mediated rejection (AMR). We herein report the incidence of both acute AMR and acute cellular rejection (ACR) during the first year posttransplantation, in a cohort of kidney transplant recipients (KTR) of deceased donor (DD) grafts, according to their DSA status. Pretransplant DSA do not preclude DD-KT in negative CDC-XM recipients at our center. Patients and methods 246 KT were performed at our center between 01/2012 and 12/2015 and 100 KTR obtained from a DD were analyzed; 24% harbored DSA by SAB assay, MFI values > 500 were considered positive. All recipients received thymoglobulin induction and generic tacrolimus-based maintenance therapy. Graft biopsies were performed by protocol on months 3 and 12 as well as per indication. The incidence of AMR and ACR was correlated with the existence of pretransplant DSA. Results Overall, 34% of patients developed an acute rejection episode, 54.2% in the DSA group versus 27.6% in the non-DSA group (p = 0.032), and most of these events were detected as subclinical conditions in protocol biopsies. AMR events developed in 33.3% and 19.7% (p = 0.176) in the DSA and the non-DSA groups, respectively. ACR events were found in 16.6% and 6.6% (p = 0.127) in the DSA and non-DSA groups, respectively. Graft function was similar between groups at the end of the 1st year posttransplant and no immunological graft loss occurred. Conclusion Despite the use of depleting induction therapy and adequate tacrolimus trough levels along with MMF and steroids, a high rate of rejection events was observed during the first year post-transplantation. [ABSTRACT FROM AUTHOR]

The virtual crossmatch, which is based on single antigen bead technology, is used in the prediction of crossmatch results. However, this assay differs in sensitivity and specificity from crossmatch methods. In our study, the results of physical crossmatches, performed with three different methods, were assessed against virtual crossmatch results. The aim was to determine the potential cut-off values for donor specific antibodies (DSA) that would predict the crossmatch results obtained by different methods. The results of different crossmatch techniques were correlated with the virtual crossmatch. The receiver operating characteristic (ROC) analysis revealed the Flow cytometric crossmatch (FCXM) and Luminex crossmatch (LXM) to be the most accurate, with area under curve (AUC) values of 0.861 and 0.805, respectively. While we found that the virtual crossmatch correlated well with all the crossmatch results, FCXM produced the best results (83% of the DSA detected). LXM outperformed the other tests in terms of the accuracy in separating class II DSA. [ABSTRACT FROM AUTHOR]

The detrimental impact of preformed anti-HLA donor-specific antibodies (DSA) is well defined, contrarily to non-donor-specific antibodies (NDSA). We sought to evaluate their clinical impact in a cohort of 724 kidney graft recipients in whom anti-HLA antibodies were thoroughly screened and identified in pre-transplant sera by solid-phase assays. NDSA or DSA were detected in 100 (13.8%) and 47 (6.5%) recipients respectively, while 577 (79.7%) were non-allosensitized (NaS). Incidence of antibody-mediated rejection at 1-year was 0.7%, 4.0% and 25.5% in NaS, NDSA and DSA patients, respectively (NaS vs. NDSA P = 0.004; NaS vs. DSA P < 0.001; NDSA vs. DSA P < 0.001). Graft survival was lowest in DSA (78.7%), followed by NDSA (88.0%) and NaS (93.8%) recipients (NaS vs. NDSA P = 0.015; NaS vs. DSA P < 0.001; NDSA vs. DSA P = 0.378). Multivariable competing risk analysis confirmed both NDSA (sHR = 2.19; P = 0.025) and DSA (sHR = 2.87; P = 0.012) as significant predictors of graft failure. The negative effect of NDSA and DSA on graft survival was significant in patients receiving no induction ( P = 0.019) or an anti-IL-2 receptor antibody ( P < 0.001), but not in those receiving anti-thymocyte globulin ( P = 0.852). The recognition of the immunological risk associated with preformed DSA but also NDSA have important implications in patients’ risk stratification, and may impact clinical decisions at transplant. [ABSTRACT FROM AUTHOR]

In contrast to other types of organ transplantation, liver-transplant recipients used to be considered highly resistant to donor-specific antibodies (DSAs). Consequently, most transplant programs did not consider the presence of DSAs at transplantation or during the follow-up. However, since the early 1990s, antibody-mediated pathological lesions have been recognized in ABO-incompatible liver-transplant recipients. Recent data confirm the detrimental effect of preformed and de novo DSAs in ABO-compatible liver transplantation, with inferior clinical outcomes in patients presenting with circulating antibodies. Acute antibody-mediated rejection (AMR), plasma-cell hepatitis, biliary stricture, but also long-term complications, such as chronic rejection, liver ductopenia, and graft fibrosis, are now recognized to be associated with DSAs. Moreover, some non-HLA DSAs are suspected to induce graft dysfunction. Clinical, biological, and histological patterns within AMR need to be clarified. Treatment of these complications has yet to be defined. This article summarizes recent advances concerning the impact of preformed and de novo DSAs in liver transplantation, it defines the complications associated with DSAs, and discusses the potential strategies to manage patients with such complications. [ABSTRACT FROM AUTHOR]

Presensitized kidney transplant recipients are at high-risk for early antibody-mediated rejection. We studied the impact of pre- and post-transplant donor-specific human leukocyte antigen (HLA) antibodies (DSA) and T-cell-activation on the occurrence of antibody-mediated rejection episodes (AMR) and graft loss (AMR-GL) in a unique cohort of 80 desensitized high-risk kidney transplant recipients. Patients with pre-transplant DSA demonstrated more AMR episodes than patients without DSA, but did not show a significantly increased rate of AMR-GL. The rates of AMR and AMR-GL were not significantly increased in patients with complement split product (C1q)-binding pre-transplant DSA. Pre-transplant C1q-DSA became undetectable post-transplant in 11 of 13 (85%) patients; 2 (18%) of these 11 patients showed AMR but no AMR-GL. In contrast, the post-transplant presence of C1q-DSA was associated with significantly higher rates of AMR (86 vs 33 vs 0%; P<0.001) and AMR-GL (86 vs 0 vs 0%; log-rank P<0.001) compared with post-transplant DSA without C1q-binding or the absence of DSA. Patients with both pre-transplant DSA and evidence of pre-transplant T-cell-activation as indicated by soluble CD30-positivity showed a significantly increased risk for AMR-GL [HR=11.1, 95% confidence interval (CI)=1.68-73.4; log-rank P=0.013]. In these high-risk patients, AMR-GL was associated with total DSA in combination with T-cell-activation pre-transplant, and de novo or persistent C1q-binding DSA post-transplant. [ABSTRACT FROM AUTHOR]

Background The diagnosis of antibody-mediated rejection (AMR) in the lung transplant is still an area under investigation. We performed a blinded multicenter study to determine if any statistically significant histologic findings in transbronchial biopsy specimens from lung transplant patients correlate with the presence of donor-specific antibodies (DSAs). Methods We asked 9 pathologists with experience in lung transplantation to evaluate 161 lung transplant biopsy specimens for various histologic parameters. The findings were correlated with antibody status positive for DSAs, positive for non-DSAs, and no antibodies (NABs) present. The significance of each histologic variable was reviewed. Results We found no statistically significant association with acute cellular rejection, airway inflammation, or bronchiolitis obliterans and the presence or absence of antibodies. However, biopsy specimens with DSAs had a statistically significant difference vs NABs in the setting of acute lung injury, with or without diffuse alveolar damage ( p = 0.0008), in the presence of capillary neutrophilic inflammation ( p = 0.0014), and in samples with endotheliitis ( p = 0.0155). In samples with complement 4d staining, there was a trend but no statistically significant difference between specimens associated with DSAs and specimens with NABs. Conclusions Capillary inflammation, acute lung injury, and endotheliitis significantly correlated with DSAs. The infrequently observed diffuse staining for complement 4d limits the usefulness of this stain. [ABSTRACT FROM AUTHOR]

Objective De novo donor-specific anti-human leukocyte antigen antibodies develop in a high proportion of lung transplant recipients early after lung transplantation. We recently showed that de novo donor-specific antibodies (DSA) occurrence is associated with significantly increased mortality. Here, we studied the efficacy of a preemptive treatment protocol. Methods A retrospective observational study was conducted on all lung transplantations at Hanover Medical School between January 2009 and May 2013. Results Among the 500 transplant recipients, early DSA developed in 86 (17%). Of these, 56 patients (65%; Group A) received therapeutic plasma exchange, and 30 patients (35%; Group B) did not. Among Group A patients, 51 also received rituximab. Between groups, there was no statistically significant difference in mortality, incidence of pulsed steroid therapies, rejections diagnosed by biopsy specimen, incidence of bronchitis obliterans syndrome (BOS), or infections requiring hospitalization at 1 year and 3 years. Also, there were no statistically significant differences after matching 21 Group A with 21 Group B patients through propensity score analysis. Significantly more Group A patients (65%) than Group B patients (34%) cleared DSA at hospital discharge ( p = 0.01). At the last control after transplantation (median, 14 months; interquartile range, 5–24 months), 11 Group A (22%) and 9 Group B patients (33%) still showed DSA ( p = 0.28). Conclusions Preemptive treatment with therapeutic plasma exchange and rituximab led to improved elimination of DSA early after lung transplantation ( p = 0.01). However, spontaneous elimination in untreated Group B patients also occurred frequently. This treatment protocol was not associated with significantly improved outcome. [ABSTRACT FROM AUTHOR]

Background Bronchiolitis obliterans syndrome (BOS) is the major cause of late graft failure after lung transplantation. The objective was to determine whether de novo donor human leukocyte antigen (HLA)-specific antibodies (DSA) are associated with the development of BOS or patient survival. Data were analyzed from 188 lung transplant recipients with a follow-up period up to 8 years. Methods HLA antibody monitoring was performed at 3-month intervals post-transplant at routine outpatient clinic attendances and during the investigation of any acute deterioration. HLA antibody data were available for 148 patients; 66 (45%) had produced HLA antibodies after transplant, of which 38 (26%) were DSA and 28 (19%) non–donor-specific HLA antibodies. Results De novo DSA was associated with development of BOS Stage 1 (BOS1; hazard ratio [HR] = 2.302, p = 0.0015), BOS2 (HR = 3.627, p < 0.0001) and BOS3 (HR = 5.736, p < 0.0001). De novo persistent DSA correlated strongly with shorter time to onset of BOS3 (HR = 6.506, p = 0.0001). There was a significant reduction in patient survival associated with de novo DSA (HR = 1.886, p = 0.047). In multivariable analyses, de novo DSA was an independent predictor for development of all stages of BOS as well as an independent predictor of poor patient survival. Conclusions De novo DSA is a major risk factor for progression to BOS and shorter patient survival. Treatments to remove antibodies or limit antibody-mediated damage could be considered when DSA are first detected. However, a randomized, controlled trial of treatment options would enable a clearer understanding of the benefits, if any, of antibody-removal therapies. [ABSTRACT FROM AUTHOR]

Background The impact of Luminex-detected HLA antibodies on outcomes after lung transplantation is unclear. Herein we have undertaken a retrospective study of pre-transplant sera from 425 lung transplants performed between 1991 and 2003. Methods Pre-transplant sera, originally screened by complement-dependent cytotoxicity (CDC) assays, were retrospectively tested for the presence of HLA-specific antibodies using HLA-coated Luminex beads and C4d deposition on Luminex beads. The results were correlated with graft survival at 1 year. Results Twenty-seven patients were retrospectively identified as having been transplanted against donor-specific HLA antibodies (DSA) and 36 patients against non–donor-specific HLA antibodies (NDSA). DSA-positive patients had 1-year survival of 51.9% compared with 77.8% for NDSA and 71.8% for antibody-negative patients ( p = 0.029). One-year survival of patients with complement-fixing DSA was 12.5% compared with 62.5% for non–complement-fixing DSA, 75.8% for non–complement-fixing NDSA and 71.8% for antibody-negative patients ( p < 0.0001). DSA-positive patients with mean fluorescence intensity (MFI) >5,000 had 1-year survival of 33.3% compared with 71.4% for MFI 2,000 to 5000 and 62.5% for MFI <2,000 ( p = 0.0046). Multivariable analysis revealed DSA to be an independent predictor of poor patient survival within 1 year ( p = 0.0010, hazard ratio [HR] = 3.569) as well as complement-fixing DSA ( p < 0.0001, HR = 11.083) and DSA with MFI >5,000 ( p = 0.0001, HR = 5.512). Conclusions Pre-formed DSA, particularly complement-fixing DSA, and high MFI are associated with poor survival within the first year after lung transplantation. Risk stratification according to complement fixation or MFI levels may allow for increased transplantation in sensitized patients. [ABSTRACT FROM AUTHOR]

Background Extracorporeal photopheresis (ECP) has been used to treat chronic rejection after lung transplantation (LTx). We investigated the effect of ECP on several immune parameters that have been associated with poor lung function, including donor-specific antibodies (DSA) to human leukocyte antigen (HLA), antibodies against the lung-associated self-antigens (SAg), Kα1-tubulin (Kα1T), collagen I and V, and circulating levels of pro-inflammatory and anti-inflammatory cytokines. Methods Sera were collected from post-LTx patients diagnosed with bronchiolitis obliterans before and 6 months after initiation of ECP. DSA and cytokine levels were measured by Luminex (Invitrogen, Carlsbad, CA). Changes in lung function over the 6 months preceding and after the initiation of ECP were measured by retrospective analysis of spirometry performed at routine clinic visits. Results ECP was associated with a significant decline in DSA levels as well as antibodies to lung-associated SAg. ECP also reduced circulating levels of pro-inflammatory cytokines and increased levels of anti-inflammatory cytokines. These immunologic changes were associated with a significant 63% reduction in the rate of decline in forced expiratory volume in 1 second over a 1-year period. Though statistically insignificant, a higher rate of clearance of antibodies to lung-associated SAg was strongly associated with better response to ECP. Conclusions ECP is associated with a reduction in the levels of circulating DSA, antibodies to lung-associated SAg (Kα1T, collagen I, and collagen V), and circulating levels of several pro-inflammatory cytokines. We propose that these changes contribute to the beneficial effect of ECP in reducing the decline in lung function. [ABSTRACT FROM AUTHOR]

Donor-specific alloantibodies (DSA) to HLA-DP may cause antibody-mediated rejection (AMR), especially in re-transplants. We describe the immunization history of a patient who received 3 kidney transplants; the 3rd kidney was completely matched except at DPA1 and DPB1. Prior to the 3rd transplant, single antigen bead analysis (SAB) showed DSA reactivity against DPA1 shared by the 1st and 3rd donors, but B and T flow crossmatch (FXM) results were negative. Within 11days the 3rd transplant underwent acute C4d+ AMR which coincided with the presence of complement (C1q)-binding IgG1 DSA against donor DPA1 and DPB1. Using HLAMatchmaker and SAB, we provide evidence that eplet (epitope) spreading on DPA1 and eplet sharing on differing DPB1 alleles of the 1st and 3rd transplants was associated with AMR. Since weak DSA to DPA1/DPB1 may induce acute AMR with negative FXM, donor DPA1/DPB1 high resolution typing should be considered in sensitized patients with DP-directed DSA. [ABSTRACT FROM AUTHOR]

Abstract: Presensitization against a broad array of HLA is associated with prolonged waiting times and inferior kidney allogaft survival. Although the use of solid phase assay (SPA) for the detection and characterization of anti-HLA antibodies provides greater sensitivity than complement-dependent lymphocytotoxicity (CDC) assay, it often detects donor specific antibodies (DSA) which turn out to be clinically irrelevant. Our data reinforce the concept that these two types of assays should be used in parallel for pre-and post-transplantation monitoring of anti-HLA antibodies in recipients of solid organ allografts. [Copyright &y& Elsevier]

Abstract: Objectives: The clinical relevance of pre-transplant “low-level” donor specific anti-HLA antibodies (DSAs) in crossmatch negative kidney transplant recipients remains unclear. To determine what level of DSA associates with antibody mediated rejection (AMR) could be the way to measure the clinical relevance of pre-transplant “low-level” donor specific anti-HLA antibodies (DSAs) in crossmatch negative kidney transplant recipients. Design and methods: A retrospective analysis of 221 patients from October 2008 to December 2009 was included in this study. Sera were obtained pre-transplant and two weeks post-transplant and tested for DSA using LABScreen single antigen beads. Results: Among the 221 patients, 11 experienced AMR within 200days after transplant (5%). Pre-transplant DSA was associated with AMR at multiple mean fluorescence intensity (MFI) cutoffs (500, 1000, 2000, 3000, 5000; p=0.003, 0.001, 0.007, 0.003, and 0.003, respectively). No correlation was seen between acute T-cell mediated rejection (CMR) and pre-transplant DSA at any of the same MFI cutoffs. There was an increased risk of AMR with higher levels of pre-transplant DSA. Finally, an increase in DSA MFI from pre- to two weeks post-transplant was indicative of a higher probability of AMR. Conclusion: Overall, this data supports using the single antigen bead to detect “low-level” DSA both pre- and post- as having a positive and persistent DSA may be predictive of higher AMR rates and poorer graft survival. [Copyright &y& Elsevier]

Background: Humoral immune responses during heart transplantation may result in antibody-mediated rejection (AMR), which is now taken into account on endomyocardial biopsy (EMB) specimens and ranked according to the pathologic AMR (pAMR) grades of the International Society for Heart and Lung Transplantation classification. This classification might benefit from new immunohistological markers and validation by others biomarkers, namely donor-specific antibodies (DSA). Methods: From the 293 protocol EMBs performed in 113 patients in our institution during a 1-year period for this prospective study, 280 EMB specimens were available with both histology and immunohistochemistry. C4d and labeling of intravascular cells by cluster of differentiation (CD) 68 were performed on paraffin sections. Available sera (n = 150) concomitant of EMB specimens were tested for the presence of DSA. All of the pAMR+ EMB specimens, along with a set of randomized pAMR0 EMB specimens, were immunolabeled for mammalian target of rapamycin (mTOR) effectors, phosphorylated 70 S6-kinase (p70S6K) and phosphorylated S6 ribosomal protein (pS6RP). Results: AMR was diagnosed in 37 EMB specimens (13.2%): 1 pAMR1(I+), 27 pAMR1(H+), and 9 pAMR2. The proportion of DSA-positive EMB varied according to the pAMR grade, with pAMR0, pAMR1(H+), and pAMR2 EMB presenting 17.6%, 77.3%, and 100% of DSA-positivity, respectively. Among the 30 pAMR+ specimens with available DSA testing and the 30 pAMR0 randomized specimens, mTOR pathway immunohistochemistry showed endothelial cell positivity for p70S6K in 17 pAMR+ EMB specimens (56.7%) and in 1 pAMR0 EMB specimen (3.3%). pS6RP was detected in 8 pAMR+ EMB specimens (26.7%) and in 1 pAMR0 EMB specimen (3.3%). Conclusions: p70S6K and pS6RP immunohistochemistry afford new markers of AMR on EMB specimens because their expression is correlated with microcirculation inflammation and DSA. The correlation of DSA with pAMR grade suggests that this grading system is valid. [Copyright &y& Elsevier]

Background: Lung transplantation is limited by chronic lung allograft dysfunction. Acute cellular rejection (ACR) is a risk factor for allograft dysfunction; however, the role of antibody-mediated rejection (AMR) is not well characterized. Methods: This was a retrospective review from 2007 to 2011 of lung transplant recipients with human leukocyte antigen (HLA) antibody testing using Luminex (Luminex Corp, Austin, TX) single-antigen beads. Statistics included Fisher’s exact test for significance. Results: Donor-specific antibodies (DSA) developed in 13 of 44 patients. Of the 13 with DSA, 12 had cystic fibrosis compared with 18 of 31 in the non-DSA group (p = 0.035). Of those with DSAs, 23.1% occurred within the first year, and 69.2% occurred between 1 and 3 years. Twelve of 13 DSA patients had anti-HLA DQ specificity compared with 2 of 31 non-DSA patients (p = 0.0007). AMR developed in 10 of the 13 DSA patients compared with 1 of 31 non-DSA patients (p = 0.0001). The DSA group experienced 2.6 episodes/patient of cellular rejection vs 1.7 episodes/patient in the non-DSA group (p = 0.059). Bronchiolitis obliterans syndrome developed in 11 of 13 in the DSA group vs 10 of 31 in the non-DSA group (p = 0.0024). In the DSA group, 11.5% HLAs matched compared with 20.4% in the non-DSA group (p = 0.093). AMR developed in 11 of 22 patients in the non-DSA HLA group compared with 0 of 22 in the group without non-DSA HLA antibodies (p = 0.002). Survival at 1 and 3 years was 92% and 36% in the DSA group, respectively, and 97% and 65% in the non-DSA group. Conclusions: DSAs and non-DSAs occur frequently after lung transplantation. DSAs are prevalent in the cystic fibrosis population and are associated with AMR, bronchiolitis obliterans syndrome, and possibly, ACR. [ABSTRACT FROM AUTHOR]

Abstract: Objective: Due to increasing waiting times for deceased donor kidneys, living donor kidney transplantation is increasingly performed in the presence of donor-specific antibodies (DSA). Methods: Twenty-three patients with Luminex-detected DSA were successfully desensitized by anti-CD20 therapy and immunoadsorption (N = 19) or plasmapheresis (N = 4) and received a kidney transplant from a living donor. Twelve of the 23 patients (52%) had a positive CDC and/or ELISA crossmatch result before desensitization. Six patients were negative in CDC as well as ELISA screening but positive in Luminex for DSA. Results: The 23 patients received a median of 8 apheresis treatments before and 5 treatments after transplantation. Induction therapy was performed with either thymoglobulin (N = 11) or basiliximab (N = 12). The 2-year graft survival rate was 100%. At last follow up, a median of 12 months after transplantation, median serum creatinine was 1.42 mg/dL, median MDRD-GFR 59.5 mL/min/1.73 m2, and median urinary protein-to-creatinine ratio 0.12. Ten out of fourteen patients (71%) who had completed the first year after transplantation by the time of analysis had no DSA by day 360. Acute T-cell mediated rejection was diagnosed in one patient (4%), and antibody-mediated changes were found in 5 patients (22%). Four out of these 5 patients showed evidence of persistent (N = 2) or reemerging plus/minus de novo DSA (N = 2) on day 360, and the 2 patients with persistent DSA lost their allograft subsequently on days 750 and 810, respectively. Infectious complications were infrequent. Conclusions: Our previously described treatment algorithm for desensitization of living donor kidney transplant recipients with DSA results in good graft outcomes with a low rate of side effects. [Copyright &y& Elsevier]

Abstract: Previous studies had shown that donor-specific anti-HLA antibodies may highly influence the survival rate of corneal allografts, although the anterior chamber generally represents an immune‐privileged compartment of the eye. We postulated that the introduction of a novel crossmatch procedure for the detection of donor-specific anti-HLA antibodies in recipients awaiting a corneal graft would be adequate to investigate their influence on the outcome of the graft survival. The Antibody Monitoring System (AMS) HLA class I & II crossmatch ELISA was adapted for the use of material from the outer scleral rim instead of blood lymphocytes to isolate the donors'' HLA molecules. In case of detectable donor-specific anti-HLA class I and/or class II antibodies (DSA) this result was confirmed using an identification ELISA to specify the detectable recipient''s anti-HLA antibodies. PCR-based genetic tissue typing of the donors was performed also using their outer scleral rims. 45 recipients of corneal grafts were analyzed for DSA prior to or after grafting, respectively. 75% of the recipients with preformed DSA exhibited immunological complications up to the complete graft loss in four cases during the first two months. In contrast 77% of the recipients without DSA did not show any complications during the follow up period of averagely 18months. Only two cases of graft loss were observed in this group after 17 and 23months, respectively. The results demonstrate the impact of preventing donor-specific anti-HLA antibodies which are for the first time reliably detectable in any laboratory''s daily work using the adapted AMS-ELISA. [Copyright &y& Elsevier]

Abstract: Heart transplantation has become an important therapeutic option for the treatment of end-stage heart disease. Over the past 40 years, much has been learned about the immunologic responses to the allograft heart, including elucidation of the mechanisms of acute cellular rejection and antibody-mediated rejection (AMR). In AMR, damage to the allograft vasculature including capillaries and coronary arteries causes graft dysfunction and decreased survival. The recent recognition of AMR in cardiac transplantation has led to an evolution in the evaluation of the biopsy and graft surveillance. The use of serologic testing and both histologic and immunohistochemical criteria have improved the detection of AMR. [Copyright &y& Elsevier]

Abstract: B cell responses to minor histocompatibility antigens (mHags) have not been extensively studied after allogeneic hematopoietic stem cell transplantation (HSCT). Glutathione S-transferase T1 (GSTT1) is a drug metabolizing enzyme encoded by a single gene that is highly expressed in liver and kidney. Anti-GSTT1 antibodies have been described in the context of antibody-mediated rejection in kidney and liver transplantation, due to a mismatch between donor and recipient. The aim of the present study was to investigate the specific immune response against GSTT1 in HSCT with production of antibodies and their influence in the development of hepatic graft-versus-host-disease (GVHD). Forty patients and their respective donors were included in the study. The median follow-up time was 35.6months (range 0.6–76months) and a total of 349 serum samples were tested for the presence of anti-GSTT1 antibodies by ELISA test. Statistical analysis was performed by defining the GSTT1 null donor/positive recipient as mismatch compared with the other three genetic combinations regarded as GSTT1-matched. Antibodies were found in three patients within the group of null donor/positive recipient and one within the null/null group. Development of liver GVHD, particularly its acute form, was highly associated with the GSTT1-mismatch (P =0.0178) and with the presence of post-transplant anti-GSTT1 antibodies (P =0.0076). We conclude that GSTT1 could be considered as a new mHag in hepatic GVHD. The fact that three donors were parous females and the rapid production of antibodies after HSCT suggests the existence in the graft of memory B-cells specific for the GSTT1 antigen. [Copyright &y& Elsevier]

Abstract: It has been known for 40 years that cytotoxic human leukocyte antigen (HLA) antibodies are associated with graft rejection. However, the complement-dependent cytotoxicity assay (CDC) used to define these clinically deleterious antibodies suffers from a lack of sensitivity and specificity. Recently, methods exploiting immunoglobulin G (IgG) antibody binding to HLA single antigen beads (SAB) have overcome sensitivity and specificity drawbacks but introduced a new dilemma: which of the much broader set of antibodies defined by these methods are clinically relevant. To address this, we developed a complement-fixing C1q assay on the HLA SAB that combines sensitivity, specificity, and functional potential into one assay. We compared the CDC, IgG, and C1q assays on 96 sera having 2,118 defined antibodies and determined that CDC detects only 19% of complement-fixing antibodies detected by C1q, whereas C1q detects only 47% of antibodies detected by IgG. In the same patient, there is no predictability by IgG mean fluorescence intensity (MFI) as to which of the antibodies will bind C1q because fixation is independent of MFI values. In 3 clinical studies, C1q+ antibodies appear to be more highly correlated than those detected by IgG alone for antibody-mediated rejection in hearts as well as for kidney transplant glomerulopathy and graft failure. [Copyright &y& Elsevier]

Abstract: Antibodies against HLA antigens offer a window to the response of transplant recipients to the challenge of allografts. HLA alleles produced by recombinant technology and attached to color-coded polystyrene microspheres are now in widespread use. They offer antibody detection by flow cytometry at a high level of sensitivity. Methods used in the author''s laboratory for the interpretation of these tests are described. They include a correction for variable antigen density, and the binding of normal immunoglobulins to the conjugated beads. The ‘normalized ratio’ provides a good method of scoring results. Antibodies specific for the antigens of the donors were associated with kidney and heart allograft loss. Antibodies against crossreactive antigens, without reactivity against donor HLA, did not appear to increase the risk of developing chronic rejection. Antibody production by peripheral blood B cells was analyzed. The idea of sequestration of HLA antibodies in the graft as a major cause for their absence in the blood after transplantation is discussed. IgM antibodies against donor HLA antigens were found before transplantion and also were produced both early and late in the post-transplant course. In addition, IgM antibodies against donor HLA appeared to be associated with decreased survival of kidney and heart allografts. [Copyright &y& Elsevier]

Abstract: Donor-specific HLA antibodies have been associated with acute and chronic rejection. Such antibodies may sometimes not be detected in recipient serum. In an attempt to learn about possible mechanisms, we investigated antibody production by recipient B lymphocytes in vitro. Peripheral blood B cells were obtained from 36 subjects, including 16 allograft recipients, 12 sensitized patients, three multiparous women with serum HLA antibodies, and five healthy non-transfused male subjects. Purified B cells were cultured with a cell line expressing CD40 ligand. Culture supernatants were screened for HLA antibodies, and positive samples were analyzed using single antigen beads to determine antibody specificity. HLA antibody–producing B cells were detected in persons known to be sensitized. In 13 of 16 allograft recipients, IgG antibodies against mismatched donor HLA antigens were observed, and donor-specific antibodies were sometimes produced in B-cell cultures when serum reactions were negative. IgM antibodies against HLA antigens were also identified in cultures from some transplant recipients. In two patients tested, the majority of antibody-producing B cells developed from CD27+ memory B cells. Our results suggest that analysis of B cells producing antibodies specific for donor antigens may be a useful tool for identifying and monitoring the humoral immune response in organ transplant recipients. [Copyright &y& Elsevier]

Antibody-mediated rejection is a major cause of graft failure in organ transplantation. For this reason, B cell responses are of particular interest to transplantation research. Rats are important model organisms for transplant studies, but B cell alloimmune assays and B cell subset markers are poorly established in rats. We alloimmunized rats by donor blood injection using the high responder rat strain combination Brown Norway (donor) and Lewis (recipient) rats. Using splenocytes from alloimmunized and control rats, we established assays to assess allospecific B cell proliferation and the capacity to generate allospecific B memory cells and alloantibody-secreting cells after antigenic rechallenge in vitro using a mixed lymphocyte reaction. Furthermore, we defined a simple gating and sorting strategy for pre- and post-germinal center follicular B cells, as well as non-switched and switched plasmablasts. Our protocols for assessing B cell alloresponses and B cell subsets in rats may help to accelerate research into the role of B cells and manipulation of humoral alloresponses in transplant research. [ABSTRACT FROM AUTHOR]

The 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. We 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. On 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. According 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. [ABSTRACT FROM AUTHOR]

The screening for IgG subclass donor-specific antibodies (DSAs) in allograft recipients uses IgG1-4 subclass-specific monoclonal antibodies (mAbs) that should be mono-specific. The cross-reactivity discrepancies reported for IgG subclass-specific mAbs warranted a critical cross-reactivity pattern analysis of the IgG subclass-specific mAbs most commonly used to detect DSAs. We tested the reactivity of 2 anti-IgG1-, 3 anti-IgG2-, 1 anti-IgG3-, and 2 anti-IgG4-specific PE-conjugated mAbs against microbeads coated with IgG1-4 proteins separately. Each IgG subclass protein was coated at three densities on the beads (0.5, 1, and 2 μg of protein per 106 beads), and the PE-conjugated mAbs were titrated from 0.04 μg/mL to 5 μg/mL. The IgG subclass reactivity of the sample was acquired on the Luminex multiplex platform. Among the IgG subclass-specific mAbs, only the anti-IgG3 (clone: HP6050) mAb was mono-specific. All other mAbs tested were binding to IgG subclass proteins other than their respective immunogen, thereby being cross-reactive. IgG subclass cross-reactivity patterns were dependent on the concentration of both IgG subclass-specific mAbs and IgG1–4 protein targets coated onto the beads. With the current IgG subclass mAbs available, 3 of the 15 possible combinations of IgG1–4 subclass protein could be identified. While the remaining 12 unique combinations cannot be distinguished clearly, 6 groups that corresponded to two different unique combinations of IgG1–4 subclass protein could be identified. The dilution of serum samples and IgG subclass-specific mAbs, other than the anti-IgG3 (clone: HP6050), must be further optimized before their implementation in IgG subclass DSA screening in allograft recipients. • Monoclonal antibodies used to detect IgG subclass HLA antibodies were crossreactive. • Only anti-IgG3 monoclonal antibody was mono-specific. • Crossreactivity pattern was dependent on monoclonal antibody concentration. • Crossreactivity pattern was dependent on IgG1-4 protein target concentration. • Nine reactivity patterns were observed instead of 15 with confirmed monospecificty. [ABSTRACT FROM AUTHOR]

Background Donor-specific antibodies of the IgG isotype are measured routinely for diagnostic purposes in renal transplant recipients and are associated with antibody-mediated rejection and long-term graft loss. Objective This study aimed to investigate whether MHC-specific antibodies of the IgE isotype are induced during allograft rejection. Methods Anti-MHC/HLA IgE levels were measured in sera of mice grafted with skin or heart transplants from various donor strains and in sera of kidney transplant patients with high levels of HLA IgG. Mediator release was triggered in vitro by stimulating basophils that were coated with murine or human IgE-positive serum, respectively, with specific recombinant MHC/HLA antigens. Kidney tissue samples obtained from organ donors were analyzed by using flow cytometry for cells expressing the high-affinity receptor for IgE (FcεRI). Results Donor MHC class I– and MHC class II–specific IgE was found on acute rejection of skin and heart grafts in several murine strain combinations, as well as during chronic antibody-mediated heart graft rejection. Anti-HLA IgE, including donor HLA class I and II specificities, was identified in a group of sensitized transplant recipients. Murine and human anti-MHC/HLA IgE triggered mediator release in coated basophils on stimulation with specific MHC/HLA antigens. HLA-specific IgE was not linked to atopy, and allergen-specific IgE present in allergic patients did not cross-react with HLA antigens. FcεRI+ cells were found in the human renal cortex and medulla and provide targets for HLA-specific IgE. Conclusion These results demonstrate that MHC/HLA-specific IgE develops during an alloresponse and is functional in mediating effector mechanisms. Graphical abstract [ABSTRACT FROM AUTHOR]