De Novo Donor-Specific HLA Antibodies Research Articles

Solid-Phase C1q/C3d Fixing Readouts Correlate with High Median Fluorescence Intensity (MFI) De Novo Donor-Specific HLA Antibodies and C4d⁺ Antibody-Mediated Rejection in Kidney Transplant Recipients.

BackgroundSolid-phase assays to investigate the complement-activating capacity of HLA antibodies have been utilized to optimize organ allocation and improve transplant outcomes. The clinical utility of C1q/C3d-binding characteristics of de novo donor-specific anti-HLA antibodies (dnDSA) associated with C4d-positive antibody-mediated rejection (C4d+ AMR) in kidney transplants (KTx) has not been defined.Material/MethodsSera from 120 KTx recipients that had dnDSA concurrent with protocol/cause biopsy (median 3.8 years after transplantation) were screened for C1q and C3d-binding dnDSA. The difference in the incidence of C4d+ AMR between recipients with and without C1q/C3d-binding dnDSA was assessed.ResultsOver 86% of dnDSAs were class II antibodies. The immunodominant dnDSAs characterized by the highest median fluorescence intensity (MFI) in most recipients were HLA-DQ antibodies (67%). Most recipients (62%, n=74) had either C1q+ (56%), C3d+ (48%), or both C1q+C3d+ (41.2%) dnDSA, while the remaining 38% were negative for both C1q and C3d. Of those with C1q+/C3d+ dnDSA, 87% had high-MFI IgG (MFI=14144±5363 and 13932±5278, respectively), while 65% of C1q−C3d− dnDSA had low-MFI IgG (MFI=5970±3347). The incidence of C4d+ AMR was significantly higher in recipients with C1q+ (66%), C3d+ (74%), and C1q+C3d+ (72%) dnDSA than in those with C1q−C3d− dnDSA (30%) recipients. Recipients with C3d+/C1q+ dnDSA had higher C4d+ scores on biopsy.ConclusionsC1q+/C3d+ dnDSA were associated with C4d+ AMR and high-IgG MFI. Our data call into question the predictive utility of C1q/C3d-binding assays in identifying KTx recipients at risk of allograft failure. In conclusion, IgG MFI is sufficient for clinical management, and the C1q/C3d-assays with added cost do not provide any additional information.

Development of De Novo Donor-specific HLA Antibodies and AMR in Renal Transplant Patients Depends on CYP3A5 Genotype.

Background.The single-nucleotide polymorphism CYP3A5 rs776746 is related to a reduction in the metabolizing activity of the CYP3A5 enzyme. People carrying at least one copy of the wild-type allele, defined as CYP3A5 expressers, exhibit higher clearance and lower trough concentrations of tacrolimus than homozygous nonexpressers, and this difference may affect alloimmunization and allograft function.Methods.We retrospectively studied 400 kidney transplant recipients treated with a tacrolimus-based immunosuppression regimen to detect CYP3A5 genotype, de novo formation of HLA antibodies and donor-specific antibodies (DSAs), and clinical outcome up to 5 y after transplant.Results.We found that 69 (17%) of the 400 patients were CYP3A5 expressers. During the first 3 y after transplant, CYP3A5 expressers tended to have lower tacrolimus trough levels than nonexpressers, although their tacrolimus dosage was as much as 80% higher. De novo DSAs were found more frequently in CYP3A5 expressers than in nonexpressers (13/69 [19%] versus 33/331 [10%], P = 0.02). De novo DSA-free survival rates (P = 0.02) were significantly lower for expressers than for nonexpressers. CYP3A5 genotype had no effect on allograft failure, but CYP3A5 expressers exhibited a significantly higher frequency of antibody-mediated rejection. CYP3A5 expresser status was an independent risk factor for the development of de novo DSAs (relative risk, 2.34, P = 0.01).Conclusions.Early detection of CYP3A5 expressers, enabling genotype-based dose adjustment of tacrolimus immediately after renal transplant, may be a useful strategy for reducing the risk of de novo DSA production and antibody-mediated rejection.

CLINICAL SIGNIFICANCE OF PRETRANSPLANT DONOR-SPECIFIC HLA ANTIBODIES IN KIDNEY TRANSPLANT RECIPIENTS OF HISPANIC POPULATION

Introduction: Donor-specific HLA antibodies (DSA) before kidney transplantation (KT) is one of the major risk factors for humoral rejection and lower graft survival (1-2). In order to identify prognostic factors and develop follow up strategies in this high risk population, it is required for transplant center to share their outcome data (3-4). In addition, in most previous studies the Hispanic population is poorly represented. Materials and Methods: Retrospective cohort and comparative study in KT recipients with pretransplant DSA measured by Luminex, negative AHG-CDC-XM (living and deceased donor) and negative flow cross match (living donor). Our main objective was to determine graft survival and describe the incidence of acute rejection (AR) and renal function (eGFR by CKD-EPI). Results: Between September 2006 and March 2017, 411 patients received KT in the INCMNSZ, 60 were identified with pretransplant DSA and compared with 60 KT recipients without pretransplant DSA, paired by donor type, induction and maintenance immunosuppression. Median age was 33 (range 27-44) years, 55 (45%) were women, 55 (45.8%) had unknown etiology of ESRD, months on dialysis [24 (range 12-48)], 10.7% received second KT, 63.3% were living donor receptor, 75% had thymoglobulin induction, 96% were with tacrolimus, mycofenolate mofetil and steroids as maintenance immunosupression. The incidence of AR was increased in the pretransplant DSA group (35.5% vs. 15.2%, p=0.011) and the median time between KT and AR episodes was lower in the pretransplant DSA group [12.8 (8.3-23.6) vs 32.1 (25.9-40.6), (p<0.0001)] months.In the pretransplant DSA group 52.4% of the biopsies with AR were protocol biopsies and 29% were secondary to graft disfunction.Incidence of De Novo DSA were similar between groups [30 (53.6%) vs 29 (56.8%), (p=0.732)].After 37.4 (range 29.2-52.3) months of follow up, eGFR was similar between groups 65.0 ± 22.0 vs 69.8 ± 21.4 ml/min/1.73m2 (p=0.19) and there were no difference in graft survival (87.7% vs 96.7% p=0.240) between pretransplant DSA and without pretransplant DSA groups, respectively.Discussion and conclusions: Although there is a higher incidence of rejection, KT recipients with pretransplant DSA could be considered candidates for KT. One limitation of this study is the small sample size and short follow-up time. However, we acknowledge that in this population of high immunological risk, induction therapy with lymphocyte depletors and powerful maintenance immunosuppression, combined with follow-up strategies as protocol biopsies with early treatment of subclinical rejection could provide similar graft survival compared to KT receptors without pretransplant DSA.

Mid-Term Outcomes After Treatment for Antibody-Mediated Rejection by De Novo Donor-Specific HLA Antibody in Renal Transplant Recipients: Does Early Treatment Lead to Better Outcomes?

BackgroundDe novo donor-specific HLA antibody (DSA) and antibody-mediated rejection (ABMR) are strongly associated with late allograft loss in renal transplant recipients. However, the impact of therapeutic intervention with the current treatment options for ABMR remains unclear. This study aimed to elucidate the efficacy of treatment for ABMR. MethodsSixty-seven patients who had de novo DSAs underwent diagnostic biopsy for ABMR, and these patients were classified into 3 groups: ABMR-free group (n = 40), clinical ABMR group (n = 15), and subclinical ABMR group (n = 12). The ABMR-positive groups were treated mainly with double-filtration plasmapheresis followed by rituximab and corticosteroid pulse. The patient characteristics and graft outcomes were compared between groups. ResultsThe clinical and subclinical ABMR groups were younger and had a higher number and mean fluorescence intensity (MFI) of de novo DSAs than the ABMR-free group. The graft survival in the clinical ABMR group was significantly lower than that in the ABMR-free group, but the subclinical ABMR group had a surprisingly good graft survival rate compared to the ABMR-free group (43.3% vs 100% vs 94.2% 5 years after diagnostic biopsy in the clinical ABMR, subclinical ABMR, and ABMR-free groups, respectively, P < .001). ConclusionsOur findings indicated that early therapeutic intervention for patients with de novo DSAs may improve graft survival.

A diagnostic 'C' saw: the ups and downs of C1q testing.

The present review will focus on recently published data of solid organ allograft recipients reporting that patients with de-novo donor-specific HLA antibodies (DSA) that fix complement in vitro have a significantly higher risk for antibody-mediated rejection (AMR) and/or graft loss compared to patients whose de-novo DSA do not fix complement or patients who present with preexisting complement fixing DSA. HLA DSAs that fix complement in vitro appear to be a key indicator for rejection and failure of kidney, heart, and lung allografts from studies performed around the world. The majority of these studies are population based and retrospective in nature. Although these studies seemingly indicate that in-vitro complement activating DSAs represent a higher clinical risk than noncomplement fixing DSAs, the majority have not accounted for false-negative reactions attributable to the so-called prozone/interference phenomenon. In the limited number of published studies addressing that concern, high mean fluorescence intensity (MFI) value noncomplement fixing DSAs correlate as well as complement fixing DSAs with AMR and graft loss. Combined with the cost of additional testing, these observations bring into question whether there is sufficient clinical applicability to warrant routine testing for complement fixing antibodies. Complement fixing DSAs are clearly associated with AMR and/or loss of transplanted allografts. However, under appropriate testing conditions, complement fixing capability typically correlates with MFI values of the DSAs. As such, the routine implementation of in-vitro assays to determine whether DSAs fix complement is of questionable value especially when considering additional issues such as cost of testing, logistics, and whether the test results factor into individualized patient care.

Anti-perlecan antibodies and acute humoral rejection in hypersensitized patients without forbidden HLA specificities after kidney transplantation

BackgroundThe improvement in the definition of serum anti-HLA antibodies (HLA-Abs) profiles after Luminex-assay implementation in transplant patients follow-up is clear. This success has permitted the development of hypersensitized-recipient allocation and donor-paired exchange programs improving the access to transplantation. However, non-HLA Abs have been described in transplanted patients but their effect in hypersensitized transplanted recipients is unclear. MethodsTwenty-seven HLA hypersensitized patients awaiting for kidney transplantation (KT) were studied and 11 of them were followed after KT. The HLA Abs profile was confirmed in serum by Single Antigen Luminex assay and panel reactive of antigens >98% was achieved in all patients. Subsequently, the ability to fix complement by C1q test was also assessed. Serum non-HLA Abs before and 1 month after transplantation were measured in the 11 hypersensitized recipients. Results95.2% of the hypersensitized on waiting list had concomitant serum anti-HLA and non-HLA Abs. The more frequent specificity in non-HLA Abs were found against Glutathione S-transferase theta-1 (GSST-1) (in 62%) and C-terminal fragment of perlecan (LG3) (in 52%). Four out of 11 transplanted patients presented early antibody-mediated rejection (ABMR) confirmed by biopsy and had serum anti-LG3 antibodies, two of them with concomitant anti-anti-angiotensin II type I receptor. Only one patient developed de novo-donor specific HLA antibodies. ConclusionsThe incidence of non-HLA antibodies in patients in the waiting list is largely underestimated. The concomitance anti-HLA and non-HLA Abs in hypersensitized patients is very common and the detection of non-HLA Abs in this population could allow to identify patients with an increased risk of humoral rejection.

Clinical impact of complement (C1q, C3d) binding De Novo donor-specific HLA antibody in kidney transplant recipients.

IntroductionComplement binding activity of donor-specific HLA antibodies (DSA) has been suggested as a new tool to stratify immunologic risk in kidney transplantation (KT). The objective of this study was to evaluate the clinical implication of C1q/C3d binding activity of de novo DSA (dnDSA) in KT recipients.Material and methodsA total of 161 pretransplant DSA-negative recipients were monitored for dnDSA at the time of biopsy. C1q/C3d binding activities of dnDSA were assessed using C1qScreen assay (One lambda, USA) and Lifecodes C3d detection assay (Immucor, USA), respectively. Clinical outcomes including biopsy-proven antibody mediated rejection (AMR), C4d detection and post-biopsy graft survival were investigated.ResultsDe-novo DSAs were detected in fifty-four (33.5%) patients (HLA class I only, n = 19; class II only, n = 29; both class I and II, n = 6). Of them, complement binding activities were detected in 26 (48.1%) patients, including 17 C1q+ and 24 C3d+ patients. Both C1q and C3d positivity were associated with increased mean fluorescence intensity values of dnDSA. Complement binding activity of dnDSA enhanced the incidence of AMR (25.0% in C1q-C3d-, 36.4% in C1q+/C3d- or C1q-/C3d+, and 60.0% in C1q+/C3d+ patients) (P <0.001). The incidence of AMR was not different between patients with C1q+ and those with C3d+ dnDSA (64.7%, 11/17 versus 45.8%, 11/24, P = 0.238). In comparison between C1q and C3d assay according to HLA specificity, C1q+ HLA class I ± II dnDSA was the best predictor for AMR (odds ratio: 27.2). C1q+/C3d+ dnDSA was associated with more C4d deposition in allograft tissue and inferior post-biopsy graft survival. Clinical outcomes were not significantly different between C1q+ and C3d+ dnDSA-positive patients.ConclusionDetection of complement binding activity using both C1q and C3d assays can be a further prognostic marker for predicting AMR and allograft outcome in dnDSA+ kidney transplant patients.

Immunosuppression Related to De Novo Donor-Specific HLA Antibody Production after Renal Transplantation

Aim One of the major factors influencing graft loss is antibody-mediated rejection caused by the production of donor-specific HLA antibodies (DSA). The optimization of multidrug combination therapy using immunosuppressants is required in order to prevent DSA production. We herein evaluated the relationships among clinical variants including immunosuppressant levels and the prediction of DSA production after renal transplantation (Tx). Methods Living renal transplant patients (n=160) at our institute who received standard immunosuppressant protocols with basiliximab, tacrolimus (Tac), mycophenolate mofetil (MMF), and steroids between July 2004 and September 2013 were enrolled. The production of serum anti-HLA antibodies at one-year post-Tx was analyzed by solid-phase bead-based assays using Luminex. The HLA specificity of DSA was evaluated based on the data of donor HLA typing. In the present study, preformed DSA and de novo DSA were defined as those detected pre-Tx and at one-year post-Tx, and those detected at one-year post-Tx only, respectively. Risk factors for DSA production were statistically evaluated using clinical variables including Tac trough levels and the incidence of virus infections. Results DSA production at one-year post-Tx was observed in 15 patients including 9 with preformed DSA and 7 with de novo DSA. Preformed and de novo DSA were detected in one patient. De novo DSA production was more likely to occur in patients with low serum Tac levels (cut-off value: 3.2 ng/mL), in those who ceased MMF medication, and in those who acquired CMV infection within one-year post-Tx (p=0.008, 0.044, and 0.050, respectively). Conclusions The appropriate control of Tac trough levels, continuation of MMF intake, and prevention of CMV infection were found to be important for avoiding de novo DSA production at one-year post-Tx in the present study.

Effect of Steroid Withdrawal on the Appearance of De Novo Donor-Specific HLA Antibodies in Kidney Transplant Recipients

Effect of Steroid Withdrawal on the Appearance of De Novo Donor-Specific HLA Antibodies in Kidney Transplant Recipients

Donor-Recipient Matching Based on Predicted Indirectly Recognizable HLA Epitopes Independently Predicts the Incidence of De Novo Donor-Specific HLA Antibodies Following Renal Transplantation.

De novo donor-specific HLA antibodies (dnDSA) are recognized as a risk factor for premature allograft failure. Determinants of DSA specificity are generated via the indirect allorecognition pathway. Here, we present supportive data for the relevance of predicted indirectly recognizable HLA epitopes (PIRCHE) to predict dnDSA following kidney transplantation. A total of 2787 consecutive kidney transplants performed between 1995 and 2015 without preformed DSA have been analyzed. De novo DSA were detected by single antigen bead assay. HLA epitope mismatches were determined by the HLAMatchmaker and PIRCHE approach and correlated in uni- and multivariate analyses with 10-year allograft survival and incidence of dnDSA. The PIRCHE-II score moderately predicted allograft survival. However, the predictive value of elevated PIRCHE-II scores >9 for the incidence of dnDSA was statistically significant (p < 0.001). In a multivariate Cox regression analysis adjusted for antigen mismatch and HLAMatchmaker epitopes, the PIRCHE-II score could be identified as an independent risk factor for dnDSA. The PIRCHE-II score independently from the antigen mismatch and HLAMatchmaker epitopes could be revealed as being a strong predictor for dnDSA. PIRCHE may help to identify acceptable mismatches with decreased risk of dnDSA and thus improve long-term renal allograft survival.

De Novo Donor-Specific HLA Antibodies Developing Early or Late after Transplant Are Associated with the Same Risk of Graft Damage and Loss in Nonsensitized Kidney Recipients.

De novo posttransplant donor-specific HLA-antibody (dnDSA) detection is now recognized as a tool to identify patients at risk for antibody-mediated rejection (AMR) and graft loss. It is still unclear whether the time interval from transplant to DSA occurrence influences graft damage. Utilizing sera collected longitudinally, we evaluated 114 consecutive primary pediatric kidney recipients grafted between 2002 and 2013 for dnDSA occurrence by Luminex platform. dnDSAs occurred in 39 patients at a median time of 24.6 months. In 15 patients, dnDSAs developed within 1 year (early-onset group), while the other 24 seroconverted after the first posttransplant year (late-onset group). The two groups were comparable when considering patient- and transplant-related factors, as well as DSA biological properties, including C1q and C3d complement-binding ability. Only recipient age at transplant significantly differed in the two cohorts, with younger patients showing earlier dnDSA development. Late AMR was diagnosed in 47% of the early group and in 58% of the late group. Graft loss occurred in 3/15 (20%) and 4/24 (17%) patients in early- and late-onset groups, respectively (p = ns). In our pediatric kidney recipients, dnDSAs predict AMR and graft loss irrespective of the time elapsed between transplantation and antibody occurrence.

Kidney Intragraft Homing of De Novo Donor-Specific HLA Antibodies Is an Essential Step of Antibody-Mediated Damage but Not Per Se Predictive of Graft Loss.

Donor-specific HLA antibody (DSA)-mediated graft injury is the major cause of kidney loss. Among DSA characteristics, graft homing has been suggested as an indicator of severe tissue damage. We analyzed the role of de novo DSA (dnDSA) graft homing on kidney transplantation outcome. Graft biopsy specimens and parallel sera from 48 nonsensitized pediatric kidney recipients were analyzed. Serum samples and eluates from graft biopsy specimens were tested for the presence of dnDSAs with flow bead technology. Intragraft dnDSAs (gDSAs) were never detected in the absence of serum dnDSAs (sDSAs), whereas in the presence of sDSAs, gDSAs were demonstrated in 72% of biopsy specimens. A significantly higher homing capability was expressed by class II sDSAs endowed with high mean fluorescence intensity and C3d- and/or C1q-fixing properties. In patients with available sequential biopsy specimens, we detected gDSAs before the appearance of antibody-mediated rejection. In sDSA-positive patients, gDSA positivity did not allow stratification for antibody-mediated graft lesions and graft loss. However, a consistent detection of skewed unique DSA specificities was observed over time within the graft, likely responsible for the damage. Our results indicate that gDSAs could represent an instrumental tool to identify, among sDSAs, clinically relevant antibody specificities requiring monitoring and possibly guiding patient management.

OR26 Taking the hla-specific memory b cell elispot to the next level: assaying the full donor hla repertoire

Aim Pre-existing or de-novo donor-specific HLA antibodies (DSA) represent an important risk factor affecting transplant outcome. Patients with a history of immunization but lacking serum DSA may harbor dormant memory B cells which can rapidly produce DSA upon re-encounter with the same HLA. Current methods to detect memory B cells mainly utilize synthetic monomeric/tetrameric HLA molecules which generally do not represent the complete HLA repertoire of an individual. Here, we present a donor-specific HLA-ELISPOT assay enabling the screening for HLA-specific memory B cells in peripheral blood of immunized individuals using cell lysates as a natural source of both HLA class I and II. Methods Peripheral blood mononuclear cells (PBMC) or splenocytes were treated with non-ionic detergents to obtain HLA-containing lysates. Human B cell hybridomas producing specific HLA antibodies were tested against these lysates for validation purposes (n = 10). Next, polyclonally activated peripheral blood B cells from women with a history of pregnancy were tested for the presence of HLA-specific memory B cells against paternal PBMC lysates as the source of HLA (n = 22). Autologous lysates served as negative controls. Results For all hybridomas tested, we detected spot formation against the lysates that contain the corresponding HLA specificities whereas no spots were observed against lysates with irrelevant HLA, indicating the specificity of the assay. Comparable number of spots in total IgG and HLA-ELISPOT assays assured that all antibody-secreting cells were detected. In women with a history of pregnancy, we found a median of 14 (range: 0–802 spots) and 9 spots (range: 0–764) per million IgG producing cells for class I and II, respectively. Conclusion This novel lysate-based ELISPOT assay allows for the first time to quantify all donor HLA class I and II-specific memory B cells and may predict anamnestic responses in transplant recipients with a history of immunization.

The Influence of Immunosuppressive Agents on the Risk of De Novo Donor-Specific HLA Antibody Production in Solid Organ Transplant Recipients

Production of de novo donor-specific antibodies (dnDSA) is a major risk factor for acute and chronic antibody-mediated rejection and graft loss after all solid organ transplantation. In this article, we review the data available on the risk of individual immunosuppressive agents and their ability to prevent dnDSA production. Induction therapy with rabbit antithymocyte globulin may achieve a short-term decrease in dnDSA production in moderately sensitized patients. Rituximab induction may be beneficial in sensitized patients, and in abrogating rebound antibody response in patients undergoing desensitization or treatment for antibody-mediated rejection. Use of bortezomib for induction therapy in at-risk patients is of interest, but the benefits are unproven. In maintenance regimens, nonadherent and previously sensitized patients are not suitable for aggressive weaning protocols, particularly early calcineurin inhibitor withdrawal without lymphocyte-depleting induction. Early conversion to mammalian target of rapamycin inhibitor monotherapy has been reported to increase the risk of dnDSA formation, but a combination of mammalian target of rapamycin inhibitor and reduced-exposure calcineurin inhibitor does not appear to alter the risk. Early steroid therapy withdrawal in standard-risk patients after induction has no known dnDSA penalty. The available data do not demonstrate a consistent effect of mycophenolic acid on dnDSA production. Risk minimization for dnDSA requires monitoring of adherence, appropriate risk stratification, risk-based immunosuppression intensity, and prospective DSA surveillance.

C1q Binding Activity of De Novo Donor-specific HLA Antibodies in Renal Transplant Recipients With and Without Antibody-mediated Rejection.

Complement fixation by donor-specific HLA antibodies (DSA) is a primary mechanism for antibody-mediated damage of organ allografts. Using a recently developed kit that measures C1q binding to distinguish complement fixing and nonfixing antibodies, studies showed that C1q + DSAs have a higher risk of rejection and graft loss compared to C1q-DSA. The objective of this study was to assess the ability of the C1q-binding assay to identify clinically significant de novo DSA in renal transplant recipients and to define the properties of DSA that confer C1q binding ability. The DSA-positive sera from 34 kidney recipients, 19 with biopsy-proven antibody-mediated rejection (AMR) + and 15 who were AMR-, were assayed in C1q-binding assays (C1q Screen; One Lambda, Inc. Canoga Park, CA). The correlation between C1q-binding activity, presence of AMR, DSA mean fluorescence intensity (MFI) values, and immunoglobulin G isotype was determined. Fifty-three percent (10/19) of sera from AMR+ patients had C1q + DSA, whereas only 13% (2/15) of sera from AMR- patients contained C1q + DSA. C1q + DSA exhibited significantly higher MFI values regardless of whether they were from AMR+ or AMR- patients (16,118 ± 6698 vs 6429 ± 4003; P < 0.0001). C1q + DSA converted to C1q - when diluted to a comparable MFI level as the C1q - DSA from AMR- patients, and some C1q - antibodies converted to C1q + when concentrated to MFI levels comparable to those observed for AMR+/C1q + sera. The C1q binding activity by de novo DSA in patients with AMR largely reflects differences in antibody strength. The C1q assay does not appear to distinguish functionally distinct DSA with clinical significance.

68-P: DONOR-SPECIFIC CLASS II HLA-DQ COMPLEMENT BINDING ANTIBODY IS ASSOCIATED WITH SEVERE REJECTION IN LUNG TRANSPLANTATION

Aim The significance of donor-specific HLA antibodies (DSA) in lung transplantation (LTx) has emerged with the introduction of solid phase assays for antibody (Ab) detection. In the present study we evaluated 1) frequency of DSA in LTx using Luminex (L)-based methods for specificity and functional characterization and 2) the impact of de novo DSA on LTx outcome. Methods LTx from 2011 to 2012 and screened prospectively with L-PRA beads were included in the analysis. Testing was done pre- and post-Tx at 1 month and then every 2 months with surveillance and clinical bronchoscopies. DSA specificity/strength (MFI) determined with single antigen beads, and complement binding with the L-C1q assay, was associated with cellular (ACR) and Ab mediated rejection (AMR). Results 161 consecutive LTx with minimum follow-up of 6 months and negative CDC T- and B-cell crossmatch were studied. 10% had DSA pre-Tx and 19% had de-novo DSA. 54% of LTx had high grade ACR; de novo DSA was higher in ACR (27/87, 31%) vs. no ACR (10/74, 13.5%). The most frequent (67%) de novo DSA were HLA-DQ including Ab towards DQA,DQB/DQA pair and DQB (fig). In LTx with persistent, severe ACR (n=4), increased levels of de novo DSA with C1q reactivity were associated with AMR that required treatment with PP/IVIG and Ritux. In contrast, LTx (n=9) with persistent ACR and low level or declining DSA with transient C1q reactivity did not develop AMR. Similarly, no AMR was diagnosed in LTx who had one or more ACR and transient DSA. DSA detected without ACR were also low level, transient and without C1q reactivity. Conclusions Persistent and high grade ACR with C1q binding class II HLA-DQ specific DSA was associated with AMR. [figure1]

De Novo Donor-Specific HLA Antibodies Decrease Patient and Graft Survival in Liver Transplant Recipients

The role of de novo donor-specific HLA antibodies (DSA) in liver transplantation remains unknown as most of the previous studies have only focused on preformed HLA antibodies. To understand the significance of de novo DSA, we designed a retrospective cohort study of 749 adult liver transplant recipients with pre- and posttransplant serum samples that were analyzed for DSA. We found that 8.1% of patients developed de novo DSA 1 year after transplant; almost all de novo DSAs were against HLA class II antigens, and the majority were against DQ antigens. In multivariable modeling, the use of cyclosporine (as opposed to tacrolimus) and low calcineurin inhibitor levels increased the risk of de novo DSA formation, while a calculated MELD score >15 at transplant and recipient age >60 years old reduced the risk. Multivariable analysis also demonstrated that patients with de novo DSA at 1-year had significantly lower patient and graft survival. In conclusion, we demonstrate that de novo DSA development after liver transplantation is an independent risk factor for patient death and graft loss.

Increased Frequency of Class II HLA-DQ Donor-Specific Antibodies Is Associated with Mixed Cellular and Humoral Rejection in Lung Transplantation

Purpose The significance of donor-specific HLA antibodies (DSA) in lung transplantation (LTx) has emerged with the introduction of solid phase assays for antibody detection. Using ELISA based screening we have shown that 14% of LTx had de novo DSA and most (78%) developed chronic allograft rejection vs. 24% in patients without DSA. Further evaluation of DSA with Luminex (L) based methods for screening, specificity and functional analysis is needed to determine the impact of pre-formed and de-novo DSA on outcome. Methods and Materials Patients who were transplanted January 2011 to July 2012 and were screened prospectively for DSA with L-PRA were included in the analysis. The screening included samples pre-Tx and post LTx at 1 month and every 2 months in conjunction with surveillance and clinical bronchoscopies. The specificity of DSA and strengths (MFI) determined with single antigen beads (L-IgG) and complement binding with L-C1q- assay (L-C1q) was associated with cellular rejection and antibody mediated rejection. Results This study was done on 149 consecutive LTx with a minimum follow-up of 3 months and adequate DSA screening. All had negative CDC T and B cell crossmatches. 5% had DSA pre-Tx that persisted post-Tx and 23% had de-novo DSA within 3-6 months post LTx. Overall, 55% of LTx had high grade ACR; de-novo DSA was higher in ACR (22/82, 27%) vs. no ACR (5/67, 7%) (p 70%) de-novo DSA were HLA-DQ specific including Ab towards DQB, DQA and combination of DQB/DQA pairs. Severe ACR (n=4) with DSA and C1q reactivity was treated with plasmapheresis, IVIG and Rituximab for AMR. Patients with ACR and low level or declining DSA that had a transient or no C1q reactivity (n=19) were not treated for AMR. DSA in LTx without ACR were also low level, transient and without C1q reactivity. Conclusions The frequency of de-novo DSA is higher than previously reported. Persistent, high grade ACR with C1q binding class II HLA-DQ DSA was associated with AMR.

Time from Development of De Novo Donor-Specific HLA Antibodies to Rejection or Graft Loss in Pediatric Cardiac Transplantation

Purpose There is increasing evidence that de novo donor-specific HLA antibodies (DSA) are associated with poor outcome following cardiac transplantation. Management of these remains problematic and there is little data on lag time from DSA development to an adverse event. We aimed to determine average time from first detection of DSA to rejection, cardiac allograft vasculopathy(CAV) or graft loss in a pediatric cohort. Methods and Materials Luminex screens and single antigen tests were performed on stored samples taken at 3, 6, 12 months and then annually in patients transplanted age Results 108 patients, mean age at transplant 7.4 (0.1-15.9) years were studied, mean follow-up 8.2 (1.9-15.7) years. All had a CDC-negative crossmatch and retrospective serum analysis confirmed they had no DSA prior to transplant. 43/108 (40%) patients had DSA with median time from transplant to DSA development of 2 (0.25-11) years. Freedom from DSA was 91% at 3 months, 86% at 6 months, 84% at 1 year, 69% at 5 years, 58% at 10 years and 48% at 15 years post-transplant. 9 patients with DSA (21%) developed CAV, and in 6 (67%), DSA were detected prior to CAV diagnosis with median lag time of 2.2 (1-4.9) years. 14 patients (33%) had a rejection episode, 9 (64%) had DSA prior to rejection with median lag time time of 2.8 (0.2-7.2) years. 10 patients (23%) died or were re-transplanted, with median time from development of DSA to graft loss of 1.7 (0.1-4.8) years. 28/33 (85%) patients with an adverse outcome had Class II DSA with anti-DQ the most prevalent. Conclusions In patients with DSA in whom an adverse event occurred (CAV, Rejection or Graft loss) DSA were detected in 25/33 (76%) prior to the adverse outcome. The majority of patients with an adverse outcome in this study had Class II DSA detected prior to that event. These findings suggest that once DSAs have developed increased surveillance for adverse events should be implemented.

Posttransplant De Novo Donor-Specific HLA Antibodies Identify Pediatric Kidney Recipients at Risk for Late Antibody-Mediated Rejection

The emerging role of humoral immunity in the pathogenesis of chronic allograft damage has prompted research aimed at assessing the role of anti-HLA antibody (Ab) monitoring as a tool to predict allograft outcome. Data on the natural history of allografts in children developing de novo Ab after transplantation are limited. Utilizing sera collected pretransplant, and serially posttransplant, we retrospectively evaluated 82 consecutive primary pediatric kidney recipients, without pretransplant donor-specific antibodies (DSA), for de novo Ab occurrence, and compared results with clinical-pathologic data. At 4.3-year follow up, 19 patients (23%) developed de novo DSA whereas 24 had de novo non-DSA (NDSA, 29%). DSA appeared at a median time of 24 months after transplantation and were mostly directed to HLA-DQ antigens. Among the 82 patients, eight developed late/chronic active C4d+ antibody-mediated rejection (AMR), and four C4d-negative AMR. Late AMR correlated with DSA (p < 0.01), whose development preceded AMR by 1-year median time. Patients with DSA had a median serum creatinine of 1.44 mg/dL at follow up, significantly higher than NDSA and Ab-negative patients (p < 0.005). In our pediatric cohort, DSA identify patients at risk of renal dysfunction, AMR and graft loss; treatment started at Ab emergence might prevent AMR occurrence and/or progression to graft failure.