Early Graft Losses in Paired Kidney Exchange: Experience from 10 Years of the National Kidney Registry.

Cooperative kidney paired donation (KPD) networks account for an increasing proportion of all living donor kidney transplants in the United States. There is sparse data on the rate of primary non-function (PNF) losses and their consequences within KPD networks. We studied National Kidney Registry (NKR) transplants (2/14/2009-12/31/2017) and quantified PNF, graft loss within 30 days of transplantation, and graft losses in the first year post-transplant and assessed potential risk factors. Of 2364 transplants, there were 38 (1.6%) grafts lost within the first year, 13 (0.5%) with PNF. When compared to functioning grafts, there were no clinically significant differences in blood type compatibility, degree of HLA mismatch, number of veins/arteries, cold ischemia, and travel times. Of 13 PNF cases, 2 were due to early venous thrombosis, 2 to arterial thrombosis, and 2 to failure of desensitization and development of AMR. Given the low rate of PNF, the NKR created a policy to allocate chain-end kidneys to recipients with PNF following event review and attributable to surgical issues of donor nephrectomy. It is expected that demonstration of low incidence of poor early graft outcomes and the presence of a 'safety net' would further encourage program participation in national KPD. (link)

Genomic Mismatch at LIMS1 Locus and Kidney Allograft Rejection

BACKGROUND: In the context of kidney transplantation, genomic incompatibilities between donor and recipient may lead to allosensitization against new antigens. We hypothesized that recessive inheritance of gene-disrupting variants may represent a risk factor for allograft rejection.
METHODS : We performed a two-stage genetic association study of kidney allograft rejection. In the first stage, we performed a recessive association screen of 50 common gene-intersecting deletion polymorphisms in a cohort of kidney transplant recipients. In the second stage, we replicated our findings in three independent cohorts of donor–recipient pairs. We defined genomic collision as a specific donor–recipient genotype combination in which a recipient who was homozygous for a gene-intersecting deletion received a transplant from a nonhomozygous donor. Identification of alloantibodies was performed with the use of protein arrays, enzyme-linked immunosorbent assays, and Western blot analyses.
RESULTS : In the discovery cohort, which included 705 recipients, we found a significant association with allograft rejection at the LIMS1 locus represented by rs893403 (hazard ratio with the risk genotype vs. nonrisk genotypes, 1.84; 95% confidence interval [CI], 1.35 to 2.50; P=9.8×10−5). This effect was replicated under the genomic-collision model in three independent cohorts involving a total of 2004 donor–recipient pairs (hazard ratio, 1.55; 95% CI, 1.25 to 1.93; P=6.5×10−5). In the combined analysis (discovery cohort plus replication cohorts), the risk genotype was associated with a higher risk of rejection than the nonrisk genotype (hazard ratio, 1.63; 95% CI, 1.37 to 1.95; P=4.7×10−8). We identified a specific antibody response against LIMS1, a kidney-expressed protein encoded within the collision locus. The response involved predominantly IgG2 and IgG3 antibody subclasses.
CONCLUSIONS : We found that the LIMS1 locus appeared to encode a minor histocompatibility antigen. Genomic collision at this locus was associated with rejection of the kidney allograft and with production of anti-LIMS1 IgG2 and IgG3. (Funded by the Columbia University Transplant Center and others.) (read more)

Should Epitope-Based HLA Compatibility Be Used in the Kidney Allocation System?

The new kidney allocation system (KAS) still applies donor-recipient HLA compatibility at the antigen level and some four-digit alleles have been added recently. This system is used to record unacceptable mismatches for sensitized transplant candidates with serum HLA antibodies. Since the reactivities of such antibodies are specifically associated with epitopes rather than HLA antigens, a more scientifically accurate assessment of mismatch acceptability could be based on epitopes. HLA class I and class II epitope specificity analyses can now be readily performed with serum antibody assays with single allele panels. This report describes an epitope-based HLA compatibility system for KAS and involves recipient and donor HLA typing at the four-digit allele level. It focuses on sensitized patients who have serum antibodies specific for HLA epitopes that can be entered as unacceptable mismatches in the transplant candidate database. Newly developed software programs could readily identify compatible HLA types (read more)

Acquired down-regulation of donor-specific antibody production after ABO-incompatible kidney transplantation

The mechanism of long-term B cell immunity against donor blood group antigens in recipients who undergo ABO-incompatible (ABOi) living-donor kidney transplantation (LKTx) is unknown. To address this question, we evaluated serial anti-A and anti-B antibody titers in 50 adult recipients. Donor-specific antibody titers remained low (≤1:4) in 42 recipients (84%). However, antibodies against nondonor blood group antigens were continuously produced in recipients with blood type O. We stimulated recipients’ peripheral blood mononuclear cells in vitro to investigate whether B-cells produced antibodies against donor blood group antigens in the absence of graft adsorption in vivo. Antibodies in cell culture supernatant were measured using specific enzyme-linked immunosorbent assays (ELISA). Thirty-five healthy volunteers and 57 recipients who underwent ABO-compatible LKTx served as controls. Antibody production in vitro against donor blood group antigens by cells from ABOi LKTx patients was lower than in the control groups. Immunoglobulin deposits were undetectable in biopsies of grafts of eight recipients with low antibody titers (≤1:4) after ABOi LKTx. One patient with blood type A1 who received a second ABOi LKTx from a type B donor did not produce B-specific antibodies. These findings suggest diminished donor-specific antibody-production function in the setting of adult ABOi LKTx (read more)

Pretransplantation donor-recipient pair seroreactivity against BK polyomavirus predicts viremia and nephropathy after kidney transplantation

Kidney transplant donors are currently not implicated in predicting BK polyomavirus (BKPyV) infection in kidney transplant recipients. It has been postulated, however, that BKPyV infection originates from the kidney allograft. Since BKPyV-seroreactivity correlates with BKPyV-replication and, therefore, might mirror the infectious load, we investigated whether BKPyV-seroreactivity of the donor predicts viremia and BKPyV-associated nephropathy (BKPyVAN) in the recipient. In a retrospective cohort of 407 living kidney donor-recipient pairs pretransplantation donor and recipient sera were tested for BKPyV IgG-levels and correlated with the occurrence of recipient BKPyV viremia and BKPyVAN within one year posttransplantation. As such, donor BKPyV IgG-level was strongly associated with BKPyV viremia and BKPyVAN (p < 0.001), while recipient BKPyV-seroreactivity showed a non-significant inverse trend. Pairing of high BKPyV-seroreactive donors with low seroreactive recipients resulted in a 10-fold increased risk for BKPyV viremia (HR 10.1, 95% CI: 3.5 – 29.0, p < 0.001). In multivariate analysis, donor BKPyV-seroreactivity was the strongest pretransplantation factor associated with viremia (p < 0.001) and BKPyVAN (p = 0.007). The proportional relation between donor BKPyV-seroreactivity and recipient infection suggests that donor BKPyV-seroreactivity reflects the infectious load of the kidney allograft, and calls for the use of pretransplantation BKPyV-serological testing of (potential) donors and recipients (read more)

The Risk of Transplant Failure With HLA Mismatch in First Adult Kidney Allografts From Deceased Donors

Background: Since the beginning of the technology, there has been active debate about the role of human leucocyte antigen (HLA) matching in kidney allograft survival. Recent studies have reported diminishing importance of HLA matching, which have, in turn, been challenged by reports that suggest the continuing importance of these loci. Given the controversies, we examined the effect of HLA compatibility on kidney allograft survival by studying all first adult kidney transplants in the United States from a deceased donor.
Methods: Using the United Network for Organ Sharing data, we identified first deceased donor kidney transplants between October 1, 1987, and December 31, 2013. Recipients were classified by their number of HLA mismatches. Cox multivariate regression analyses adjusting for recipient and donor transplant characteristics were performed to determine the impact of HLA compatibility on kidney allograft survival.
Results: Study cohort included 189 141 first adult kidney alone transplants, with a total of 994 558 years of kidney allograft follow-up time. Analyses adjusted for recipient and donor characteristics demonstrated a 13% higher risk (hazard ratio, 1.13; 95% confidence interval, 1.06-1.21) with 1 mismatch and a 64% higher risk (hazard ratio, 1.64, 95% confidence interval, 1.56-1.73) with 6 mismatches. Dividing the mismatch categories into 27 ordered permutations, and testing their 57 within mismatch category differences, demonstrated that all but 1 were equal, independent of locus.
Conclusions: A significant linear relationship of hazard ratios was associated with HLA mismatch and affects allograft survival even during the recent periods of increasing success in renal transplantation (read more)

A reliable method for avoiding false negative results with Luminex single antigen beads; evidence of the prozone effect.

Luminex single antigen bead (SAB) assays have become an essential tool in monitoring the status of antibody to the Human Leucocyte Antigen (HLA) of patients both before and after transplantation. In addition SAB data is used to aid risk stratification to assess immunological risk of humoral rejection in solid organ transplantation (CTAG/BTAG guidelines). Increasingly laboratories are reporting false negative results at high antibody titre due to a prozone effect. Here we report a case study where the prozone effect led to a false negative antibody result that could have resulted in adverse outcome. We describe a method to reliably remove the prozone effect through heat inactivation and the addition of Ethylenediaminetetraacetic acid (EDTA) to the Luminex wash buffer (read more)

Flow Cytometry Crossmatch Reactivity With Pronase-Treated T Cells Induced by Non-HLA Autoantibodies in Human Immunodeficiency Virus-Infected Patients.

Pronase treatment is used in the flow cytometry crossmatch (FCXM) to prevent nonspecific antibody binding on B cells. However, we have observed unexpected positive results with pronase-treated T cells in human immunodeficiency virus (HIV)-infected patients. In this study, 25 HIV-infected patients without HLA antibodies were tested with pronase-treated and nontreated cells. HIV-positive sera were pretreated with reducing agents and preabsorbed with pronase-treated and nontreated T or B cells before crossmatching. All patients displayed FCXM reactivity with pronase-treated T cells but not with nontreated T cells. None of the patients exhibited FCXM reactivity with pronase-treated and nontreated B cells. These patients displayed FCXM reactivity with pronase-treated CD4+ and CD8+ T cells but not with their nontreated counterparts. Preabsorption with pronase-treated T cells reduced the T cell FCXM reactivity. Preabsorption with pronase-treated B cells or nontreated T and B cells did not have any effect on the T cell FCXM reactivity. Pretreatment with reducing agents did not affect the T cell FCXM reactivity. 15 of 21 HIV-infected kidney allograft recipients with pronase-treated T cell FCXM reactivity display long-term graft survival (1,193 ± 631 days). These data indicate that HIV-infected patients have nondeleterious autoantibodies recognizing cryptic epitopes exposed by pronase on T cells. (read more)

New Classification of Donation after Circulatory Death Donors Definitions and Terminology

In the face of a crisis in organ donation, the transplant community are increasingly utilising donation after circulatory death (DCD) donors. Over the last 10 years, with the increasing usage of DCD donors, we have seen the introduction in a number of new terms and definitions. We report the results of the 6th International Conference in Organ Donation held in Paris in 2013 and report a consensus agreement of an established expert European Working Group on the definitions and terminology regarding DCD donation, including refinement of the Maastricht definitions. This document forms part of a special series where recommendations are presented for uncontrolled and controlled DCD donation and organ specific guidelines for kidney, pancreas, liver and lung transplantation. An expert panel formed a consensus on definitions and terms aiming to establish consistent usage of terms in DCD donation (read more)

Deciphering IgM interference in IgG anti-HLA antibody detection with flow beads assays

In flow beads assays, the interference of IgM for IgG anti-HLA antibodies detection is not precisely understood. Using the screening flow beads assay for class I HLA antibodies, we analyzed the binding of two IgG mAbs, the anti-class I HLA W6/32 and an anti-beta-2-microglobulin, in the presence of an anti-beta-2-microglobulin IgM mAb. In neat serum, the IgM mAb impaired the detection of both IgG. In EDTA-treated serum, the interference was stronger for the anti-beta-2-microglobulin IgG than for W6/32, in agreement with the finding in surface plasmon resonance that this IgM competed with the anti-beta-2-microglobulin IgG but not with W6/32. The IgM interference was higher in neat than in EDTA-treated serum for both IgG mAbs. The IgM interference was also analyzed with class II single antigen flow beads and sera from two kidney recipients containing IgG and IgM donor specific antibodies. Anti-HLA IgG detection was partially corrected by EDTA, and restored by IgM inactivation with DTT, confirming the results observed with the mAbs. Therefore, three mechanisms can explain the IgM interference for IgG anti-HLA antibodies in flow beads assays: direct competition for antigen, steric hindrance and complement activation (read more)

Abdominal Wall Transplantation: A Sentinel Marker for Rejection

Abdominal wall (AW) transplantation (AWTX) has revolutionized difficult abdominal closure after intestinal transplantation (ITX). More importantly, the skin of the transplanted AW may serve as an immunological tool for differential diagnosis of bowel dysfunction post-transplant. Between 08/2008 and 10/2014, 29 small bowel transplantations were performed in 28 patients (16male;12female; 41±13years). Two groups were identified: The SOT (solid organ transplant)-group (n=15; 12ITX and 3MMVTX) and the SOT+AWTX-group (n=14; 12ITX and 2MMVTX), the latter included one ITX+AWTX retransplantation. Two doses of alemtuzumab were used for induction (30mg, 6 and 24hours post-reperfusion) and tacrolimus (trough levels 8–12ng/ml) was used for maintenance immunosuppression. Patient survival was similar in both groups (67%vs.61%). However, the SOT+AWTX-group showed faster post-transplant recovery, a better intestinal graft survival (79%vs.60%), a lower intestinal rejection rate (7%vs.27%) and a lower rate of misdiagnoses, where viral infection was mistaken and treated for rejection (14%vs.33%). The skin component of the AW may serve as an immune modulator and sentinel marker for immunological activity in the host. This can be a vital tool for timely prevention of intestinal graft rejection and more importantly avoidance of over-immunosuppression in cases of bowel dysfunction not related to graft rejection (read more).

The Role of Lymphoid Neogenesis in Allografts

De novo induction of organized lymphoid aggregates at non-lymphoid sites has been observed in many chronic inflammatory conditions where foreign antigens such as infectious agents, auto- or alloantigens, persist. The prevailing opinion in the field of transplantation is that lymphoid neogenesis within allografts is detrimental to the establishment of immune tolerance. These structures, commonly referred to as tertiary lymphoid organs (TLOs), are thought to contribute to graft rejection by generating and propagating local alloimmune responses. However, recent studies have shown that TLOs rich in regulatory Foxp3+ cells are present in long term accepting allografts. The notion that TLOs can contribute to the local downregulation of immune responses has been corroborated in other chronic inflammation models. These findings suggest that contrary to previous suggestions that the induction of TLOs in allografts is necessarily harmful, the induction of “tolerogenic” TLOs may prove advantageous. In this review, we discuss our current understanding of how TLOs are induced and how they regulate immune responses with a particular focus on alloimmunity (read more)

Renal Transplantation with Final Allocation Based on the Virtual Crossmatch

Solid phase immunoassays (SPI) are now routinely used to detect HLA antibodies. However the flow cytometric crossmatch (FCXM) remains the established method for assessing final donor-recipient compatibility. Since 2005 we have followed a protocol whereby the final allocation decision for renal transplantation is based on SPI (not the FCXM). Here we report long term graft outcomes for 508 consecutive kidney transplants using this protocol. All recipients were negative for donor specific antibody by SPI. Primary outcomes are graft survival and incidence of acute rejection within one year (AR<1yr) for FCXM+ (n=54) and FCXM- (n=454) recipients. Median follow up is 7.1 years. FCXM+ recipients were significantly different from FCXM- recipients for the following risk factors: living donor (24% vs 39%, p=0.03), duration of dialysis (31.0 months vs 13.5 months, p=0.008), retransplants (17% vs 7.3%, p=0.04), % sensitized (63% vs 19%, p=0.001) and PRA>80% (20% vs 4.8%, p=0.001). Despite these differences, 5 year actual graft survival rates are 87 and 84% respectively. AR<1yr occurred in 13% FCXM+ and 12% FCXM- recipients. Crossmatch status was not associated with graft outcomes in any univariate or multivariate model. Renal transplantation can be performed successfully, using SPI as the definitive test for donor-recipient compatibility (read more)

ABH-glycan microarray characterizes ABO subtype antibodies: fine specificity of immune tolerance after ABO-incompatible transplantation

Organ transplantation from ABO blood group-incompatible (ABOi) donors requires accurate detection, effective removal and subsequent surveillance of anti-donor antibodies. Because ABH antigen subtypes are expressed differently in various cells and organs, measurement of antibodies specific for the antigen subtypes in the graft is essential. Erythrocyte agglutination, the century-old assay used clinically, does not discriminate subtype-specific ABO antibodies and provides limited information on antibody isotypes. We designed and created an ABO-glycan microarray and demonstrate here the precise assessment of the presence and, importantly, the absence of donor-specific antibodies in an international study of pediatric heart transplant patients. Specific IgM, IgG and IgA isotype antibodies to non-self ABH-subtypes were detected in controls and recipients of ABO-compatible (ABOc) transplants. Conversely, in children who received ABOi transplants, antibodies specific for A-subtype-II and/or B-subtype-II antigens, the only ABH antigen subtypes expressed in heart tissue, were absent, demonstrating the fine specificity of B-cell tolerance to donor/graft blood group antigens. In contrast to the hemagglutination assay, the ABO-glycan microarray allows detailed characterization of donor-specific antibodies necessary for effective transplant management, representing a major step forward in precise ABO antibody detection (read more)

Humanized Mouse Models for Transplant Immunology

Our understanding of the molecular pathways that control immune responses, particularly immunomodulatory molecules that control the extent and duration of an immune response, have led to new approaches in the field of transplantation immunology to induce allograft survival. These molecular pathways are being defined precisely in murine models and translated into clinical practice; however, many of the newly available drugs are human-specific reagents. Furthermore, many species-specific differences exist between mouse and human immune systems. Recent advances in the development of humanized mice, namely, immunodeficient mice engrafted with functional human immune systems, have led to the availability of a small animal model for the study of human immune responses. Humanized mice represent an important preclinical model system for evaluation of new drugs and identification of the mechanisms underlying human allograft rejection without putting patients at risk. This review highlights recent advances in the development of humanized mice and their use as preclinical models for the study of human allograft responses (read more).

A2/A2B to B Renal Transplantation: Past, Present, and Future Directions

One component of the new national kidney allocation system (KAS) in the United States that was implemented on December 4, 2014, was the allocation of kidneys from A2 and A2B (A, non-A1 and AB, non-A1B) deceased donors into blood group B candidates (A2/A2B to B). In so far as this is an important component of the new KAS that has the potential to further increase the access to transplantation for blood group B candidates on the waiting list, most of whom are minority candidates, we will review the body of evidence and historical perspectives that led to its inclusion in the new KAS. This review will also describe prospects for more widespread use of A2/A2B to B transplantation and a novel mechanism of humoral immunosuppression in B patients before and after transplantation with an A2 or A2B kidney (read more)

EpViX: A cloud-based tool for epitope reactivity analysis and epitope virtual crossmatching to identify low immunologic risk donors for sensitized recipients

One of the challenges facing solid organ transplantation programs globally is the identification of low immunological risk donors for sensitized recipients by HLA allele genotype. Because recognition of donor HLA alleles by host antibodies is at the core of organ rejection, the objective of this work was to develop a new version of the EpHLA software, named EpViX, which uses an HLAMatchmaker algorithm and performs automated epitope virtual crossmatching at the initiation of the organ donation process. EpViX is a free, web-based application developed for use over the internet on a tablet, smartphone or computer. This program was developed using the Ruby programming language and the Ruby-on-Rails framework. To improve the user experience, the EpViX software interface was developed based on the best human–computer interface practices. To simplify epitope analysis and virtual crossmatching, the program was integrated with important available web-based resources, such as OPTN, IMGT/HLA and the International HLA Epitope Registry. We successfully developed a program that allows people to work collaboratively and effectively during the donation process by accurately predicting negative crossmatches, saving time and other resources (read more)

New Immunosuppressive Cell Therapy to Prolong Survival of Induced Pluripotent Stem Cell–Derived Allografts

Background: Induced pluripotent stem cell (iPSC) technology provides new opportunities in regenerative medicine to generate grafts from donors for transplantation. However, particularly when allogeneic iPSCs are used, immune suppression is required to avoid rejection of iPSC-derived grafts. In this study, we examine a concept that protection of iPSCs-derived allografts can be achieved when transplantation is accompanied with the administration of immunosuppressive cells generated from the same iPSCs resource.
Methods: Mouse iPSCs were differentiated into immunosuppressive cells by a culture protocol using granulocyte macrophage-colony-stimulating factor, macrophage-colony-stimulating factor, IL-4, and lipopolysaccharide. Adherent clusters were collected and examined for the ability to suppress allogeneic T- and B-cell responses, as well as for the contribution to prolonged allogeneic graft survival in transplantation models.
Results: Myeloid cells with immunosuppressive features were successfully induced from iPSCs, and thus referred to as iPSC-derived suppressor cells (iPS-SCs). The iPS-SCs resemble macrophages in terms of cell surface molecules and gene expressions. Furthermore, iPS-SCs efficiently suppressed allogeneic T- and B-cell proliferation in a nitric oxide–dependent manner, and iPS-SCs were found to suppress alloantibody production and prolong substantially the survival of iPSC-derived grafts, such as embryoid bodies and cardiomyocytes, in in vivo allogeneic transplantation models.
Conclusions: A certain fraction of macrophage-like cells with immunosuppressive functions can be generated from donor iPSCs, which contribute to the prolonged survival of grafts derived from the same iPSCs in allogeneic recipients. These results suggest a new immunosuppressive strategy of combined donor iPSC-derived graft and immunosuppressive cell transplantation in regenerative medicine using iPSCs (read more)

Predicted Indirectly Recognizable HLA Epitopes Presented by HLA-DRB1 Are Related to HLA Antibody Formation During Pregnancy

Pregnancy can prime maternal immune responses against inherited paternal HLA of the fetus, leading to the production of child-specific HLA antibodies. We previously demonstrated that donor-specific HLA antibody formation after kidney transplantation is associated with donor-derived HLA epitopes presented by recipient HLA class II (predicted indirectly recognizable HLA epitopes presented by HLA class II [PIRCHE-II]). In the present study, we evaluated the role of PIRCHE-II in child-specific HLA antibody formation during pregnancy. A total of 229 mother–child pairs were HLA typed. For all mismatched HLA class I molecules of the child, we subsequently predicted the number of HLA epitopes that could be presented by maternal HLA class II molecules. Child-specific antigens were classified as either immunogenic or nonimmunogenic HLA based on the presence of specific antibodies and correlated to PIRCHE-II numbers. Immunogenic HLA contained higher PIRCHE-II numbers than nonimmunogenic HLA. Moreover, the probability of antibody production during pregnancy increased with the number of PIRCHE-II. In conclusion, our data suggest that the number of PIRCHE-II is related to the formation of child-specific HLA antibodies during pregnancy. Present confirmation of the role of PIRCHE-II in antibody formation outside the transplantation setting suggests the PIRCHE-II concept is universal (read more and editorial)

Discarded Human Thymus Is a Novel Source of Stable and Long-Lived Therapeutic Regulatory T Cells

Regulatory T cell (Treg)–based therapy is a promising approach to treat many immune-mediated disorders such as autoimmune diseases, organ transplant rejection, and graft-versus-host disease (GVHD). Challenges to successful clinical implementation of adoptive Treg therapy include difficulties isolating homogeneous cell populations and developing expansion protocols that result in adequate numbers of cells that remain stable, even under inflammatory conditions. We investigated the potential of discarded human thymuses, routinely removed during pediatric cardiac surgery, to be used as a novel source of therapeutic Tregs. Here, we show that large numbers of FOXP3+ Tregs can be isolated and expanded from a single thymus. Expanded thymic Tregs had stable FOXP3 expression and long telomeres, and suppressed proliferation and cytokine production of activated allogeneic T cells in vitro. Moreover, expanded thymic Tregs delayed development of xenogeneic GVHD in vivo more effectively than expanded Tregs isolated based on CD25 expression from peripheral blood. Importantly, in contrast to expanded blood Tregs, expanded thymic Tregs remained stable under inflammatory conditions. Our results demonstrate that discarded pediatric thymuses are an excellent source of therapeutic Tregs, having the potential to overcome limitations currently hindering the use of Tregs derived from peripheral or cord blood (read more)