Balancing Equity and HLA Matching in Deceased-Donor Kidney Allocation with Eplet Mismatch.

Background: Prioritization of HLA antigen-level matching in the US kidney allocation system intends to improve post-transplant survival but causes racial disparities and thus has been substantially de-emphasized. Recently, molecular matching based on eplets has been found to improve risk stratification compared to antigen matching. Methods: To assign eplets unambiguously, we utilized a cohort of 5193 individuals with high resolution allele-level HLA genotypes from the National Kidney Registry. Using repeated random sampling to simulate donor-recipient genotype pairings based on the ethnic composition of the historical US deceased donor pool, we profiled the percentage of well-matched donors for candidates by ethnicity. Results: The percentage of well-matched donors with zero-DR/DQ eplet mismatch was 3-fold less racially disparate for Black and Asian candidates than percentage of donors with zero-ABDR antigen mismatches, and 2-fold less racially disparate for Latino candidates. For other HLA antigen and eplet mismatch thresholds, the percentage of well-matched donors was more similar across candidate ethnic groups. Conclusions: Compared to the current zero-ABDR antigen mismatch, prioritizing a zero-DR/DQ eplet mismatch in allocation would decrease racial disparities and increase the percentage of well-matched donors. High resolution HLA deceased donor genotyping would enable unambiguous assignment of eplets to operationalize molecular mismatch metrics in allocation. Key Points: Question: What is the impact of prioritizing low molecular mismatch transplants on racial and ethnic disparities in US deceased-donor kidney allocation, compared to the current prioritization of antigen-level matching?Findings: The lowest-risk eplet mismatch approach decreases racial disparities up to 3-fold compared to lowest-risk antigen mismatch and identifies a larger number of the lowest allo-immune risk donors.Meaning: Prioritizing eplet matching in kidney transplant allocation could both improve outcomes and reduce racial disparities compared to the current antigen matching.

Integrative multi-omics profiling in human decedents receiving pig heart xenografts.

In a previous study, heart xenografts from 10-gene-edited pigs transplanted into two human decedents did not show evidence of acute-onset cellular- or antibody-mediated rejection. Here, to better understand the detailed molecular landscape following xenotransplantation, we carried out bulk and single-cell transcriptomics, lipidomics, proteomics and metabolomics on blood samples obtained from the transplanted decedents every 6 h, as well as histological and transcriptomic tissue profiling. We observed substantial early immune responses in peripheral blood mononuclear cells and xenograft tissue obtained from decedent 1 (male), associated with downstream T cell and natural killer cell activity. Longitudinal analyses indicated the presence of ischemia reperfusion injury, exacerbated by inadequate immunosuppression of T cells, consistent with previous findings of perioperative cardiac xenograft dysfunction in pig-to-nonhuman primate studies. Moreover, at 42 h after transplantation, substantial alterations in cellular metabolism and liver-damage pathways occurred, correlating with profound organ-wide physiological dysfunction. By contrast, relatively minor changes in RNA, protein, lipid and metabolism profiles were observed in decedent 2 (female) as compared to decedent 1. Overall, these multi-omics analyses delineate distinct responses to cardiac xenotransplantation in the two human decedents and reveal new insights into early molecular and immune responses after xenotransplantation. These findings may aid in the development of targeted therapeutic approaches to limit ischemia reperfusion injury-related phenotypes and improve outcomes.

Cellular dynamics in pig-to-human kidney xenotransplantation.

BACKGROUND: Xenotransplantation of genetically engineered porcine organs has the potential to address the challenge of organ donor shortage. Two cases of porcine-to-human kidney xenotransplantation were performed, yet the physiological effects on the xenografts and the recipients' immune responses remain largely uncharacterized. METHODS: We performed single-cell RNA sequencing (scRNA-seq) and longitudinal RNA-seq analyses of the porcine kidneys to dissect xenotransplantation-associated cellular dynamics and xenograft-recipient interactions. We additionally performed longitudinal scRNA-seq of the peripheral blood mononuclear cells (PBMCs) to detect recipient immune responses across time. FINDINGS: Although no hyperacute rejection signals were detected, scRNA-seq analyses of the xenografts found evidence of endothelial cell and immune response activation, indicating early signs of antibody-mediated rejection. Tracing the cells' species origin, we found human immune cell infiltration in both xenografts. Human transcripts in the longitudinal bulk RNA-seq revealed that human immune cell infiltration and the activation of interferon-gamma-induced chemokine expression occurred by 12 and 48 h post-xenotransplantation, respectively. Concordantly, longitudinal scRNA-seq of PBMCs also revealed two phases of the recipients' immune responses at 12 and 48-53 h. Lastly, we observed global expression signatures of xenotransplantation-associated kidney tissue damage in the xenografts. Surprisingly, we detected a rapid increase of proliferative cells in both xenografts, indicating the activation of the porcine tissue repair program. CONCLUSIONS: Longitudinal and single-cell transcriptomic analyses of porcine kidneys and the recipient's PBMCs revealed time-resolved cellular dynamics of xenograft-recipient interactions during xenotransplantation. These cues can be leveraged for designing gene edits and immunosuppression regimens to optimize xenotransplantation outcomes. FUNDING: This work was supported by NIH RM1HG009491 and DP5OD033430.

Specificity of HLA monoclonal antibodies and their use to determine HLA expression on lymphocytes and peripheral blood stem cells.

HLA Class I and II expression are known to differ locus-to-locus, however, HLA expression on the cell-surface is frequently reported as the total amount of HLA Class I or II antigens. This is despite evidence that indicates the differential expression of HLA can influence patient outcomes post-transplantation. Although numerous commercially available HLA monoclonal antibodies (mAbs) exist to characterize HLA expression, there is currently a lack of detailed information regarding their reactivities to HLA specificities. The specificities of locus-specific HLA mAbs (nine Class I and four Class II mAbs) were evaluated by two solid-phase Luminex single antigen bead assays. The reactivity patterns of these mAbs were then confirmed by flow cytometry using lymphocytes and PBSCs (peripheral blood stem cells). Out of the 13 HLA mAbs tested, only four (one Class I and three Class II mAbs) displayed intra-locus reactivity without also reacting to inter-locus specificities. Epitope analysis revealed the presence of shared epitopes across numerous HLA loci, explaining much of the observed inter-locus reactivity. The specificity of the HLA mAbs seen in solid-phase assays was confirmed against PBSCs and lymphocytes by flow cytometry. Using this method, we observed differences in the cell surface expression of HLA-C, HLA-DR, HLA-DQ, and HLA-DP between PBSCs and lymphocytes. Our results emphasize the need to characterize the reactivity patterns of HLA mAbs using solid-phase assays before their use on cells. Through understanding the reactivity of these HLA mAbs, the cellular expression of HLA can be more accurately assessed in downstream assays.

Heart transplantation: advances in expanding the donor pool and xenotransplantation.

Approximately 65 million adults globally have heart failure, and the prevalence is expected to increase substantially with ageing populations. Despite advances in pharmacological and device therapy of heart failure, long-term morbidity and mortality remain high. Many patients progress to advanced heart failure and develop persistently severe symptoms. Heart transplantation remains the gold-standard therapy to improve the quality of life, functional status and survival of these patients. However, there is a large imbalance between the supply of organs and the demand for heart transplants. Therefore, expanding the donor pool is essential to reduce mortality while on the waiting list and improve clinical outcomes in this patient population. A shift has occurred to consider the use of organs from donors with hepatitis C virus, HIV or SARS-CoV-2 infection. Other advances in this field have also expanded the donor pool, including opt-out donation policies, organ donation after circulatory death and xenotransplantation. We provide a comprehensive overview of these various novel strategies, provide objective data on their safety and efficacy, and discuss some of the unresolved issues and controversies of each approach.

Immune response after pig-to-human kidney xenotransplantation: a multimodal phenotyping study.

BACKGROUND: Cross-species immunological incompatibilities have hampered pig-to-human xenotransplantation, but porcine genome engineering recently enabled the first successful experiments. However, little is known about the immune response after the transplantation of pig kidneys to human recipients. We aimed to precisely characterise the early immune responses to the xenotransplantation using a multimodal deep phenotyping approach. METHODS: We did a complete phenotyping of two pig kidney xenografts transplanted to decedent humans. We used a multimodal strategy combining morphological evaluation, immunophenotyping (IgM, IgG, C4d, CD68, CD15, NKp46, CD3, CD20, and von Willebrand factor), gene expression profiling, and whole-transcriptome digital spatial profiling and cell deconvolution. Xenografts before implantation, wild-type pig kidney autografts, as well as wild-type, non-transplanted pig kidneys with and without ischaemia-reperfusion were used as controls. FINDINGS: The data collected from xenografts suggested early signs of antibody-mediated rejection, characterised by microvascular inflammation with immune deposits, endothelial cell activation, and positive xenoreactive crossmatches. Capillary inflammation was mainly composed of intravascular CD68+ and CD15+ innate immune cells, as well as NKp46+ cells. Both xenografts showed increased expression of genes biologically related to a humoral response, including monocyte and macrophage activation, natural killer cell burden, endothelial activation, complement activation, and T-cell development. Whole-transcriptome digital spatial profiling showed that antibody-mediated injury was mainly located in the glomeruli of the xenografts, with significant enrichment of transcripts associated with monocytes, macrophages, neutrophils, and natural killer cells. This phenotype was not observed in control pig kidney autografts or in ischaemia-reperfusion models. INTERPRETATION: Despite favourable short-term outcomes and absence of hyperacute injuries, our findings suggest that antibody-mediated rejection in pig-to-human kidney xenografts might be occurring. Our results suggest specific therapeutic targets towards the humoral arm of rejection to improve xenotransplantation results. FUNDING: OrganX and MSD Avenir.

Pig-to-human heart xenotransplantation in two recently deceased human recipients.

Genetically modified xenografts are one of the most promising solutions to the discrepancy between the numbers of available human organs for transplantation and potential recipients. To date, a porcine heart has been implanted into only one human recipient. Here, using 10-gene-edited pigs, we transplanted porcine hearts into two brain-dead human recipients and monitored xenograft function, hemodynamics and systemic responses over the course of 66 hours. Although both xenografts demonstrated excellent cardiac function immediately after transplantation and continued to function for the duration of the study, cardiac function declined postoperatively in one case, attributed to a size mismatch between the donor pig and the recipient. For both hearts, we confirmed transgene expression and found no evidence of cellular or antibody-mediated rejection, as assessed using histology, flow cytometry and a cytotoxic crossmatch assay. Moreover, we found no evidence of zoonotic transmission from the donor pigs to the human recipients. While substantial additional work will be needed to advance this technology to human trials, these results indicate that pig-to-human heart xenotransplantation can be performed successfully without hyperacute rejection or zoonosis.

One-year immunologic outcomes of lung transplantation utilizing hepatitis C-viremic donors.

Little is known about the effects of hepatitis C viremia on immunologic outcomes in the era of direct-acting antivirals. We conducted a prospective, single-arm trial of lung transplantation from hepatitis C-infected donors into hepatitis C-naïve recipients (n = 21). Recipients were initiated on glecaprevir-pibrentasvir immediately post-transplant and were continued on therapy for a total of 8 weeks. A control group of recipients of hepatitis C-negative lungs were matched 1:1 on baseline variables (n = 21). The primary outcome was the frequency of acute cellular rejection over 1-year post-transplant. Treatment with glecaprevir-pibrentasvir was well tolerated and resulted in viremia clearance after a median of 16 days of therapy (IQR 10-24 days). At one year, there was no difference in incidence of acute cellular rejection (71.4% vs. 85.7%, P = .17) or rejection requiring treatment (33.3% vs. 57.1%, P = .12). Mean cumulative acute rejection scores were similar between groups (.46 [SD ± .53] vs. .52 [SD ± .37], P = .67). Receipt of HCV+ organs was not associated with acute rejection on unadjusted Cox regression analysis (HR .55, 95% CI .28-1.11, P = .09), or when adjusted for risk factors known to be associated with acute rejection (HR .57, 95% CI .27-1.21, P = .14). Utilization of hepatitis C infected lungs with immediate treatment leads to equivalent immunologic outcomes at 1 year.

Results of Two Cases of Pig-to-Human Kidney Xenotransplantation.

BACKGROUND: Xenografts from genetically modified pigs have become one of the most promising solutions to the dearth of human organs available for transplantation. The challenge in this model has been hyperacute rejection. To avoid this, pigs have been bred with a knockout of the alpha-1,3-galactosyltransferase gene and with subcapsular autologous thymic tissue. METHODS: We transplanted kidneys from these genetically modified pigs into two brain-dead human recipients whose circulatory and respiratory activity was maintained on ventilators for the duration of the study. We performed serial biopsies and monitored the urine output and kinetic estimated glomerular filtration rate (eGFR) to assess renal function and xenograft rejection. RESULTS: The xenograft in both recipients began to make urine within moments after reperfusion. Over the 54-hour study, the kinetic eGFR increased from 23 ml per minute per 1.73 m2 of body-surface area before transplantation to 62 ml per minute per 1.73 m2 after transplantation in Recipient 1 and from 55 to 109 ml per minute per 1.73 m2 in Recipient 2. In both recipients, the creatinine level, which had been at a steady state, decreased after implantation of the xenograft, from 1.97 to 0.82 mg per deciliter in Recipient 1 and from 1.10 to 0.57 mg per deciliter in Recipient 2. The transplanted kidneys remained pink and well-perfused, continuing to make urine throughout the study. Biopsies that were performed at 6, 24, 48, and 54 hours revealed no signs of hyperacute or antibody-mediated rejection. Hourly urine output with the xenograft was more than double the output with the native kidneys. CONCLUSIONS: Genetically modified kidney xenografts from pigs remained viable and functioning in brain-dead human recipients for 54 hours, without signs of hyperacute rejection. (Funded by Lung Biotechnology.).

Immunologic risk stratification of pediatric heart transplant patients by combining HLAMatchmaker and PIRCHE-II.

BACKGROUND: Molecular-level human leukocyte antigen (HLA) mismatch is a powerful biomarker of rejection; however, few studies have explored its use in heart transplant recipients, and none have attempted to use the results of separate algorithms synergistically. Here we tested the hypothesis that a combination of HLAMatchmaker and Predicted Indirectly Recognizable HLA Epitopes (PIRCHE-II) can be used to identify more patients at low risk of rejection. METHODS: We studied 274 recipient/donor pairs enrolled in the Clinical Trials in Organ Transplantation in Children (CTOTC) performing class I and II HLA genotyping by next-generation sequencing to determine eplet mismatch (epMM) load and PIRCHE-II score. Correlation with clinical outcomes was performed on 131 cases. RESULTS: Of the 131 patients, 100 without pre-formed donor specific antibody (DSA) were used to identify cutoffs for the Class I, HLA-DR, and HLA-DQ epMM load and PIRCHE-II score for risk of developing post-transplant DSA (epMM: Class I/DR/DQ = 9/9/6; PIRCHE-II: 141/116/111) and antibody-mediated rejection (ABMR) (epMM: 9/8/8; PIRCHE-II: 157/80/201). Patients with above cut-off epMM load appear to be less likely to develop DSA and ABMR if their PIRCHE-II score is below cut-off (high epMM/high PIRCHE-II: 12.3%-20.3% DSA and 9%-13.5% ABMR vs high epMM/low PIRCHE-II: 4%-10% DSA and 0%-2% ABMR). CONCLUSION: For the first time in a pediatric heart transplant cohort, immunologic risk cut-offs for DSA and ABMR have been established. When used together, epMM load and PIRCHE-II score allow us to reclassify a portion of cases with high epMM load as having a lower risk for developing DSA and ABMR.

Impact of carfilzomib-based desensitization on heart transplantation of sensitized candidates.

BACKGROUND: Allosensitization in heart transplant candidates is associated with longer transplant wait times and post-transplant complications. We summarize our experience with desensitization using carfilzomib, an irreversible proteasome inhibitor that causes plasma cell apoptosis. METHODS: One cycle of desensitization consisted of plasmapheresis and carfilzomib 20 mg/m2 on days 1, 2, 8, 9, 15, and 16 with intravenous immune globulin 2 g/kg after carfilzomib on day 16. Patients underwent repeat cycles as indicated. We compare calculated panel-reactive antibody (cPRA) for neat combined Class I and II IgG and C1q pre- and post-treatment using a cutoff for cPRA entry of ≥ 4000 and 500 MFI, respectively. RESULTS: From June 2013 to October 2019, 9 patients underwent 20 cycles of carfilzomib-based desensitization. Each cycle resulted in an average cPRA decrease of 24% (95% CI: 6-42) for IgG and 36% (95% CI: 17-55) for C1q. From treatment start to finish, mean cPRA fell from 76% to 40% (p = 0.01) for IgG and 56% to 4% (p = 0.017) for C1q. Six of 9 patients have been transplanted with 5 of the transplanted hearts crossing preoperative donor-specific antibodies. During a median follow-up of 35.1 months, all transplanted patients have survived with only 1 occurrence of treated rejection. Side effects of desensitization included acute kidney injury (67%) and thrombocytopenia (33%) with all episodes self-resolving. CONCLUSIONS: A carfilzomib-based desensitization strategy among heart transplant candidates reduces the level of HLA antibodies and complement binding, facilitates successful transplantation, and is associated with excellent outcomes at 3 years.

Approaching the sensitized lung patient: risk assessment for donor acceptance.

The presence of HLA antibodies is widely recognized as a barrier to solid organ transplantation, and for lung transplant candidates, it has a significant negative impact on both waiting time and waiting list mortality. Although HLA antibodies have been associated with a broad spectrum of allograft damage, precise characterization of these antibodies in allosensitized candidates may enhance their accessibility to transplant. The introduction of Luminex-based single antigen bead (SAB) assays has significantly improved antibody detection sensitivity and specificity, but SAB alone is not sufficient for risk-stratification. Functional characterization of donor-specific antibodies (DSA) is paramount to increase donor accessibility for allosensitized lung candidates. We describe here our approach to evaluate sensitized lung transplant candidates. By employing state-of-the-art technologies to assess histocompatibility and determine physiological properties of circulating HLA antibodies, we can provide our Clinical Team a better risk assessment for lung transplant candidates and facilitate a "road map" to transplant. The cases presented in this paper illustrate the "individualized steps" taken to determine calculated panel reactive antibodies (cPRA), titer and complement-fixing properties of each HLA antibody present in circulation. When a donor is considered, we can better predict the risk associated with potentially crossing HLA antibodies, thereby allowing the Clinical Team to approach allosensitized lung patients with an individualized medicine approach. To facilitate safe access of sensitized lung transplant candidates to potential donors, a synergy between the histocompatibility laboratory and the Clinical Team is essential. Ultimately, donor acceptance is a decision based on several parameters, leading to a risk-stratification unique for each patient.

Donor-specific antibody characteristics, including persistence and complement-binding capacity, increase risk for chronic lung allograft dysfunction.

BACKGROUND: Chronic lung allograft dysfunction (CLAD) is the major complication limiting long-term survival in lung transplant recipients (LTRs), with those developing donor-specific anti-human leukocyte antigen (HLA) antibodies (DSAs) previously found to have increased risk for CLAD. However, as DSA responses vary in timing of development, specificity, breadth, persistence, and complement-binding capacity, we hypothesized that these characteristics would impact CLAD and survival outcomes. METHODS: We retrospectively analyzed DSA characteristics and outcomes in a single-center cohort of 582 LTRs who had serum samples collected prospectively from 2010 to 2016. Luminex-based single antigen bead assays were performed to assess DSA. RESULTS: DSAs were detected in 247 LTRs (42%), of which 124 (21.3%) were de novo DSAs and 53 (9.1%) were complement-binding (C1q+). CLAD developed in 208 LTRs (35.7%) during the follow-up period, with 67.8% determined as bronchiolitis obliterans syndrome phenotype and 32.2% as restrictive allograft syndrome phenotype. We found a shorter time to CLAD in LTRs with persistent DSAs (p = 0.04) and HLA-DQ-specific DSAs (p = 0.03). LTRs who developed C1q+ DSAs had significantly shorter time to CLAD (p < 0.001), with 100% of C1q+ DSAs being persistent and no differences between CLAD phenotypes. CLAD-free survival was significantly reduced in LTRs who developed C1q+ DSAs (p = 0.001), HLA-DQ-specific DSAs (p = 0.03), and multiple DSAs (p = 0.02). CONCLUSIONS: Together, our findings demonstrate that DSA characteristics of persistence, HLA-DQ specificity, and C1q+ DSAs are associated with shorter time to CLAD. Additionally, C1q+, HLA-DQ-specific, and multiple DSAs are associated with decreased CLAD-free survival. These characteristics may improve DSA risk stratification for deleterious outcomes in LTRs.

Therapeutic Human IgG Preparations Contain Mixture of HLA Antibodies to Native HLA Antigens and Cryptic Epitopes With Little Clinical Significance.

BACKGROUND: Human immunoglobulins (H-Ig) are widely used in solid organ transplantation for immunoglobulin G (IgG) replacement and for desensitization and treatment of antibody-mediated rejection. They are obtained from plasma pools and may contain HLA antibodies that can be detrimental to transplant recipients. The goal of this study was to evaluate HLA antibodies in multiple lots of 2 commercial H-Ig preparations by Luminex single-antigen bead (SAB) and cell-based crossmatch assays. METHODS: Thirty lots of 2 commercial H-Ig products (CSL Behring, King of Prussia, PA) were evaluated: 6 Hizentra and 24 Privigen. All were adsorbed and diluted 1:10 before testing. HLA IgG antibodies were determined by 2 Luminex SAB kits and C1q screen for complement-binding capability. Lots were tested for the presence of antibody to denatured vs. intact class I HLA alleles using acid-treated SAB. Surrogate T and B-cell flow cytometry crossmatches (FCXM) were performed with peripheral blood lymphocytes from 2 healthy donors. RESULTS: Twenty-two (73%) lots at 1:10 showed SAB reactivity with mean fluorescent intensity of 2000 or greater for HLA class I, 67% (20/30 lots) for class II. The reactivity pattern was similar using both SAB kits. Acid treatment revealed antibodies to denatured class I: the majority of HLA-C, half of HLA-B and few HLA-A alleles. No C1q reactivity was observed. Surrogate flow cytometry crossmatch results were positive (>150 median channel shift), but were fourfold to eightfold lower than expected. CONCLUSIONS: The H-Ig products tested consisted of low titer, non-complement-binding HLA class I and class II antibodies; most of the observed class I HLA reactivity was toward denatured HLA antigens.

Immune Responses of HLA Highly Sensitized and Nonsensitized Patients to Genetically Engineered Pig Cells.

BACKGROUND: We investigated in vitro whether HLA highly sensitized patients with end-stage renal disease will be disadvantaged immunologically after a genetically engineered pig kidney transplant. METHODS: Blood was drawn from patients with a calculated panel-reactive antibody (cPRA) 99% to 100% (Gp1, n = 10) or cPRA 0% (Gp2, n = 12), and from healthy volunteers (Gp3, n = 10). Serum IgM and IgG binding was measured (i) to galactose-α1-3 galactose and N-glycolylneuraminic acid glycans by enzyme-linked immunosorbent assay, and (ii) to pig red blood cell, pig aortic endothelial cells, and pig peripheral blood mononuclear cell from α1,3-galactosyltransferase gene-knockout (GTKO)/CD46 and GTKO/CD46/cytidine monophosphate-N-acetylneuraminic acid hydroxylase-knockout (CMAHKO) pigs by flow cytometry. (iii) T-cell and B-cell phenotypes were determined by flow cytometry, and (iv) proliferation of T-cell and B-cell carboxyfluorescein diacetate succinimidyl ester-mixed lymphocyte reaction. RESULTS: (i) By enzyme-linked immunosorbent assay, there was no difference in IgM or IgG binding to galactose-α1-3 galactose or N-glycolylneuraminic acid between Gps1 and 2, but binding was significantly reduced in both groups compared to Gp3. (ii) IgM and IgG binding in Gps1 and 2 was also significantly lower to GTKO/CD46 pig cells than in healthy controls, but there were no differences between the 3 groups in binding to GTKO/CD46/CMAHKO cells. (iii and iv) Gp1 patients had more memory T cells than Gp2, but there was no difference in T or B cell proliferation when stimulated by any pig cells. The proliferative responses in all 3 groups were weakest when stimulated by GTKO/CD46/CMAHKO pig peripheral blood mononuclear cell. CONCLUSIONS: (i) End-stage renal disease was associated with low antipig antibody levels. (ii) Xenoreactivity decreased with increased genetic engineering of pig cells. (iii) High cPRA status had no significant effect on antibody binding or T-cell and B-cell response.

Short-term adverse effects of early subclinical allograft inflammation in kidney transplant recipients with a rapid steroid withdrawal protocol.

The impact of subclinical inflammation (SCI) noted on early kidney allograft biopsies remains unclear. This study evaluated the outcome of SCI noted on 3-month biopsy. A total of 273/363 (75%) kidney transplant recipients with a functioning kidney underwent allograft biopsies 3-months posttransplant. Among those with stable allograft function at 3 months, 200 biopsies that did not meet the Banff criteria for acute rejection were identified. These were Group I: No Inflammation (NI, n = 71) and Group II: Subclinical Inflammation (SCI, n = 129). We evaluated differences in kidney function at 24-months and allograft histology score at 12-month biopsy. SCI patients had a higher serum creatinine (1.6 ± 0.7 vs 1.38 ± 0.45; P = .02) at 24-months posttransplant, and at last follow-up at a mean of 42.5 months (1.69 ± 0.9 vs 1.46 ± 0.5 mg/dL; P = .027). The allograft chronicity score (ci + ct + cg + cv) at 12-months posttransplant was higher in the SCI group (2.4 ± 1.35 vs 1.9 ± 1.2; P = .02). The incidence of subsequent rejections within the first year in SCI and NI groups was 24% vs 10%, respectively (P = .015). De novo donor-specific antibody within 12 months was more prevalent in the SCI group (12/129 vs 1/71, P = .03). SCI is likely not a benign finding and may have long-term implications for kidney allograft function.

Significance of Anti-HLA Antibodies on Adult and Pediatric Heart Allograft Outcomes.

As methods for human leukocyte antigens (HLA) antibody detection have evolved and newer solid phase assays are much more sensitive, the last 15 years has seen a renewed focus on the importance of HLA antibodies in solid organ transplant rejection. However, there is still much controversy regarding the clinical significance of antibody level as depicted by the mean fluorescence intensity of a patient's neat serum. Emerging techniques, including those that identify antibody level and function, show promise for the detection of individuals at risk of allograft rejection, determination of the effectiveness of desensitization prior to transplant, and for monitoring treatment of rejection. Here, we review current publications regarding the relevance of donor-specific HLA antibodies (DSA) in adult and pediatric heart transplantation (HT) with graft survival, development of antibody-mediated rejection and cardiac allograft vasculopathy (CAV). The negative impact of DSA on patient and allograft survival is evident in adult and pediatric HT recipients. Many questions remain regarding the most appropriate frequency of assessment of pre- and posttransplant DSA as well as the phenotype of DSA memory vs. true de novo antibody using large multicenter adult and pediatric cohorts and state-of-the-art methodologies for DSA detection and characterization.

Inhibitors of XIAP sensitize CD40-activated chronic lymphocytic leukemia cells to CD95-mediated apoptosis.

Patients with chronic lymphocytic leukemia (CLL) treated with adenovirus CD154 (Ad-CD154, CD40 ligand [CD40L]) gene therapy experienced rapid reductions in leukemia cell counts and lymph node size associated with the induced expression of Fas (CD95). However, CLL cells initially resist CD95-mediated apoptosis within the first 3 days after CD40 ligation in vitro. Thereafter, they become sensitive, which is associated with the CD40-induced expression of the proapoptotic protein B-cell leukemia 2 homology 3 (BH3) interacting domain death agonist (Bid). We hypothesized that the initial resistance to CD95-mediated apoptosis may be due to the high-level expression of X-linked inhibitor of apoptosis protein (XIAP) by CLL cells. Consistent with this, CLL cells from patients 1 day after treatment with autologous Ad-CD154-transduced CLL cells became sensitive to CD95-mediated apoptosis following treatment with a novel XIAP inhibitor, 1540-14. Similarly, 1540-14 specifically enhanced CD95-mediated apoptosis of CLL cells following CD40 ligation in vitro. Immunoblot analyses demonstrated that treatment with 1540-14 allowed CD40-stimulated CLL cells to experience high-level activation of caspases-8 and -3 and cleavage of poly(adenosine diphosphate [ADP]-ribose) polymerase following CD95 ligation. This study demonstrates that distal apoptosis regulators contribute to the initial resistance of CD40-activated CLL cells to CD95-mediated apoptosis and suggests that XIAP inhibitors might enhance the effectiveness of immune-based treatment strategies that target CD40, such as CD154 gene therapy.

Cell detachment and apoptosis induction of immortalized human prostate epithelial cells are associated with early accumulation of a 45 kDa nuclear isoform of clusterin.

Clusterin, ubiquitously distributed in mammalians, was cloned and identified as the most potently induced gene during rat prostate involution following androgen deprivation. Also found to be involved in many other patho-physiological processes, its biological significance is still controversial, particularly with regard to apoptosis. We previously showed that transient over-expression of clusterin blocked cell cycle progression of simian-virus-40-immortalized human prostate epithelial cell lines PNT1A and PNT2. We show in the present study that the accumulation of an intracellular 45 kDa clusterin isoform was an early event closely associated with death of PNT1A cells caused by cell detachment followed by apoptosis induction (anoikis). Cell morphological changes, decreased proliferation rate and cell cycle arrest at G0/G1-S-phase checkpoint were all strictly associated with the production and early translocation to the nucleus of a 45 kDa clusterin isoform. Later, nuclear clusterin was found accumulated in detached cells and apoptotic bodies. These results suggest that a 45 kDa isoform of clusterin, when targeted to the nucleus, can decrease cell proliferation and promotes cell-detachment-induced apoptosis, suggesting a possible major role for clusterin as an anti-proliferative gene in human prostate epithelial cells.

Expression of CD10 by B-chronic lymphocytic leukemia cells undergoing apoptosis in vivo and in vitro.

BACKGROUND AND OBJECTIVES: B-cell chronic lymphocytic leukemia (B-CLL) is an accumulating disease of slowly proliferating cells. CD10 is not normally expressed on the surface of B-CLL cells. The aim of this study was to ascertain whether B-CLL cells, induced into apoptosis, expressed surface CD10, since a correlation between apoptosis and CD10 expression has been demonstrated. DESIGN AND METHODS: Peripheral blood cells from 31 untreated B-CLL patients were induced into apoptosis by etoposide, fludarabine or Ga(mu)-Ab treatment and tested for CD10 expression by flow cytometry. Normal CD5+ B cells were also induced into apoptosis and tested for CD10 expression. RESULTS: CD10 positive cells were absent in B-CLL cell suspensions, but were detected following in vitro culture, and their appearance paralleled that of apoptotic cells. Treatment with etoposide, fludarabine or Ga(mu)-Ab enhanced both apoptosis and CD10 expression. Inhibition of apoptosis by VAD-fmk or Ga(delta)-Ab prevented CD10 expression. Cell separation tests following induction of apoptosis demonstrated that CD10+ cells were apoptotic. CD10+ cells were observed in the peripheral blood of two patients within a few hours following fludarabine infusion. In another patient, who failed to respond, no CD10+ cells were seen. Expression of CD10 was observed also in normal CD5+ B cells when these were induced into apoptosis. INTERPRETATION AND CONCLUSIONS: This study demonstrates that B-CLL cells, as well as normal CD5+ B cells, become CD10+ following apoptosis induction in vitro. Some of the data obtained also suggest a use for CD10 to monitor apoptosis of B-CLL in a clinical setting.