Comparative Assessment of Anti-HLA Antibodies Using Two Commercially Available Luminex-Based Assays.

BACKGROUND: Allospecific anti-HLA antibodies (Abs) are associated with rejection of solid organ grafts. The 2 main kits to detect anti-HLA Ab in patient serum are commercialized by Immucor and One Lambda/ThermoFisher. We sought to compare the performance of both platforms. METHODS: Background-adjusted mean fluorescence intensity (MFI) values were used from both platforms to compare sera collected from 125 pretransplant and posttransplant heart and lung transplant recipients. RESULTS: Most HLA class I (94.5%) and HLA class II (89%) Abs with moderate to high MFI titer (≥4000) were detected by both assays. A modest correlation was observed between MFI values obtained from the 2 assays for both class I (r = 0.3, r2 = 0.09, P < 0.0001) and class II Ab (r = 0.707, r2 = 0.5, P < 0.0001). Both assays detected anti-class I and II Ab that the other did not; however, no specific HLA allele was detected preferentially by either of the 2 assays. For a limited number of discrepant sera, dilution resulted in comparable reactivity profiles between the 2 platforms. CONCLUSIONS: Immucor and One Lambda/ThermoFisher assays have a similar, albeit nonidentical, ability to detect anti-HLA Ab. Although the correlation between the assays was present, significant variances exist, some of which can be explained by a dilution-sensitive "prozone" effect.

Ventricular assist device elicits serum natural IgG that correlates with the development of primary graft dysfunction following heart transplantation.

BACKGROUND: Pre-transplant sensitization is a limiting factor in solid-organ transplantation. In heart transplants, ventricular assist device (VAD) implantation has been associated with sensitization to human leukocyte antigens (HLA). The effect of VAD on non-HLA antibodies is unclear. We have previously shown that polyreactive natural antibodies (Nabs) contribute to pre-sensitization in kidney allograft recipients. Here we assessed generation of Nabs after VAD implantation in pre-transplant sera and examined their contribution to cardiac allograft outcome. METHODS: IgM and IgG Nabs were tested in pre-transplant serum samples collected from 206 orthotopic heart transplant recipients, including 128 patients with VAD (VAD patients) and 78 patients without VAD (no-VAD patients). Nabs were assessed by testing serum reactivity to apoptotic cells by flow cytometry and to the generic oxidized epitope, malondialdehyde, by enzyme-linked immunosorbent assay. RESULTS: No difference was observed in serum levels of IgM Nabs between VAD and no-VAD patients. However, serum IgG Nabs levels were significantly increased in VAD compared with no-VAD patients. This increase was likely due to the presence of the VAD, as revealed by lower serum IgG Nabs levels before implantation. Elevated pre-transplant IgG Nabs level was associated with development of primary graft dysfunction (PGD). CONCLUSIONS: Our study demonstrates that VAD support elicits IgG Nabs reactive to apoptotic cells and oxidized epitopes. These findings further support broad and non-specific B-cell activation by VAD, resulting in IgG sensitization. Moreover, the association of serum IgG Nabs levels with development of PGD suggests a possible role for these antibodies in the inflammatory reaction accompanying this complication.

Donor-specific anti-HLA antibodies with antibody-mediated rejection and long-term outcomes following heart transplantation.

BACKGROUND: Donor-specific anti-HLA antibodies (DSA) are common after heart transplantation and are associated with rejection, cardiac allograft vasculopathy, and mortality. A noninvasive diagnostic test for pathologic antibody-mediated rejection (pAMR) does not exist. METHODS: From January 1, 2010, through August 31, 2013, 221 consecutive adult patients underwent heart transplantation and were followed through October 1, 2015. The primary objective was to determine whether the presence of DSA could detect AMR at the time of pathologic diagnosis. Secondary analyses included association of DSA (stratified by major histocompatibility complex class and de novo status) during AMR with new graft dysfunction, graft loss (mortality or retransplantation), and development of cardiac allograft vasculopathy. RESULTS: During the study period, 69 patients (31.2%) had DSA (24% had de novo DSA), and there were 74 episodes of pAMR in 38 patients. Sensitivity of DSA at any mean fluorescence intensity to detect concurrent pAMR was only 54.3%. The presence of any DSA during pAMR increased the odds of graft dysfunction (odds ratio = 5.37; 95% confidence interval [CI], 1.34-21.47; p = 0.018), adjusting for age, sex, and timing of AMR. Circulating class II DSA after transplantation increased risk of future pAMR (hazard ratio = 2.97; 95% CI, 1.31-6.73; p = 0.009). Patients who developed de novo class II DSA had 151% increased risk of graft loss (contingent on 30-day survival) compared with patients who did not have DSA (95% CI, 1.11-5.69; p = 0.027). CONCLUSIONS: DSA were inadequate to diagnose pAMR. Class II DSA provided prognostic information regarding future pAMR, graft dysfunction with pAMR, and graft loss.

The effect of timing and graft dysfunction on survival and cardiac allograft vasculopathy in antibody-mediated rejection.

BACKGROUND: Antibody-mediated rejection (AMR) has been associated with increased death and cardiac allograft vasculopathy (CAV). Early studies suggested that late AMR was rarely associated with graft dysfunction, whereas recent reports have demonstrated an association with increased mortality. We investigated the timing of AMR and its association with graft dysfunction, death, and CAV. METHODS: This retrospective cohort study identified all adult orthotopic heart transplant (OHT) recipients (N = 689) at Columbia University Medical Center from 2004 to 2013. There were 68 primary cases of AMR, which were stratified by early (< 1 year post-OHT) or late (> 1 year post-OHT) AMR. Kaplan-Meier survival analysis and modeling was performed with multivariable logistic regression and Cox proportional hazards regression. RESULTS: From January 1, 2004, through October 1, 2015, early AMR (median 23 days post-OHT) occurred in 43 patients and late AMR (median 1,084 days post-OHT) occurred in 25. Graft dysfunction was less common with early compared with late AMR (25.6% vs 56%, p = 0.01). Patients with late AMR had decreased post-AMR survival compared with early AMR (1 year: 80% vs 93%, 5 years: 51% vs 73%, p < 0.05). When stratified by graft dysfunction, only those with late AMR and graft dysfunction had worse survival (30 days: 79%, 1 year: 64%, 5 years: 36%; p < 0.006). The association remained irrespective of age, sex, donor-specific antibodies, left ventricular assist device use, reason for OHT, and recovery of graft function. Similarly, those with late AMR and graft dysfunction had accelerated development of de novo CAV (50% at 1 year; hazard ratio, 5.42; p = 0.009), whereas all other groups were all similar to the general transplant population. CONCLUSIONS: Late AMR is frequently associated with graft dysfunction. When graft dysfunction is present in late AMR, there is an early and sustained increased risk of death and rapid development of de novo CAV despite aggressive treatment.

ILT3.Fc inhibits the production of exosomes containing inflammatory microRNA in supernatants of alloactivated T cells.

Immune activation needs to be tightly regulated to control immune-mediated tissue damage. Inhibitory pathways serve to terminate an immune response and resolve inflammation. Persistent exposure to antigens can drive development of adaptive regulatory cells. Similarly exposure of activated T cells to the recombinant ILT3-Fc molecule during priming triggers the differentiation of CD8 T suppressor cells and the induction of CD4 T helper anergy. Ts express high levels of immunoregulatory signature genes together with low levels of microRNA which control their function. Analysis of microRNA contained by exosomes from cultures in which T cells were alloactivated in the presence or absence of ILT3.Fc, demonstrated that this agent inhibits the release of inflammatory microRNA. The source of such inflammatory microRNA was found to reside in alloactivated CD4 T cells, since exosomes from MLC primed CD4 T cells were shown to diminish the suppressive activity of ILT3-Fc-induced CD8(+) Ts at high effector to suppressor T cell ratios. This indicates that inflammatory exosomes can swing the balance between effector and regulatory T cells in favor of immunity. These data suggest that isolation and characterization of micro-RNA containing exosomes in patients' circulation may be of use for treatment, prevention and monitoring of immune activation.

Detection of donor-specific-antibodies by solid phase assay and its relevance to complement-dependent-lymphocytotoxicity cross-matching in kidney transplantation.

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

HLA antibodies in pediatric heart transplantation.

We have analyzed the impact of anti-HLA antibodies present in the patients' circulation prior and/or following heart transplantation in a population of 108 pediatric recipients. Anti-HLA class I and class II antibodies were monitored by traditional CDC using donor and panel T and B lymphocytes and by SPA for detection of DSA. There was a highly significant correlation between the development of AMR and presence of CDC- or SPA-detected DSA. However, the fraction of the transplant population which remained AMR-free was much higher among patients with SPA-detected compared to CDC-detected DSA. Furthermore, long-term graft survival was negatively affected only by cytotoxic, complement-fixing anti-HLA class I antibodies developing following transplantation. Anti-HLA class I or class II antibodies detected by SPA had no effect on long-term survival rates.

Membrane and soluble ILT3 are critical to the generation of T suppressor cells and induction of immunological tolerance.

The tolerogenic phenotype of human dendritic cells is characterized by high cell surface expression of the inhibitory receptor ILT3. ILT3 signals both intracellularly inhibiting tyrosine phosphorylation, NF-kappaB and MAPK p38 activity, transcription of certain co-stimulatory molecules, secretion of cytokines and chemokines, and extracellularly into the T cells with which the dendritic cells interact. Both ILT3(high) tolerogenic dendritic cells and soluble ILT3 induce CD4 Th anergy and differentiation of antigen specific CD8 T suppressor cells. Recombinant ILT3-Fc protein has important immunotherapeutic potential acting directly on activated T cells and promoting the induction of immunological tolerance.

Comparative outcome analysis of ABO-incompatible and positive crossmatch renal transplantation: a single-center experience.

BACKGROUND: ABO-incompatible (ABOi) and positive crossmatch (XM) renal transplants pose special immunologic challenges. It is important to compare outcomes, study resource utilization, and attempt to risk stratify patients in these higher risk transplant settings. METHODS: We compared apheresis utilization and transplant outcomes in ABOi, XM, and combined ABOi-XM renal transplants. We also analyzed multiple parameters, including patient and laboratory variables, to identify predictors of transplant outcome. RESULTS: Incidences of early (< or =30 days posttransplant) antibody-mediated rejection (AMR) and acute cellular rejection (ACR) were similar among the three incompatible groups whereas they differed in allograft rejection for late (>30 days posttransplant) AMR and ACR. Notably, there were no episodes of late AMR among ABOi patients. Patients treated with more than four pretransplant plasmapheresis/intravenous immunoglobulin (PP/IVIg) had a greater likelihood of experiencing early AMR. The median number of posttransplant PP/IVIg treatments was greater than twofold higher in ABOi-XM and XM patients compared to ABOi patients. Patients who required more than five posttransplant PP/IVIg procedures and those with one or more prior renal transplants had higher incidences of late ACR. CONCLUSIONS: Our analysis aids in defining apheresis resource utilization and helps in risk stratification of incompatible renal transplantation. It also aids in predicting allograft rejection and provides an opportunity for preemptive monitoring and treatment.

Suppression of xenogeneic graft-versus-host disease by treatment with immunoglobulin-like transcript 3-Fc.

Allogeneic hematopoietic cell transplantation represents an important therapy for certain malignant and nonmalignant diseases. However, graft-versus-host disease (GVHD) is a major cause of mortality and morbidity. The search for agents that can efficiently suppress GVHD has been going on for more than half a century. GVHD is particularly strong in xenogeneic donor-recipient combinations, given the unlimited number of potentially immunogenic antigens donor lymphocytes encounter in the host. Using a hu-nonobese diabetic/severe combined immunodeficiency (hu-NOD/SCID) gamma-null model of xenogeneic GVHD, we have demonstrated that treatment with recombinant immunoglobulin-like transcript 3-Fc protein induces the differentiation of CD8(+) T suppressor cells and blocks the cellular and humoral arm of the GVH reaction.

Relevance of different antibody detection methods for the prediction of antibody-mediated rejection and deceased-donor kidney allograft survival.

Presensitizing alloantibodies may represent a grave danger in organ transplantation, increasing the risk of antibody-mediated rejection (AMR) and graft loss. However, not all antibodies are harmful to the graft. In our study of a cohort of 325 deceased-donor renal allograft recipients, the patients were determined eligible to receive an allograft based on a negative complement-dependent cytotoxicity (CDC) crossmatch (XM). Yet at the time of transplantation, many candidates displayed donor-specific antibodies (DSA) by more sensitive methods, such as solid-phase assays (SPA, Luminex) or flow cytometry crossmatch (FCXM). The majority of the patients who were DSA positive by either SPA (67%) or FCXM (66%) presented an AMR-free clinical course posttransplantation. Among the patients who developed AMR (N = 29), 76% proved clinically manageable and did not lose the graft. Analysis of the DSA mean fluorescence intensities (MFI) of Luminex indicated no statistically significant difference between patients who experienced AMR episodes and those who did not. Importantly, many of the patients with AMR did not test positive for DSA by SPA (20/29) or FCXM (14/29). Despite false-positive and false-negative results, the detection of DSA by SPA or FCXM was positively associated with AMR, but not with actuarial graft survival. The field of organ transplantation has always struggled to reconcile two opposing goals: improving transplantation outcome while increasing access to transplantation. SPA and FCXM appear to be oversensitive and defining patients as "sensitized" according to these methods would block access to transplantation for many candidates who would otherwise benefit greatly from receiving the allograft. Nevertheless, SPA and FCXM are invaluable tools, assisting clinicians in gauging AMR risk and tailoring immunosuppression of the posttransplantation immunological monitoring accordingly.

The relationship of nodular endocardial infiltrates (Quilty lesions) to survival, patient age, anti-HLA antibodies, and coronary artery disease following heart transplantation.

BACKGROUND: Quilty lesions are mononuclear cell infiltrates identified in human heart transplant biopsies. The biologic significance of Quilty lesions remains undetermined. METHODS: We monitored acute rejection by biopsy and lymphocyte growth assay (LGA) as well as transplant-related coronary artery disease (TRCAD) by yearly angiogram in 285 recipients of primary heart allografts. Patients showing Quilty lesions on biopsies during the first year posttransplant were compared with patients without such lesions. Recipients' sera were obtained at the time of biopsy and tested for anti-HLA Class I and II antibodies. RESULTS: The actuarial survival of patients who developed Quilty lesions was significantly better than those who did not (P=.0074). Patients with Quilty lesions were younger and more likely to have a biopsy diagnosis of acute rejection (P=.002) and positive LGA (P<.0001) during the first posttransplant year. Among patients who do not form anti-HLA Class II antibodies, those with Quilty lesions were more likely than patients without Quilty lesions to develop TRCAD 5 years posttransplantation (P=.04). There was no correlation of Quilty status with the number of HLA donor-recipient mismatches or posttransplant development of anti-HLA antibodies. CONCLUSIONS: Quilty formers showed improved survival and are more likely to be diagnosed with acute rejection on biopsy and have positive LGAs. Allograft recipients who do not form anti-HLA Class II antibodies but do form Quilty lesions are more likely to develop TRCAD by 5 years posttransplantation than those who do not form Quilty lesions.

Acute and hyperacute humoral rejection in kidney allograft recipients treated with anti-human thymocyte antibodies.

Equine and rabbit antihuman thymocyte globulins (ATGs) have been used in renal transplantation for prevention and treatment of acute rejection. We now report that hyperacute and acute antibody-mediated rejection of renal allografts occurred in three newly transplanted patients who received ATG for induction therapy. Antibody studies performed using complement-dependent cytotoxicity, flow cytometry, enzyme-linked immunosorbent assay, and Luminex yielded negative results for antilymphocytic and antiendothelial cell antibodies in the pretransplant sera obtained from these patients. ATG treatment was initiated at the time of transplantation. One of the patients experienced hyperacute rejection and required transplant nephrectomy within 24 h of transplantation. The other two patients developed acute antibody-mediated rejection within 14 days after transplantation. None of the patients developed antihuman leukocyte antigen antibodies when humoral rejection occurred. However, xenoantibodies that strongly bound to human lymphocytes and, importantly, to activated endothelial cells, were identified in the sera obtained at the time of humoral rejection. Hence, our results strongly implicate ATG in the induction of antibody-mediated rejection of kidney allografts. Flow cytometry testing of ATG reactivity to endothelial cells may be useful in identifying and discarding the ATG lots containing xenoantibodies that can bind to activated endothelial cells of the transplant.

Novel single nucleotide polymorphisms in the human immune inhibitory immunoglobulin-like T cell receptor type 4.

The capacity of antigen presenting cells to induce anergy in T helper cells and elicit the generation of T suppressor cells is regulated by a variety of positive and negative signals. Antigen-specific CD8(+)CD28(-) and CD4(+)CD25(+) T suppressor/regulatory cells induce the upregulation of inhibitory receptors expressed by antigen-presenting cells (APC) belonging to the family of immunoglobulin-like transcripts (ILTs) and downregulation of costimulatory molecules in APC. Immunoglobulin-like immune inhibitory receptor (ILT4), one of the inhibitory receptors expressed by tolerogenic APC, interacts with human leukocyte antigen A, B, and G molecules and transmits negative signals that interfere with the activation of monocytes and dendritic cells. Reported is the identification of two single nucleotide polymorphisms within domain 1 (IgD1) of ILT4 at positions 113 and 144. Domain 1 is part of the distal membrane portion of ILT4, which is engaged in protein-protein interactions between APC and T cells.

Anti-HLA antibodies in heart transplantation.

We have analyzed the relationship between the development of transplant-related coronary artery disease (TRCAD) and the following potential risk factors: (a). number of HLA mismatches between recipient and donor; (b). production of anti-HLA antibodies; (c). growth of lymphocytes infiltrating the graft; and (d). frequency of biopsy proven episodes of acute rejection. The study population consisted of 285 adult heart allograft recipients who were monitored over a period of two years or more. The results demonstrate a significant correlation between TRCAD, generation of anti-HLA class II antibodies and potential of lymphocytes infiltrating the graft to proliferate ex-vivo in medium containing IL-2. Humoral and cellular immune responses to HLA-DR antigens expressed by the graft seem to underlie the development of TRCAD.