Harnessing Scientific and Technological Advances to Improve Equity in Kidney Allocation Policies.

We reported that current assignment of HLA-DQ is a barrier to organ allocation. Here we simulated the impact of incorporating HLA-DQ antigens and antibodies as A/B and αß allelic variants, respectively, on calculated panel reactive antibody (cPRA) and probability of finding potential compatible donors (PCD). A cohort of 1224 donors and 2075 sensitized candidates was analyzed using HLA-DQαß allelic (study) versus serologic (current practice) nomenclature. A significant (p < 10-4 ) decrease in cPRA was observed with higher impact for male versus female, and first transplant versus retransplant (p < 10-4 ), affecting mostly patients with moderate cPRA (30-80%). Consequently, the number of patients qualifying for 100% cPRA points according to the United Network for Organ Sharing-Kidney Allocation System decreased by 37%. More critically, by using allelic versus serologic nomenclature for HLA-DQ, the number of PCDs for all patients was increased, with male and first-transplant patients showing a higher expansion compared with female and retransplants. Patients of blood group O showed the highest benefit. The goal of reporting unacceptable antigens is to improve accuracy of virtual crossmatching and increase the likelihood of finding immunologically compatible donors. Our simulation provides strong support for the need to re-evaluate the use of allele typing and how HLA-DQ antigens and antibodies are incorporated into allocation policies to ensure equity.

Hiding in Plain Sight-A New Look at HLA Epitopes: A Case Report.

"Epitope matching" has become a buzz word in solid organ transplantation. Its goal is to improve matching between donor and recipient, to minimize risk for antibody-mediated rejection and to reduce sensitization associated with graft failure. Current software allows identification and enumeration of amino acid sequence mismatches in the form of HLA eplets; however, "eplets" and "epitopes" are not interchangeable terms, and the understanding of what contributes to the antigenicity and immunogenicity of HLA B cell epitopes is still very limited and inadequate. In fact, we still do not know what constitutes an HLA epitope or how to define it in a clinically useful way. To allow for judicious implementation of epitope matching, it is critical to explore the full spectrum of factors that affect allorecognition. In exploring antibody-binding patterns, we have uncovered a potential tool-currently hidden in plain sight-that may shed light on some aspects of epitope characteristics.

Donor-Specific HLA Antibodies in Living Versus Deceased Donor Liver Transplant Recipients.

With less ischemia, improved donor selection and controlled procedures, living donor liver transplantation (LDLT) might lead to less HLA donor-specific antibody (DSA) formation or fewer adverse outcomes than deceased donor liver transplantation (DDLT). Using the multicenter A2ALL (Adult-to-Adult Living Donor Liver Transplantation Cohort Study) biorepository, we compared the incidence and outcomes of preformed and de novo DSAs between LDLT and DDLT. In total, 129 LDLT and 66 DDLT recipients were identified as having serial samples. The prevalence of preformed and de novo DSAs was not different between DDLT and LDLT recipients (p = 0.93). There was no association between patient survival and the timing (preformed vs. de novo), class (I vs. II) and relative levels of DSA between the groups; however, preformed DSA was associated with higher graft failure only in DDLT recipients (p = 0.01). De novo DSA was associated with graft failure regardless of liver transplant type (p = 0.005) but with rejection only in DDLT (p = 0.0001). On multivariate analysis, DSA was an independent risk factor for graft failure regardless of liver transplant type (p = 0.017, preformed; p = 0.002, de novo). In conclusion, although similar in prevalence, DSA may have more impact in DDLT than LDLT recipients. Although our findings need further validation, future research should more robustly test the effect of donor type and strategies to mitigate the impact of DSA.

Nonchimeric HLA-Identical Renal Transplant Tolerance: Regulatory Immunophenotypic/Genomic Biomarkers.

We previously described early results of a nonchimeric operational tolerance protocol in human leukocyte antigen (HLA)-identical living donor renal transplants and now update these results. Recipients given alemtuzumab, tacrolimus/MPA with early sirolimus conversion were multiply infused with donor hematopoietic CD34(+) stem cells. Immunosuppression was withdrawn by 24 months. Twelve months later, operational tolerance was confirmed by rejection-free transplant biopsies. Five of the first eight enrollees were initially tolerant 1 year off immunosuppression. Biopsies of three others after total withdrawal showed Banff 1A acute cellular rejection without renal dysfunction. With longer follow-up including 5-year posttransplant biopsies, four of the five tolerant recipients remain without rejection while one developed Banff 1A without renal dysfunction. We now add seven new subjects (two operationally tolerant), and demonstrate time-dependent increases of circulating CD4(+) CD25(+++) CD127(-) FOXP3(+) Tregs versus losses of Tregs in nontolerant subjects (p < 0.001). Gene expression signatures, developed using global RNA expression profiling of sequential whole blood and protocol biopsy samples, were highly associative with operational tolerance as early as 1 year posttransplant. The blood signature was validated by an external Immune Tolerance Network data set. Our approach to nonchimeric operational HLA-identical tolerance reveals association with Treg immunophenotypes and serial gene expression profiles.

HLA epitopes as viewed by antibodies: what is it all about?

The need for new approaches to define HLA antibodies, in the context of organ transplantation, is intensely debated among HLA professionals. In this review, we sought to provide background and perspective to current understanding of the immunogenicity of HLA mismatches with respect to the humoral alloimmune response and the definition of B cell epitopes. Initial data suggest that epitope matching not only assists in defining better matches for the current transplant, but also minimizes the risk of developing de novo HLA-donor-specific-antibodies posttransplant. In other words, other than lowering the risk of current graft rejection, epitope matching is likely to lower overall future sensitization levels and thus increases the likelihood of finding a compatible donor when the need for a retransplantation arises. More detailed knowledge of epitopes makes it possible to investigate what constitutes permissible versus non-permissible HLA mismatches. The currently available evidence suggest that epitope matching is the most rational way to decrease the risk of HLA-linked transplant rejection. This review is aimed at stimulating further and more intense collaborative effort in this field.

Unintended Consequences of the New National Kidney Allocation Policy in the United States.

The new national Kidney Allocation System of the Organ Procurement and Transplantation Network (OPTN), effective as of December 4, 2014, was designed to improve the chances of transplanting the most highly sensitized patients on the waitlist, those with calculated panel reactive antibody values of 98%, 99% and 100%. Recently, it was suggested that these highly sensitized patients will experience inequitable access, given the reported high prevalence of antibodies to HLA-DP, and the fact that only about 1/3 of deceased donors are typed for HLA-DP antigens. Here we report that 320/2948 flow cytometric crossmatches performed for the Northwestern transplant program over the past 28 months were positive solely due to HLA-DP donor-specific antibodies (11%; 16.5% of patients with HLA antibodies-sensitized patients). We further show that 58/207 (12%) HLA-DR serologically matched donor-recipient pairs had a positive B cell flow crossmatch due to donor-specific HLA class II antibodies, and 2/34 (6%) serologic zero-HLA-A-B-DR mismatch had a positive flow crossmatch due to HLA-DSA. We therefore provide information regarding the necessity and importance of complete donor HLA typing including both chains of the HLA-DP antigen (encoded by HLA-DPA1 and HLA-DPB1) at the time of organ offer.

Assessing Antibody Strength: Comparison of MFI, C1q, and Titer Information.

The presence of donor-specific HLA antibodies before or after transplantation may have different implications based on the antibody strength. Yet, current approaches do not provide information regarding the true antibody strength as defined by antigen-antibody dissociation rate. To assess currently available methods, we compared between neat mean fluorescence intensity (MFI) values, C1q MFI values, ethylenediaminetetraacetic acid (EDTA)-treated samples, as well as titration studies and peak MFI values of over 7000 Luminex-based single-antigen HLA antibody data points. Our results indicate that neat MFI values do not always accurately depict antibody strength. We further showed that EDTA treatment (6%) does not always remove all inhibitory factors compared with C1q or titration studies. In this study of patients presenting with multiple antibody specificities, a prozone effect was observed in 71% of the cohort (usually not affecting all antibody specificities within a single serum sample, though). Similar to titration studies, the C1q assay was able to address the issue of potential inhibition; however, its limitation is its low sensitivity and inability to detect the presence of weak antibodies. Titration studies are the only method among the approaches used in this study to provide information suggesting antigen-antibody dissociation rates and are, therefore, likely to provide better indication of true antibody strength.

Should HLA mismatch acceptability for sensitized transplant candidates be determined at the high-resolution rather than the antigen level?

Defining HLA mismatch acceptability of organ transplant donors for sensitized recipients has traditionally been based on serologically defined HLA antigens. Now, however, it is well accepted that HLA antibodies specifically recognize a wide range of epitopes present on HLA antigens and that molecularly defined high resolution alleles corresponding to the same low resolution antigen can possess different epitope repertoires. Hence, determination of HLA compatibility at the allele level represents a more accurate approach to identify suitable donors for sensitized patients. This approach would offer opportunities for increased transplant rates and improved long term graft survivals.

Comprehensive assessment and standardization of solid phase multiplex-bead arrays for the detection of antibodies to HLA.

Solid phase multiplex-bead arrays for the detection and characterization of HLA antibodies provide increased sensitivity and specificity compared to conventional lymphocyte-based assays. Assay variability due to inconsistencies in commercial kits and differences in standard operating procedures (SOP) hamper comparison of results between laboratories. The Clinical Trials in Organ Transplantation Antibody Core Laboratories investigated sources of assay variation and determined if reproducibility improved through utilization of SOP, common reagents and normalization algorithms. Ten commercial kits from two manufacturers were assessed in each of seven laboratories using 20 HLA reference sera. Implementation of a standardized (vs. a nonstandardized) operating procedure greatly reduced MFI variation from 62% to 25%. Although laboratory agreements exceeded 90% (R(2) ), small systematic differences were observed suggesting center specific factors still contribute to variation. MFI varied according to manufacturer, kit, bead type and lot. ROC analyses showed excellent consistency in antibody assignments between manufacturers (AUC > 0.9) and suggested optimal cutoffs from 1000 to 1500 MFI. Global normalization further reduced MFI variation to levels near 20%. Standardization and normalization of solid phase HLA antibody tests will enable comparison of data across laboratories for clinical trials and diagnostic testing.

Perception versus reality?: Virtual crossmatch--how to overcome some of the technical and logistic limitations.

The goal of this work was to evaluate concordance between (a) actual flow cytometric crossmatch (FCXM) that is performed by the OPO laboratory servicing our transplant center and (b) virtual XM (vXM) prediction based on antibody identification by solid-phase methods performed in our laboratory. A total of 1586 FCXM, performed between June 2007 and September 2008, between all potential deceased donors in our region and sera from patients awaiting kidney or kidney-pancreas transplant, listed at Northwestern Memorial Hospital were evaluated. A key finding of this analysis was the understanding that a thorough vXM cannot be performed in some donor/recipient pairs due to the lack of certain antibody profile data specific to the donor in question. Obtaining more in depth and stringent information regarding antibody specificities, we demonstrate an excellent sensitivity and specificity of the vXM assays- 86.1% and 96.8%, respectively, with a positive likelihood ratio and negative likelihood ratios of 26.9 and 0.14, respectively. The vXM can serve as an outstanding tool to predict HLA compatibility between donor and recipient, with the caveat that the presence/absence of all antibodies against the potential donor and their strength have been thoroughly investigated.

Immunology of the central nervous system.

Immunology of the central nervous system (CNS) is a growing field of study. Until recently the brain was considered an 'immunologically privileged' site. It is increasingly apparent that the CNS has a significant but tightly regulated capability to mount an inflammatory and immune response. This article serves as an introduction to the special section at the start of this issue on neuroimmunology. We also focus on several immunological concepts that are particularly relevant in the context of neuroimmunology-cross-reactivity, the immunological synapse and the nature of the immune response to transplantation in the CNS. We conclude that the fundamental concepts are common to all branches of immunology. Better understanding of the basic mechanisms will blur the borders between the different areas of immunology.

Differential sensitivity of resting and IL-2 activated NK cells to R-verapamil.

Natural killer (NK) cells are the first lymphoid population to reconstitute the peripheral blood compartment of immunologically compromised bone marrow transplant (BMT) recipients. Recent data suggest that, among patients transplanted for leukemia, NK cells can prevent or delay disease relapse by mediating a cytotoxic graft vs leukemia (GvL) response. Although the major mechanism by which NK cells mediate target cell lysis involves degranulation and release of cytolytic effector molecules (granzymes, proteoglycans, perforin), accumulating evidence suggests that NK cells possess additional pathways to mediate target cell killing. In fact, it is well recognized that recombinant cytokines such as IL-2 enhance the in vitro cytolytic activity of NK cells. In this study, we observed that the lytic activity mediated by resting and IL-2 activated NK cells against the same target cell appears to occur via two distinct pathways, as distinguished by their differential response to R-verapamil. Specifically, we observed that 25 microM R-verapamil inhibited the lytic activity of resting NK cells against K562 targets by approximately 50%. However, the lytic activity of IL-2 activated NK cells was unaffected by this concentration of R-verapamil. Additional studies suggested that the inhibitory effect of R-verapamil on NK cytotoxic activity was associated with its ability to prevent degranulation of cytotoxic granules. Specifically, R-verapamil inhibited BLT esterase release from resting but not IL-2 activated NK cells. These data suggest that IL-2 activated NK cells can promote target cell lysis by a pathway (possibly degranulation independent) distinct from that used by resting NK cells. We speculate that the target of R-verapamil on resting NK cells is P-glycoprotein (Pgp), an ABC transporter that we recently reported was expressed on NK cells and whose functional activity is known to be inhibited by R-verapamil.

Role of cytokine gene polymorphism in hepatitis C recurrence and allograft rejection among liver transplant recipients.

BACKGROUND: Cytokines play a key role in the regulation of immune responses. The maximal capacity of cytokine production varies between individuals and was shown to correlate with polymorphism in cytokine gene promoters. The objective of this study was to analyze the role of cytokine allelic variations in susceptibility to early graft rejection episodes and recurrence of hepatitis C infection in liver transplant (LTx) recipients. METHODS: The genetic profile of five cytokines was studied in 68 LTx recipients and 49 controls using polymerase chain reaction sequence specific primers. All individuals were genotyped as high or low producers of TNF-alpha and IL-6 and high, intermediate, or low producers of transforming growth factor beta (TGF-beta), interferon gamma (IFN-gamma), and interleukin 10 (IL-10) based on single nucleotide substitutions. RESULTS: No statistically significant differences were observed between patients with or without early rejection episodes. A significant proportion of patients more prone to rejection were genotyped as having a low production profile of IL-10 compared with the control population (P=0.04). These data are in accordance with reports regarding other solid-organ transplant recipients. Patients with no recurrence of hepatitis C had the inherent ability to produce higher TGF-beta levels than did patients with recurrent disease (P=0.042). Among nonrecurrent patients, the percentage of genetically low IL-10 producers was higher than among recurrent patients (P=0.07). Furthermore, a genetic tendency to produce higher levels of IFN-gamma was noted among LTx recipients with nonrecurrent hepatitis C than among those with recurrent hepatitis C. CONCLUSIONS: While no significant correlation was detected between particular cytokine profile and early rejection episodes, our data strongly suggest an association between cytokine gene polymorphism of TGF-beta, IL-10, and INF-gamma and recurrence of hepatitis C in LTx recipients.

Flow cytometric detection of HLA-specific antibodies as a predictor of heart allograft rejection.

BACKGROUND: Historically, panel reactive antibody (PRA) analysis to detect HLA antibodies has been performed using cell-based complement-dependent cytotoxicity (CDC) techniques. Recently, a flow cytometric procedure (FlowPRA) was introduced as an alternative approach to detect HLA antibodies. The flow methodology, using a solid phase matrix to which soluble HLA class I or class II antigens are attached is significantly more sensitive than CDC assays. However, the clinical relevance of antibodies detected exclusively by FlowPRAhas not been established. In this study of cardiac allograft recipients, FlowPRA was performed on pretransplant sera with no detectable PRA activity as assessed by CDC assays. FlowPRA antibody activity was then correlated with clinical outcome. METHODS: PRA analysis by anti-human globulin enhanced (AHG) CDC and FlowPRA was performed on sera corresponding to final cross-match specimens from 219 cardiac allograft recipients. In addition, sera collected 3-6 months posttransplant from 91 patients were evaluated. The presence or absence of antibodies was correlated with episodes of rejection and patient survival. A rejection episode was considered to have occurred based on treatment with antirejection medication and/or histology. RESULTS: By CDC, 12 patients (5.5%) had pretransplant PRA >10%. In contrast, 72 patients (32.9%) had pretransplant anti-HLA antibodies detectable by FlowPRA (34 patients with only class I antibodies; 7 patients with only class II antibodies; 31 patients with both class I and class II antibodies). A highly significant association (P<0.001) was observed between pretransplant HLA antibodies detected by FlowPRA and episodes of rejection that occurred during the first posttransplant year. Fifteen patients died within the first year posttransplant. Of nine retrospective flow cytometric cross-matches that were performed, two were in recipients who had no pretransplant antibodies detectable by FlowPRA. Both of these cross-matches were negative. In contrast, five of seven cross-matches were positive among recipients who had FlowPRA detectable pretransplant antibodies. Posttransplant serum specimens from 91 patients were also assessed for antibodies by FlowPRA. Among this group, 58 patients had FlowPRA antibodies and there was a trend (although not statistically significant) for a biopsy documented episode of rejection to have occurred among patients with these antibodies. CONCLUSIONS: Collectively, our data suggest that pre- and posttransplant HLA antibodies detectable by FlowPRA and not AHG-CDC identify cardiac allograft recipients at risk for rejection. Furthermore, a positive donor reactive flow cytometric cross-match is significantly associated with graft loss. Thus, we believe that detection and identification of HLA-specific antibodies can be used to stratify patients into high and low risk categories. An important observation of this study is that in the majority of donor:recipient pairs, pretransplant HLA antibodies were not directed against donor antigens. We speculate that these non-donor-directed antibodies are surrogate markers that correspond to previous T cell activation. Thus, the rejection episodes that occur in these patients are in response to donor-derived MHC peptides that share cryptic determinants with the HLA antigens that initially sensitized the patient.

IL-4 inhibits P-glycoprotein in normal and malignant NK cells.

Patients presenting with a natural killer (NK) cell leukemia generally have a poor prognosis. NK cell tumors are generally resistant to numerous chemotherapeutic drugs and even combination chemotherapy usually results in only short term remissions. The drug resistance of NK cell leukemias may be at least partially explained by their expression of the multidrug resistant transporter, P-glycoprotein (Pgp). In this study, we demonstrate that the expression and function of Pgp activity on NK cells (leukemic and normal) can be reversed with IL-4.