Cardiovascular Effects of Autologous Bone Marrow-Derived Mesenchymal Stromal Cell Therapy With Early Tacrolimus Withdrawal in Renal Transplant Recipients: An Analysis of the Randomized TRITON Study

BackgroundAfter renal transplantation, there is a need of immunosuppressive regimens that effectively prevent allograft rejection while minimizing cardiovascular complications. This substudy of the TRITON trial evaluated the cardiovascular effects of autologous bone marrow–derived mesenchymal stromal cells (MSCs) in renal transplant recipients.Methods and ResultsRenal transplant recipients were randomized to MSC therapy, infused at weeks 6 and 7 after transplantation, with withdrawal at week 8 of tacrolimus or standard tacrolimus dose. Fifty‐four patients (MSC group=27; control group=27) underwent transthoracic echocardiography at weeks 4 and 24 after transplantation and were included in this substudy. Changes in clinical and echocardiographic variables were compared. The MSC group showed a benefit in blood pressure control, assessed by a significant interaction between changes in diastolic blood pressure and the treatment group (P=0.005), and a higher proportion of patients achieving the predefined blood pressure target of ConclusionsMSC strategy is associated with improved blood pressure control, regression of left ventricular hypertrophy, and prevention of progressive diastolic dysfunction at 24 weeks after transplantation.</p

Implementation of molecular matching in transplantation requires further characterization of both immunogenicity and antigenicity of individual HLA epitopes

Over the past decade, high HLA epitope mismatch scores have been associated with inferior transplant outcomes using several tools, of which HLAMatchmaker is most well-known. This software uses theoretically defined polymorphic amino acid configurations, called eplets, for HLA compatibility analysis. Although consideration of eplet mismatch loads has potential for immunological risk stratification of transplant patients, the use of eplet matching in organ allocation algorithms is hindered by lacking knowledge of the immunogenicity of individual eplets, and the possibility that single mismatched amino acids, rather than complete eplets, are responsible for HLA antibody induction.There are several approaches to define eplet immunogenicity, such as antibody verification of individual eplets, and data-driven approaches using large datasets that correlate specific eplet mismatches to donor specific antibody formation or inferior transplant outcomes. Data-driven approaches can also be used to define whether single amino acid mismatches may be more informative than eplet mismatches for predicting HLA antibody induction.When using epitope knowledge for the assignment of unacceptable antigens, it important to realize that alleles sharing an eplet to which antibodies have formed are not automatically all unacceptable since multiple contact sites determine the binding strength and thus biological function and pathogenicity of an antibody, which may differ between reactive alleles.While the future looks bright for using HLA epitopes in clinical decision making, major steps need to be taken to make this a clinical reality.(c) 2021 The Author(s). Published by Elsevier Inc. on behalf of American Society for Histocompatibility and Immunogenetics. This is an open access article under the CC BY-NC-ND license (http://creativecommons. org/licenses/by-nc-nd/4.0/)

A Comprehensive Evaluation of the Antibody-Verified Status of Eplets Listed in the HLA Epitope Registry

Matching strategies based on HLA eplets instead of HLA antigens in solid organ transplantation may not only increase the donor pool for highly sensitized patients, but also decrease the incidence of de novo donor-specific antibody formation. However, since not all eplets are equally capable of inducing an immune response, antibody verification is needed to confirm their ability to be bound by antibodies, such that only clinically relevant eplets are considered. The HLA Epitope Registry has documented all theoretically defined HLA eplets along with their antibody verification status and has been the foundation for many clinical studies investigating eplet mismatch in transplantation. The verification methods for eplets in the Registry range from polyclonal sera from multi- and uni-parous women to murine and human monoclonal antibodies (mAbs), and antibodies purified by adsorption and elution from sera of HLA immunized individuals. The classification of antibody verification based on different methods for validation is problematic, since not all approaches represent the same level of evidence. In this study, we introduce a classification system to evaluate the level of evidence for the antibody-verified status of all eplets in the HLA Epitope Registry. We demonstrate that for a considerable number of eplets, the antibody-verified status is solely based on polyclonal serum reactivity of multiparous women or on reactivity of murine mAbs. Furthermore, we noted that a substantial proportion of patient sera analyses and human mAb data presented in the HLA Epitope Registry Database has never been published in a peer-reviewed journal. Therefore, we tested several unpublished human HLA-specific mAbs by luminex single antigen beads assay to analyze their HLA reactivity for eplet antibody verification. Although the majority of analyzed mAbs indeed verified their assigned eplets, this was not the case for a number of eplets. This comprehensive overview of evidence for antibody verification of eplets in the HLA Epitope Registry is instrumental for future investigations towards eplet immunogenicity and clinical studies considering antibody-verified eplet mismatch in transplantation and warrants further standardization of antibody verification using high quality data

Left Atrial Structural and Functional Response in Kidney Transplant Recipients Treated With Mesenchymal Stromal Cell Therapy and Early Tacrolimus Withdrawal

Background: Autologous bone marrow–derived mesenchymal stromal cell (MSC) therapy and withdrawal of calcineurin inhibitors (CNIs) has been shown to improve systemic blood pressure control and left ventricular hypertrophy regression in kidney transplant recipients. In the current subanalysis, we aimed to evaluate the impact of this novel immunosuppressive regimen on the longitudinal changes of left atrial (LA) structure and function after kidney transplantation. Methods: Kidney transplant recipients randomized to MSC therapy—infused at weeks 6 and 7 after transplantation, with complete discontinuation at week 8 of tacrolimus (MSC group)—or standard tacrolimus dose (control group) were evaluated with transthoracic echocardiography at weeks 4 and 24 after kidney transplantation. The changes in echocardiographic parameters were compared between the randomization arms using an analysis of covariance model adjusted for baseline variable. Results: Fifty-four participants (MSC therapy = 27; tacrolimus therapy = 27) were included. There was no significant interaction between the allocated treatment and the changes of indexed maximal LA volume (LAVImax) over the study period. Conversely, between 4 and 24 weeks post-transplantation, an increase in indexed minimal LA volume (LAVImin) was observed in control subjects, while it remained unchanged in the MSC group, leading to a significant difference between groups (P = .021). Additionally, patients treated with MSC therapy showed a benefit in LA function, assessed by a significant interaction between changes in LA emptying fraction and LA reservoir strain and the randomization arm (P = .012 and P = .027, respectively). Conclusions: The combination of MSC therapy and CNIs withdrawal prevents progressive LA dilation and dysfunction in the first 6 months after kidney transplantation. LAVImin and LA reservoir strain may be more sensitive markers of LA reverse remodeling, compared with LAVImax

HLA-DQ eplet mismatch load may identify kidney transplant patients eligible for tacrolimus withdrawal without donor-specific antibody formation after mesenchymal stromal cell therapy

Recently, the randomized phase-II Triton study demonstrated that mesenchymal stromal cell (MSC) therapy facilitated early tacrolimus withdrawal in living donor kidney transplant recipients. The current sub-study analyzed formation of de novo donor-specific HLA antibodies (dnDSA) in the context of the degree of HLA eplet mismatches. At the time of protocol biopsy at 6 months, 7/29 patients (24%) in the MSC group and 1/27 patient (3.7%) in the control group had developed dnDSA. In the MSC group, all dnDSA were anti-HLA-DQ; two patients had anti-DQ alone and five patients combined with anti-class I, HLA-DR or -DP. Despite excess dnDSA formation in the MSC-arm of the study, the evolution of eGFR (CKD-EPI) and proteinuria were comparable 2 years posttransplant. All dnDSA were complement-binding and three patients had antibody-mediated rejection in the protocol biopsy, but overall rejection episodes were not increased. Everolimus had to be discontinued in nine patients because of toxicity, and tacrolimus was reintroduced in six patients because of dnDSA formation. The HLA-DQ eplet mismatch load independently associated with dnDSA (adjusted hazard ratio = 1.07 per eplet mismatch, p = 0.008). A threshold of ≥11 HLA-DQ eplet mismatches predicted subsequent dnDSA in all 11 patients in the MSC group, but specificity was low (44%). Further research is warranted to explore HLA molecular mismatch load as a biomarker to guide personalized maintenance immunosuppression in kidney transplantation

Population pharmacokinetics of subcutaneous alemtuzumab in kidney transplantation

Aim: Alemtuzumab is a monoclonal antibody used as induction immunosuppressive therapy in kidney transplantation. It targets CD52 on lymphocytes, inducing profound immune cell depletion upon administration. Owing to its off-label status in kidney transplantation, its pharmacokinetic characteristics are largely unknown in this setting, and its current fixed dosing algorithm originates from other populations. We developed a population pharmacokinetic model for alemtuzumab in kidney transplant recipients and investigated the potential of personalized alemtuzumab therapy. Methods: In total, 362 pharmacokinetic observations drawn 0-165 days after transplantation were available from 61 adult kidney transplant recipients who received two consecutive doses of 15 mg alemtuzumab subcutaneously. A population pharmacokinetic model was developed using nonlinear mixed-effects modelling and applied to simulate various dosing regimens. Results: The alemtuzumab concentration-time data were best described by a two-compartmental model with first-order absorption and parallel first-order and time-varying concentration-dependent elimination, with between-subject variability on the first-order elimination (39.6%) and central distribution volume (39.6%). Alemtuzumab pharmacokinetics varied with body size, rendering lighter individuals exposed to lympholytic alemtuzumab concentrations (>0.1 mg/L) for prolonged durations as compared to their heavier peers. This between-subject variability could be reduced through lean bodyweight-adjusted dosing, showing a twofold to threefold reduction in the slope of the median alemtuzumab exposure over the bodyweight range. Conclusion: Alemtuzumab displays substantial pharmacokinetic variability in kidney transplant recipients, which may warrant a personalized treatment strategy. Lean bodyweight-adjusted dosing poses an option for individualized dosing, but further evaluation of its potential clinical benefit is warranted

Two Human Monoclonal HLA-Reactive Antibodies Cross-React with Mamu-B*008, a Rhesus Macaque MHC Allotype Associated with Control of Simian Immunodeficiency Virus Replication

MHC class I molecules play an important role in adaptive immune responses against intracellular pathogens. These molecules are highly polymorphic, and many allotypes have been characterized. In a transplantation setting, a mismatch between MHC allotypes may initiate an alloimmune response. Rhesus macaques (Macaca mulatta, Mamu) are valuable as a preclinical model species in transplantation research as well as to evaluate the safety and efficacy of vaccine candidates. In both lines of research, the availability of nonhuman primate MHC-reactive mAbs may enable in vitro monitoring and detection of presence of particular Mamu molecules. In this study, we screened a collection of thoroughly characterized HLA class I–specific human mAbs for cross-reactivity with rhesus macaque MHC class I allotypes. Two mAbs, OK4F9 and OK4F10, recognize an epitope that is defined by isoleucine (I) at amino acid position 142 that is present on the Indian rhesus macaque Mamu-B*008:01 allotype, which is an allotype known to be associated with elite control of SIV replication. The reactive pattern of a third mAb, MUS4H4, is more complex and includes an epitope shared on Mamu-A2*05:01 and -B*001:01–encoded Ags. This is the first description, to our knowledge, of human HLA-reactive mAbs that can recognize Mamu allotypes, and these can be useful tools for in vitro monitoring the presence of the relevant allelic products. Moreover, OK4F9 and OK4F10 can be powerful mAbs for application in SIV-related research. The Journal of Immunology, 2021, 206: 1957–1965

Two Human Monoclonal HLA-Reactive Antibodies Cross-React with Mamu-B*008, a Rhesus Macaque MHC Allotype Associated with Control of Simian Immunodeficiency Virus Replication

MHC class I molecules play an important role in adaptive immune responses against intracellular pathogens. These molecules are highly polymorphic, and many allotypes have been characterized. In a transplantation setting, a mismatch between MHC allotypes may initiate an alloimmune response. Rhesus macaques (Macaca mulatta, Mamu) are valuable as a preclinical model species in transplantation research as well as to evaluate the safety and efficacy of vaccine candidates. In both lines of research, the availability of nonhuman primate MHC-reactive mAbs may enable in vitro monitoring and detection of presence of particular Mamu molecules. In this study, we screened a collection of thoroughly characterized HLA class I–specific human mAbs for cross-reactivity with rhesus macaque MHC class I allotypes. Two mAbs, OK4F9 and OK4F10, recognize an epitope that is defined by isoleucine (I) at amino acid position 142 that is present on the Indian rhesus macaque Mamu-B*008:01 allotype, which is an allotype known to be associated with elite control of SIV replication. The reactive pattern of a third mAb, MUS4H4, is more complex and includes an epitope shared on Mamu-A2*05:01 and -B*001:01–encoded Ags. This is the first description, to our knowledge, of human HLA-reactive mAbs that can recognize Mamu allotypes, and these can be useful tools for in vitro monitoring the presence of the relevant allelic products. Moreover, OK4F9 and OK4F10 can be powerful mAbs for application in SIV-related research. The Journal of Immunology, 2021, 206: 1957–1965

Two Human Monoclonal HLA-Reactive Antibodies Cross-React with Mamu-B*008, a Rhesus Macaque MHC Allotype Associated with Control of Simian Immunodeficiency Virus Replication

Transplantation and autoimmunit

HLA antibody affinity determination: From HLA-specific monoclonal antibodies to donor HLA specific antibodies (DSA) in patient serum

Organs transplanted across donor-specific HLA antibodies (DSA) are associated with a variety of clinical outcomes, including a high risk of acute kidney graft rejection. Unfortunately, the currently available assays to determine DSA characteristics are insufficient to clearly discriminate between potentially harmless and harmful DSA. To further explore the hazard potential of DSA, their concentration and binding strength to their natural target, using soluble HLA, may be informative. There are currently a number of biophysical technologies available that allow the assessment of antibody binding strength. However, these methods require prior knowledge of antibody concentrations. Our objective within this study was to develop a novel approach that combines the determination of DSA-affinity as well as DSA-concentration for patient sample evaluation within one assay. We initially tested the reproducibility of previously reported affinities of human HLA-specific monoclonal antibodies and assessed the technology-specific precision of the obtained results on multiple platforms, including surface plasmon resonance (SPR), bio-layer interferometry (BLI), Luminex (single antigen beads; SAB), and flow-induced dispersion analysis (FIDA). While the first three (solid-phase) technologies revealed comparable high binding-strengths, suggesting measurement of avidity, the latter (in-solution) approach revealed slightly lower binding-strengths, presumably indicating measurement of affinity. We believe that our newly developed in-solution FIDA-assay is particularly suitable to provide useful clinical information by not just measuring DSA-affinities in patient serum samples but simultaneously delivering a particular DSA-concentration. Here, we investigated DSA from 20 pre-transplant patients, all of whom showed negative CDC-crossmatch results with donor cells and SAB signals ranging between 571 and 14899 mean fluorescence intensity (MFI). DSA-concentrations were found in the range between 11.2 and 1223 nM (median 81.1 nM), and their measured affinities fall between 0.055 and 24.7 nM (median 5.34 nM; 449-fold difference). In 13 of 20 sera (65%), DSA accounted for more than 0.1% of total serum antibodies, and 4/20 sera (20%) revealed a proportion of DSA even higher than 1%. To conclude, this study strengthens the presumption that pre-transplant patient DSA consists of various concentrations and different net affinities. Validation of these results in a larger patient cohort with clinical outcomes will be essential in a further step to assess the clinical relevance of DSA-concentration and DSA-affinity.ISSN:2059-231