Memory B-cell derived donor-specific antibodies do not predict outcome in sensitized kidney transplant recipients: a retrospective single-center study.

Background: Repeated exposure to sensitizing events can activate HLA-specific memory B cells, leading to the production of donor-specific memory B cell antibodies (DSAm) that pose a risk for antibody-mediated rejection (ABMR) in kidney transplant recipients (KTRs). This single-center retrospective study aimed to identify DSAm and assess their association with outcomes in a cohort of KTRs with pretransplant serum donor-specific antibodies (DSA). Methods: We polyclonally activated pretransplant peripheral blood mononuclear cells (PBMCs) from 60 KTRs in vitro, isolated and quantified IgG from the culture supernatant using ELISA, and analyzed the HLA antibodies of eluates with single antigen bead (SAB) assays, comparing them to the donor HLA typing for potential DSAm. Biopsies from 41 KTRs were evaluated for rejection based on BANFF 2019 criteria. Results: At transplantation, a total of 37 DSAm were detected in 26 of 60 patients (43%), of which 13 (35%) were found to be undetectable in serum. No significant association was found between pretransplant DSAm and ABMR (P=0.53). Similar results were observed in a Kaplan-Meier analysis for ABMR within the first year posttransplant (P=0.29). Additionally, MFI levels of DSAm showed no significant association with ABMR (P=0.28). Conclusion: This study suggests no significant association between DSAm and biopsy-proven clinical ABMR. Further prospective research is needed to determine whether assessing DSAm could enhance existing immunological risk assessment methods for monitoring KTRs, particularly in non-sensitized KTRs.

The role of RBC antigen transgene integration sites on RBC biology in mice.

BACKGROUND: Transgenic mice expressing RBC specific antigens are widely used in mechanistic studies of RBC alloimmunization. Existing RBC donor strains have random transgene integration, potentially disrupting host elements that can confound biological interpretation. STUDY DESIGN AND METHODS: Integration site and genomic alterations were characterized by both targeted locus amplification and congenic backcrossing in the five most commonly used RBC alloantigen donor strains (KEL-K2hi , KEL-K2med , and KEL-K2lo , and KEL-K1). A targeted transgenic approach was developed to allow RBC specific transgene expression from a safe harbor locus (ROSA26). Alloimmune responses were assessed by transfusing alloantigen expressing RBCs into wild-type recipients and measuring alloantibodies by flow cytometry. RESULTS/FINDINGS: Four of the five analyzed strains had at least one gene disrupted by the transgene integration but none of the disrupted genes are known to be involved in RBC biology. The integration of KEL-K2med potentially altered the immunological properties of RBCs, although the biological significance of the observed changes is unclear. The ROSA26 targeted approach resulted in a single copy of the transgene that maintains RBC specific expression without random disruption of genomic elements. CONCLUSION: These findings provide a detailed characterization of genomic disruption by transgene integration found in commonly used RBC donor strains that is relevant to numerous previous publications as well as future studies. With the possible exception of KEL-K2med , transgene integration is not predicted to affect RBC biology in existing models, and new models can avoid this concern using the described targeted transgenic approach.

Non-Invasive Monitoring for Rejection in Kidney Transplant Recipients After SARS-CoV-2 mRNA Vaccination.

Introduction: Studies have shown reduced antiviral responses in kidney transplant recipients (KTRs) following SARS-CoV-2 mRNA vaccination, but data on post-vaccination alloimmune responses and antiviral responses against the Delta (B.1.617.2) variant are limited. Materials and methods: To address this issue, we conducted a prospective, multi-center study of 58 adult KTRs receiving mRNA-BNT162b2 or mRNA-1273 vaccines. We used multiple complementary non-invasive biomarkers for rejection monitoring including serum creatinine, proteinuria, donor-derived cell-free DNA, peripheral blood gene expression profile (PBGEP), urinary CXCL9 mRNA and de novo donor-specific antibodies (DSA). Secondary outcomes included development of anti-viral immune responses against the wild-type and Delta variant of SARS-CoV-2. Results: At a median of 85 days, no KTRs developed de novo DSAs and only one patient developed acute rejection following recent conversion to belatacept, which was associated with increased creatinine and urinary CXCL9 levels. During follow-up, there were no significant changes in proteinuria, donor-derived cell-free DNA levels or PBGEP. 36% of KTRs in our cohort developed anti-wild-type spike antibodies, 75% and 55% of whom had neutralizing responses against wild-type and Delta variants respectively. A cellular response against wild-type S1, measured by interferon-γ-ELISpot assay, developed in 38% of KTRs. Cellular responses did not differ in KTRs with or without antibody responses. Conclusions: SARS-CoV-2 mRNA vaccination in KTRs did not elicit a significant alloimmune response. About half of KTRs who develop anti-wild-type spike antibodies after two mRNA vaccine doses have neutralizing responses against the Delta variant. There was no association between anti-viral humoral and cellular responses.

Prevention of Fetal/Neonatal Alloimmune Thrombocytopenia in Mice: Biochemical and Cell Biological Characterization of Isoforms of a Human Monoclonal Antibody.

Maternal alloantibodies toward paternally inherited Ags on fetal platelets can cause thrombocytopenia and bleeding complications in the fetus or neonate, referred to as fetal and neonatal alloimmune thrombocytopenia (FNAIT). This is most commonly caused by Abs against the human platelet Ag (HPA)-1a in Caucasians, and a prophylactic regimen to reduce the risk for alloimmunization to women at risk would be beneficial. We therefore aimed to examine the prophylactic potential of a fully human anti-HPA-1a IgG1 (mAb 26.4) with modified Fc region or altered N-glycan structures. The mAb 26.4 wild-type (WT) variants all showed efficient platelet clearance capacity and ability to mediate phagocytosis independent of their N-glycan structure, compared with an effector silent variant (26.4.AAAG), although the modified N-glycan variants showed differential binding to FcγRs measured in vitro. In an in vivo model, female mice were transfused with platelets from transgenic mice harboring an engineered integrin ß3 containing the HPA-1a epitope. When these preimmunized mice were bred with transgenic males, Abs against the introduced epitope induced thrombocytopenia in the offspring, mimicking FNAIT. Prophylactic administration of the mAb 26.4.WT, and to some extent the mAb 26.4.AAAG, prior to platelet transfusion resulted in reduced alloimmunization in challenged mice and normal platelet counts in neonates. The notion that the effector silent variant hampered alloimmunization demonstrates that rapid platelet clearance, as seen with mAb 26.4.WT, is not the sole mechanism in action. Our data thus successfully demonstrate efficient Ab-mediated immunosuppression and prevention of FNAIT by anti-HPA-1a monoclonal variants, providing support for potential use in humans.

Monitoring B cell alloresponses in rats.

Antibody-mediated rejection is a major cause of graft failure in organ transplantation. For this reason, B cell responses are of particular interest to transplantation research. Rats are important model organisms for transplant studies, but B cell alloimmune assays and B cell subset markers are poorly established in rats. We alloimmunized rats by donor blood injection using the high responder rat strain combination Brown Norway (donor) and Lewis (recipient) rats. Using splenocytes from alloimmunized and control rats, we established assays to assess allospecific B cell proliferation and the capacity to generate allospecific B memory cells and alloantibody-secreting cells after antigenic rechallenge in vitro using a mixed lymphocyte reaction. Furthermore, we defined a simple gating and sorting strategy for pre- and post-germinal center follicular B cells, as well as non-switched and switched plasmablasts. Our protocols for assessing B cell alloresponses and B cell subsets in rats may help to accelerate research into the role of B cells and manipulation of humoral alloresponses in transplant research.

SARS Cov-2 vaccination induces de novo donor-specific HLA antibodies in a renal transplant patient on waiting list: A case report.

The ability of COVID-19 vaccination to induce anti-HLA antibodies (Abs) formation in renal transplant candidates is not well studied. A 42-year-old man on a renal transplant waitlist, with no sensitization history, was tested for DSA before and after COVID-19 vaccination. Patient has consistently tested negative for COVID-19 virus. Eighteen days after receiving first dose of mRNA-based vaccine, flow cytometry crossmatch (FCXM) was strongly positive with de novo donor-specific Ab (dnDSA) against B57 and de novo non-DSA against B58. Before vaccination, preliminary FCXM was negative with no anti-HLA Abs. This event prompted the transplant team to cancel the surgery. COVID-19 vaccination could be associated with anti-HLA Abs formation in renal patients on waitlists that could affect future transplantability.

The utility of 'home-made' reagent red blood cells for antibody screening during pre-transfusion compatibility testing in Uganda.

BACKGROUND: The WHO recommends that pre-transfusion testing should include ABO/RhD grouping followed by screening for red blood cell (RBC) alloantibodies using the indirect antiglobulin test (IAT). However, in Uganda, current practice does not include RBC alloantibody screening. OBJECTIVE: To assess the utility of 'home-made' reagent RBCs in alloantibody screening. MATERIALS AND METHODS: In a laboratory-based study, group O RhD positive volunteer donors were recruited and their extended phenotype performed for C, c, E, e, K, Fya, Fyb Jkb, S and s antigens. These 'home-made' reagent RBCs were preserved using Alsever's solution and alloantibody detection tests performed. For quality assurance, repeat alloantibody screening of patients' samples was done at Bloodworks Northwest Laboratory in Seattle, United States. RESULTS: A total of 36 group O RhD positive individuals were recruited as reagent RBC donors (median age, 25 years; range, 21 - 58 years; male-to-female ratio, 1.6:1). Out of the 311 IATs performed, 32 (10.3%) were positive. Confirmatory IAT testing in the United States was in agreement with the findings in Uganda. CONCLUSION: Use of 'home-made' reagent RBCs during pre-transfusion testing in Uganda is feasible. We recommend the introduction of pre-transfusion IAT alloantibody screening in Uganda using 'home-made' reagent RBCs to improve transfusion safety.

Blockade of IL-6/IL-6R Signaling Attenuates Acute Antibody-Mediated Rejection in a Mouse Cardiac Transplantation Model.

Acute antibody-mediated rejection (AAMR) is an important cause of cardiac allograft dysfunction, and more effective strategies need to be explored to improve allograft prognosis. Interleukin (IL)-6/IL-6R signaling plays a key role in the activation of immune cells including B cells, T cells and macrophages, which participate in the progression of AAMR. In this study, we investigated the effect of IL-6/IL-6R signaling blockade on the prevention of AAMR in a mouse model. We established a mouse model of AAMR for cardiac transplantation via presensitization of skin grafts and addition of cyclosporin A, and sequentially analyzed its features. Tocilizumab, anti-IL-6R antibody, and recipient IL-6 knockout were used to block IL-6/IL-6R signaling. We demonstrated that blockade of IL-6/IL-6R signaling significantly attenuated allograft injury and improved survival. Further mechanistic research revealed that signaling blockade decreased B cells in circulation, spleens, and allografts, thus inhibiting donor-specific antibody production and complement activation. Moreover, macrophage, T cell, and pro-inflammatory cytokine infiltration in allografts was also reduced. Collectively, we provided a highly practical mouse model of AAMR and demonstrated that blockade of IL-6/IL-6R signaling markedly alleviated AAMR, which is expected to provide a superior option for the treatment of AAMR in clinic.

Prognostic Value Evaluation of HLA-DRB1*07:01, *10, *12, *13:01 Alleles for Alloimmunization in Transfusion-Dependent Thalassemia.

BACKGROUND: Transfusion is a lifesaving treatment for lots of patients. However, in chronic blood recipients such as thalassemia patients, there are high concerns about alloantibody production that affects the quality of their life. Therefore, research on risk factors of alloimmunization has been started and followed. This study aimed at the determination of correlation probability between some HLADRB1 alleles and alloimmunization in Iranian thalassemia patients. MATERIALS AND METHODS: The present study was conducted on 60 alloimmunized and 60 non-alloimmunized transfusion-dependent thalassemia patients. Antibody screening and identification tests were carried out using the tube method, and HLA-DRB1 genotyping was done using a single specific primer-polymerase chain reaction (PCRSSP). RESULTS: The results of antibody screening showed that the most prevalent alloantibodies were Anti-K (46.7 %), and followed by Anti-E (32.5 %), Anti-C (15.8 %), Anti-D (13.3 %), Anti-e (8 %), and Anti-c (5.8 %), respectively. DRB1*07:01 was detected more in non-responder patients (28.3 %). However, data analysis showed that there is no significant relationship between DRB1*07:01, *10, *13:01 frequency among responder and non-responder groups (p = 0·195, 0.648, 0.402, respectively). There was not any significant correlation between HLA-DRB1*10 and *13:01 allele and alloimmunization. There was a significant association between HLA-DRB1*12 and alloimmunization (p < 0·05, OR = 2.071, CI: 1.716-2.501). CONCLUSION: The findings of this study represented that there is a significant relationship between HLADRB1*12 and Kell and E alloantibodies. Although more developed studies on other HLA alleles are demanded, these findings can be valuable in determining important alloimmunization risk factors to better management of transfusion complications.

Alloimmunization in transfused patients with constitutional anemias in Norway.

BACKGROUND AND OBJECTIVES: The status of red blood cell alloimmunization in patients with constitutional anemias including hemoglobinopathies is not known in Norway. The study objective was to investigate the impact of a strategy based on phenotype-matching for C, c, E, e, K, Jka, Jkb, Fya, Fyb, S and s on alloimmunization. MATERIALS AND METHODS: We reviewed transfusions of 40 patients retrospectively using the computerized blood bank management system and medical records; including diagnosis, age at start of transfusion therapy, gender, number and age of packed red blood cell units transfused, follow-up time, phenotypes of the donors and patients, antigen-negative patients exposed to antigen-positive packed red blood cell units, transfusion reactions and alloantibody specificities. RESULTS: Forty patients received 5402 packed red blood cell units. Alloimmunization frequency was 20 % for the whole group, being 7%, 25 % and 30 % in patients with sickle cell disease (n = 14), thalassemia (n = 16) and other conditions (n = 10), respectively. The alloantibodies detected were anti-E, -c, -C, -Cw, -K, -Jka and -Lua. CONCLUSION: Good communication between the clinicians and the transfusion services is essential for successful management of patients with constitutional anemias. Providing full phenotype-matched units was not always possible. Extended pheno-/genotyping before the first transfusion and providing antigen-negative units for antigen-negative patients for at least C, c, E and K in every red cell transfusion would probably have reduced the alloimmunization rate. Non-phenotype-matched transfusions seem to be the main reason for alloimmunization. Finding markers for identifying responders prone to alloimmunization will ensure targeted transfusion strategies.

New Perspectives on Desensitization in the Current Era - An Overview.

Blood group and tissue incompatibilities remain significant barriers to achieving transplantation. Although no patient should be labeled "un-transplantable" due to blood group or tissue incompatibility, all candidates should be provided with individualized and realistic counseling regarding their anticipated wait times for deceased donor or kidney paired donation matching, with early referral to expert centers for desensitization when needed. Vital is the careful selection of patients whose health status is such that desensitizing treatment is less likely to cause serious harm and whose anti-HLA antibody status is such that treatment is likely to accomplish the goal of increasing organ offers with an acceptable final crossmatch. Exciting new developments have re-energized the interest and scope of desensitization in the times ahead.

Hidden Patterns of Anti-HLA Class I Alloreactivity Revealed Through Machine Learning.

Detection of alloreactive anti-HLA antibodies is a frequent and mandatory test before and after organ transplantation to determine the antigenic targets of the antibodies. Nowadays, this test involves the measurement of fluorescent signals generated through antibody-antigen reactions on multi-beads flow cytometers. In this study, in a cohort of 1,066 patients from one country, anti-HLA class I responses were analyzed on a panel of 98 different antigens. Knowing that the immune system responds typically to "shared" antigenic targets, we studied the clustering patterns of antibody responses against HLA class I antigens without any a priori hypothesis, applying two unsupervised machine learning approaches. At first, the principal component analysis (PCA) projections of intra-locus specific responses showed that anti-HLA-A and anti-HLA-C were the most distantly projected responses in the population with the anti-HLA-B responses to be projected between them. When PCA was applied on the responses against antigens belonging to a single locus, some already known groupings were confirmed while several new cross-reactive patterns of alloreactivity were detected. Anti-HLA-A responses projected through PCA suggested that three cross-reactive groups accounted for about 70% of the variance observed in the population, while anti-HLA-B responses were mainly characterized by a distinction between previously described Bw4 and Bw6 cross-reactive groups followed by several yet undocumented or poorly described ones. Furthermore, anti-HLA-C responses could be explained by two major cross-reactive groups completely overlapping with previously described C1 and C2 allelic groups. A second feature-based analysis of all antigenic specificities, projected as a dendrogram, generated a robust measure of allelic antigenic distances depicting bead-array defined cross reactive groups. Finally, amino acid combinations explaining major population specific cross-reactive groups were described. The interpretation of the results was based on the current knowledge of the antigenic targets of the antibodies as they have been characterized either experimentally or computationally and appear at the HLA epitope registry.

Nox2-deficient Tregs improve heart transplant outcomes via their increased graft recruitment and enhanced potency.

Nox2 is a ROS-generating enzyme, deficiency of which increases suppression by Tregs in vitro and in an in vivo model of cardiac remodeling. As Tregs have emerged as a candidate therapy in autoimmunity and transplantation, we hypothesized that Nox2 deficiency in Tregs in recipient mice may improve outcomes in a heart transplant model. We generated a potentially novel B6129 mouse model with Treg-targeted Nox2 deletion (Nox2fl/flFoxP3Cre+ mice) and transplanted with hearts from CB6F1 donors. As compared with those of littermate controls, Nox2fl/flFoxP3Cre+ mice had lower plasma levels of alloantibodies and troponin-I, reduced levels of IFN-γ in heart allograft homogenates, and diminished cardiomyocyte necrosis and allograft fibrosis. Single-cell analyses of allografts revealed higher absolute numbers of Tregs and lower CD8+ T cell infiltration in Nox2-deficient recipients compared with Nox2-replete mice. Mechanistically, in addition to a greater suppression of CD8+CD25- T effector cell proliferation and IFN-γ production, Nox2-deficient Tregs expressed higher levels of CCR4 and CCR8, driving cell migration to allografts; this was associated with increased expression of miR-214-3p. These data indicate that Nox2 deletion in Tregs enhances their suppressive ability and migration to heart allografts. Therefore, Nox2 inhibition in Tregs may be a useful approach to improve their therapeutic efficacy.

Anti-D alloimmunization in Rh(D) negative adults with severe traumatic injury.

INTRODUCTION: Widely varying rates of alloimmunization associated with transfusing uncrossmatched RBC products to trauma patients as part of hemostatic resuscitation have been reported. We characterized the rates of RBC alloimmunization in our severely injured Rh(D) negative trauma population who received uncrossmatched Rh(D) positive RBC products. METHODS: In a 10-year retrospective analysis to assess Rh(D) alloimmunization risks, Rh(D) negative adult trauma patients initially requiring uncrossmatched group O Rh(D) positive RBC products with either RBC units or low titer group O whole blood as part of massive transfusion protocol (MTP) activation were identified. Only those Rh(D) negative patients whose initial antibody screenings were negative were included. Duration of serologic follow-up from date of MTP activation to either date of anti-D detection or most recent negative antibody screening was calculated. RESULTS: There were 129 eligible Rh(D) negative trauma patients identified. Median injury severity score was 25. Anti-D was detected in 10 (7.8%) patients after a median of 161.5 days; the median duration of serologic follow-up in those who did not have anti-D detected was 220 days. Patients who had anti-D detected were less severely injured and received fewer Rh(D) positive RBC products versus those who did not. DISCUSSION: In our severely injured adult trauma patients with MTP activation requiring uncrossmatched group O Rh(D) positive RBC products, the rate of anti-D detection was low. Additional studies are necessary to determine generalizability of these findings and fully characterize alloimmunization risks in trauma patients with varying extents of injury.

Rate of RhD-alloimmunization after the transfusion of multiple RhD-positive primary red blood cell-containing products.

INTRODUCTION: Early transfusion reduces mortality in bleeding patients. In this setting, RhD-positive blood products might be transfused. This study determined the association between the RhD-alloimmunization rate and the number of RhD-positive products transfused. METHODS: RhD-negative patients between 13 and 50 years who were transfused with ≥1 RhD-positive red blood cell (RBC) or whole blood units between January 1, 2000 and December 31, 2019 in a healthcare network were identified. Study patients had to have had at least one antibody detection test performed ≥14 days after the index RhD-positive transfusion and not receive RhIg. Patients were stratified into groups that received 1, 2, 3-5, 6-10, 11-20, and >20 RhD-positive transfusions and the RhD-alloimmunization rate was determined for each group. RESULTS: There were 335 patients included; 52/335 (15.5%) were females. Overall, there were 117/335 (34.9%, CI: 29.8%-40.3%) recipients who became RhD-alloimmunized. There was no significant dosage effect in the RhD-alloimmunization rates as the exposure to RhD-positive units increased from one RhD-positive unit to more than 20 RhD-positive units (p = .270 for non-parametric trend test). In an exploratory analysis, patients who received 100% of their RhD-positive transfusions within 72 h of the index transfusion had a significantly higher rate of RhD-alloimmunization compared to those who were transfused over a longer period of time (42.3% vs. 21.4%, respectively; p = .001). CONCLUSION: These results suggest that there may not be an increased RhD-alloimmunization risk with transfusing multiple RhD-positive units after one RhD-positive unit has been transfused. These findings need confirmation in larger studies.

Highly Sensitized Patients Are Well Served by Receiving a Compatible Organ Offer Based on Acceptable Mismatches.

Highly sensitized kidney patients accrue on the transplant waiting list due to their broad immunization against non-self Human Leucocyte Antigens (HLA). Although challenging, the best option for highly sensitized patients is transplantation with a crossmatch negative donor without any additional therapeutic intervention. The Eurotransplant Acceptable Mismatch (AM) program was initiated more than 30 years ago with the intention to increase the chance for highly sensitized patients to be transplanted with such a compatible donor. The AM program allows for enhanced transplantation to this difficult to transplant patient group by allocating deceased donor kidneys on the basis of a match with the recipient's own HLA antigens in combination with predefined acceptable antigens. Acceptable antigens are those HLA antigens towards which the patients has never formed antibodies, as determined by extensive laboratory testing. By using this extended HLA phenotype for allocation and giving priority whenever a compatible donor organ becomes available, organ offers are made for roughly 80% of patients in this program. Up till now, more than 1700 highly sensitized patients have been transplanted through the AM program. Recent studies have shown that the concept of acceptable mismatches being truly immunologically acceptable holds true for both rejection rates and long-term graft survival. Patients that were transplanted through the AM program had a similar rejection incidence and long-term graft survival rates identical to non-sensitized patients transplanted through regular allocation. However, a subset of patients included in the AM program does not receive an organ offer within a reasonable time frame. As these are often patients with a rare HLA phenotype in comparison to the Eurotransplant donor population, extension of the donor pool for these specific patients through further European collaboration would significantly increase their chances of being transplanted. For those patients that will not benefit from such strategy, desensitization is the ultimate solution.

Non-transfusion dependent thalassemia is independently associated with higher alloimmunization risk than transfusion dependent thalassemia and would benefit the most from extended red cell antigen-matching.

BACKGROUND: Alloimmunization prevalence is conventionally used to identify RBCs alloimmunization risk factors among thalassemia patients, but it may be confounded by differences in transfusion exposure especially between non-transfusion dependent thalassemia (NTDT) and transfusion dependent thalassemia (TDT) patients. To better identify thalassemia patients with high alloimmunization risks, we used cumulative incidence of first alloimmunization as a function of RBCs transfused to compare alloimmunization risks between TDT and NTDT and to evaluate other risk factors. We also proposed practical strategies to prevent alloimmunization in thalassemia. STUDY DESIGN AND METHODS: Adult TDT and NTDT patients who had received ≥2 transfusions and no alloimmunization before their first transfusion were included. Alloimmunization was defined as the development of clinically significant alloantibodies. We estimated the first alloimmunization incidence from transfusion by Kaplan-Meier analysis with the horizontal axis expressed as cumulative non-antigen-matched RBC units transfused. We compared this incidence between TDT and NTDT, and analyzed for other alloimmunization risk factors and the alloantibody specificities/frequencies. RESULTS: The alloimmunization prevalence was similar between TDT and NTDT (27% vs. 30% respectively, p = .726). However, for the same transfusion exposure, NTDT had higher alloimmunization incidence than TDT (hazard ratio 8.59, 95% confidence interval [2.25-32.74], p = .002), independent of age at first transfusion and last follow-up, gender, and splenectomy. Anti-E, anti-c, anti-Mia , and anti-Jka were most frequent. DISCUSSION: NTDT has the highest alloimmunization risk and would benefit the most from extended RBC antigen-matching, especially C, c, E, and e. Other blood group antigen-matching should be guided by the patient/donor disparities and alloantibody frequencies in different populations.

Allo-Specific Humoral Responses: New Methods for Screening Donor-Specific Antibody and Characterization of HLA-Specific Memory B Cells.

Antibody-mediated allograft rejection (AMR) causes more kidney transplant failure than any other single cause. AMR is mediated by antibodies recognizing antigens expressed by the graft, and antibodies generated against major histocompatibility complex (MHC) mismatches are especially problematic. Most research directed towards the management of clinical AMR has focused on identifying and characterizing circulating donor-specific HLA antibody (DSA) and optimizing therapies that reduce B-cell activation and/or block antibody secretion by inhibiting plasmacyte survival. Here we describe a novel set of reagents and techniques to allow more specific measurements of MHC sensitization across different animal transplant models. Additionally, we have used these approaches to isolate and clone individual HLA-specific B cells from patients sensitized by pregnancy or transplantation. We have identified and characterized the phenotypes of individual HLA-specific B cells, determined the V(D)J rearrangements of their paired H and L chains, and generated recombinant antibodies to determine affinity and specificity. Knowledge of the BCR genes of individual HLA-specific B cells will allow identification of clonally related B cells by high-throughput sequence analysis of peripheral blood mononuclear cells and permit us to re-construct the origins of HLA-specific B cells and follow their somatic evolution by mutation and selection.

Allo-anti-M: Detection peaks around 2 years of age, but may be attenuated by red blood cell transfusion.

BACKGROUND: Anti-M is frequently observed as a naturally occurring antibody of little clinical significance. Naturally occurring anti-M is often found in children although the specific triggers of production, persistence, and evanescence of anti-M have yet to be elucidated. METHODS: In a retrospective, multicenter, nationwide cohort survey conducted from 2001 to 2015, alloantibody screening was performed before and after transfusion in 18,944 recipients younger than 20 years. Recipients were categorized into six cohorts based on their age at transfusion; within and among these cohorts, allo-anti-M was analyzed in regard to its production, persistence, and evanescence. RESULTS: In 44 patients, anti-M detected before and/or after transfusion was an age-related phenomenon, with a median age of 2 years and an interquartile range of 1-3 years; anti-M was most frequently detected in a cohort of children 1 to <5 years (0.77%, 31 of 4035). At least five patients were presumed to have concurrent infections. Among 1575 adolescents/young adults (15 to <20 years), no anti-M was detected. Of 29 patients with anti-M prior to transfusion, the antibody fell to undetectable levels in 17 recipients (89.5%, of whom at least 13 received only M-negative red cells) after anywhere from 5 days to 5.8 years; anti-M persisted in 2, and was not tested in 10. Only 15 recipients (0.08%) produced new anti-M after transfusion. CONCLUSION: Naturally occurring anti-M is a phenomenon of younger ages, predominantly between 1 and 3 years. After transfusion, it often falls to undetectable levels.