Donor-specific antibody responses against human leukocyte antigen (HLA) proteins mismatched between transplant donors and recipients cause allograft loss, yet the structural HLA epitopes targeted by alloreactive B cells and antibodies remain largely unresolved. We profiled the HLA-A∗01:01-specific B cell response in the transplanted kidney and blood of a recipient undergoing antibody-mediated rejection and identified immunodominant B cell and antibody responses that emerged early in the alloimmune response. These responses were focused on topographically exposed mismatched HLA residues located in the α helices along the peptide-binding groove of HLA-A∗01:01. We demonstrated that the anti-HLA-A∗01:01 B cell alloresponse converged and was maintained on this same immunodominant HLA subregion, which comprises only 20% of the HLA molecule, in a diverse group of HLA-A∗01:01-mismatched transplant recipients. Thus, the B cell and antibody alloresponses appear tightly focused on a topographically defined region on the HLA-A∗01:01 crown that is conserved across individuals expressing distinct constellations of self-HLA-A.

Pig-to-human xenotransplantation is rapidly approaching the clinical arena; however, it is unclear which immunomodulatory regimens will effectively control human immune responses to pig xenografts. We transplanted a gene-edited pig kidney into a brain-dead human recipient on pharmacologic immunosuppression and studied the human immune response to the xenograft using spatial transcriptomics and single-cell RNA sequencing. Human immune cells were uncommon in the porcine kidney cortex early after xenotransplantation and consisted of primarily myeloid cells. Both the porcine resident macrophages and human infiltrating macrophages expressed genes consistent with an alternatively activated, anti-inflammatory phenotype. No significant infiltration of human B or T cells into the porcine kidney xenograft was detected. Altogether, these findings provide proof of concept that conventional pharmacologic immunosuppression is sufficient to restrict infiltration of human immune cells into the xenograft early after compatible pig-to-human kidney xenotransplantation.

About half of patients with Crohn's disease (CD) develop selective serum IgG response to flagellin proteins of the Lachnospiraceae family. Here, we identified a dominant B cell peptide epitope in CD, locating in the highly conserved "hinge region" between the D0 and D1 domains at the amino-terminus of Lachnospiraceae flagellins. Serum IgG reactive to this epitope is present at an elevated level in adult CD patients and in pediatric CD patients at diagnosis. Most importantly, high levels of serum IgG to the hinge epitope were found in most infants from 3 different geographic regions (Uganda, Sweden, and the USA) at one year of age. This vigorous homeostatic response decrements with age as it is not present in healthy adults. These data identify a distinct subset of CD patients, united by a shared reactivity to this dominant flagellin epitope that may represent failure of a homeostatic response beginning in infancy.

Donor-specific antibody (DSA) responses against human leukocyte antigen (HLA) proteins mismatched between kidney transplant donors and recipients cause allograft loss. The rules governing the immunogenicity of non-self donor HLA are poorly understood. Using single-cell, molecular, structural, and proteomic techniques, we profiled the HLA-specific B cell response in the kidney and blood of a transplant recipient with antibody-mediated rejection (AMR). We observed an immunodominant B cell antibody response focused on topographically exposed, solvent-accessible mismatched HLA residues along the peptide-binding groove - a subregion comprising only 20% of the HLA molecule. We further demonstrated that, even within a diverse cohort of transplant recipients, the B cell alloresponse consistently converges on this same immunodominant subregion on the crown of the HLA molecule. Based on these findings, we propose that B cell immunodominance in transplant rejection relies on antigenic topography, and we suggest that this link could be exploited for organ matching and therapeutics.

Chronic lung allograft dysfunction (CLAD) is the most common cause of long-term lung allograft failure. Several factors, including respiratory virus infection (RVI), have been associated with CLAD development, but the underlying mechanisms of these associations are not well understood. We hypothesize that RVI in lung transplant recipients elicits the development of donor-specific antibodies (DSAs), thus providing a mechanistic link between RVI and CLAD development. To test this hypothesis, we retrospectively evaluated for the presence of HLA antibodies in a cohort of lung transplant recipients with symptomatic RVI within the first four months post-transplant using sera at two time points (at/directly after the transplant and following RVI) and time-matched controls without RVI (post-transplant). We found a trend toward the development of de novo DSAs in those with symptomatic RVI versus controls [6/21 (29%) vs. 1/21 (5%), respectively, p = 0.09]. No cases or controls had DSA at baseline. We also found increased rates of CLAD and death among those who developed class II DSA versus those who did not (CLAD: 5/7 (71.4%) vs. 19/34 (54.3%), death: 5/7 (71.4%) vs. 17/35 (48.6%)). Prospective studies evaluating the temporal development of DSA after RVI in lung transplant patients and the subsequent outcomes are warranted.

B cells can be divided into distinct subsets based on origin, activation history and functional attributes. Most human studies, which have characterized bulk polyclonal populations of circulating B cells, suggest heterogeneity between the different B cell subsets. Indeed, we showed that memory B (Bmem) and plasma cells from the blood of the same donor exhibit distinct patterns of isotype distribution, mutational burden and have relatively limited clonal sharing. However, this heterogeneity may not extend to comparisons between the same B cell subset found within different tissues or cells that share the same reactivity. To test this, we examined influenza hemagglutinin (HA)-specific Bmem in multiple donor-matched tissues. We first generated a high-throughput bead array displaying HAs from the seasonal (H1, H3) and pandemic (H2, H5, H7, H9) flu strains and screened the serum from >80 brain-dead human tissue donors. All the donors exhibited high serum IgG reactivity against the circulating strains. We also identified serum reactivity in some donors for HAs that are not circulating in humans, suggesting that these donors likely have HA-specific IgG that are potentially broadly reactive and may have cross-reactive B cells. To directly identify B cells with cross-reactive potential, we enumerated H1 (CA09) and H2 (NETH1999) specific Bmem by flow cytometry. We show that Bmem specific for H1 or H2 are found in the spleen and blood whereas Bmem specific for both H1 and H2 are present in the mediastinal lymph node (LN), spleen and mesenteric LN. The data suggest that the repertoire of HA specific Bmem likely differ between tissues. Going forward, we will characterize the repertoire and molecular signatures of these HA-specific Bmem populations. Supported by grants from NIH (U19 AI142737,)

High-throughput customizable CBA automated script-based analysis The utility of high-throughput systems to evaluate antigen-specific antibody (Ab) has been highlighted by the SARS-CoV-2 pandemic. Pathogen-specific Ab levels are often used to assess protection following vaccination and, in the case of novel pathogens, an indication of prior exposure. Several platforms exist to visualize antigen-specific Ab, however most are not quantitative and are difficult to scale for population level studies. Additionally, the sensitivity across platforms differs making direct comparisons between studies difficult. Cytometric bead arrays (CBA) are an attractive platform for antigen-specific Ab measurements as they can be used to assess Ab reactivity against several antigens and of several isotypes to be performed simultaneously. Additionally, CBAs exhibit high sensitivity and can be designed to provide quantitative measurements. Using commercially available particles, a biotin-Streptavidin bead loading strategy, and the inclusion of indirect standards, we describe a flexible system that can be modified to include a variety of antigens. Here we describe two arrays, focused on antigens derived from either β-coronaviruses or influenza virus. To support the high-throughput capacity of this system, we developed a suit of automated software tools, the CBA Toolbox, to process raw data into antigen-reactive IgM, IgA, and IgG concentrations. We describe quality control requirements, assay performance, and normalizations to accurately quantitate antigen-specific Ig.

Antibody (Ab) responses against human leukocyte antigen (HLA) proteins mismatched between donor and recipient are leading cause of allograft loss in kidney transplantation. However, therapies targeting alloreactive B cell and Ab-secreting cell (ASC) are lacking, motivating the need to understand how to prevent and abrogate these alloresponses. Using molecular, structural, and proteomic techniques, we profiled the B cell response in a kidney transplant recipient with antibody-mediated rejection and graft loss. We found that this response spanned the rejected organ and peripheral blood, stimulated the differentiation of multiple B cell subsets, and produced a high-affinity, donor-specific, anti-HLA response. We found epitopic immunodominance that relied on highly exposed, solvent-accessible mismatched HLA residues as well as structural and biomolecular evidence of autoreactivity against the recipient’s self-HLA allele. These alloreactive and autoreactive signatures converged in the recipient’s circulating donor-specific Ab repertoire, suggesting that rejection requires both the recognition of non-self and breaches of tolerance to lead to alloinjury and graft loss.

Although rare, infection and vaccination can result in antibodies to human leukocyte antigens (HLA). We analyzed the effect of SARS-CoV-2 infection or vaccination on HLA antibodies in waitlisted renal transplant candidates. Specificities were collected and adjudicated if the calculated panel reactive antibodies (cPRA) changed after exposure. Of 409 patients, 285 (69.7 %) had an initial cPRA of 0 %, and 56 (13.7 %) had an initial cPRA > 80 %. The cPRA changed in 26 patients (6.4 %), 16 (3.9 %) increased, and 10 (2.4 %) decreased. Based on cPRA adjudication, cPRA differences generally resulted from a small number of specificities with subtle fluctuations around the borderline of the participating centers' cutoff for unacceptable antigen listing. All five COVID recovered patients with an increased cPRA were female (p = 0.02). In summary, exposure to this virus or vaccine does not increase HLA antibody specificities and their MFI in approximately 99 % of cases and 97 % of sensitized patients. These results have implications for virtual crossmatching at the time of organ offer after SARS-CoV-2 infection or vaccination, and these events of unclear clinical significance should not influence vaccination programs.

A radical solution is needed for the organ supply crisis, and the domestic pig is a promising organ source. In preparation for a clinical trial of xenotransplantation, we developed an in vivo pre-clinical human model to test safety and feasibility tenets established in animal models. After performance of a novel, prospective compatible crossmatch, we performed bilateral native nephrectomies in a human brain-dead decedent and subsequently transplanted two kidneys from a pig genetically engineered for human xenotransplantation. The decedent was hemodynamically stable through reperfusion, and vascular integrity was maintained despite the exposure of the xenografts to human blood pressure. No hyperacute rejection was observed, and the kidneys remained viable until termination 74 h later. No chimerism or transmission of porcine retroviruses was detected. Longitudinal biopsies revealed thrombotic microangiopathy that did not progress in severity, without evidence of cellular rejection or deposition of antibody or complement proteins. Although the xenografts produced variable amounts of urine, creatinine clearance did not recover. Whether renal recovery was impacted by the milieu of brain death and/or microvascular injury remains unknown. In summary, our study suggests that major barriers to human xenotransplantation have been surmounted and identifies where new knowledge is needed to optimize xenotransplantation outcomes in humans.

Alloimmune responses in kidney transplant (KT) patients previously hospitalized with COVID-19 are understudied. We analyzed a cohort of 112 kidney transplant recipients who were hospitalized following a positive SARS-CoV-2 test result during the first 20 months of the COVID-19 pandemic. We found a cumulative incidence of 17% for the development of new donor-specific antibodies (DSA) or increased levels of pre-existing DSA in hospitalized SARS-CoV-2-infected KT patients. This risk extended 8 months post-infection. These changes in DSA status were associated with late allograft dysfunction. Risk factors for new or increased DSA responses in this KT patient cohort included the presence of circulating DSA pre-COVID-19 diagnosis and time post-transplantation. COVID-19 vaccination prior to infection and remdesivir administration during infection were each associated with decreased likelihood of developing a new or increased DSA response. These data show that new or enhanced DSA responses frequently occur among KT patients requiring admission with COVID-19 and suggest that surveillance, vaccination, and antiviral therapies may be important tools to prevent alloimmunity in these individuals.

The coronavirus disease 2019 (COVID-19) pandemic has highlighted the urgent need for effective prophylactic vaccination to prevent the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Intranasal vaccination is an attractive strategy to prevent COVID-19 as the nasal mucosa represents the first-line barrier to SARS-CoV-2 entry. The current intramuscular vaccines elicit systemic immunity but not necessarily high-level mucosal immunity. Here, we tested a single intranasal dose of our candidate adenovirus type 5-vectored vaccine encoding the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein (AdCOVID) in inbred, outbred, and transgenic mice. A single intranasal vaccination with AdCOVID elicited a strong and focused immune response against RBD through the induction of mucosal IgA in the respiratory tract, serum neutralizing antibodies, and CD4+ and CD8+ T cells with a Th1-like cytokine expression profile. A single AdCOVID dose resulted in immunity that was sustained for over six months. Moreover, a single intranasal dose completely protected K18-hACE2 mice from lethal SARS-CoV-2 challenge, preventing weight loss and mortality. These data show that AdCOVID promotes concomitant systemic and mucosal immunity and represents a promising vaccine candidate.

Memory B cells play an important role in immunity to pathogens as these cells are poised to rapidly differentiate into antibody-secreting cells upon antigen re-encounter. Memory B cells also develop over the course of HLA-sensitization during pregnancy and transplantation. In this review, we discuss the potential contribution of memory B cells to pregnancy sensitization as well as the impact of these cells on transplant candidacy and outcomes. We start by summarizing how B cell subsets are altered in pregnancy and discuss what is known about HLA-specific B cell responses given our current understanding of fetal antigen availability in maternal secondary lymphoid tissues. We then review the molecular mechanisms governing the generation and maintenance of memory B cells during infection – including the role of T follicular helper cells - and discuss the experimental evidence for the development of these cells during pregnancy. Finally, we discuss how memory B cells impact access to transplantation and transplant outcomes for a range of transplant recipients.

Background: Aeromedical retrieval is an essential component of contemporary emergency care systems. However, in many locations, ground emergency medical services are dispatched to the scene of an incident first to assess the patient and then call for a helicopter if needed. The time to definitive care therefore includes the helicopter's flight to the scene, flight to the trauma center, and nonflying time. Mission ground time (MGT) includes the time required to get the helicopter airborne, as well as time spent at the scene, packaging and loading the casualty into the aircraft. Estimates of MGT typically vary from 10 to 30 min. The impact of MGT duration on population coveragedthe number of residents that could be taken to a trauma center within a set timedis not known. The aim of this study was to compare population coverage for different durations of MGT in a single state. Methods: Coverage was calculated using elliptical coverage areas ("isochrones") based on the location of helicopter bases and Level I and Level II trauma centers. The calculations were performed using Microsoft Excel, assuming a cruising speed of 133 knots (246 km/h), and mapped using arcGIS. The access time threshold was set at 60 min, and we evaluated MGTs of 10, 15, 20, 25, and 30 min. Results: MGT has a marked impact on population coverage. The effect is, furthermore, not linear. When considering the state's three Level I trauma centers, decreasing MGT from 30 to 10 min increased population coverage from 61.2% to 84.2%. When also considering Level II centers, decreasing MGT from 30 min to 10 min increased coverage by 20%. Conclusions: Elliptical isochrones, with allowance for MGT, provide realistic estimates of population coverage. MGT significantly impacts the proportion of the population that can be taken to a Level I and/or Level II Trauma Center within a set time. The impact is not linear, reflecting the uneven distribution of the population. Consideration should be given to minimizing MGT to preserve the benefits of aeromedical retrieval. (C) 2020 Elsevier Inc. All rights reserved.