Browsing by Author "Jackson, Annette M"
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Item Open Access A cell-based multiplex immunoassay platform using fluorescent protein-barcoded reporter cell lines.(Communications biology, 2021-11) Song, Shengli; Manook, Miriam; Kwun, Jean; Jackson, Annette M; Knechtle, Stuart J; Kelsoe, GarnettMultiplex immunoassays with acellular antigens are well-established based on solid-phase platforms such as the Luminex® technology. Cell barcoding by amine-reactive fluorescent dyes enables analogous cell-based multiplex assays, but requires multiple labeling reactions and quality checks prior to every assay. Here we describe generation of stable, fluorescent protein-barcoded reporter cell lines suitable for multiplex screening of antibody to membrane proteins. The utility of this cell-based system, with the potential of a 256-plex cell panel, is demonstrated by flow cytometry deconvolution of barcoded cell panels expressing influenza A hemagglutinin trimers, or native human CCR2 or CCR5 multi-span proteins and their epitope-defining mutants. This platform will prove useful for characterizing immunity and discovering antibodies to membrane-associated proteins.Item Open Access Allo-Specific Humoral Responses: New Methods for Screening Donor-Specific Antibody and Characterization of HLA-Specific Memory B Cells.(Frontiers in immunology, 2021-01) Song, Shengli; Manook, Miriam; Kwun, Jean; Jackson, Annette M; Knechtle, Stuart J; Kelsoe, GarnettAntibody-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.Item Embargo Evaluating Humoral Immune Responses Against HLA and Cytomegalovirus in Human Lung Transplantation(2024) Harnois, MelissaIn the past 30 years, the global prevalence of chronic respiratory diseases has increased by 40%. Importantly, lung transplantation remains the only definitive treatment for patients with advanced lung disease. Despite the rapidly growing need for lung transplantation, lung transplant recipients have by far the worst survival rates among all solid organ transplant types, with a current median survival of only 6 years post transplantation. Chronic lung allograft dysfunction (CLAD) is the leading cause of limited long-term survival of lung transplant recipients, affecting approximately 50% of patients within 5 years post-transplantation. Previous studies have identified humoral alloimmune responses against mismatched donor HLA and cytomegalovirus (CMV) infection as primary independent risk factors for chronic deterioration of the transplanted allograft and the fatal diagnosis of CLAD. This dissertation is comprised of work that aims to address critical gaps in knowledge in the fields of transplant immunology and infectious disease. The first component focuses on CMV-specific humoral immunity in naturally infected healthy blood donors (Chapter 2) and in CMV seropositive lung transplant recipients (Chapter 3). The second area of study focuses on humoral alloimmune responses and current methods used to evaluate and characterize donor HLA-specific alloantibodies (Chapter 4), as well as factors contributing to HLA-DQ immunogenicity (Chapter 5). These projects utilized clinical data and longitudinally collected human plasma samples from the completed multicenter Clinical Trials in Organ Transplantation (CTOT)-20 and CTOT-22 consortium studies. The findings from Chapter 2 contribute to our knowledge of naturally acquired immunity against CMV, which informs strategies for antibody-based therapeutics to help treat CMV infection as well as CMV vaccine development. The results from Chapter 3 indicate that CMV-specific antibodies have the potential to be used as novel biomarkers for viremia risk, possibly in combination with other known risk factors for viremia including CMV-specific T cell responses. These tools may help inform antiviral treatment duration in clinical practice. Chapter 4 of this dissertation represents the first in-depth technical comparison across traditional and modified single antigen bead Luminex assays using IgG enriched sera for donor-HLA specific post-transplant monitoring. These findings may be useful for clinical workflows to help improve efficiency and cost by stratifying patient samples that may benefit from additional testing in modified assays. Finally, Chapter 5 of this dissertation examines the specificity and timing of donor HLA-specific antibodies (DSA), as well as other risk factors for DSA development. In this project, we detail novel strategies for calculating HLA-DQ mismatch by accounting for trans-encoded DQ heterodimer formation. The results from this study also reveal elevated risk for developing DSA among patients with high-risk epitope mismatches and suggest that different organs may require different HLA mismatch calculation strategies. Overall, the results from these projects advance our understanding of the human immune response to alloantigens and cytomegalovirus in lung transplantation and push the field forward to help improve patient outcomes.
Item Open Access HLA Loci and Recurrence of Focal Segmental Glomerulosclerosis in Pediatric Kidney Transplantation.(Transplantation direct, 2021-10) Shaw, Brian I; Ochoa, Alejandro; Chan, Cliburn; Nobuhara, Chloe; Gbadegesin, Rasheed; Jackson, Annette M; Chambers, Eileen TRecurrent focal segmental glomerulosclerosis (FSGS) after kidney transplantation accounts for the majority of allograft failures in children with primary FSGS. Although current research focuses on FSGS pathophysiology, a common etiology and mechanisms of disease recurrence remain elusive.Methods
We performed a retrospective review of the Scientific Registry of Transplant Recipients to determine the association of specific HLA recurrence of FSGS. Kidney transplants recipients under the age of 19 who were diagnosed with FSGS, who were transplanted after January 1, 2000, and who had complete HLA data were included in the study. We performed simple logistic regression on all HLA A, B, C, DR, and DQ represented in the dataset and FSGS recurrence and then determined those associated with recurrence using the Benjamini-Hochberg method for multiple comparisons. For those HLAs that were associated with recurrence, we further determined the effect of matching recipient and donor HLA with recurrence.Results
HLA DR7, DR53, DQ2, DR52, and DQ7 were associated with increased or decreased risk of recurrent disease after transplantation. We identified a risk haplotype consisting of HLA-DR7, DR53, and DQ2 that was consistently associated with an increased risk of recurrence (odds ratio 1.91; 95% confidence interval, 1.44-2.54, P < 0.001). We also found that donor/recipient concordance for HLA-DQ7 was associated with a decreased risk of recurrence (odds ratio 0.42; 95% confidence interval, 0.37-0.53, P = 0.009).Conclusions
HLA profiles may be used for risk stratification of recurrence of FSGS in pediatric kidney transplant recipients and deserves further study.