Utilizing principal component analysis in the identification of clinically relevant changes in patient HLA single antigen bead solid phase testing patterns.

BACKGROUND: HLA antibody testing is essential for successful solid-organ allocation, patient monitoring post-transplant, and risk assessment for both solid-organ and hematopoietic transplant patients. Luminex solid-phase testing is the most common method for identifying HLA antibody specificities, making it one of the most complex immunoassays as each panel contains over 90 specificities for both HLA class I and HLA class II with most of the analysis being performed manually in the vendor-provided software. Principal component analysis (PCA), used in machine learning, is a feature extraction method often utilized to assess data with many variables. METHODS & FINDINGS: In our study, solid organ transplant patients who exhibited HLA donor-specific antibodies (DSAs) were used to characterize the utility of PCA-derived analysis when compared to a control group of post-transplant and pre-transplant patients. ROC analysis was utilized to determine a potential threshold for the PCA-derived analysis that would indicate a significant change in a patient's single antigen bead pattern. To evaluate if the algorithm could identify differences in patterns on HLA class I and HLA class II single antigen bead results using the optimized threshold, HLA antibody test results were analyzed using PCA-derived analysis and compared to the clinical results for each patient sample. The PCA-derived algorithm had a sensitivity of 100% (95% CI, 73.54%-100%), a specificity of 75% (95% CI, 56.30%-92.54%), with a PPV of 65% (95% CI, 52.50%-83.90%) and an NPV of 100%, in identifying new reactivity that differed from the patients historic HLA antibody pattern. Additionally, PCA-derived analysis was utilized to assess the potential over-reactivity of single antigen beads for both HLA class I and HLA class II antibody panels. This assessment of antibody results identified several beads in both the HLA class I and HLA class II antibody panel which exhibit over reactivity from 2018 to the present time. CONCLUSIONS: PCA-derived analysis would be ideal to help automatically identify patient samples that have an HLA antibody pattern of reactivity consistent with their history and those which exhibit changes in their antibody patterns which could include donor-specific antibodies, de novo HLA antibodies, and assay interference. A similar method could also be applied to evaluate the over-reactivity of beads in the HLA solid phase assays which would be beneficial for lot comparisons and instructive for transplant centers to better understand which beads are more prone to exhibiting over-reactivity and impact patient care.

Bilineal evolution of a U2AF1-mutated clone associated with acquisition of distinct secondary mutations.

Rare hematologic malignancies display evidence of both myeloid and lymphoid differentiation. Here, we describe such a novel bilineal event discovered in an adult woman with B-lymphoblastic leukemia (BLL). At the time of BLL diagnosis, the patient had a normal karyotype and a bulk sequencing panel identified pathogenic variants in BCOR, EZH2, RUNX1, and U2AF1, a genotype more typical of myeloid neoplasia. Additionally, the patient was noted to have 3-year history of cytopenias, and morphologic dyspoiesis was noted on post-treatment samples, raising the possibility of an antecedent hematologic disorder. To investigate the clonal architecture of her disease, we performed targeted sequencing on fractionated samples enriched for either B-lymphoblasts or circulating granulocytes. These studies revealed a truncal founder mutation in the spliceosome gene U2AF1 in both fractions, while distinct secondary mutations were present only in B-lymphoblasts (BCOR, NRAS) or myeloid cells (ASXL1, EZH2, RUNX1). These results indicate that both processes evolved from a common U2AF1-mutated precursor, which then acquired additional mutations during a process of divergent evolution and bilineal differentiation. Our findings highlight an atypical mechanism of BLL leukemogenesis and demonstrate the potential utility of fractionated sequencing in the characterization of acute leukemia.

Next-generation sequencing and clinical histocompatibility testing.

Histocompatibility testing is essential for donor identification and risk assessment in solid organ and hematopoietic stem cell transplant. Additionally, it is useful for identifying donor specific alleles for monitoring donor specific antibodies in post-transplant patients. Next-generation sequence (NGS) based human leukocyte antigen (HLA) typing has improved many aspects of histocompatibility testing in hematopoietic stem cell and solid organ transplant. HLA disease association testing and research has also benefited from the advent of NGS technologies. In this review we discuss the current impact and future applications of NGS typing on clinical histocompatibility testing for transplant and non-transplant purposes.

Influence of Germline Genetics on Tacrolimus Pharmacokinetics and Pharmacodynamics in Allogeneic Hematopoietic Stem Cell Transplant Patients.

Tacrolimus exhibits high inter-patient pharmacokinetics (PK) variability, as well as a narrow therapeutic index, and therefore requires therapeutic drug monitoring. Germline mutations in cytochrome P450 isoforms 4 and 5 genes (CYP3A4/5) and the ATP-binding cassette B1 gene (ABCB1) may contribute to interindividual tacrolimus PK variability, which may impact clinical outcomes among allogeneic hematopoietic stem cell transplantation (HSCT) patients. In this study, 252 adult patients who received tacrolimus for acute graft versus host disease (aGVHD) prophylaxis after allogeneic HSCT were genotyped to evaluate if germline genetic variants associated with tacrolimus PK and pharmacodynamic (PD) variability. Significant associations were detected between germline variants in CYP3A4/5 and ABCB1 and PK endpoints (e.g., median steady-state tacrolimus concentrations and time to goal tacrolimus concentration). However, significant associations were not observed between CYP3A4/5 or ABCB1 germline variants and PD endpoints (e.g., aGVHD and treatment-emergent nephrotoxicity). Decreased age and CYP3A5*1/*1 genotype were independently associated with subtherapeutic tacrolimus trough concentrations while CYP3A5*1*3 or CYP3A5*3/*3 genotypes, myeloablative allogeneic HSCT conditioning regimen (MAC) and increased weight were independently associated with supratherapeutic tacrolimus trough concentrations. Future lines of prospective research inquiry are warranted to use both germline genetic and clinical data to develop precision dosing tools that will optimize both tacrolimus dosing and clinical outcomes among adult HSCT patients.

HLA alleles and haplotypes observed in 263 US families.

The 17th International HLA and Immunogenetics Workshop (IHIW) conducted a project entitled "The Study of Haplotypes in Families by NGS HLA". We investigated the HLA haplotypes of 1017 subjects in 263 nuclear families sourced from five US clinical immunogenetics laboratories, primarily as part of the evaluation of related donor candidates for hematopoietic stem cell and solid organ transplantation. The parents in these families belonged to five broad groups - African (72 parents), Asian (115), European (210), Hispanic (118) and "Other" (11). High-resolution HLA genotypes were generated for each subject using next-generation sequencing (NGS) HLA typing systems. We identified the HLA haplotypes in each family using HaplObserve, software that builds haplotypes in families by reviewing HLA allele segregation from parents to children. We calculated haplotype frequencies within each broad group, by treating the parents in each family as unrelated individuals. We also calculated standard measures of global linkage disequilibrium (LD) and conditional asymmetric LD for each ethnic group, and used untruncated and two-field allele names to investigate LD patterns. Finally we demonstrated the utility of consensus DNA sequences in identifying novel variants, confirming them using HLA allele segregation at the DNA sequence level.

Characteristics, properties, and potential applications of circulating cell-free dna in clinical diagnostics: a focus on transplantation.

The initial discovery of cell-free DNA (cfDNA) in 1948 by Mandel and Metais has led to numerous investigations evaluating the role of cfDNA in various disease states. cfDNA has been characterized in various patient populations with similar results. cfDNA are typically 150 bp of double-stranded DNA that are thought to be released from nucleosomes during apoptosis and necrosis. They are found in circulation as monomers, dimers, and trimers. Different specimen types yield significantly different amounts cfDNA. While serum yields the highest amount of cfDNA, it contains the most genomic DNA contamination compared to Streck and plasma specimen types. The utility of cfDNA as a biomarker was advanced by the completion of the Human Genome Project and enabled interrogation of tumor markers in cancer patients. While tumor genetics may have been the initial application of cfDNA, the most successful application of cfDNA as a clinical biomarker is noninvasive prenatal testing (NIPT). CfDNA has become the gold-standard for NIPT testing, allowing for high sensitivity while maintaining specificity for aneuploidy. Because prenatal testing is essentially mixed genome analysis, application of cfDNA analysis to solid organ transplantation is a clear diagnostic target. There have been several studies examining the role of cfDNA in solid organ transplantation. These studies identified cfDNA as a surrogate marker for rejection with a high level of concordance with biopsies. While the data thus far are promising, there is still a need for more prospective studies to determine the clinical utility of cfDNA in solid organ transplant rejection.

The Past, Present, and Future of HLA Typing in Transplantation.

The HLA region is the most polymorphic genes in the human genome and is associated with an increasing number of disease states. Historically, HLA typing methodology has been governed by phenotypic determination. This practice has evolved into the use of molecular methods such as real-time PCR, sequence-specific oligonucleotides, and sequencing-based methods. Numerous studies have identified HLA matching as a key determinate to improve patient outcomes from transplantation. Solid-organ transplants focus on HLA-DRB1 in renal organ allocation while hematopoietic cell transplants focus on HLA-A, -B, -C, -DRB1 matching. The role of HLA typing in the future will be driven by HLA expression, understanding of HLA haplotypes, and rapid HLA typing.

HLAProfiler utilizes k-mer profiles to improve HLA calling accuracy for rare and common alleles in RNA-seq data.

BACKGROUND: The human leukocyte antigen (HLA) system is a genomic region involved in regulating the human immune system by encoding cell membrane major histocompatibility complex (MHC) proteins that are responsible for self-recognition. Understanding the variation in this region provides important insights into autoimmune disorders, disease susceptibility, oncological immunotherapy, regenerative medicine, transplant rejection, and toxicogenomics. Traditional approaches to HLA typing are low throughput, target only a few genes, are labor intensive and costly, or require specialized protocols. RNA sequencing promises a relatively inexpensive, high-throughput solution for HLA calling across all genes, with the bonus of complete transcriptome information and widespread availability of historical data. Existing tools have been limited in their ability to accurately and comprehensively call HLA genes from RNA-seq data. RESULTS: We created HLAProfiler ( https://github.com/ExpressionAnalysis/HLAProfiler ), a k-mer profile-based method for HLA calling in RNA-seq data which can identify rare and common HLA alleles with > 99% accuracy at two-field precision in both biological and simulated data. For 68% of novel alleles not present in the reference database, HLAProfiler can correctly identify the two-field precision or exact coding sequence, a significant advance over existing algorithms. CONCLUSIONS: HLAProfiler allows for accurate HLA calls in RNA-seq data, reliably expanding the utility of these data in HLA-related research and enabling advances across a broad range of disciplines. Additionally, by using the observed data to identify potential novel alleles and update partial alleles, HLAProfiler will facilitate further improvements to the existing database of reference HLA alleles. HLAProfiler is available at https://expressionanalysis.github.io/HLAProfiler/ .

Performance Characteristics and Validation of Next-Generation Sequencing for Human Leucocyte Antigen Typing.

High-resolution human leukocyte antigen (HLA) matching reduces graft-versus-host disease and improves overall patient survival after hematopoietic stem cell transplant. Sanger sequencing has been the gold standard for HLA typing since 1996. However, given the increasing number of new HLA alleles identified and the complexity of the HLA genes, clinical HLA typing by Sanger sequencing requires several rounds of additional testing to provide allele-level resolution. Although next-generation sequencing (NGS) is routinely used in molecular genetics, few clinical HLA laboratories use the technology. The performance characteristics of NGS HLA typing using TruSight HLA were determined using Sanger sequencing as the reference method. In total, 211 samples were analyzed with an overall accuracy of 99.8% (2954/2961) and 46 samples were analyzed for precision with 100% (368/368) reproducibility. Most discordant alleles were because of technical error rather than assay performance. More important, the ambiguity rate was 3.5% (103/2961). Seventy-four percentage of the ambiguities were within the DRB1 and DRB4 loci. HLA typing by NGS saves approximately $6000 per run when compared to Sanger sequencing. Thus, TruSight HLA assay enables high-throughput HLA typing with an accuracy, precision, ambiguity rate, and cost savings that should facilitate adoption of NGS technology in clinical HLA laboratories.

Clinical validation of NGS technology for HLA: An early adopter's perspective.

Clinical validation of NGS for HLA typing has been a topic of interest with many laboratories investigating the merits. NGS has proven effective at reducing ambiguities and costs while providing more detailed information on HLA genes not previously sequenced. The ability of NGS to multiplex many patients within a single run presents unique challenges and sequencing new regions of HLA genes requires application of our knowledge of genetics to accurately determine HLA typing. This review represents my laboratory's experience in validation of NGS for HLA typing. It describes the obstacles faced with validation of NGS and is broken down into pre-analytic, analytic, and post-analytic challenges. Each section includes solutions to address them.

Acute and chronic B cell depletion disrupts CD4+ and CD8+ T cell homeostasis and expansion during acute viral infection in mice.

B cells provide humoral protection against pathogens and promote cellular immunity through diverse nonclassical effector functions. To assess B cell function in promoting T cell homeostasis, mature B cells were either acutely or chronically depleted in mice using CD20 mAb. Acute B cell depletion in either 2- or 4-mo-old mice significantly reduced spleen and lymph node CD4(+) and CD8(+) T cell numbers, including naive, activated, and Foxp3(+)CD25(+)CD4(+) regulatory T cell subsets. The numbers of IFN-γ- and TNF-α-producing T cells were also significantly reduced. Chronic B cell depletion for 6 mo in aged naive mice resulted in a 40-70% reduction in activated CD4(+) and CD8(+) T cell numbers and 20-50% reductions in IFN-γ-producing T cells. Therefore, B cells were necessary for maintaining naive CD4(+) and CD8(+) T cell homeostasis for subsequent optimal T cell expansion in young and old mice. To determine the significance of this finding, a week of B cell depletion in 4-mo-old mice was followed by acute viral infection with lymphocytic choriomeningitis virus Armstrong. Despite their expansion, activated and cytokine-producing CD4(+) and CD8(+) T cell numbers were still significantly reduced 1 wk later. Moreover, viral peptide-specific CD4(+) and CD8(+) T cell numbers and effector cell development were significantly reduced in mice lacking B cells, whereas lymphocytic choriomeningitis virus titers were dramatically increased. Thus, T cell function is maintained in B cell-depleted mice, but B cells are required for optimal CD4(+) and CD8(+) T cell homeostasis, activation, and effector development in vivo, particularly during responses to acute viral infection.

Regulatory B cell (B10 Cell) expansion during Listeria infection governs innate and cellular immune responses in mice.

Pathogens use numerous methods to subvert host immune responses, including the modulation of host IL-10 production by diverse cell types. However, the B cell sources of IL-10 and their overall influence on innate and cellular immune responses have not been well characterized during infections. Using Listeria as a model pathogen, infection drove the acute expansion of a small subset of regulatory B cells (B10 cells) that potently suppress inflammation and autoimmunity through the production of IL-10. Unexpectedly, spleen bacteria loads were 92-97% lower in B10 cell-deficient CD19(-/-) mice, in mice depleted of mature B cells, and in mice treated with CD22 mAb to preferentially deplete B10 cells before infection. By contrast, the adoptive transfer of wild-type B10 cells reduced bacterial clearance by 38-fold in CD19(-/-) mice through IL-10-dependent pathways. B10 cell depletion using CD22 mAb significantly enhanced macrophage phagocytosis of Listeria and their production of IFN-γ, TNF-α, and NO ex vivo. Accelerated bacteria clearance following B10 cell depletion significantly reduced Ag-specific CD4(+) T cell proliferation and cytokine production, but did not alter CD8(+) T cell responses. B10 cell regulatory function during innate immune responses was nonetheless dependent on cognate interactions with CD4(+) T cells because B10 cells deficient in IL-10, MHC-II, or IL-21R expression did not influence Listeria clearance. Thus, Listeria manipulates immune responses through a strategy of immune evasion that involves the preferential expansion of endogenous B10 cells that regulate the magnitude and duration of both innate and cellular immune responses.

Immunization of young African green monkeys with OprF epitope 8-OprI-type A- and B-flagellin fusion proteins promotes the production of protective antibodies against nonmucoid Pseudomonas aeruginosa.

There is currently no approved vaccine against Pseudomonas aeruginosa, the major cause of morbidity and mortality in cystic fibrosis (CF) patients and a major pathogen in ventilated and burn patients. In a previous study, we demonstrated the immunization of mice with OprF(311-341)-OprI-type A- and B-flagellin fusion proteins dramatically enhanced clearance of nonmucoid P. aeruginosa. The goal of the current study was to evaluate the ability of OprF(311-341)-OprI-flagellins to elicit the production of protective IgG in young (4-6 months old) African green monkeys. Intramuscular immunization of African green monkeys with 1, 3, 10, or 30mug of OprF(311-341)-OprI-flagellins generated robust antigen-specific IgG responses. In addition, immunization with OprF(311-341)-OprI-flagellins elicited high-affinity anti-flagellins, OprI, and OprF IgG that individually promoted extensive deposition of complement component C3 on P. aeruginosa and synergized to facilitate maximal C3 deposition. Passive immunization of mice with plasma from OprF(311-341)-OprI-flagellins immunized monkeys significantly reduced lung bacterial burden three days post-challenge compared to mice that received pre-immunization plasma. Based on our results, OprF(311-341)-OprI-A- and B-flagellin fusion proteins are highly effective in mice and nonhuman primates and thus merit additional development as a potential vaccine for use in humans.

A fusion protein vaccine containing OprF epitope 8, OprI, and type A and B flagellins promotes enhanced clearance of nonmucoid Pseudomonas aeruginosa.

Although chronic Pseudomonas aeruginosa infection is the major cause of morbidity and mortality in cystic fibrosis (CF) patients, there is no approved vaccine for human use against P. aeruginosa. The goal of this study was to establish whether a multivalent vaccine containing P. aeruginosa type A and B flagellins as well as the outer membrane proteins OprF and OprI would promote enhanced clearance of P. aeruginosa. Intramuscular immunization with flagellins and OprI (separate) or OprI-flagellin fusion proteins generated significant antiflagellin immunoglobulin G (IgG) responses. However, only the fusions of OprI with type A and type B flagellins generated OprI-specific IgG. Immunization with a combination of OprF epitope 8 (OprF(311-341)), OprI, and flagellins elicited high-affinity IgG antibodies specific to flagellins, OprI, and OprF that individually promoted extensive deposition of C3 on P. aeruginosa. Although these antibodies exhibited potent antibody-dependent complement-mediated killing of nonmucoid bacteria, they were significantly less effective with mucoid isolates. Mice immunized with the OprF(311-341)-OprI-flagellin fusion had a significantly lower bacterial burden three days postchallenge and cleared the infection significantly faster than control mice. In addition, mice immunized with the OprF(311-341)-OprI-flagellin fusion had significantly less inflammation and lung damage throughout the infection than OprF-OprI-immunized mice. Based on our results, OprF(311-341)-OprI-flagellin fusion proteins have substantial potential as components of a vaccine against nonmucoid P. aeruginosa, which appears to be the phenotype of the bacterium that initially colonizes CF patients.