Eric T. Weimer

CARD9-Dependent Neutrophil Recruitment Protects against Fungal Invasion of the Central Nervous System.

Candida is the most common human fungal pathogen and causes systemic infections that require neutrophils for effective host defense. Humans deficient in the C-type lectin pathway adaptor protein CARD9 develop spontaneous fungal disease that targets the central nervous system (CNS). However, how CARD9 promotes protective antifungal immunity in the CNS remains unclear. Here, we show that a patient with CARD9 deficiency had impaired neutrophil accumulation and induction of neutrophil-recruiting CXC chemokines in the cerebrospinal fluid despite uncontrolled CNS Candida infection. We phenocopied the human susceptibility in Card9 -/- mice, which develop uncontrolled brain candidiasis with diminished neutrophil accumulation. The induction of neutrophil-recruiting CXC chemokines is significantly impaired in infected Card9 -/- brains, from both myeloid and resident glial cellular sources, whereas cell-intrinsic neutrophil chemotaxis is Card9-independent. Taken together, our data highlight the critical role of CARD9-dependent neutrophil trafficking into the CNS and provide novel insight into the CNS fungal susceptibility of CARD9-deficient humans.

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

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

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.

Glomerular C4d deposits can mark structural capillary wall remodelling in thrombotic microangiopathy and transplant glomerulopathy: C4d beyond active antibody-mediated injury: a retrospective study

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 laboratorys 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.

Clinically focused exome sequencing identifies an homozygous mutation that confers DOCK8 deficiency.

Peritubular capillary C4d (ptc‐C4d) usually marks active antibody‐mediated rejection, while pseudolinear glomerular capillary C4d (GBM‐C4d) is of undetermined diagnostic significance, especially when seen in isolation without concurrent ptc‐C4d. We correlated GBM‐C4d with structural GBM abnormalities and active antibody‐mediated rejection in 319 renal transplant and 35 control native kidney biopsies. In kidney transplants, ptc‐C4d was associated with GBM‐C4d in 97% by immunofluorescence microscopy (IF) and 61% by immunohistochemistry (IHC; P < 0.001). Transplant glomerulopathy correlated with GBM‐C4d (P < 0.001) and presented with isolated GBM‐C4d lacking ptc‐C4d in 69% by IF and 40% by IHC. Strong isolated GBM‐C4d was found post year‐1 in repeat biopsies with transplant glomerulopathy. GBM‐C4d staining intensity correlated with Banff cg scores (rs = 0.45, P < 0.001). Stepwise exclusion and multivariate logistic regression corrected for active antibody‐mediated rejection showed significant correlations between GBM duplication and GBM‐C4d (P = 0.001). Native control biopsies with thrombotic microangiopathies demonstrated GBM‐C4d in 92% (IF, P < 0.001) and 35% (IHC). In conclusion, pseudolinear GBM‐C4d staining can reflect two phenomena: (i) structural GBM changes with duplication in native and transplant kidneys or (ii) active antibody‐mediated rejection typically accompanied by ptc‐C4d. While ptc‐C4d is a dynamic ‘etiologic’ marker for active antibody‐mediated rejection, isolated strong GBM‐C4d can highlight architectural glomerular remodelling.

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

Live (viral and mycobacterial) vaccines are generally contraindicated in immunocompromised individuals (10). However, as rotavirus vaccination is recommended to be started at the age of 2 months, many SCID patients have already received it at the time of diagnosis (7). TREC-based NBS for SCID has been found efficient for early diagnosis of the disease and could thus prevent the administration of live vaccines to those infants (2, 3). NBS for SCID has become nationally recommended in the USA in 2010 (2). It is not yet carried out in Europe, although trials for implementing population-wide NBS for SCID are currently ongoing in several European countries (3). Delfien Bogaert; Kristof Van Schil; Tom Taghon; Victoria Bordon; Carolien Bonroy; Melissa Dullaers; Elfride De Baere & Filomeen Haerynck Clinical Immunology Research Lab, Department of Pulmonary Medicine, Ghent University Hospital, Ghent, Belgium; Department of Pediatric Immunology and Pulmonology, Ghent University Hospital, Ghent, Belgium; Center for Medical Genetics, Ghent University, Ghent University Hospital, Ghent, Belgium; Department of Clinical Chemistry, Microbiology and Immunology, Ghent University, Ghent, Belgium; Department of Pediatric Hematology and Oncology, Ghent University Hospital, Ghent, Belgium; Department of Clinical Chemistry, Microbiology and Immunology, Laboratory of Clinical Biology, Ghent University Hospital, Ghent, Belgium E-mail: filomeen.haerynck@uzgent.be DOI:10.1111/pai.12455

The past, present, and future of HLA typing in transplantation

BackgroundThe 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.ResultsWe 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.ConclusionsHLAProfiler 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/.

P151 Eliminating false positive flow cytometric crossmatches due to monoclonal antibody therapy

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.

Buccal swab genomic DNA fragmentation predicts likelihood of successful HLA genotyping by next-generation sequencing

Aim Biologics are increasingly used in autoimmune, cancer, and transplant patients. Unfortunately they can also interfere in laboratory tests. Monoclonal antibodies, such as Rituximab, are well known to cause positive flow cytometry crossmatches (FCM) independent of donor specific HLA antibody (DSA). We assessed the ability of blocking antibodies to Rituximab (RTX) and Alemtuzumab (ALM) to negate interference by these biologics in the FCM. Methods FCM was performed with serum from patients receiving RTX and/or ALM. Some sera were pretreated with anti-Rituximab ( α RTX) and/or anti-Alemtuzumab ( α ALM) blocking antibodies (Bio-Rad, Inc). HLA DSA was determined using multiplex bead arrays (LabScreen Single Antigen, Thermofisher, Inc.). FCM channel shifts (CS) were compared between treated sera and sera treated with PBS as a control. Results Blocking antibodies were titrated in serum to determine the working dilution to use in subsequent studies. Sera from patients receiving RTX and/or ALM were treated with the predetermined dilution of blocking antibody. Blocking antibody pre-treatment reduced FCM CS with surrogate donors to levels comparable to PBS controls: RTX blocking: pre-treatment CS = 23 T and 302 B; post-treatment CS = 35 T and 29 B; ALM blocking: pre-treatment CS = 262 T and 236 B; post-treatment CS = 34 T and 2 B. A highly sensitized (cPRA = 99%; DSA ;= B35, Cw4, DR17, DR52, DQ5) pediatric renal transplant candidate (RTX and ALM treated) was crossmatched with their living donor using pre-transplant and 4 day post-transplant (post-RTX and ALM treatment) sera. Post-treatment CS were increased without blocking but reduced with blocking antibody treatment. Blocked FCM CS results correlated better with reduced DSA MFI summed values in contrast to the unblocked serum (Table 1). Conclusions Antibodies to RTX and ALM effectively block interference by these biologics in FCM resulting in the ability to provide useful FCM data for patient management.

Rapid and quality controlled screening assay for the HLA-B57 antigen by flow cytometry

Many clinical human leukocyte antigen (HLA) laboratories are adopting next-generation sequencing (NGS) technology for HLA genotyping. There have been several reports of the cost-benefit and reduction in turn-around-time provided by NGS. Ninety-six percent of buccal swabs and peripheral blood samples had reportable HLA genotyping by NGS. The HLA loci most likely to fail genotyping from buccal swabs were DQB1, DPB1, and DPA1. Successful buccal swab samples had significantly less genomic DNA fragmentation compared to buccal swab samples that were unsuccessful. Increasing sequencing depth of coverage for heavily fragmented samples rescued HLA genotyping. This information provides laboratories with a quality assurance parameter that reduces the amount of repeat NGS needed to achieve high-resolution HLA genotyping. This information should further reduce laboratory and patient costs for HLA genotyping.