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In Duchenne muscular dystrophy (DMD), the right ventricle (RV) tends to be relatively well preserved, but characterization remains difficult due to its complex architecture. Tissue phase mapping (TPM) is a phase contrast cine MRI technique that allows for multidirectional assessment of myocardial velocities.To use TPM to elucidate relationships between myocardial structure, function, and clinical variables in DMD.Retrospective.A total of 20 patients with muscular dystrophy (median age: 16 years); 18 age-matched normal controls (median age: 15 years).Three-directional velocity encoded cine gradient echo sequence (TPM) at 1.5 T, balanced steady-state free procession (bSSFP), T1 mapping with extracellular volume (ECV), and late gadolinium enhancement (LGE).TPM in basal, mid, and apical short-axis planes was performed as part of a standard MRI study with collection of clinical data. Radial, circumferential, and longitudinal velocities (Vr, Vφ, and Vz, respectively) and corresponding time to peak (TTP) velocities were quantified from TPM and used to calculate RV twist as well as intraventricular and interventricular dyssynchrony. The correlations between TPM velocities, myocardial structure/function, and clinical variables were assessed.Unpaired t-test, Wilcoxon rank-sum test, Bland-Altman analyses were used for comparisons between DMD patients and controls and between DMD subgroups. Pearson's test was used for correlations (r). Significance level: P 0.05.Compared to controls, DMD patients had preserved RV ejection fraction (RVEF 53% ± 8%) but significantly increased interventricular dyssynchrony (Vφ: 0.49 ± 0.21 vs. 0.72 ± 0.17). Within the DMD cohort, RV dyssynchrony significantly increased with lower LV ejection fraction (intraventricular Vr and Vz: r = -0.49; interventricular Vz: r = 0.48). In addition, RV intraventricular dyssynchrony significantly increased with older age (Vz: r = 0.67).RV remodeling in DMD occurs in the context of preserved RVEF. Within DMD, this abnormal RV deformation is associated with older age and decreased LVEF.4.Stage 2.

Next generation sequencing (NGS) assays are state of the art for HLA genotyping. To sequence on an Illumina sequencer, the DNA of interest must be enriched, fragmented, and bookended with known oligonucleotide sequences, a process known as library construction. Many HLA genotyping assays enrich the target loci by long-range PCR (LR-PCR), prior to fragmentation. This PCR step has been reported to introduce errors in the DNA to be sequenced, including inaccurate replication of repeated sequences, and the in vitro recombination of alleles encoded on separate chromosomes. An alternative library construction method involves fragmentation of genomic DNA, followed by hybrid-capture (HC) enrichment of target HLA loci. This HC-based method involves PCR, but with far fewer cycles. Consequently, the HC method had significantly fewer PCR-induced errors, including more faithful replication of repeated sequences, and the near elimination of recombinant sequences. These improvements likely produce more accurate NGS sequencing data of HLA loci.

The molecular interactions that regulate chronic inflammation underlying metabolic disease remain largely unknown. Since the CD24-Siglec interaction regulates inflammatory response to danger-associated molecular patterns (DAMPs), we have generated multiple mouse strains with single or combined mutations of Cd24 or Siglec genes to explore the role of the CD24-Siglec interaction in metaflammation and metabolic disorder. Here, we report that the CD24-Siglec-E axis, but not other Siglecs, is a key suppressor of obesity-related metabolic dysfunction. Inactivation of the CD24-Siglec-E pathway exacerbates, while CD24Fc treatment alleviates, diet-induced metabolic disorders, including obesity, dyslipidemia, insulin resistance, and nonalcoholic steatohepatitis (NASH). Mechanistically, sialylation-dependent recognition of CD24 by Siglec-E induces SHP-1 recruitment and represses metaflammation to protect against metabolic syndrome. A first-in-human study of CD24Fc (NCT02650895) supports the significance of this pathway in human lipid metabolism and inflammation. These findings identify the CD24-Siglec-E axis as an innate immune checkpoint against metaflammation and metabolic disorder and suggest a promising therapeutic target for metabolic disease.

Percutaneous balloon pulmonary valvuloplasty (PBPV) is the treatment of choice for isolated pulmonary valve stenosis. While this procedure is highly efficacious and has an excellent safety profile, as currently practiced, patients are obligatorily exposed to the secondary risks of ionizing radiation and contrast media. To mitigate these risks, we developed a protocol which utilized echo guidance for portions of the procedure which typically require fluoroscopy and/or angiography. Ten cases of echo-guided pulmonary valvuloplasty (EG-PBPV) for isolated pulmonary stenosis in children less than a year of age were compared to a historical cohort of nineteen standard cases using fluoroscopy/angiography alone, which demonstrated equivalent procedural outcomes and safety, while achieving a median reduction in radiation (total dose area product) and contrast load of 80% and 84%, respectively. Our early experience demonstrates that EG-PBPV in neonates and infants has results equivalent to standard valvuloplasty but with less radiation and contrast.

Heteroclitic antibodies bind to a related antigen with higher affinity than to the immunizing antigen to which they were generated. This uncommon phenomenon is not well characterized for antibodies to HLA antigens. Here we analyzed allosera reactivity from two transplant recipients sensitized to mismatched donor alleles DQB1*06:01 and DQB1*06:02 respectively. Epitope analysis demonstrated the reactivity of both sera was restricted to DQB1*04, 05, and 06 alleles, with a specificity associated with the 55R eplet. Serum from one of these subjects (TE) was significantly more reactive with DQB1*04 alleles than the immunizing DQB1*06:01 or other alleles, a pattern not present in serum from the other patient. Antibody absorption/elution experiments using B cell lines expressing DQB1*06:01 or DQB1*04:02 alleles confirmed that the heteroclitic TE antibody eluted from cells carrying DQB1*06:01 was significantly more reactive with beads carrying the DQB1*04 alleles than with the DQB1*06 or other alleles. The significantly higher reactivity of the heteroclitic alloantibody with DQB1*04 specificity was explained structurally by variations of amino acid residues within 3.5 A of 55R. These findings have important implications for the interpretation of DQ alloantibody cross-reactivity frequently observed in transplant recipients.

We describe a unique ABO compatible and 9/10 HLA-matched case of successful allogeneic hematopoietic cell transplantation (HCT) after effective desensitization of a strong anti-HLA-A24 donor-specific antibody (DSA) with mean fluorescence intensity of approximately 18 000. Due to absence of a suitable matched unrelated donor the patient sibling was considered the best available donor, and it was decided to desensitize patient prior to transplant. The strength of HLA-A24 DSA slowly decreased over the course of treatment, necessitating a total of 23 sessions of therapeutic plasma exchange in order to bring the DSA strength to undetectable levels, followed by a successful transplant. In summary, the outcome of this case shows effective application of desensitization treatment to remove strong DSA in HCT patients.

Platelets are a vital part of the hemostatic system, providing the physical basis of primary hemostasis and the surface for formation of the secondary hemostatic fibrin clot, in addition to important roles in inflammatory and immune signaling and response. Platelets express antigens from several important antigen systems: blood group ABO, human leukocyte antigen (HLA), and human platelet antigen (HPA). Antibodies to all of these systems can cause immune clearance of incompatible platelets. We discuss the various alloimmune thrombocytopenia syndromes: posttransfusion purpura, passive alloimmune thrombocytopenia, fetal/neonatal alloimmune thrombocytopenia, transplantation-associated alloimmune thrombocytopenia, and alloimmune platelet transfusion refractoriness. Platelet transfusion refractoriness is the most commonly encountered alloimmune thrombocytopenia condition, with the others being less common (though likely underrecognized). Antibodies to all three antigen systems (ABO, HLA, and HPA) are associated with immune platelet transfusion refractoriness, with HLA generally being the most important. In contrast, fetal/neonatal alloimmune thrombocytopenia, posttransfusion purpura (PTP), passive alloimmune thrombocytopenia, and transplantation-associated alloimmune thrombocytopenia are all caused by antibodies to the HPA system. PTP is unique among the alloimmune thrombocytopenia syndromes in its combined immune attack against allogeneic platelets and autologous platelets.

A limitation of solid-phase human leukocyte antigen (HLA) antibody assays is the falsely low/negative result of samples with high-titer antibodies, a phenomenon known as the prozone effect. Here we compared the efficacy of ethylenediaminetetraacetic acid (EDTA) and dithiothreitol (DTT) treatment of serum samples in overcoming the prozone effect. A total of 21 serum samples were treated with either EDTA or DTT before HLA single antigen bead assay. The efficacy of prozone effect reversal, compared with untreated samples, was examined on fourfold, serially diluted samples, from neat to 1:256, using PBS as diluent. EDTA reversed the prozone effect in all tested samples, with an efficiency of greater than 84%, estimated by the ratio of undiluted sample mean fluorescence intensity (MFI) to peak MFI, for any given dilution. In contrast, the efficiency of DTT treatment was as low as 47%. These results show superior prozone effect reversal with EDTA treatment, compared with DTT.

The 17(th) 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 1,017 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, and confirming them using HLA allele segregation at the DNA sequence level.

Celiac disease is a common inflammatory disease triggered by dietary gluten in genetically susceptible individuals. The strongest and best-characterized genetic susceptibilities in celiac disease are class II human leukocyte antigen (HLA) genes known as HLA-DQ2 and DQ8. HLA genetic testing is available through a number of commercial and academic laboratories and is used in the evaluation of celiac disease and to identify at-risk family members. Importantly, HLA genetic testing has a high negative predictive value for celiac disease, but a low positive predictive value. Therefore, for a practicing clinician, it is important to understand when to order HLA genetic testing, what test to order, and how to interpret the result. This review provides a practical primer on HLA genetics in celiac disease.

The use of 3D printed models of the right ventricular outflow tract (RVOT) for surgical and interventional planning is growing and often requires image segmentation of cardiac magnetic resonance (CMR) images. Segmentation results may vary based on contrast, image sequence, signal threshold chosen by the operator, and manual post-processing. The purpose of this study was to determine potential biases and post-processing errors in image segmentation to enable informed decisions. Models of the RVOT and pulmonary arteries from twelve patients who had contrast enhanced CMR angiography with gadopentetate dimeglumine (GPD), gadofosveset trisodium (GFT), and a post-GFT inversion-recovery (IR) whole heart sequence were segmented, trimmed, and aligned by three operators. Geometric agreement and minimal RVOT diameters were compared between sequences and operators. To determine the contribution of threshold, interoperator variability was compared between models created by the same two operators using the same versus different thresholds. Geometric agreement by Dice between objects was high (intraoperator: 0.89-0.95; interoperator: 0.95-0.97), without differences between sequences. Minimal RVOT diameters differed on average by - 1.9 to - 1.3 mm (intraoperator) and by 0.4 to 1.4 mm (interoperator). The contribution of threshold to interoperator geometric agreement was not significant (same threshold: 0.96 ± 0.06, different threshold: 0.93 ± 0.05; p = 0.181), but minimal RVOT diameters were more variable with different versus constant thresholds (- 9.12% vs. 2.42%; p

The HLA genes are the most polymorphic of the human genome, and novel HLA alleles are continuously identified, often by clinical Sanger sequencing-based typing (SBT) assays. Introduction of next-generation sequencing (NGS) technologies for clinical HLA typing may significantly improve this process. Here we compare four cases of novel HLA alleles identified and characterized by both SBT and NGS. The tested NGS system sequenced broader regions of the HLA loci, and identified novel polymorphisms undetected by SBT. Subsequent characterization of the novel alleles in isolation of coencoded alleles by SBT required custom-designed primers, while the NGS system was able to sequence both alleles in phase. However, the tested assay was unable to amplify buccal cell DNA for subsequent NGS sequencing, presumably due to the lower quality of these samples. While NGS assays will undoubtedly increase novel allele identification, more stringent DNA sample requirements may be necessary for this new technology.

Several key findings from the late 1960s to mid-1970s regarding thyroid hormone metabolism and circulating thyroglobulin composition converged with studies pertaining to the role of T lymphocytes in autoimmune thyroiditis. These studies cemented the foundation for subsequent investigations into the existence and antigenic specificity of thymus-derived natural regulatory T cells (nTregs). These nTregs prevented the development of autoimmune thyroiditis, despite the ever-present genetic predisposition, autoantigen (thyroglobulin), and thyroglobulin-reactive T cells. Guided by the hypothalamus-pituitary-thyroid axis as a fixed set-point regulator in thyroid hormone metabolism, we used a murine model and compared at key junctures the capacity of circulating thyroglobulin level (raised by thyroid-stimulating hormone or exogenous thyroglobulin administration) to strengthen self-tolerance and resist autoimmune thyroiditis. The findings clearly demonstrated an essential role for raised circulating thyroglobulin levels in maintaining the dominance of nTreg function and inhibiting thyroid autoimmunity. Subsequent identification of thyroglobulin-specific nTregs as CD4+CD25+Foxp3+ in the early 2000s enabled the examination of probable mechanisms of nTreg function. We observed that whenever nTreg function was perturbed by immunotherapeutic measures, opportunistic autoimmune disorders invariably surfaced. This review highlights the step-wise progression of applying insights from endocrinologic and immunologic studies to advance our understanding of the clonal balance between natural regulatory and autoreactive T cells. Moreover, we focus on how tilting the balance in favor of maintaining peripheral tolerance could be achieved. Thus, murine autoimmune thyroiditis has served as a unique model capable of closely simulating natural physiologic conditions.

Thyroglobulin (Tg), a homodimer of 660 kD comprising 2748 amino acids, is the largest autoantigen known. The prevalence of autoimmune thyroid disease, including Hashimoto's thyroiditis and Graves' disease, has provided the impetus for identifying pathogenic T cell epitopes from human Tg over two decades. With no known dominant epitopes, the search has long been a challenge for investigators. After identifying HLA-DRB1∗03:01 (HLA-DR3) and H2E(b) as susceptibility alleles for Tg-induced experimental autoimmune thyroiditis in transgenic mouse strains, we searched for naturally processed T cell epitopes with MHC class II-binding motif anchors and tested the selected peptides for pathogenicity in these mice. The thyroiditogenicity of one peptide, hTg2079, was confirmed in DR3 transgenic mice and corroborated in clinical studies. In H2E(b)-expressing transgenic mice, we identified three T cell epitopes from mouse Tg, mTg179, mTg409 and mTg2342, based on homology to epitopes hTg179, hTg410 and hTg2344, respectively, which we and others have found stimulatory or pathogenic in both DR3- and H2E-expressing mice. The high homology among these peptides with shared presentation by DR3, H2E(b) and H2E(k) molecules led us to examine the binding pocket residues of these class II molecules. Their similar binding characteristics help explain the pathogenic capacity of these T cell epitopes. Our approach of using appropriate human and murine MHC class II transgenic mice, combined with the synthesis and testing of potential pathogenic Tg peptides predicted from computational models of MHC-binding motifs, should continue to provide insights into human autoimmune thyroid disease.

Aim Turnaround time (TAT) for HLA typing is critical for transplantation. We sought to establish a faster protocol for low-resolution HLA typing using the rSSOP methodology. Methods The LABType™ rSSOP method (One Lambda) was altered to reduce hybridization time. Modifications included centrifugation and incubation duration reductions, and reagent volume adjustments. Single parameters were first changed and tested. Successful modifications were used in the next round of tests. All modifications were tested with a set of PCR amplicons prepared from three samples, which contained previously HLA-typed high quality DNA. Following a series of rSSOP typings for these samples at HLA-A, B, C, DRB1, and DQB1/DQA1 loci, an accelerated protocol was established. Discovery phase concluded with protocol validation at all loci including HLA-DRB3/4/5 and DPB1/DPA1. Full validation confirmed intra-operator, inter- and intra-assay, as well as multiple DNA source reproducibility. DNA was extracted from different samples including ACD blood (n = 4), EDTA blood (n = 4), cord segment (n = 5), buccal swab (n = 5) and mouth-wash samples (n = 4). Results The original centrifugation of 3000 rpm for 2 min (total 2 min 22 s) was changed to a quick spin (up to 3000 rpm; 33 s), reducing each centrifugation step by 1 min 50 s. Additionally, one centrifugation and one transfer of plate step were eliminated. The duration of incubation time was changed for denaturation (from 10 min to 2.5 min), hybridization (from 15 min to 5 min), and streptavidin-PE (SAPE, from 5 min to 2.5 min). The reagent volume modifications includes the reduction of washing buffer (from 100 μl to 50 μl per well) and increase in SAPE (from 25 μl to 50 μl per well). Compared to the original protocol, the accelerated protocol reduced the hybridization time approximately 30 min. This method allowed us to obtain consistent and accurate typing results for all tested HLA loci among different operators, intra- and inter-assays, and a variety of DNA samples. Conclusions Fulfilling requests for faster TAT may be achieved by adaptation of current methodologies, which may help improving patient management. The accelerated rSSOP protocol is an effective method for reducing testing time, while still giving robust results for HLA class I and class II typing. (RLU and JJX contributed equally).

A female patient presented for follow-up care approximately ten years post-cardiac transplantation. HLA antibody testing revealed multiple class I and class II donor specific antibodies (DSA), including anti-HLA A1 (MFI > 2500), DQ6, and DQ7/DQ8/DQ9 antibodies (DQ antibodies have MFIs >15,000). Per our current testing protocols, the sample was reflexed to C1q to determine if any DSAs were complement- fixing. As expected, based on antibody strength, the class II DSAs were C1q positive, while anti-HLA A1 was C1q negative. Interestingly, C1q testing revealed reactivity for anti-HLA B8 which was not previously detected by our standard IgG testing methodology. While anti-HLA B8 was not a DSA to the previous transplant, we wanted to determine the underlying cause for the positive reactivity seen with C1q testing: prozone effect or IgM? Since our laboratory routinely treats all sera tested for HLA antibodies with EDTA prior to testing, we thought prozone was unlikely as previous studies performed in our lab demonstrated the removal of this effect. Nonetheless, to rule out prozone with another method, IgG testing was repeated using a series of dilutions (Neat, 1:4, 1:16, 1:64, and 1:256). This testing did not yield positive reactivity for anti-HLA B8, and therefore prozone effect was excluded, indicating that EDTA methodology is robust to overcome prozone effect. To confirm the isotype of this antibody, IgM testing was conducted in three sera using the same assays as the IgG testing which produced positive results. In conclusion, anti-HLA B8 was determined to be complement-fixing and of the IgM isotype. Unexpectedly, our results supported the absence of an IgM-IgG isotype class switch for the period of testing performed in our lab spanning 17 months. *RLU and JJX contributed equally to this work.

Medullary thymic epithelial cells (mTECs) play a critical role in thymic negative selection of autoreactive thymocytes, especially for thymocytes specific for peripheral tissue-restricted self-antigens (TRA). Deficiency in lymphotoxin b receptor (LTbetaR) is associated with peripheral tissue inflammation, but whether this is caused by defective negative selection has been unclear; the significance of the LTbetaR pathway for negative selection is evident in some models but not others. Here, we revisit the data and clarify the role of LTbetaR in mTEC development and function and thymic TRA expression. These processes are discussed as potential mechanisms for LTbetaR-mediated control of negative selection.

Coinhibitory pathways are thought to act in later stages of an adaptive immune response, but whether coinhibition contributes to early innate immunity is unclear. We show that engagement of the newly discovered coinhibitory receptor B and T lymphocyte attenuator (BTLA) by herpesvirus entry mediator (HVEM) is critical for negatively regulating early host immunity against intracellular bacteria. Both HVEM−/− and BTLA−/−, but not LIGHT−/−, mice are more resistant to listeriosis compared with wild-type mice, and blockade of the BTLA pathway promotes, while engagement inhibits, early bacterial clearance. Differences in bacterial clearance were seen as early as 1 day postinfection, implicating the initial innate response. Therefore, innate cell function in BTLA−/− mice was studied. We show that innate cells from BTLA−/− mice secrete significantly more proinflammatory cytokines upon stimulation with heat-killed Listeria. These results provide the first evidence that a coinhibitory pathway plays a critical role in regulating early host innate immunity against infection.

Murine experimental autoimmune thyroiditis (EAT) is a model for Hashimoto's thyroiditis, an organ-specific autoimmune disease characterized by mononuclear cell infiltration and destruction of the thyroid gland. Susceptibility to EAT is MHC-linked, and influenced by CD4(+)CD25(+)Foxp3(+) regulatory T cells (Tregs). Treg depletion enables thyroiditis induction with mouse thyroglobulin (mTg) in traditionally-resistant mice and mTg-induced, Treg-mediated tolerance protects against EAT induction in genetically-susceptible mice. Here, we demonstrate the existence of naturally-existing CD4(+)CD25(+)Foxp3(+) Tregs (nTregs) influencing thyroiditis development in naive susceptible mice and that induction of thyroiditis in these mice involves overcoming peripheral homeostatic immune suppression by nTregs. Additionally we demonstrate that nTregs are required for induction of antigen-specific tolerance, indicating that induced EAT tolerance is a result of activation of naturally-existing nTregs rather than de novo generation of induced Tregs (iTregs). Examination of several potential costimulatory molecules previously described as involved in peripheral activation of Tregs demonstrates a critical role indeed for CTLA-4 in the activation of nTregs leading to development of EAT tolerance and providing a mechanism for mTg-induced Treg activation during tolerance induction. Together, these data reinforce the important role of Tregs in mediating self-tolerance, and illuminate a potential mechanism for their therapeutic expansion in induced tolerance.

A retrospective review of 33 patients comparing community-associated methicillin-resistant Staphylococcus aureus retropharyngeal abscess (RPA) with community-associated methicillin-susceptible S. aureus RPA from 2002-2013 at Texas Children's Hospital revealed most cases of S. aureus RPA have been due to community-associated methicillin-resistant S. aureus, which appears to be associated with a more complicated clinical course than RPA caused by community-associated methicillin-susceptible S. aureus.

Due to the expanded genomic coverage of many next-generation sequencing (NGS) assays for HLA typing, and their recent introduction into immunogenetics laboratories, there has been an increase in the identification of novel alleles. While the majority of novel alleles are simple, single nucleotide polymorphisms (SNP), others require an extensive knowledge of the NGS analysis software in order to be suitably identified. Herein, we describe a case of gene conversion, a primary way in which HLA diversity is maintained, in a novel DPB1 allele. An optimized NGSgo protocol (GenDx) was used for sequencing the DPB1 locus, with analysis by NGSengine (GenDx). Initially analyzed using default parameters, including both coding and non-coding regions of the amplicon, a typing assignment of DPB1∗02:01:19, and a novel allele, similar to DPB1∗512:01, with an SNP in exon 2 was obtained. Using a lab-developed process, the data was reanalyzed using exon 2 only, with a resulting assignment of DPB1∗40:01, without mismatches. Utilizing the full amplicon and manually aligned to DPB1∗40:01, the sample was reanalyzed, revealing that the novel allele matched DPB1∗40:01 in exons 1 and 2. However, multiple mismatches were present in the remaining sequence. This result indicated that, while the 5′ end of the novel allele likely derived from DPB1∗40:01, the 3′ end may have been derived from another allele. In order to determine which DPB1 allele may have served as the template for this gene conversion event, known DPB1 alleles were manually aligned to the sample, with the finding that the sequence for DPB1∗06:01:01 was identical to the sample sequence at exons 3–5. We concluded that the novel allele likely resulted from a gene conversion event between the DPB1∗40:01 and 06:01:01 alleles. With the increased adoption of NGS assays for HLA typing, the detection of novel alleles due to gene conversion will also likely increase. With the aid of a skilled analyst, and using the comprehensive process described here, the proper identification of such alleles may be simplified and reported appropriately to the clinician.

Aim DNA sequencing has long been the gold standard for HLA typing, offering the ability to interrogate each base pair of a targeted region. Sanger sequencing-based typing assays are known to suffer from allele dropout, whereby one of two encoded alleles is less represented in the results, due to poor amplification. Next-generation sequencing (NGS) includes several distinct technologies, defined by their ability to sequence clonal DNA fragments in a high-throughput manner. One such technology, provided by Illumina, sequences thousands of short DNA fragments, with read depth orders of magnitude higher than Sanger sequencing, which may allow for the identification of poorly-amplified alleles. Here we sought to determine the rate of allele dropout in an Illumina-based HLA typing assay. Methods NGSgo (GenDx) was used to produce libraries for the HLA-A, B, C, DRB1, DRB3/4/5, DQA1, DQB1, DPA1, and DPB1 loci for sequencing on a MiSeq (Illumina). NGSengine (GenDx) was used to analyze the data and type the loci. 85 unique samples were run in parallel by a combination of previously-validated assays (SBT, SSP, rSSOP) and by NGS. Results The NGS assay resulted in very high read depths (>100 for our clinical results) and a very low error rate. Use of NGSengine software allowed detection of both alleles at heterozygous loci when the alleles were unevenly represented (imbalanced). In cases of extreme allele imbalance, data visualization by NGSengine allowed detection of the minor allele during manual review. Of the 811 patient loci tested by the previously-validated assays, 584 were heterozygous, 108 were homozygous and 119 were absent; the NGS results were 100% concordant. Conclusions In our experience with a clinical NGS typing assay, we found the Illumina sequencing technology was able to successfully identify all alleles present in tested samples, including poorly-amplified alleles. While the possibility of rare allele dropout still exists with this technology, due to alleles with novel polymorphisms at a primer-binding site, we found that with an appropriate combination of NGS library construction, sequencing technology, and analysis software, allele dropout is a low frequency, to non-existent, event. This reduces the need for extra testing to confirm homozygous loci during routine clinical HLA typing.

A 71 year old female was initially diagnosed with myelodysplastic syndrome (del 5q) which progressed to AML one year later. Patient had pancytopenia, experienced bruising, and needed blood transfusions. Allogeneic stem cell transplant was recommended and she presented to the University of Chicago Medicine for allo-stem cell transplant work up. This patient had one potential related donor (sibling), who was a 9/10 match (mismatch at HLA-A). When examining the patient’s HLA antibodies, it was found that she was highly sensitized, and anti-HLA-A24 was a donor specific antibody (DSA). A search for matched unrelated donors was conducted but no suitable donors were found. It was decided to accept her sibling and desensitize the patient prior to transplant. The desensitization protocol consisted of plasmapheresis (PP), with IVIG (100 mg/kg) given after each PP. The initial MFI of the DSA was >18,000 and showed evidence of rebound after each PP. As a result, PP was performed daily or every other day, with A24 MFI measurement before and after each PP (Figure). While the strength of A24 decreased over the course, a total of 18 sessions of PP were needed to decrease the strength to Download : Download high-res image (161KB) Download : Download full-size image

Aim Next-generation sequencing (NGS) assays for HLA typing are being rapidly adopted by clinical laboratories due to their ability to result in unambiguous, high-resolution typing in the vast majority of cases. Analysis of the NGS data involves specialized software algorithms to align hundreds of short oligonucleotide reads to the allele sequences managed by the IMGT/HLA. There is significant variability in the sequence length of alleles in the IMGT/HLA database, as an allele can be named by providing sequence from as little as one exon, while many alleles have full-locus sequences. This variability is problematic for the matching algorithms, which compute scores for alignment to the entire locus. To overcome this limitation, software vendors use imputed sequences, filling in the sequences missing from IMGT/HLA with sequence from a variety of sources. Since the imputed sequence may not match the patient alleles, use of imputed data can lead to mistypings. Here we describe our attempt to overcome the limitation of matching to imputed data. Methods NGSengine (GenDx) was used to analyze Illumina sequencing data from HLA-A, B, C, DRB1, DRB3/4/5, DQA1, DQB1, DPA1, and DPB1 amplicons. Potential mistypings were identified by further analysis of loci for which NGSengine did not report a typing without exonic mismatches. Results 28 samples were identified without typings that matched exonic data. 25 were determined to be novel alleles. 3 samples were mistyped based on mismatches to imputed data in NGSengine. A process was developed to analyze all potential novel alleles by restricting analysis to the peptide binding domains (Exons 2 and 3 for Class I, and 2 for class II). With this method, true novel alleles were successfully discriminated from mistyped alleles. Conclusions Compared to legacy HLA typing assays, NGS assays often sequence larger portions of the HLA loci, with full-locus coverage for many HLA genes. This presents a challenge to the analysis software, which must align patient data to the incomplete IMGT/HLA database. The use of imputed data helps with the computational algorithms, but can introduce its own problems when correctly-typed alleles are excluded due to mismatches to imputed data. The process described here overcomes this limitation, ensuring the correct typing of all HLA alleles present in the IMGT/HLA.

Aim Our laboratory has recently reported that serum treatment with EDTA is sufficient to overcome the prozone effect. However, it has been reported that in rare cases, specimens are partially resistant to EDTA treatment, and serum dilutions are still necessary. Although we have not encountered such case in our laboratory, we sought to determine if differences in the technical aspects of the assay may contribute to EDTA’s effectiveness. Methods We screened samples sent to our laboratory for HLA antibody testing for suspected prozone effect, based on MFI values of >15,000. Selected samples were tested in three protocols. Protocol #1: to determine the presence of prozone effect, serum samples were frozen, thawed, centrifuged (14,000 rpm for 10 min), and tested at neat, 1:4, 1:16, 1:64, and 1:264 by single antigen (SA) bead assays (One Lambda). Protocol #2: to test the clinically-validated method used by our lab, samples were frozen, thawed, centrifuged, treated with EDTA (4 mM), and tested at neat only; incubation and washing was performed in a 1 μM filter plate on a vacuum plate-washing system (Biotek ELx50). Protocol #3: to test the effect of EDTA in fresh samples with the manual washing method, fresh serum samples were centrifuged, treated with EDTA, and then tested at neat only; incubation and washing was performed in a traditional round-bottom plate. Data were acquired on a Luminex-200 and analyzed with HLAFusion software (One Lambda). Results We identified prozone effects in 18 patients’ samples: 8 patients’ class I antibodies, 8 patients’ class II antibodies, and 2 patients’ class I and II antibodies. Following EDTA treatment, fresh and frozen samples showed similar MFI values for the corresponding assignments, including 138 class I and 103 class II specificities. In addition, all determined specificities showed higher MFI values in EDTA-treated samples than the peak MFI value in sample with dilution only, regardless of which technical protocol was used. Conclusions Samples with prozone effect resistant to EDTA treatment were not detected in our study, independent of technical variability for sample processing. EDTA treatment fully overcomes prozone effect in our testing system. Factors causing low efficacy of EDTA in rare cases remain unknown.

Aim Autoimmunity introduces a variety of difficulties in interpretation of solid phase HLA antibody testing due to autoantibodies present in patient serum. However, because autoantibodies may be present without obvious symptoms or a clear diagnosis, this information is always available when interpreting HLA test results. The current study sought to establish a mechanism for detecting these patients and generate an algorithm for handling samples with autoantibodies. Methods We retrospectively searched the electronic medical record to identify patients with autoimmune disease who were tested by our lab for HLA alloantibodies, and further cataloged any issues encountered during testing of these patients’ samples. This information was subsequently used to formulate an algorithm for testing autoimmune samples. Results A total of 214 patients with diagnosed autoimmunity were identified, including patients with organ-specific autoimmune conditions, most commonly type 1 diabetes mellitus (n = 43, 20%), and systemic autoimmune conditions, most commonly systemic lupus erythematosus (n = 29, 13.6%) and Sarcoidosis (n = 6, 2.8%). The most common issues encountered during testing were found to be high background on solid-phase antibody testing (n = 38, 17.8%), difficulties in assigning Ab specificities (n = 10, 4.7%), many allele specific antibody assignments (n = 5, 2.3%); and antibodies against self HLA antigens (n = 1, 0.5%). A workflow was designed to detect these patients and handle their samples (Fig. 1). Conclusions Using patients with diagnosed autoimmunity, we identified a high rate of interference of autoantibodies with solid phase HLA antibody assays. Extrapolating these results to a testing algorithm, we hope to be able to more readily detect and document patient samples that require special handling for autoantibody interference, even in the absence of an official autoimmunity diagnosis. Download : Download high-res image (170KB) Download : Download full-size image

Both pathogen- and tissue damage-associated molecular patterns induce inflammation through toll-like receptors (TLRs), while sialic acid-binding immunoglobulin superfamily lectin receptors (Siglecs) provide negative regulation. Here we report extensive and direct interactions between these pattern recognition receptors. The promiscuous TLR binders were human SIGLEC-5/9 and mouse Siglec-3/E/F. Mouse Siglec-G did not show appreciable binding to any TLRs tested. Correspondingly, Siglece deletion enhanced dendritic cell responses to all microbial TLR ligands tested, while Siglecg deletion did not affect the responses to these ligands. TLR4 activation triggers Neu1 translocation to cell surface to disrupt TLR4:Siglec-E interaction. Conversely, sialidase inhibitor Neu5Gc2en prevented TLR4 ligand-induced disruption of TLR4:Siglec E/F interactions. Absence of Neu1 in hematopoietic cells or systematic treatment with sialidase inhibitor Neu5Gc2en protected mice against endotoxemia. Our data raised an intriguing possibility of a broad repression of TLR function by Siglecs and a sialidase-mediated de-repression that allows positive feedback of TLR activation during infection. DOI: http://dx.doi.org/10.7554/eLife.04066.001, eLife digest Many living things have an immune system that is able to detect invading bacteria, viruses and other pathogens and trigger a response targeted against the threat before it causes lasting damage. Cells employ a number of different receptors that can detect these pathogens or the molecules that they produce. In animals, toll-like receptors (or TLRs) are a type of protein that recognizes patterns or structures that are found in many different types of pathogen, known as pathogen-associated molecular patterns (or PAMPs). Injured cells release proteins that are also recognized by toll-like receptors and are called danger associated molecular patterns (or DAMPs). An immune response is triggered when PAMPs and DAMPs are recognized, but the response must be properly controlled. If it goes awry, it can result in an over-activation of the immune cells that can lead to life-threatening conditions, one of which is called sepsis. Siglecs are proteins that bind to a sugar molecule, which is found attached to many other proteins, and are known to inhibit the immune response. However, it remained unclear how Siglecs do this and if they can interact directly with toll-like receptors. Chen et al. now show that most (although not all) Siglecs bind to TLRs, and that deleting the gene for a Siglec protein that can bind to multiple TLRs boosted the response of the immune cells to a range of microbial PAMPs. Deleting the gene for another Siglec that did not bind to any TLRs had no effect on the immune response. Chen et al. suggest that the Siglec proteins that interact with toll-like receptors act a bit like a brake that slows down the activation of the receptors. However, when an immune cell detects a foreign molecule through a TLR, an enzyme called Neu1 is relocated from the inside of the cell to the cell's surface, where it removes the sugar molecules from the TLRs. This disrupts the interaction between the TLRs and the Siglecs, thus activating the receptors and triggering an immune response against the invading pathogen or damaged cells. This represents a newly discovered mechanism that can regulate the signaling of TLRs. Chen et al. also show that a chemical compound that stops the function of the Neu1 enzyme prevents the toll-like receptors—and hence the immune cells—from becoming overly activated. Mice treated with this compound are protected against sepsis triggered by the presence of a bacterial PAMP. These results suggest that the Neu1 enzyme may be a promising new target for treating sepsis; further work will now be required to assess the potential side effects caused by inhibiting this enzyme. DOI: http://dx.doi.org/10.7554/eLife.04066.002

Perivascular adipose tissue (PVAT), long assumed to be nothing more than vessel-supporting connective tissue, is now understood to be an important, active component of the vasculature, with integral roles in vascular health and disease. PVAT is an adipose tissue with similarities to both brown and white adipose tissue, although recent evidence suggests that PVAT develops from its own precursors. Like other adipose tissue depots, PVAT secretes numerous biologically active substances that can act in both autocrine and paracrine fashion. PVAT has also proven to be involved in vascular inflammation. Although PVAT can support inflammation during atherosclerosis via macrophage accumulation, emerging evidence suggests that PVAT also has antiatherosclerotic properties related to its abilities to induce nonshivering thermogenesis and metabolize fatty acids. We here discuss the accumulated knowledge of PVAT biology and related research on models of hypertension and atherosclerosis.

Aim The development of de novo donor-specific antibodies (DSA) post-transplant is an area of interest for the transplant community. There are several challenges in detecting DSA, including the use of recombinant HLA antigens vs. affinity-purified antigens from a phenotype, and the lack of donor typing at many loci. Herein, we describe a case where surrogate flow-cytometric crossmatch (FCXM) was used in conjunction with solid-phase immunoassays for the detection of DSA in a heart transplant recipient. Methods Post-transplant serum was tested by solid-phase immunoassays for the detection of DSA to the following donor antigens: A2,23; B7,8; Bw6; Cw7; DR15,17; DR52; DQ2,6. Confirmation of antibody specificity was obtained using FCXM using three surrogate donor cells. Results Initial testing by One Lambda LABScreen® PRA and Single Antigen assays provided negative results for HLA class I, and inconclusive results for HLA class II, suggesting possible reactivity to HLA-DQ2, HLA-DQA1∗05:01/DQB1∗02:01, or false-positive reactivity. In order to confirm the possible specificities obtained, surrogate crossmatching was performed utilizing pronased cells from donors with the following HLA types: Donor 1- DQB1∗02:XX, 03:XX (DQ9); DQA1∗02:01; Donor 2- DQB1∗02:01; DQA1∗05:01; Donor 3- DQB1∗02:01, 03:01 (DQ7); DQA1∗05:01, with recipient serum at dilutions of Neat, 1:2, 1:4, and 1:16. T cell crossmatch results at all dilutions were negative for each donor cell tested. Donor 1 tested B cell negative at all dilutions tested, while donor 2 tested positive at serum dilutions of Neat, 1:2, and 1:4, and donor 3 tested positive at serum dilutions of Neat and 1:2. This evidence allowed for us to assign a strong antibody, defined by our laboratory as predicted to yield a positive FCXM, to the DQA1∗05:01/DQB1∗02:01 heterodimer. Since donor HLA-DQA1 typing was not available, it is inconclusive whether the antibody detected should be considered donor-specific. Conclusion We conclude that while solid-phase immunoassays are useful tools for the detection of HLA antibodies, there are several challenges present which could require the use of additional assays for confirmation of test results. In addition, the lack of complete donor typing often makes it difficult for the laboratory to accurately interpret the findings of such results.

Aim Next-generation sequencing (NGS) assays for HLA typing are rapidly being introduced into clinical practice. For common NGS assays, sequenced libraries consist of a mixture of labeled DNA fragments from multiple amplicons and samples. During our validation of a commercially-available NGS assay, we found sample and locus representation varied considerably, compromising data quality. In order to ensure even amplicon and sample loading, we optimized the locus and sample pooling steps. Methods The NGSgo HLA typing kit (GenDx) was used to type the HLA-A, B, C, DRB1, DRB3/4/5, DQB1, DQA1, DPB1 and DPA1 loci, with modifications to the manufacturer’s protocol described below. After PCR amplification, each amplicon was quantified using Quantifluor One dsDNA dye (Promega). Equimolar amounts of each amplicon from a single sample were pooled, and fragmentation, adapter ligation, and indexing steps were performed per the manufacturer’s protocol. Before pooling to a single library, each patient sample was quantified by two Methods KAPA Library Quantification (Kapa Biosystems) for the number of viable DNA libraries, and TapeStation gel electrophoresis (Agilent) for fragment size measurement. These two metrics were used to estimate equimolar pooling of each patient sample into a final library pool before being loaded onto a MiSeq (Ilumina) for sequencing. Results After introduction of quantification of all amplicons and equimolar pooling, locus representation on a per-sample basis was much more even; loci ranged from 0.24 to 3.94 times ideal representation using the manufacture’s protocol, but only 0.70 to 1.5 times ideal representation after equimolar pooling. Likewise, sample-level variation in pooling was extreme using the manufacturer’s protocol (ranging from 0.26 to 3.39 times ideal representation), and was much more even after addition of KAPA and TapeStation quantification of individual samples (ranging from 0.76 to 1.19 times ideal representation). Conclusions Using the modifications described here, we were able to obtain evenly-loaded samples, which allowed for the use of smaller, less expensive flow cells, or the use of a shared portion of a larger flow cell. Together, these modifications enhance the data quality for all patient samples while reducing the overall cost of the NGS assay.

Aim The United Network for Organ Sharing (UNOS), the sole contractor for deceased-donor transplantation in the US, requires HLA type in serologic nomenclature. Currently, however, the vast majority of patients and donors are typed by molecular methods. The 2008 HLA dictionary (hla.alleles.org/dictionary), which lists serologic assignments for many HLA-A, B, C, DRB1, DRB3/4/5 and DQB1 alleles, is used to “translate” molecular typing to serologic equivalents. However, many HLA alleles have multiple or conflicting entries in the HLA dictionary, while many rare or newly-identified alleles are not present. Moreover, UNOS accepts many “serologic” designations that do not appear in the HLA dictionary. To overcome these limitations, we created an algorithm to compile an internal document for UNOS-specific HLA type. Methods The 2008 HLA dictionary and Organ Procurement and Transplantation Network standards were used as primary sources for HLA typing information. Predicted amino acid sequence was used to assign Bw4/w6 (Tissue Antigens 2001: 57: 474). Results The algorithm, summarized below, was used to determine serologic assignments, which we termed “UNOS equivalents.” Expressed DRB3, DRB4 or DRB5 alleles were listed as DR52, DR53, or DR51, respectively, regardless of HLA dictionary assignments. Similarly, HLA-C alleles with first-field numbers greater than 08 were assigned UNOS equivalents corresponding to their first-field numbers, regardless of HLA dictionary assignments. For other alleles, the “Expert” assignment was given precedence over the “WHO” assignment listed in the HLA dictionary. Alleles not appearing in the HLA dictionary were assigned UNOS equivalents based on the first-field number. For alleles with multiple or conflicting entries, an algorithm was written to result in a single UNOS equivalent. For B∗15 alleles with unknown Bw4/w6 assignments, a published algorithm was used. Conclusions The algorithm described here resulted in a document that standardizes the “translation” of HLA alleles to list in UNOS, so that a single UNOS equivalent can be obtained from any HLA-A, B, C, DRB1, DRB3/4/5, or DQB1 type. As subsequent serologic studies are published, this document can serve as a framework for determining typings to enter into UNOS, to facilitate efficient sharing of deceased donor organs.

SummaryAnti-HER2/neu antibody therapy is reported to mediate tumor regression by interrupting oncogenic signals and/or inducing FcR-mediated cytotoxicity. Here, we demonstrate that the mechanisms of tumor regression by this therapy also require the adaptive immune response. Activation of innate immunity and T cells, initiated by antibody treatment, was necessary. Intriguingly, the addition of chemotherapeutic drugs, although capable of enhancing the reduction of tumor burden, could abrogate antibody-initiated immunity leading to decreased resistance to rechallenge or earlier relapse. Increased influx of both innate and adaptive immune cells into the tumor microenvironment by a selected immunotherapy further enhanced subsequent antibody-induced immunity, leading to increased tumor eradication and resistance to rechallenge. This study proposes a model and strategy for anti-HER2/neu antibody-mediated tumor clearance.

Murine experimental autoimmune thyroiditis (EAT) is a model for Hashimoto's thyroiditis that has served as a prototype of T cell-mediated autoimmunity for more than three decades. Key roles for MHC restriction and autoantigen influence on susceptibility to autoimmunity have been demonstrated in EAT. Moreover, it has served a unique role in investigations of self tolerance. In the early 1980s, self tolerance and resistance to EAT induction could be enhanced by increasing circulating levels of the autoantigen, thyroglobulin (Tg), by exogenous addition as well as endogenous release. This observation, directly linking circulating self antigen to self tolerance, led to subsequent investigations of the role of regulatory T cells (Tregs) in self tolerance. These studies revealed that protection against autoimmunity, in both naive and tolerized mice, was mediated by thymically-derived CD4(+)CD25(+)Foxp3(+) Tregs. Moreover, these naturally-existing Tregs required proper costimulation, in context with autoantigen presentation, to maintain and enhance self tolerance. In particular was the selected use of MHC- and heterologous Tg-restricted models from both conventional and transgenic mice. These models helped to elucidate the complex interplay between autoantigen presentation and MHC class II-mediated T cell selection in the development of Treg and autoreactive T cell repertoires determining susceptibility to autoimmunity. Here we describe these investigations in further detail, providing a context for how EAT has helped shape our understanding of self tolerance and autoimmunity.

Susceptibility and resistance to experimental autoimmune thyroiditis is encoded by MHC H2A genes. We reported that traditionally resistant B10 (H2b) mice permit thyroiditis induction with mouse thyroglobulin (mTg) after depleting regulatory T cells (Tregs), supporting Ab presentation to thyroiditogenic T cells. Yet, Eak transgenic mice, expressing Ab and normally absent Eb molecules (E+B10 mice), are susceptible to thyroiditis induction without Treg depletion. To explore the effect of Eb expression on mTg presentation by Ab, seven putative Ab-binding, 15–16-mer peptides were synthesized. Five were immunogenic for both B10 and E+B10 mice. The effect of Eb expression was tested by competition with an Eα52-68 peptide, because Eα52-68 occupies ∼15% of Ab molecules in E+B10 mice, binding with high affinity. Eα52-68 competitively reduced the proliferative response to mTg, mTg1677, and mTg2342 of lymph node cells primed to each Ag. Moreover, mTg1677 induced mild thyroiditis in Treg-depleted B10 mice, and in E+B10 mice without the need for Treg depletion. Eα52-68 competition with mTg-derived peptides may impede clonal deletion of pathogenic, mTg-specific T cells in the thymus.

The A-E+ transgenic mouse is highly susceptible to human thyroglobulin (hTg)-induced thyroiditis, but strongly tolerant to a challenge by mouse thyroglobulin (mTg), in stark contrast to traditionally susceptible strains, wherein mTg induces stronger thyroiditis. To identify mouse thyroid epitopes recognized by destructive, hTg-primed T cells, we selected the three hTg epitopes known to be presented by H2E(b), as the basis for synthesizing potential mTg epitopes. One 15-mer peptide, mTg409, did prime T cells, elicit Ab, and induce thyroiditis. Moreover, cells primed with corresponding, pathogenic hTg410 cross-reacted with mTg409, and vice versa. mTg409 contained 4/4 anchor residues, similar to the corresponding hTg peptide. Based on this finding, a second mTg epitope, mTg179, was subsequently identified. These mTg autoepitopes, identified by using thyroiditogenic hTg epitopes, help to explain the severe thyroiditis seen in this novel A-E+ transgenic model.

Aim One of the limitations of solid-phase HLA antibody assays is the false low/negative reading in a subset of samples with high titer/strength HLA antibodies, a phenomenon known as the prozone effect. The goal of this study was to compare the efficacy of ethylenediaminetetraacetic acid (EDTA) vs. dithiothreitol (DTT) treatment of serum samples in overcoming the prozone effect in solid-phase HLA antibody assays on the Luminex platform. Methods A total of 21 serum samples were tested by single-antigen bead assays (One Lambda), including 14 previously identified prozone samples (3 class I, 11 class II), 6 previously identified non-prozone samples with HLA antibodies (3 each for class I and II), and 1 negative sample. Serum was treated either by adding EDTA to a final concentration of 5 mM, or by incubating with 5 mM DTT for 30 min at 37 °C. To evaluate the efficacy of prozone effect reversal on HLA single antigen beads assay, fourfold serial dilutions, from neat to 1:256, were tested for each positive sample, using PBS as a diluent. Results Prozone effects were reduced in all 14 samples by either EDTA or DTT treatment. Results revealed sharp increases of MFI values in treated serum samples at neat, compared to non-treated samples. However, EDTA and DTT treatment were not equally efficient in overcoming the prozone effect. EDTA had nearly 100% reversal of prozone effect in all the tested samples, estimated by the ratio of MFI at neat to peak MFI at any given dilution. In contrast, the efficacy by DTT treatment ranged from 47% to 100%. The difference between the treatments was particularly notable in samples with strong prozone effects, defined by peak MFI values beyond a 1:16 dilution with untreated samples. Consequently, EDTA-treated serum samples showed virtually no further MFI increase when diluted, while some DTT-treated serum samples still had MFI increases when diluted, an indicator of residual prozone effect. For the negative control sample as well as non-prozone samples, comparable results were obtained from EDTA, DTT and non-treated serum. Conclusions Our study indicates that EDTA is more efficient in reversing the prozone effect, compared to DTT treatment. Furthermore, the stability of EDTA and the simplicity and robustness of serum treatment provide additional advantages over DTT for routine clinical testing.

Aim Sanger sequencing-based typing (SBT) requires multiple steps to sequence novel HLA alleles, as allele-specific (AS) primers are needed to separate novel from other encoded alleles. Next-generation sequencing (NGS) includes phase information, and has the potential to sequence novel alleles without AS primers. Here we compare the process of novel allele identification and sequencing using SBT and NGS assays. Methods Novel alleles were identified in peripheral blood during routine clinical testing using Atria SBT kits (Abbot Molecular). Germline encoding was verified by typing buccal samples. For Sanger sequencing of novel alleles, AS amplification primers were designed to take advantage of nucleotide polymorphisms between encoded alleles, and patients with homozygous loci were used to verify primer specificity. Amplified products were Sanger sequenced using custom or commercially-available primers. For NGS sequencing, the Holotype X4 (Omixon) kit was used in its default configuration. Results Four novel, germline-encoded HLA alleles were identified in three patients. Novel HLA-A and B alleles were sequenced using AS amplification primers and Sanger sequencing. A novel DQB1 allele previously identified but not resolved by SBT was sequenced using the NGS assay. A novel DQB1 allele not identified by SBT, as the polymorphism was outside the covered region, was identified and sequenced by NGS. The names HLA-A∗66:22, B∗07:238, DQB1∗03:180, and DQB1∗06:01:15 have been officially assigned by the WHO Nomenclature Committee. Conclusions While SBT assays used in the clinical setting are able to identify novel alleles, subsequent sequencing is a cumbersome and time-consuming process. Furthermore, SBT assays may miss novel polymorphisms outside of the exons covered by the assay. NGS systems, on the other hand, can identify and sequence novel alleles over a larger genomic region as part of the normal clinical workflow. However, we experienced issues with the Omixon NGS kit, including the inability to sequence buccal cell DNA, and difficulties amplifying the DQB1 locus, which could have impacted clinical workflow had NGS been the primary method of testing. As the emerging technology of NGS is introduced to complement established SBT assays, it is important to evaluate the strengths and weaknesses of each.

Co-inhibitory pathways are thought to act in later stages of an adaptive immune response, but whether co-inhibition contributes to early innate immunity is unclear. We show that engagement of the newly discovered co-inhibitory receptor B and T lymphocyte attenuator (BTLA) by herpesvirus entry mediator (HVEM) is critical for negatively regulating early host immunity against intracellular bacteria. Both HVEM-/- and BTLA-/-, but not LIGHT-/-, mice are more resistant to listeriosis compared to wild type mice, and blockade of the BTLA pathway promotes, while engagement inhibits, early bacterial clearance. Differences in bacterial clearance were seen as early as 1 day post-infection, implicating the initial innate response. Therefore, innate cell function in BTLA-/- mice was studied. We show that innate cells from BTLA-/- mice have a lower threshold for activation, and secrete significantly more pro-inflammatory cytokines upon stimulation with listeria. These results provide the first evidence that a co-inhibitory pathway plays a critical role in regulating early host immunity against infection. (Supported by CA115540, AI062026, and DK58897 to YXF and T32AI007090 to NKB)