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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.

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.

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.

Tu et al. generated a novel CD160-deficient mouse and showed impaired NK cell–mediated tumor elimination and IFN-γ production. CD160+ NK cells are functionally distinct in secretion of IFN-γ from their CD160− NK cell counterparts., NK-derived cytokines play important roles for natural killer (NK) function, but how the cytokines are regulated is poorly understood. CD160 is expressed on activated NK or T cells in humans but its function is unknown. We generated CD160-deficient mice to probe its function. Although CD160−/− mice showed no abnormalities in lymphocyte development, the control of NK-sensitive tumors was severely compromised in CD160−/− mice. Surprisingly, the cytotoxicity of NK cells was not impaired, but interferon-γ (IFN-γ) secretion by NK cells was markedly reduced in CD160−/− mice. Functionally targeting CD160 signaling with a soluble CD160-Ig also impaired tumor control and IFN-γ production, suggesting an active role of CD160 signaling. Using reciprocal bone marrow transfer and cell culture, we have identified the intrinsic role of CD160 on NK cells, as well as its receptor on non-NK cells, for regulating cytokine production. To demonstrate sufficiency of the CD160+ NK cell subset in controlling NK-dependent tumor growth, intratumoral transfer of the CD160+ NK fraction led to tumor regression in CD160−/− tumor-bearing mice, indicating demonstrable therapeutic potential for controlling early tumors. Therefore, CD160 is not only an important biomarker but also functionally controls cytokine production by NK cells.

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.

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.

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.

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

Siglec-G/10 is broadly expressed on B cells, dendritic cells and macrophage subsets. It binds strongly to CD24, a small glycosyl-phosphatidylinositol-anchored sialoprotein, in a sialylation-dependent manner. Targeted mutation of Siglecg dramatically elevates the level of natural IgM antibodies and its producer, B1 B cells. Incorporation of Siglec-G ligands to both T-dependent and T-independent immunogens reduces antibody production and induces B-cell tolerance to subsequent antigen challenges. By interacting with CD24, Siglec-G suppresses inflammatory responses to danger (damage)-associated molecular patterns, such as heat-shock proteins and high mobility group protein 1, but not to Toll-like receptor ligands. By a CD24-independent mechanism, Siglec-G has been shown to associate with Cbl to cause degradation of retinoic acid-inducible gene 1 and reduce production of type I interferon in response to RNA virus infection. The negative regulation by Siglec-G/10 may provide a mechanism for the host to discriminate between infectious nonself and noninfectious self, as envisioned by the late Dr. Charles A. Janeway.

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.

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 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.