HLA alleles and haplotypes observed in 263 US families.

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

A Clinician's Guide to Celiac Disease HLA Genetics.

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.

Effective desensitization for a strong donor-specific HLA antibody in a case of HLA-mismatched allogeneic hematopoietic cell transplantation.

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.

CD160 is essential for NK-mediated IFN-γ production.

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.

Broad and direct interaction between TLR and Siglec families of pattern recognition receptors and its regulation by Neu1.

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.

Efficacy of HLA-DRB1∗03:01 and H2E transgenic mouse strains to correlate pathogenic thyroglobulin epitopes for autoimmune thyroiditis.

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.

Direct and indirect roles of the LTbetaR pathway in central tolerance induction.

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.

The therapeutic effect of anti-HER2/neu antibody depends on both innate and adaptive immunity.

Anti-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.

Autoimmune thyroiditis: a model uniquely suited to probe regulatory T cell function.

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.

B and T lymphocyte attenuator tempers early infection immunity.

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.

Naturally-existing CD4(+)CD25(+)Foxp3(+) regulatory T cells are required for tolerance to experimental autoimmune thyroiditis induced by either exogenous or endogenous autoantigen.

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 novel H2A-E+ transgenic model susceptible to human but not mouse thyroglobulin-induced autoimmune thyroiditis: identification of mouse pathogenic epitopes.

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.

H2E-derived Ealpha52-68 peptide presented by H2Ab interferes with clonal deletion of autoreactive T cells in autoimmune thyroiditis.

Susceptibility and resistance to experimental autoimmune thyroiditis is encoded by MHC H2A genes. We reported that traditionally resistant B10 (H2(b)) mice permit thyroiditis induction with mouse thyroglobulin (mTg) after depleting regulatory T cells (Tregs), supporting A(b) presentation to thyroiditogenic T cells. Yet, Ea(k) transgenic mice, expressing A(b) and normally absent E(b) molecules (E(+)B10 mice), are susceptible to thyroiditis induction without Treg depletion. To explore the effect of E(b) expression on mTg presentation by A(b), seven putative A(b)-binding, 15-16-mer peptides were synthesized. Five were immunogenic for both B10 and E(+)B10 mice. The effect of E(b) expression was tested by competition with an Ealpha52-68 peptide, because Ealpha52-68 occupies approximately 15% of A(b) molecules in E(+)B10 mice, binding with high affinity. Ealpha52-68 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. Ealpha52-68 competition with mTg-derived peptides may impede clonal deletion of pathogenic, mTg-specific T cells in the thymus.