IFNÉ£ but not IFNα increases recognition of insulin defective ribosomal product-derived antigen to amplify islet autoimmunity.

AIMS/HYPOTHESIS: The inflammatory milieu characteristic of insulitis affects translation fidelity and generates defective ribosomal products (DRiPs) that participate in autoimmune beta cell destruction in type 1 diabetes. Here, we studied the role of early innate cytokines (IFNα) and late immune adaptive events (IFNÉ£) in insulin DRiP-derived peptide presentation to diabetogenic CD8+ T cells. METHODS: Single-cell transcriptomics of human pancreatic islets was used to study the composition of the (immuno)proteasome. Specific inhibition of the immunoproteasome catalytic subunits was achieved using siRNA, and antigenic peptide presentation at the cell surface of the human beta cell line EndoC-ßH1 was monitored using peptide-specific CD8 T cells. RESULTS: We found that IFNγ induces the expression of the PSMB10 transcript encoding the ß2i catalytic subunit of the immunoproteasome in endocrine beta cells, revealing a critical role in insulin DRiP-derived peptide presentation to T cells. Moreover, we showed that PSMB10 is upregulated in a beta cell subset that is preferentially destroyed in the pancreases of individuals with type 1 diabetes. CONCLUSIONS/INTERPRETATION: Our data highlight the role of the degradation machinery in beta cell immunogenicity and emphasise the need for evaluation of targeted immunoproteasome inhibitors to limit beta cell destruction in type 1 diabetes. DATA AVAILABILITY: The single-cell RNA-seq dataset is available from the Gene Expression Omnibus (GEO) using the accession number GSE218316 ( https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE218316 ).

Presence of immunogenic alternatively spliced insulin gene product in human pancreatic delta cells.

AIMS/HYPOTHESIS: Transcriptome analyses revealed insulin-gene-derived transcripts in non-beta endocrine islet cells. We studied alternative splicing of human INS mRNA in pancreatic islets. METHODS: Alternative splicing of insulin pre-mRNA was determined by PCR analysis performed on human islet RNA and single-cell RNA-seq analysis. Antisera were generated to detect insulin variants in human pancreatic tissue using immunohistochemistry, electron microscopy and single-cell western blot to confirm the expression of insulin variants. Cytotoxic T lymphocyte (CTL) activation was determined by MIP-1ß release. RESULTS: We identified an alternatively spliced INS product. This variant encodes the complete insulin signal peptide and B chain and an alternative C-terminus that largely overlaps with a previously identified defective ribosomal product of INS. Immunohistochemical analysis revealed that the translation product of this INS-derived splice transcript was detectable in somatostatin-producing delta cells but not in beta cells; this was confirmed by light and electron microscopy. Expression of this alternatively spliced INS product activated preproinsulin-specific CTLs in vitro. The exclusive presence of this alternatively spliced INS product in delta cells may be explained by its clearance from beta cells by insulin-degrading enzyme capturing its insulin B chain fragment and a lack of insulin-degrading enzyme expression in delta cells. CONCLUSIONS/INTERPRETATION: Our data demonstrate that delta cells can express an INS product derived from alternative splicing, containing both the diabetogenic insulin signal peptide and B chain, in their secretory granules. We propose that this alternative INS product may play a role in islet autoimmunity and pathology, as well as endocrine or paracrine function or islet development and endocrine destiny, and transdifferentiation between endocrine cells. INS promoter activity is not confined to beta cells and should be used with care when assigning beta cell identity and selectivity. DATA AVAILABILITY: The full EM dataset is available via www.nanotomy.org (for review: http://www.nanotomy.org/OA/Tienhoven2021SUB/6126-368/ ). Single-cell RNA-seq data was made available by Segerstolpe et al [13] and can be found at https://sandberglab.se/pancreas . The RNA and protein sequence of INS-splice was uploaded to GenBank (BankIt2546444 INS-splice OM489474).

Diabetes risk loci-associated pathways are shared across metabolic tissues.

AIMS/HYPOTHESIS: Numerous genome-wide association studies have been performed to understand the influence of genetic variation on type 2 diabetes etiology. Many identified risk variants are located in non-coding and intergenic regions, which complicates understanding of how genes and their downstream pathways are influenced. An integrative data approach will help to understand the mechanism and consequences of identified risk variants. METHODS: In the current study we use our previously developed method CONQUER to overlap 403 type 2 diabetes risk variants with regulatory, expression and protein data to identify tissue-shared disease-relevant mechanisms. RESULTS: One SNP rs474513 was found to be an expression-, protein- and metabolite QTL. Rs474513 influenced LPA mRNA and protein levels in the pancreas and plasma, respectively. On the pathway level, in investigated tissues most SNPs linked to metabolism. However, in eleven of the twelve tissues investigated nine SNPs were linked to differential expression of the ribosome pathway. Furthermore, seven SNPs were linked to altered expression of genes linked to the immune system. Among them, rs601945 was found to influence multiple HLA genes, including HLA-DQA2, in all twelve tissues investigated. CONCLUSION: Our results show that in addition to the classical metabolism pathways, other pathways may be important to type 2 diabetes that show a potential overlap with type 1 diabetes.

Long RNA Sequencing and Ribosome Profiling of Inflamed ß-Cells Reveal an Extensive Translatome Landscape.

Type 1 diabetes (T1D) is an autoimmune disease characterized by autoreactive T cell-mediated destruction of the insulin-producing pancreatic ß-cells. Increasing evidence suggest that the ß-cells themselves contribute to their own destruction by generating neoantigens through the production of aberrant or modified proteins that escape central tolerance. We recently demonstrated that ribosomal infidelity amplified by stress could lead to the generation of neoantigens in human ß-cells, emphasizing the participation of nonconventional translation events in autoimmunity, as occurring in cancer or virus-infected tissues. Using a transcriptome-wide profiling approach to map translation initiation start sites in human ß-cells under standard and inflammatory conditions, we identify a completely new set of polypeptides derived from noncanonical start sites and translation initiation within long noncoding RNA. Our data underline the extreme diversity of the ß-cell translatome and may reveal new functional biomarkers for ß-cell distress, disease prediction and progression, and therapeutic intervention in T1D.

CONQUER: an interactive toolbox to understand functional consequences of GWAS hits.

Numerous large genome-wide association studies have been performed to understand the influence of genetics on traits. Many identified risk loci are in non-coding and intergenic regions, which complicates understanding how genes and their downstream pathways are influenced. An integrative data approach is required to understand the mechanism and consequences of identified risk loci. Here, we developed the R-package CONQUER. Data for SNPs of interest are acquired from static- and dynamic repositories (build GRCh38/hg38), including GTExPortal, Epigenomics Project, 4D genome database and genome browsers. All visualizations are fully interactive so that the user can immediately access the underlying data. CONQUER is a user-friendly tool to perform an integrative approach on multiple SNPs where risk loci are not seen as individual risk factors but rather as a network of risk factors.

Autoimmunity against a defective ribosomal insulin gene product in type 1 diabetes.

Identification of epitopes that are recognized by diabetogenic T cells and cause selective beta cell destruction in type 1 diabetes (T1D) has focused on peptides originating from native beta cell proteins. Translational errors represent a major potential source of antigenic peptides to which central immune tolerance is lacking. Here, we describe an alternative open reading frame within human insulin mRNA encoding a highly immunogenic polypeptide that is targeted by T cells in T1D patients. We show that cytotoxic T cells directed against the N-terminal peptide of this nonconventional product are present in the circulation of individuals diagnosed with T1D, and we provide direct evidence that such CD8+ T cells are capable of killing human beta cells and thereby may be diabetogenic. This study reveals a new source of nonconventional polypeptides that act as self-epitopes in clinical autoimmune disease.

T cell receptor reversed polarity recognition of a self-antigen major histocompatibility complex.

Central to adaptive immunity is the interaction between the αß T cell receptor (TCR) and peptide presented by the major histocompatibility complex (MHC) molecule. Presumably reflecting TCR-MHC bias and T cell signaling constraints, the TCR universally adopts a canonical polarity atop the MHC. We report the structures of two TCRs, derived from human induced T regulatory (iT(reg)) cells, complexed to an MHC class II molecule presenting a proinsulin-derived peptide. The ternary complexes revealed a 180° polarity reversal compared to all other TCR-peptide-MHC complex structures. Namely, the iT(reg) TCR α-chain and ß-chain are overlaid with the α-chain and ß-chain of MHC class II, respectively. Nevertheless, this TCR interaction elicited a peptide-reactive, MHC-restricted T cell signal. Thus TCRs are not 'hardwired' to interact with MHC molecules in a stereotypic manner to elicit a T cell signal, a finding that fundamentally challenges our understanding of TCR recognition.

Alternative splicing and differential expression of the islet autoantigen IGRP between pancreas and thymus contributes to immunogenicity of pancreatic islets but not diabetogenicity in humans.

AIMS/HYPOTHESIS: Thymic expression of self-antigens during T-lymphocyte development is believed to be crucial for preventing autoimmunity. It has been suggested that G6PC2, the gene encoding islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP), is differentially spliced between pancreatic beta cells and the thymus. This may contribute to incomplete elimination of IGRP-specific T lymphocytes in the thymus, predisposing individuals to type 1 diabetes. We tested whether specific splice variation in islets vs thymus correlates with loss of tolerance to IGRP in type 1 diabetes. METHODS: Expression of G6PC2 splice variants was compared among thymus, purified medullary thymic epithelial cells and pancreatic islets by RT-PCR. Differential immunogenicity of IGRP splice variants was tested in patients and healthy individuals for autoantibodies and specific cytotoxic T lymphocytes using radiobinding assays and HLA class I multimers, respectively. RESULTS: Previously reported G6PC2 splice variants, including full-length G6PC2, were confirmed, albeit that they occurred in both pancreas and thymus, rather than islets alone. Yet, their expression levels were profoundly greater in islets than in thymus. Moreover, three novel G6PC2 variants were discovered that occur in islets only, leading to protein truncations, frame shifts and neo-sequences prone to immunogenicity. However, autoantibodies to novel or known IGRP splice variants did not differ between patients and healthy individuals, and similar frequencies of IGRP-specific cytotoxic T lymphocytes could be detected in both patients with type 1 diabetes and healthy individuals. CONCLUSIONS/INTERPRETATION: We propose that post-transcriptional variation of tissue-specific self-proteins may affect negative thymic selection, although this need not necessarily lead to disease.

Islet-specific CTL cloned from a type 1 diabetes patient cause beta-cell destruction after engraftment into HLA-A2 transgenic NOD/scid/IL2RG null mice.

Despite increasing evidence that autoreactive CD8 T-cells are involved in both the initiation of type 1 diabetes (T1D) and the destruction of beta-cells, direct evidence for their destructive role in-vivo is lacking. To address a destructive role for autoreactive CD8 T-cells in human disease, we assessed the pathogenicity of a CD8 T-cell clone derived from a T1D donor and specific for an HLA-A2-restricted epitope of islet-specific glucose-6-phosphatase catalytic-subunit related protein (IGRP). HLA-A2/IGRP tetramer staining revealed a higher frequency of IGRP-specific CD8 T-cells in the peripheral blood of recent onset human individuals than of healthy donors. IGRP(265-273)-specific CD8 T-cells that were cloned from the peripheral blood of a recent onset T1D individual were shown to secrete IFNγ and Granzyme B after antigen-specific activation and lyse HLA-A2-expressing murine islets in-vitro. Lytic capacity was also demonstrated in-vivo by specific killing of peptide-pulsed target cells. Using the HLA-A2 NOD-scid IL2rγ(null) mouse model, HLA-A2-restricted IGRP-specific CD8 T-cells induced a destructive insulitis. Together, this is the first evidence that human HLA-restricted autoreactive CD8 T-cells target HLA-expressing beta-cells in-vivo, demonstrating the translational value of humanized mice to study mechanisms of disease and therapeutic intervention strategies.

Association analysis of myosin IXB and type 1 diabetes.

To date, seven studies have provided evidence for an association between the gene encoding for myosin IXB (MYO9B) and celiac disease (CD), and inflammatory bowel diseases, including single nucleotide polymorphisms (SNPs) rs2305767, rs1457092, and rs2305764. We investigated whether MYO9B is associated with T1D. The three SNPs were genotyped in Dutch samples from 288 T1D patients and 1615 controls. The A allele of SNP rs2305767A>G showed some evidence of association with T1D (nominal p for genotype = 0.06; OR carrier = 1.51, 95% CI = 1.04-2.19), but not in British samples from 4301 case patients and 4706 controls (p = 0.53), or when the Dutch and UK data were pooled (N patients = 4582, N controls= 6224; Mantel-Hansel p = 0.83). Furthermore, the nonsynonymous rs1545620 C>A SNP that has been associated with the inflammatory bowel disease, showed no association with T1D in British case-control set (p = 0.57). We conclude that MYO9B might not be a strong determinant of T1D, although there was some association in our initial Dutch study. Further studies are needed to evaluate the role of MYO9B in T1D.

Induction of Treg by monocyte-derived DC modulated by vitamin D3 or dexamethasone: differential role for PD-L1.

Specific therapy with modulated DC may restore immunological tolerance, thereby obviating the need for chronic immunosuppression in transplantation or autoimmunity. In this study we compared the tolerizing capacity of dexamethasone (Dex)- and 1 alpha,25-dihydroxyvitamin D3 (VD3)-modulated DC. Treatment of monocytes with either VD3 or Dex resulted in DC with stable, semi-mature phenotypes compared with standard DC, with intermediate levels of co-stimulatory and MHC class II molecules, which remained unaltered after subsequent pro-inflammatory stimulation. IL-12p70 secretion was lost by VD3- and Dex-DC, whereas IL-10 secretion was unaffected. VD3-DC distinctly produced large amounts of TNF-alpha. Both VD3- and Dex-DC possessed the capacity to convert CD4 T cells into IL-10-secreting Treg potently suppressing the proliferation of responder T cells. However, only Treg induced by VD3-DC exhibited antigen specificity. VD3-, but not Dex-, DC expressed significant high levels of PD-L1 (programmed death-1 ligand), upon activation. Blockade of PD-L1 during priming redirected T cells to produce IFN-gamma instead of IL-10 and abolished acquisition of regulatory capacity. Our findings demonstrate that both VD3- and Dex-DC possess durable but differential tolerogenic features, acting via different mechanisms. Both are potentially useful to specifically down-regulate unwanted immune responses and induce immune tolerance. These modulated DC appear suitable as adjuvant in antigen-specific clinical vaccination intervention strategies.

Evidence for natural killer cell-mediated protection from metastasis formation in uveal melanoma patients.

PURPOSE: In uveal melanoma, low human leukocyte antigen (HLA) class I expression on primary tumors is associated with a decreased risk of metastasis. Consequently, it has been suggested that natural killer (NK) cells, which detect decreased expression of HLA class I, are involved in the immune control of metastases. In this study, three novel lines of evidence were identified that support a role for NK cells. METHODS: Uveal melanoma cell lines were used to determine the expression of NK cell receptor ligands (MICA, MICB, ULBP1-3, CD112, CD155, and HLA class I) and to examine sensitivity to lysis by human NK cell lines. Because interactions between polymorphic killer immunoglobulin receptors (KIRs) and HLA regulate NK cell function, KIR and HLA genotyping was performed on 154 patients with uveal melanoma and 222 healthy control subjects. RESULTS: First, all 11 uveal melanoma cell lines tested expressed ligands for activating as well as inhibitory NK cell receptors. Second, such cell lines were lysed efficiently by human NK cells in vitro. Finally, the HLA-C genotype was related to the risk of metastasis-related death in patients with uveal melanoma: The patients carrying HLA-C alleles encoding ligands for KIR2DL1 and KIR2DL2/3 (HLA-C group 1/group 2 heterozygous patients), both inhibitory NK receptors, had a longer metastasis-free survival than did those carrying HLA-C ligands for either KIR2DL1 (HLA-C group 2 homozygotes) or KIR2DL2/3 (HLA-C group 1 homozygotes). CONCLUSIONS: Together, the data support a role for NK cells in the prevention of uveal melanoma metastases.

Defective synthesis or association of T-cell receptor chains underlies loss of surface T-cell receptor-CD3 expression in enteropathy-associated T-cell lymphoma.

Enteropathy-associated T-cell lymphoma, an often fatal complication of celiac disease, can result from expansion of aberrant intraepithelial lymphocytes in refractory celiac disease type II (RCD II). Aberrant intraepithelial lymphocytes and lymphoma cells are intracellularly CD3epsilon(+) but lack expression of the T-cell receptor (TCR)-CD3 complex on the cell surface. It is unknown what causes the loss of TCR-CD3 expression. We report the isolation of a cell line from an RCD II patient with the characteristic phenotype of enteropathy-associated T-cell lymphoma. We demonstrate that in this cell line the TCR-alpha and -beta chains as well as the CD3gamma, CD3delta, CD3epsilon, and zeta-chains are present intracellularly and that assembly of the CD3gammaepsilon, CD3deltaepsilon, and zetazeta-dimers is normal. However, dimerization of the TCR chains and proper assembly of the TCR-CD3 complex are defective. On introduction of exogenous TCR-beta chains, but not of TCR-alpha chains, assembly and functional cell surface expression of the TCR-CD3 complex were restored. Defective synthesis of both TCR chains was found to underlie loss of TCR expression in similar cell lines isolated from 2 additional patients. (Pre)malignant transformation in RCD II thus correlates with defective synthesis or defective association of the TCR chains, resulting in loss of surface TCR-CD3 expression.

MICA marks additional risk factors for Type 1 diabetes on extended HLA haplotypes: an association and meta-analysis.

The association of the HLA complex on chromosome 6 does not explain total linkage of the HLA region to Type 1 Diabetes (T1D), leading to the hypothesis that there may be additional causal genes in the HLA region for immune-related disorders. Reports on the MHC Class I chain-related A (MICA) gene as candidate for association with T1D are contradicting. We investigated whether variation in MICA is associated to T1D in a cohort of 350 unrelated individuals with juvenile-onset T1D and 540 control subjects, followed by a meta-analysis of 14 studies. We also investigated an HLA-independent association for MICA with T1D. In our case-control study, we found that the MICA*A5 variant was significantly associated with an increased risk for T1D, while MICA*A6 was significantly associated with a decreased risk that was confirmed by our meta-analysis. However, the meta-analysis did not show an association of MICA*A5 T1D. Analysis of MICA alleles conditional on T1D-associated high-risk MHC class II haplotypes revealed that MICA*A6 was associated with an increased risk for T1D when this marker co-occurred with HLA DQ2DR17 T1D-risk-haplotypes. In contrast, MICA*A6 reduced the risk from the HLA DQ8DR4 T1D-risk haplotype. Moreover, MICA*A9 showed a significant association to increased risk for T1D on DQ8DR4 haplotypes. Co-inheritance of MICA*A6 with the HLA DQ2DR17 haplotype in T1D indicates this haplotype may carry the additional genetic factors for T1D, but our study does not support an independent association between MICA variants and T1D.

Elevated levels of mannose-binding lectin at clinical manifestation of type 1 diabetes in juveniles.

Mannose-binding lectin (MBL) is a recognition molecule of the lectin pathway of complement and a key component of innate immunity. MBL polymorphisms have been described that are associated with MBL serum concentration, impaired function, and diabetic complications. We investigated 86 new-onset juvenile type 1 diabetic patients and compared these with their nondiabetic siblings and healthy unrelated control subjects. Polymorphisms of MBL exon 1 and promoter were determined, and serum concentration and MBL-complex activity were measured. Although the genetic polymorphisms of MBL were not different between patients and control subjects, MBL serum concentration as well as MBL complex activity was significantly higher in new-onset diabetic patients compared with their siblings matched for high-producing MBL genotypes (P = 0.0018 and P = 0.0005, respectively). The increase in MBL complex activity in high-MBL-producing patients could only partially be explained by high MBL production, as demonstrated by an increased MBL complex activity-to-MBL concentration ratio (P = 0.004). We conclude that MBL serum concentration and complex activity are increased in early-onset diabetic patients upon manifestation independently of genetic predisposition to high MBL production, indicating a possible role in the immunopathogenesis of type 1 diabetes, in addition to the adaptive islet autoimmunity.

Mannose binding lectin gene polymorphisms confer a major risk for severe infections after liver transplantation.

BACKGROUND & AIMS: Infection is the primary cause of death after liver transplantation. Mannose binding lectin (MBL) is a recognition molecule of the lectin pathway of complement and a key component of innate immunity. MBL variant alleles have been described in the coding region of the MBL gene, which are associated with low MBL serum concentration and impaired MBL structure and function. The aims of our study were to establish the role of the liver in production of serum MBL and to evaluate the effect of MBL variant alleles on the susceptibility to infection after liver transplantation. METHODS: We investigated 49 patients undergoing orthotopic liver transplantation. MBL exon 1 and promoter polymorphisms were determined in patients and in liver donors. MBL serum concentration was determined before and during 1 year after transplantation. The incidence of clinically significant infections during this period was assessed. RESULTS: Transplantation of MBL wildtype recipients with donor livers carrying MBL variant alleles resulted in a rapid and pronounced decrease of serum MBL levels. This serum conversion was associated with the disappearance of high molecular weight MBL. No indication for extrahepatic production of serum MBL could be obtained. The presence of MBL variant alleles in the MBL gene of the donor liver, but not in the recipient, was associated with a strongly increased incidence of clinically significant infections after transplantation. CONCLUSIONS: Serum MBL is produced by the liver under strong genetic control. After liver transplantation, the MBL genotype of the donor liver is a major risk determinant for life-threatening infections.

HLA and smoking in prediction and prognosis of small cell lung cancer in autoimmune Lambert-Eaton myasthenic syndrome.

Patients with small cell lung cancer (SCLC) survive longer if they have the antibody-mediated Lambert-Eaton myasthenic syndrome (LEMS), making this autoimmune disorder a prototype disease for studying cancer immunosurveillance. Patients with nontumor LEMS (NT-LEMS) never develop SCLC but are otherwise indistinguishable clinically. Therefore, we have compared immunogenetic factors in SCLC-LEMS and NT-LEMS and studied their role in the pathogenesis of LEMS and survival from SCLC. In 48 British and 29 Dutch Caucasian LEMS patients, we studied clinical symptoms, antibody titers, HLA types and alleles at six nearby located microsatellite loci. Highly significant associations were found in NT-LEMS, which appeared strongest with HLA-B8, but also involved HLA-DQ2, -DR3 and six flanking microsatellite alleles. SCLC-LEMS patients were not different from controls. Smoking was a strong predictor of SCLC. In contrast, HLA-B8 positivity correlated with a decreased risk of SCLC even among the smokers. Moreover, in SCLC-LEMS patients, HLA-B8 positivity correlated with prolonged survival after LEMS onset. We propose that two distinct immunopathogenetic routes can lead to one clinically and serologically indistinguishable autoimmune myasthenic syndrome. HLA-DR3-B8 is strongly associated with LEMS in nontumor patients only. In other LEMS patients, SCLC apparently provides a powerful autoimmunogenic stimulus that overrides HLA restrictions in breaking tolerance to calcium channels. Moreover, negativity for HLA-B8 combined with smoking behavior points more strongly to an underlying SCLC and predicts a worse prognosis in SCLC-LEMS patients.

KIR in type 1 diabetes: disparate distribution of activating and inhibitory natural killer cell receptors in patients versus HLA-matched control subjects.

Killer cell immunoglobulin-like receptors (KIRs) modulate natural killer cell and T-cell function by interacting with HLA class 1 ligands on target cells. Both KIR and HLA are highly polymorphic. We studied the influence of KIR and HLA class 1 genes on the susceptibility to develop type 1 diabetes. The results showed increased numbers of activating KIR genes in patients compared with control subjects (P = 0.049). The combination of the activating KIR2DS2 gene, together with its putative HLA ligand, was present more frequently in patients than in diabetes high-risk HLA-matched control subjects (P = 0.030). Moreover, our results imply that an increase in activating KIR2DS2-HLA ligand pairs combined with a lack of inhibitory KIR-HLA ligand pairs is associated with an additional risk to develop type 1 diabetes in individuals with diabetes high-risk HLA alleles (P = 0.035). We propose that the genetic imbalance between KIR and their HLA class 1 ligands may enhance the activation of T-cells with a low affinity for pancreatic self-antigens, thereby contributing to the pathogenesis of type 1 diabetes.

Susceptibility loci for complex regional pain syndrome.

An association between HLA-DR13 and patients with complex regional pain syndrome (CRPS) who progressed towards multifocal or generalized tonic dystonia was recently reported. We now report on a new locus, centromeric in HLA-class I, which was significantly associated with a spontaneous development of CRPS, suggesting an interaction between trauma severity and genetic factors conferring CRPS susceptibility. Additionally, an association with the D6S1014 locus was found, supporting the previous finding of an association with HLA-DR13.