HLA-DR genotypes in patients with systemic lupus erythematosus in Taiwan.

BACKGROUND: Different human leukocyte antigen (HLA)-DR genotypes have been known to be associated with the risk of development of systemic lupus erythematosus (SLE) in different populations, although Lu et al. have reported previously that no correlation exists between the HLA-DR genotype and disease manifestation in SLE patients in Taiwan. We investigated the effects different HLA-DR genotypes had on SLE incidence in Taiwanese patients as to whether risk alleles were associated with different clinical manifestations, and the effects risk alleles had on the age of disease onset. METHODS: Two hundred thirty-four SLE patients and 346 healthy controls were enrolled. HLA-DR genotyping was performed with the HLA FluoGene DRDQ kit for each subject. Chi-square tests and t tests were performed for statistical analysis. RESULTS: HLA-DR2 was significantly more frequently found in SLE patients than in controls (odds ratio [OR] = 2.05, 95% CI, 1.44-2.92, p < 0.001). Notably, HLA-DR6 appeared to trend toward negative correlation with SLE, whereas HLA-DR8 appeared to trend toward positive correlation. HLA-DR2 patients had an earlier onset of disease as well as a higher prevalence of oral ulcer, avascular necrosis of bone, and renal involvement (lupus nephritis). CONCLUSION: HLA-DR2 was associated with SLE susceptibility in this Taiwanese population as well as lower age of disease onset and more severe clinical manifestations.

Obesity induced gut dysbiosis contributes to disease severity in an animal model of multiple sclerosis.

Background: Multiple sclerosis (MS) is an inflammatory and demyelinating disease of the CNS. The etiology of MS is complex, and results from the interaction of multiple environmental and genetic factors. Although human leukocyte antigen-HLA alleles such as HLA-DR2 and -DR3 are considered the strongest genetic factors, the environmental factors responsible for disease predisposition are not well understood. Recently, diet and gut microbiota have emerged as an important environmental factors linked to the increased incidence of MS. Especially, western diets rich in protein and fat have been linked to the increased incidence of obesity. Numerous clinical data indicate a role of obesity and gut microbiota in MS; however, the mechanistic link between gut microbiota and obesity in the pathobiology of MS remains unclear. The present study determines the mechanisms driving MS severity in the context of obesity utilizing a high-fat diet (HFD) induced obese HLA-DR3 class-II transgenic mouse model of MS. Methods: HLA-DR3 transgenic mice were kept on a standard HFD diet or Normal Chow (NC) for eight weeks. Gut microbiota composition and functional analysis were performed from the fecal DNA of mice. Experimental autoimmune encephalomyelitis-EAE (an animal model of MS) was induced by immunization with the proteolipid protein-PLP91-110 peptide in complete Freud's Adjuvant (CFA) and pertussis toxin. Results: We observed that HFD-induced obesity caused gut dysbiosis and severe disease compared to mice on NC. Amelioration of disease severity in mice depleted of gut microbiota suggested an important role of gut bacteria in severe EAE in obese mice. Fecal microbiota analysis in HFD mice shows gut microbiota alterations with an increase in the abundance of Proteobacteria and Desulfovibrionaceae bacteria and modulation of various bacterial metabolic pathways including bacterial hydrogen sulfide biosynthetic pathways. Finally, mice on HFD showed increased gut permeability and systemic inflammation suggesting a role gut barrier modulation in obesity induced disease severity. Conclusions: This study provides evidence for the involvement of the gut microbiome and associated metabolic pathways plus gut permeability in obesity-induced modulation of EAE disease severity. A better understanding of the same will be helpful to identify novel therapeutic targets to reduce disease severity in obese MS patients.

A short HLA-DRA isoform binds the HLA-DR2 heterodimer on the outer domain of the peptide-binding site.

The human leukocyte antigen (HLA) locus encodes a large group of proteins governing adaptive and innate immune responses. Among them, HLA class II proteins form α/ß heterodimers on the membrane of professional antigen-presenting cells (APCs), where they display both, self and pathogen-derived exogenous antigens to CD4+ T lymphocytes. We have previously shown that a shorter HLA-DRA isoform (sHLA-DRA) lacking 25 amino acids can be presented onto the cell membrane via binding to canonical HLA-DR2 heterodimers. Here, we employed atomistic molecular dynamics simulations to decipher the binding position of sHLA-DRA and its structural impact on functional regions of the HLA-DR2 molecule. We show that a loop region exposed only in the short isoform (residues R69 to G83) is responsible for binding to the outer domain of the HLA-DR2 peptide-binding site, and experimentally validated the critical role of F76 in mediating such interaction. Additionally, sHLA-DRA allosterically modifies the peptide-binding pocket conformation. In summary, this study unravels key molecular mechanisms underlying sHLA-DRA function, providing important insights into the role of full-length proteins in structural modulation of HLA class II receptors.

Association of F11R polymorphisms and gene expression with primary Sjögren's syndrome patients.

AIMS: F11R gene encodes junctional adhesion molecule-A protein (JAM-A), which is expressed in various types of cells and is involved in leukocyte extravasation during inflammation. Sjögren's syndrome (SS) is a chronic systemic inflammatory disease that involves lymphocytes invasion of exocrine glands. F11R has been studied in autoimmune diseases, but any association between F11R and SS has not yet been investigated. Therefore, experiments were undertaken to examine the relationships among F11R gene polymorphism, messenger RNA (mRNA) expression and SS patients. METHODS: Three hundred and twenty-nine patients with SS, and 223 healthy controls were enrolled in their recruitment from the Kaohsiung Medical University Hospital. Genomic DNA was extracted from peripheral blood mononuclear cells and gene polymorphisms were genotyped by TaqMan real-time polymerase chain reaction (PCR). F11R mRNA expression was quantitated by quantitative real-time PCR with TaqMan Gene Expression Assay. RESULTS: Our study showed the genotype -688A/C (rs6695707) was not found in relation to SS patients. The odds ratio of -436A/G (rs12567886) genotype was notably associated with less susceptibility of SS in human leukocyte antigen (HLA)-DR2 negative and HLA-DR3 negative individuals. F11R mRNA expression was lower in SS patients than in the cells of healthy controls. CONCLUSION: The result indicated that G allele of -436A/G genotype has the potential protective effect against SS disease condition. F11R mRNA was expressed significantly lower in SS patients.

The 3A6-TCR/superagonist/HLA-DR2a complex shows similar interface and reduced flexibility compared to the complex with self-peptide.

T-cell receptor (TCR) recognition of the myelin basic protein (MBP) peptide presented by major histocompatibility complex (MHC) protein HLA-DR2a, one of the MHC class II alleles associated with multiple sclerosis, is highly variable. Interactions in the trimolecular complex between the TCR of the MBP83-99-specific T cell clone 3A6 with the MBP-peptide/HLA-DR2a (abbreviated TCR/pMHC) lead to substantially different proliferative responses when comparing the wild-type decapeptide MBP90-99 and a superagonist peptide, which differs mainly in the residues that point toward the TCR. Here, we investigate the influence of the peptide sequence on the interface and intrinsic plasticity of the TCR/pMHC trimolecular and pMHC bimolecular complexes by molecular dynamics simulations. The intermolecular contacts at the TCR/pMHC interface are similar for the complexes with the superagonist and the MBP self-peptide. The orientation angle between TCR and pMHC fluctuates less in the complex with the superagonist peptide. Thus, the higher structural stability of the TCR/pMHC tripartite complex with the superagonist peptide, rather than a major difference in binding mode with respect to the self-peptide, seems to be responsible for the stronger proliferative response.

A laboratory practice that uses the polymerase chain reaction-sequence specific priming technique to rapidly screen for HLA-DR2 allotype from germline DNA in immunology course for undergraduate medical students.

In this article, we describe an in-house polymerase chain reaction-sequence specific priming (PCR-SSP) assay designed for undergraduate medical students as part of the experimental pathogen biology and immunology (EPBI) course. It screens human leukocyte antigen (HLA)-DR2 allotype from genomic DNA samples using a rapid and single-tube PCR technique, yielding definitive typing result without conventional post-amplification step like probing or Sanger sequencing. This laboratory exercise offers the undergraduate medical students an opportunity to learn about current molecular biology techniques in HLA genotyping with limited effort and cost, in addition to a better understanding of concepts presented in the classroom lectures. Upon completing this experiment module, the students show statistically significant improvement in several key indexes, such as the knowledge about the mainstream HLA DNA typing techniques, awareness of the relevance of this knowledge for their future scientific research, immunogenetics-related basic laboratory skills they acquire, and interest and desire for mastering this assay (all p < .05). This easy to implement set of experiments is composed of a two-session lab module occupying eight teaching hours, and has been run successfully in our laboratory.

Accelerated thymopoiesis and improved T-cell responses in HLA-A2/-DR2 transgenic BRGS-based human immune system mice.

Human immune system (HIS) mouse models provide a robust in vivo platform to study human immunity. Nevertheless, the signals that guide human lymphocyte differentiation in HIS mice remain poorly understood. Here, we have developed a novel Balb/c Rag2-/- Il2rg-/- SirpaNOD (BRGS) HIS mouse model expressing human HLA-A2 and -DR2 transgenes (BRGSA2DR2). When comparing BRGS and BRGSA2DR2 HIS mice engrafted with human CD34+ stem cells, a more rapid emergence of T cells in the circulation of hosts bearing human HLA was shown, which may reflect a more efficient human T-cell development in the mouse thymus. Development of CD4+ and CD8+ T cells was accelerated in BRGSA2DR2 HIS mice and generated more balanced B and T-cell compartments in peripheral lymphoid organs. Both B- and T-cell function appeared enhanced in the presence of human HLA transgenes with higher levels of class switched Ig, increased percentages of polyfunctional T cells and clear evidence for antigen-specific T-cell responses following immunization. Taken together, the presence of human HLA class I and II molecules can improve multiple aspects of human B- and T-cell homeostasis and function in the BRGS-based HIS mouse model.

Aire is not essential for regulating neuroinflammatory disease in mice transgenic for human autoimmune-diseases associated MHC class II genes HLA-DR2b and HLA-DR4.

The human autoimmune disease-associated HLA alleles HLA-DR2b (DRB1*1501) and HLA-DR4 (DRB1*0401) are strongly linked to increased susceptibility for multiple sclerosis (MS) and rheumatoid arthritis (RA), respectively. The underlying mechanisms are not fully understood, but these MHC alleles may shape the repertoire of pathogenic T cells via central tolerance. The transcription factor autoimmune regulator (AIRE) promotes central T cell tolerance via ectopic expression of tissue-specific antigens (TSAs). Aire deficiency in humans causes autoimmune polyendocrinopathy syndrome type 1 (APS1), and Aire knockout mice (Aire-/-) develop spontaneous autoimmune pathology characterized by multi-organ lymphocytic infiltrates. Here, we asked whether impaired TSAs gene expression in the absence of Aire promoted spontaneous MS- or RA-like autoimmune pathology in the context of human HLA alleles in HLA-DR2b or HLA-DR4 transgenic (tg) mice. The results show that reduced TSAs gene expression in the thymus of Aire-deficient HLA-DR2b or HLA-DR4 tg mice corresponded to mild spontaneous inflammatory infiltrates in salivary glands, liver, and pancreas. Moreover, Aire-deficiency modestly enhanced experimental autoimmune encephalomyelitis (EAE) in HLA-DR tg mice, but the animals did not show signs of spontaneous neuroinflammation or arthritis. No significant changes were observed in CD4+ T cell numbers, T cell receptor (TCR) distribution, regulatory T cells (Treg), or antigen-induced cytokine production. Abrogating Treg function by treatment with anti-CTLA-4 or anti-CD25 mAb in Aire-deficient HLA-DR tg mice did not trigger EAE or other autoimmune pathology. Our results suggest a redundant role for Aire in maintaining immune tolerance in the context of autoimmune disease-associated human HLA alleles.

Absence of the tag polymorphism for the risk haplotype HLA-DR2 for multiple sclerosis in Wixárika subjects from Mexico.

The HLA-DRB1*15:01 allele has a demonstrated risk for the development of multiple sclerosis (MS) in most populations around the world. The single nucleotide polymorphism (SNP) rs3129934 is found in linkage disequilibrium with the risk haplotype formed by the HLA-DRB1*15:01 and HLA-DQB1*06:02 alleles, and it is considered a reliable marker of the presence of this haplotype. Native Americans have a null or low prevalence of MS. In this study, we sought to identify the frequency of rs3129934 in the Wixárika ethnic group as well as in Mestizo (mixed race) patients with MS and in controls from western Mexico. Through real-time polymerase chain reaction (PCR) using TaqMan probes, we analyzed the allele and genotype frequencies of rs3129934 in Mestizo individuals with and without MS and in 73 Wixárika subjects from the state of Jalisco, Mexico. The Wixárika subjects were homozygote for the C allele of rs3129934. The allele and genotype frequency in Mestizos with MS was similar to that of other MS populations with Caucasian ancestry. The absence of the T risk allele rs3129934 (associated with the haplotype HLA-DRB1*15:01, HLA-DQ1*06:02) in this sample of Wixárika subjects is consistent with the unreported MS in this Amerindian group, related to absence of such paramount genetic risk factor.

Gut dysbiosis breaks immunological tolerance toward the central nervous system during young adulthood.

Multiple sclerosis (MS) is an autoimmune disease targeting the central nervous system (CNS) mainly in young adults, and a breakage of immune tolerance to CNS self-antigens has been suggested to initiate CNS autoimmunity. Age and microbial infection are well-known factors involved in the development of autoimmune diseases, including MS. Recent studies have suggested that alterations in the gut microbiota, referred to as dysbiosis, are associated with MS. However, it is still largely unknown how gut dysbiosis affects the onset and progression of CNS autoimmunity. In this study, we investigated the effects of age and gut dysbiosis on the development of CNS autoimmunity in humanized transgenic mice expressing the MS-associated MHC class II (MHC-II) gene, HLA-DR2a, and T-cell receptor (TCR) genes specific for MBP87-99/DR2a that were derived from an MS patient. We show here that the induction of gut dysbiosis triggers the development of spontaneous experimental autoimmune encephalomyelitis (EAE) during adolescence and early young adulthood, while an increase in immunological tolerance with aging suppresses disease onset after late young adulthood in mice. Furthermore, gut dysbiosis induces the expression of complement C3 and production of the anaphylatoxin C3a, and down-regulates the expression of the Foxp3 gene and anergy-related E3 ubiquitin ligase genes. Consequently, gut dysbiosis was able to trigger the development of encephalitogenic T cells and promote the induction of EAE during the age window of young adulthood.

Potential molecular mimicry between the human endogenous retrovirus W family envelope proteins and myelin proteins in multiple sclerosis.

Multiple sclerosis is an autoimmune disease caused by the destruction of the myelin sheath in the central nervous system. The major target molecules for the immune response are the myelin basic protein, myelin oligodendrocyte glycoprotein and proteolipid protein but the aetiology of the disease is as yet poorly understood. The HLA Class II allele DRB1*1501 in particular as well as DRB5*0101 and the expression of human endogenous retroviral envelope proteins have been linked to multiple sclerosis but the molecular mechanisms relating these remain to be elucidated. We hypothesised that cross-reactive peptide epitopes in retroviral envelope proteins and myelin proteins that can be presented by the two Class II DR molecules may play a role in initiating multiple sclerosis. Sequence homologies between retroviral envelope and myelin proteins and in silico predictions of peptides derived from them that are able to bind to the two Class II alleles were examined to test the hypothesis. The results support the hypothesis that molecular mimicry in peptide epitopes from envelope proteins of the HERV-W family of endogenous retroviruses and myelin proteins is possible and could potentially trigger multiple sclerosis. Mimicry between syncytin-1, a HERV-W envelope protein that is expressed during placentation, and myelin proteins may also explain the higher prevalence of multiple sclerosis in women. Experiments to test the ability of the identified peptide epitopes to activate TH cells are required to confirm the present findings.

Influence of HLA-DR polymorphism and allergic sensitization on humoral immune responses to intact pneumococcus in a transgenic mouse model.

Asthma is independently associated with HLA-DR3 and increased risks of pneumococcal diseases. We aimed to determine whether HLA-DR polymorphism (HLA-DRB1*03), sensitization to house dust mite (HDM), or their interaction affects humoral immune responses to pneumococcal polysaccharide and protein antigens of intact pneumococci. Induction of serum titers of anti-pneumococcal polysaccharide and anti-surface protein IgM and IgG in response to immunization with intact pneumococci (Pn) serotype 14 was determined using humanized HLA-DR3 and DR2 transgenic mice. Transgenic mice were sensitized by injecting HDM and challenged with intranasal HDM. Mice were subsequently immunized with heat-killed Pn14 at day 24. Serum titers of anti-phosphorylcholine (PC) IgM and IgG, anti-pneumococcal polysaccharide, capsular type 14 (PPS14) IgM and IgG, and anti-pneumococcal surface protein A (PspA) IgG were measured. We included a total of 44 mice (22 DR3 and 22 DR2 mice) and half of mice in each group were sensitized with HDM (i.e. 22 HDM-sensitized and 22 control mice). HDM-sensitized mice, irrespective of HLA-DR polymorphism, had significantly lower humoral immune responses. HLA-DR3 mice, irrespective of HDM sensitization, elicited a significantly lower anti-PC IgG response. In contrast, the anti-PspA IgG response was higher in DR3 relative to DR2 mice. The effect of HDM sensitization on lowering humoral immune responses to Pn14 was observed in DR3 mice regardless of the nature of the antigen, whereas such decreases were observed only for the anti-PPS14 IgG and anti-PC IgM responses in DR2 mice. HDM sensitization lowered humoral immune responses to intact pneumococcus and this effect was significantly modified by the HLA-DR polymorphism.

Molecular dynamics at the receptor level of immunodominant myelin oligodendrocyte glycoprotein 35-55 epitope implicated in multiple sclerosis.

Multiple Sclerosis (MS) is a common autoimmune disease whereby myelin is destroyed by the immune system. The disease is triggered by the stimulation of encephalitogenic T-cells via the formation of a trimolecular complex between the Human Leukocyte Antigen (HLA), an immunodominant epitope of myelin proteins and T-cell Receptor (TCR). Myelin Oligodendrocyte Glycoprotein (MOG) is located on the external surface of myelin and has been implicated in MS induction. The immunodominant 35-55 epitope of MOG is widely used for in vivo biological evaluation and immunological studies that are related with chronic Experimental Autoimmune Encephalomyelitis (EAE, animal model of MS), inflammatory diseases and MS. In this report, Molecular Dynamics (MD) simulations were used to explore the interactions of MOG35-55 at the receptor level. A detailed mapping of the developed interactions during the creation of the trimolecular complex is reported. This is the first attempt to gain an understanding of the molecular recognition of the MOG35-55 epitope by the HLA and TCR receptors. During the formation of the trimolecular complex, the residues Arg(41) and Arg(46) of MOG35-55 have been confirmed to serve as TCR anchors while Tyr(40) interacts with HLA. The present structural findings indicate that the Arg at positions 41 and 46 is a key residue for the stimulation of the encephalitogenic T-cells.

A Central Role for HLA-DR3 in Anti-Smith Antibody Responses and Glomerulonephritis in a Transgenic Mouse Model of Spontaneous Lupus.

MHC, especially HLA-DR3 and HLA-DR2, is one of the most important genetic susceptibility regions for systemic lupus erythematosus. Human studies to understand the role of specific HLA alleles in disease pathogenesis have been hampered by the presence of strong linkage disequilibrium in this region. To overcome this, we produced transgenic mice expressing HLA-DR3 (DRß1*0301) and devoid of endogenous class II (both I-A and I-E genes, AE(0)) on a lupus-prone NZM2328 background (NZM2328.DR3(+)AE(0)). Both NZM2328 and NZM2328.DR3(+)AE(0) mice developed anti-dsDNA and glomerulonephritis, but anti-dsDNA titers were higher in the latter. Although kidney histological scores were similar in NZM2328 and NZM2328.DR3(+)AE(0) mice (7.2 ± 4.3 and 8.6 ± 5.7, respectively, p = 0.48), the onset of severe proteinuria occurred earlier in NZM2328.DR3(+)AE(0) mice compared with NZM2328 mice (median, 5 and 9 mo respectively, p < 0.001). Periarterial lymphoid aggregates, classic wire loop lesions, and occasional crescents were seen only in kidneys from NZM2328.DR3(+)AE(0) mice. Interestingly, NZM2328.DR3(+)AE(0) mice, but not NZM2328 mice, spontaneously developed anti-Smith (Sm) Abs. The anti-Sm Abs were seen in NZM2328.DR3(+)AE(0) mice that were completely devoid of endogenous class II (AE(-/) (-)) but not in mice homozygous (AE(+/+)) or heterozygous (AE(+/-)) for endogenous MHC class II. It appears that only HLA-DR3 molecules can preferentially select SmD-reactive CD4(+) T cells for generation of the spontaneous anti-Sm immune response. Thus, our mouse model unravels a critical role for HLA-DR3 in generating an autoimmune response to SmD and lupus nephritis in the NZM2328 background.

Homo-ß-amino acid containing MBP(85-99) analogs alleviate experimental autoimmune encephalomyelitis.

MBP(85-99), an immuno-dominant epitope of myelin basic protein which binds to the major histocompatibility complex haplotype HLA-DR2 is widely implicated in the pathogenesis of multiple sclerosis. J5, an antagonist of MBP(85-99), that blocks the binding of MBP(85-99) to soluble HLA-DR2b much more efficiently than glatiramer acetate (a random copolymer comprising major MHC and T-cell receptor contact residues), was transformed into analogs with superior biological half-lives and antagonistic-activities by substitution of some of its residues with homo-ß-amino acids. S18, the best analog obtained ameliorated symptoms of experimental autoimmune encephalomyelitis at least twice more effectively than glatiramer acetate or J5. S18 displayed marked resistance to proteolysis in-vitro; biological impact of which was evident in the form of delayed clinical onset of disease and prolonged therapeutic-benefits. Besides active suppression of MBP(85-99)-reactive CD4(+) T-cells in-vitro and in-vivo S18 treatment also generated IL-4 producing CD4(+) T-cell clones, through which protective effect could be transferred passively.

Breaking immune tolerance by targeting CD25+ regulatory T cells is essential for the anti-tumor effect of the CTLA-4 blockade in an HLA-DR transgenic mouse model of prostate cancer.

INTRODUCTION: Recent studies suggest that the cancer immunotherapy based on the blockade of the CTLA-4-mediated inhibitory pathway is efficacious only in select populations, predominantly for immunogenic tumors or when delivered in combination with modalities that can break immunologic tolerance to tumor antigens. METHODS: We studied the effect of CD25+ cell depletion and CTLA-4 blockade on the growth of Transgenic Mouse Adenocarcinoma of Prostate (TRAMP)-PSA tumor cells in DR2bxPSA F1 mice. In these mice, immunological tolerance to PSA was established in a context of the HLA-DRB1*1501(DR2b) allele. RESULTS: In our model, single administration of anti-CD25 antibody prior to tumor inoculation significantly increased IFN-γ production in response to the CD8 T cell epitope PSA65-73 , and delayed TRAMP-PSA tumor growth compared to mice treated with isotype control antibodies. In contrast, the anti-tumor effect of the anti-CTLA-4 antibody as a monotherapy was marginal. The combinatory treatment with anti-CD25/anti-CTLA-4 antibodies significantly enhanced anti-tumor immunity and caused more profound delay in tumor growth compared to each treatment alone. The proportion of tumor-free animals was higher in the group that received combination treatment (21%) compared to other groups (2-7%). The enhanced anti-tumor immunity in response to the CD25 depletion or CTLA-4 blockade was only seen in the immunogenic TRAMP-PSA tumor model, whereas the effect was completely absent in mice bearing poorly immunogenic TRAMP-C1 tumors. DISCUSSION: Our data suggest that breaking immunological tolerance to "self" antigens is essential for the therapeutic effect of CTLA-4 blockade. Such combinatory treatment may be a promising approach for prostate cancer immunotherapy.

Lupus nephritis susceptibility loci in women with systemic lupus erythematosus.

Lupus nephritis is a manifestation of SLE resulting from glomerular immune complex deposition and inflammation. Lupus nephritis demonstrates familial aggregation and accounts for significant morbidity and mortality. We completed a meta-analysis of three genome-wide association studies of SLE to identify lupus nephritis-predisposing loci. Through genotyping and imputation, >1.6 million markers were assessed in 2000 unrelated women of European descent with SLE (588 patients with lupus nephritis and 1412 patients with lupus without nephritis). Tests of association were computed using logistic regression adjusting for population substructure. The strongest evidence for association was observed outside the MHC and included markers localized to 4q11-q13 (PDGFRA, GSX2; P=4.5×10(-7)), 16p12 (SLC5A11; P=5.1×10(-7)), 6p22 (ID4; P=7.4×10(-7)), and 8q24.12 (HAS2, SNTB1; P=1.1×10(-6)). Both HLA-DR2 and HLA-DR3, two well established lupus susceptibility loci, showed evidence of association with lupus nephritis (P=0.06 and P=3.7×10(-5), respectively). Within the class I region, rs9263871 (C6orf15-HCG22) had the strongest evidence of association with lupus nephritis independent of HLA-DR2 and HLA-DR3 (P=8.5×10(-6)). Consistent with a functional role in lupus nephritis, intra-renal mRNA levels of PDGFRA and associated pathway members showed significant enrichment in patients with lupus nephritis (n=32) compared with controls (n=15). Results from this large-scale genome-wide investigation of lupus nephritis provide evidence of multiple biologically relevant lupus nephritis susceptibility loci.

Small molecule inhibitor of antigen binding and presentation by HLA-DR2b as a therapeutic strategy for the treatment of multiple sclerosis.

The strong association of HLA-DR2b (DRB1*1501) with multiple sclerosis (MS) suggests this molecule as prime target for specific immunotherapy. Inhibition of HLA-DR2b-restricted myelin-specific T cells has the potential to selectively prevent CNS pathology mediated by these MHC molecules without undesired global immunosuppression. In this study, we report development of a highly selective small molecule inhibitor of peptide binding and presentation by HLA-DR2b. PV-267, the candidate molecule used in these studies, inhibited cytokine production and proliferation of myelin-specific HLA-DR2b-restricted T cells. PV-267 had no significant effect on T cell responses mediated by other MHC class II molecules, including HLA-DR1, -DR4, or -DR9. Importantly, PV-267 did not induce nonspecific immune activation of human PBMC. Lastly, PV-267 showed treatment efficacy both in preventing experimental autoimmune encephalomyelitis and in treating established disease. The results suggest that blocking the MS-associated HLA-DR2b allele with small molecule inhibitors may be a promising therapeutic strategy for the treatment of MS.

Peripheral Treg count and it's determinants in unsensitized and sensitized chronic kidney disease patients.

PURPOSE: Sensitization to HLA antigens resulting in anti-HLA antibodies (panel reactive antibodies; PRA) is a major problem in chronic kidney disease (CKD) patients awaiting transplantation. Induction of anti-HLA antibodies normally occurs through blood transfusion, pregnancy and prior transplantation. However, some patients develop these antibodies for unknown immunological reasons. It is hypothesized that deviations in immune regulation may account for PRA positivity in these patients. We, therefore, investigated whether a quantitative deficiency in peripheral natural regulatory T cells (CD4(+)CD25(high)Foxp3(+); nTreg) plays a role in this phenomenon. METHODS: Peripheral blood mononuclear cells from 14 patients with positive (Class I and Class II; 10-100 %) and 25 patients with negative PRA, who had not previously been sensitized by blood transfusion, pregnancy and prior transplantation and who had not received any immunomodulatory treatment within the last year, were analyzed for absolute lymphocyte and nTreg numbers through flow cytometry. Samples from 10 healthy people were also used as control. RESULTS: Mean absolute nTreg numbers were determined to be severely reduced in CKD patients (12 ± 9; n = 39) compared with healthy individuals (53 ± 17; n = 10) (p = 0.008). However, absolute nTreg numbers were similar between PRA- (12 ± 11) and PRA+ (11 ± 8) groups. Interestingly, there was a moderate correlation between the nTreg numbers and HLADR2 genotype (n = 9, r = 0.508, p < 0.05). CONCLUSION: This is the first study to demonstrate that the quantitative peripheral nTreg deficiency in CKD patients does not show a causal relationship with the presence of anti-HLA antibodies.

Structural and dynamical insights on HLA-DR2 complexes that confer susceptibility to multiple sclerosis in Sardinia: a molecular dynamics simulation study.

Sardinia is a major Island in the Mediterranean with a high incidence of multiple sclerosis, a chronic autoimmune inflammatory disease of the central nervous system. Disease susceptibility in Sardinian population has been associated with five alleles of major histocompatibility complex (MHC) class II DRB1 gene. We performed 120 ns of molecular dynamics simulation on one predisposing and one protective alleles, unbound and in complex with the two relevant peptides: Myelin Basic Protein and Epstein Barr Virus derived peptide. In particular we focused on the MHC peptide binding groove dynamics. The predisposing allele was found to form a stable complex with both the peptides, while the protective allele displayed stability only when bound with myelin peptide. The local flexibility of the MHC was probed dividing the binding groove into four compartments covering the well known peptide anchoring pockets. The predisposing allele in the first half cleft exhibits a narrower and more rigid groove conformation in the presence of myelin peptide. The protective allele shows a similar behavior, while in the second half cleft it displays a narrower and more flexible groove conformation in the presence of viral peptide. We further characterized these dynamical differences by evaluating H-bonds, hydrophobic and stacking interaction networks, finding striking similarities with super-type patterns emerging in other autoimmune diseases. The protective allele shows a defined preferential binding to myelin peptide, as confirmed by binding free energy calculations. All together, we believe the presented molecular analysis could help to design experimental assays, supports the molecular mimicry hypothesis and suggests that propensity to multiple sclerosis in Sardinia could be partly linked to distinct peptide-MHC interaction and binding characteristics of the antigen presentation mechanism.