Autoimmune susceptible HLA class II motifs facilitate the presentation of modified neoepitopes to potentially autoreactive T cells.

Rheumatoid arthritis (RA), multiple sclerosis (MS), type 1 diabetes (T1D), and celiac disease (CD), are strongly associated with susceptible HLA class II haplotypes. The peptide-binding pockets of these molecules are polymorphic, thus each HLA class II protein presents a distinct set of peptides to CD4+ T cells. Peptide diversity is increased through post-translational modifications, generating non-templated sequences that enhance HLA binding and/or T cell recognition. The high-risk HLA-DR alleles that confer susceptibility to RA are notable for their ability to accommodate citrulline, promoting responses to citrullinated self-antigens. Likewise, HLA-DQ alleles associated with T1D and CD favor the binding of deamidated peptides. In this review, we discuss structural features that promote modified self-epitope presentation, provide evidence supporting the relevance of T cell recognition of such antigens in disease processes, and make a case that interrupting the pathways that generate such epitopes and reprogramming neoepitope-specific T cells are key strategies for effective therapeutic intervention.

Salinity Stress Alters the Secondary Metabolic Profile of M. sativa, M. arborea and Their Hybrid (Alborea).

Increased soil salinity, and therefore accumulation of ions, is one of the major abiotic stresses of cultivated plants that negatively affect their growth and yield. Among Medicago species, only Medicago truncatula, which is a model plant, has been extensively studied, while research regarding salinity responses of two important forage legumes of Medicago sativa (M. sativa) and Medicago arborea (M. arborea) has been limited. In the present work, differences between M. arborea, M. sativa and their hybrid Alborea were studied regarding growth parameters and metabolomic responses. The entries were subjected to three different treatments: (1) no NaCl application (control plants), (2) continuous application of 100 mM NaCl (acute stress) and (3) gradual application of NaCl at concentrations of 50-75-150 mM by increasing NaCl concentration every 10 days. According to the results, M. arborea maintained steady growth in all three treatments and appeared to be more resistant to salinity. Furthermore, results clearly demonstrated that M. arborea presented a different metabolic profile from that of M. sativa and their hybrid. In general, it was found that under acute and gradual stress, M. sativa overexpressed saponins in the shoots while M. arborea overexpressed saponins in the roots, which is the part of the plant where most of the saponins are produced and overexpressed. Alborea did not perform well, as more metabolites were downregulated than upregulated when subjected to salinity stress. Finally, saponins and hydroxycinnamic acids were key players of increased salinity tolerance.

Chemometric Study of Fatty Acid Composition of Virgin Olive Oil from Four Widespread Greek Cultivars.

Virgin olive oil (VOO) is one of the key components of the Mediterranean diet owing to the presence of monounsaturated fatty acids and various bioactive compounds. These beneficial traits, which are usually associated with the cultivar genotype, are highlighting the demand of identifying characteristics of olive oil that will ensure its authenticity. In this work, the fatty acid (FA) composition of 199 VOO samples from Koroneiki, Megaritiki, Amfissis, and Manaki cultivars was determined and studied by chemometrics. Olive cultivar greatly influenced the FA composition, namely, oleic acid (from 75.36% for Amfissis to 65.81% for Megaritiki) and linoleic acid (from 13.35% for Manaki to 6.70% for Koroneiki). Spearman's rho correlation coefficients revealed differences and similarities among the olive oil cultivars. The use of the forward stepwise algorithm identified the FAs arachidonic acid, gadoleic acid, linoleic acid, α-linolenic acid, palmitoleic acid, and palmitic acid as the most significant for the differentiation of samples. The application of linear and quadratic cross-validation discriminant analysis resulted in the correct classification of 100.00% and 99.37% of samples, respectively. The findings demonstrated the special characteristics of the VOO samples derived from the four cultivars and their successful botanical differentiation based on FA composition.

Discrimination of botanical origin of olive oil from selected Greek cultivars by SPME-GC-MS and ATR-FTIR spectroscopy combined with chemometrics.

BACKGROUND: Consumers today wish to know the botanical origin of the olive oil they purchase. The objective of the present study was the development of robust chemometric models based on gas chromatography-mass spectrometry (GC-MS) and attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR) for the purpose of botanical differentiation of three commercial Greek olive oil cultivars. RESULTS: Using the solid-phase microextraction technique (SPME), volatile compounds (VC) were obtained and analyzed by GC-MS. Five hydrocarbons and one ester were selected by the forward stepwise algorithm, which best discriminated the olive oil samples. From ATR-FTIR analysis, the spectral regions chosen from the forward stepwise algorithm were associated with CO stretching vibration of the esters of triglycerides and the CH bending vibrations of the CH2 aliphatic group and double bonds. Application of the supervised methods of linear and quadratic discriminant cross-validation analysis, based on VC data, provided a correct classification score of 97.4% and 100.0%, respectively. Corresponding statistical analyses were used in the mid-infrared spectra, by which 96.1% of samples were discriminated correctly. CONCLUSION: ATR-FTIR and SPME-GC-MS techniques in conjunction with the appropriate feature selection algorithm and classification methods proved to be powerful tools for the authentication of Greek olive oil. The proposed methodology could be used in an industrial setting for determination of the botanical origin of Greek olive oil. © 2020 Society of Chemical Industry.

DRB4*01:01 Has a Distinct Motif and Presents a Proinsulin Epitope That Is Recognized in Subjects with Type 1 Diabetes.

DRB4*01:01 (DRB4) is a secondary HLA-DR product that is part of the high-risk DR4/DQ8 haplotype that is associated with type 1 diabetes (T1D). DRB4 shares considerable homology with HLA-DR4 alleles that predispose to autoimmunity, including DRB1*04:01 and DRB1*04:04. However, the DRB4 protein sequence includes distinct residues that would be expected to alter the characteristics of its binding pockets. To identify high-affinity peptides that are recognized in the context of DRB4, we used an HLA class II tetramer-based approach to identify epitopes within multiple viral Ags. We applied a similar approach to identify antigenic sequences within glutamic acid decarboxylase 65 and pre-proinsulin that are recognized in the context of DRB4. Seven sequences were immunogenic, eliciting high-affinity T cell responses in DRB4+ subjects. DRB1*04:01-restricted responses toward many of these peptides have been previously described, but responses to a novel pre-proinsulin 9-28 peptide were commonly observed in subjects with T1D. Furthermore, T cells that recognized this peptide in the context of DRB4 were present at significantly higher frequencies in patients with T1D than in healthy controls, implicating this as a disease-relevant specificity that may contribute to the breakdown of ß cell tolerance in genetically susceptible individuals. We then deduced a DRB4 motif and confirmed its key features through structural modeling. This modeling suggested that the core epitope within the pre-proinsulin 9-28 peptide has a somewhat unusual binding motif, with tryptophan in the fourth binding pocket of DRB4, perhaps influencing the availability of this complex for T cell selection.

Molecular basis for increased susceptibility of Indigenous North Americans to seropositive rheumatoid arthritis.

OBJECTIVE: The pathogenetic mechanisms by which HLA-DRB1 alleles are associated with anticitrullinated peptide antibody (ACPA)-positive rheumatoid arthritis (RA) are incompletely understood. RA high-risk HLA-DRB1 alleles are known to share a common motif, the 'shared susceptibility epitope (SE)'. Here, the electropositive P4 pocket of HLA-DRB1 accommodates self-peptide residues containing citrulline but not arginine. HLA-DRB1 His/Phe13ß stratifies with ACPA-positive RA, while His13ßSer polymorphisms stratify with ACPA-negative RA and RA protection. Indigenous North American (INA) populations have high risk of early-onset ACPA-positive RA, whereby HLA-DRB1*04:04 and HLA-DRB1*14:02 are implicated as risk factors for RA in INA. However, HLA-DRB1*14:02 has a His13ßSer polymorphism. Therefore, we aimed to verify this association and determine its molecular mechanism. METHODS: HLA genotype was compared in 344 INA patients with RA and 352 controls. Structures of HLA-DRB1*1402-class II loaded with vimentin-64Arg59-71, vimentin-64Cit59-71 and fibrinogen ß-74Cit69-81 were solved using X-ray crystallography. Vimentin-64Cit59-71-specific and vimentin59-71-specific CD4+ T cells were characterised by flow cytometry using peptide-histocompatibility leukocyte antigen (pHLA) tetramers. After sorting of antigen-specific T cells, TCRα and ß-chains were analysed using multiplex, nested PCR and sequencing. RESULTS: ACPA+ RA in INA was independently associated with HLA-DRB1*14:02. Consequent to the His13ßSer polymorphism and altered P4 pocket of HLA-DRB1*14:02, both citrulline and arginine were accommodated in opposite orientations. Oligoclonal autoreactive CD4+ effector T cells reactive with both citrulline and arginine forms of vimentin59-71 were observed in patients with HLA-DRB1*14:02+ RA and at-risk ACPA- first-degree relatives. HLA-DRB1*14:02-vimentin59-71-specific and HLA-DRB1*14:02-vimentin-64Cit59-71-specific CD4+ memory T cells were phenotypically distinct populations. CONCLUSION: HLA-DRB1*14:02 broadens the capacity for citrullinated and native self-peptide presentation and T cell expansion, increasing risk of ACPA+ RA.

Differential binding of pyruvate dehydrogenase complex-E2 epitopes by DRB1*08:01 and DRB1*11:01 Is predicted by their structural motifs and correlates with disease risk.

DRB1*08:01 (DR0801) and DRB1*11:01 (DR1101) are highly homologous alleles that have opposing effects on susceptibility to primary biliary cirrhosis (PBC). DR0801 confers risk and shares a key feature with other HLA class II alleles that predispose to autoimmunity: a nonaspartic acid at beta57. DR1101 is associated with protection from PBC, and its sequence includes an aspartic acid at beta57. To elucidate a mechanism for the opposing effects of these HLA alleles on PBC susceptibility, we compared the features of epitopes presented by DR0801 and DR1101. First, we identified DR0801- and DR1101-restricted epitopes within multiple viral Ags, observing both shared and distinct epitopes. Because DR0801 is not well characterized, we deduced its motif by measuring binding affinities for a library of peptides, confirming its key features through structural modeling. DR0801 was distinct from DR1101 in its ability to accommodate charged residues within all but one of its binding pockets. In particular, DR0801 strongly preferred acidic residues in pocket 9. These findings were used to identify potentially antigenic sequences within PDC-E2 (an important hepatic autoantigen) that contain a DR0801 motif. Four peptides bound to DR0801 with reasonable affinity, but only one of these bound to DR1101. Three peptides, PDC-E2145-159, PDC-E2(249-263), and PDC-E2(629-643), elicited high-affinity T cell responses in DR0801 subjects, implicating these as likely autoreactive specificities. Therefore, the unique molecular features of DR0801 may lead to the selection of a distinct T cell repertoire that contributes to breakdown of self-tolerance in primary biliary cirrhosis, whereas those of DR1101 promote tolerance.

Oral Insulin Delay of Stage 3 Type 1 Diabetes Revisited in HLA DR4-DQ8 Participants in the TrialNet Oral Insulin Prevention Trial (TN07).

OBJECTIVE: To explore if oral insulin could delay onset of stage 3 type 1 diabetes (T1D) among patients with stage 1/2 who carry HLA DR4-DQ8 and/or have elevated levels of IA-2 autoantibodies (IA-2As). RESEARCH AND METHODS: Next-generation targeted sequencing technology was used to genotype eight HLA class II genes (DQA1, DQB1, DRB1, DRB3, DRB4, DRB5, DPA1, and DPB1) in 546 participants in the TrialNet oral insulin preventative trial (TN07). Baseline levels of autoantibodies against insulin (IAA), GAD65 (GADA), and IA-2A were determined prior to treatment assignment. Available clinical and demographic covariables from TN07 were used in this post hoc analysis with the Cox regression model to quantify the preventive efficacy of oral insulin. RESULTS: Oral insulin reduced the frequency of T1D onset among participants with elevated IA-2A levels (HR 0.62; P = 0.012) but had no preventive effect among those with low IA-2A levels (HR 1.03; P = 0.91). High IA-2A levels were positively associated with the HLA DR4-DQ8 haplotype (OR 1.63; P = 6.37 × 10-6) and negatively associated with the HLA DR7-containing DRB1*07:01-DRB4*01:01-DQA1*02:01-DQB1*02:02 extended haplotype (OR 0.49; P = 0.037). Among DR4-DQ8 carriers, oral insulin delayed the progression toward stage 3 T1D onset (HR 0.59; P = 0.027), especially if participants also had high IA-2A level (HR 0.50; P = 0.028). CONCLUSIONS: These results suggest the presence of a T1D endotype characterized by HLA DR4-DQ8 and/or elevated IA-2A levels; for those patients with stage 1/2 disease with such an endotype, oral insulin delays the clinical T1D onset.

HLA Class II (DR, DQ, DP) Genes Were Separately Associated With the Progression From Seroconversion to Onset of Type 1 Diabetes Among Participants in Two Diabetes Prevention Trials (DPT-1 and TN07).

OBJECTIVE: To explore associations of HLA class II genes (HLAII) with the progression of islet autoimmunity from asymptomatic to symptomatic type 1 diabetes (T1D). RESEARCH DESIGN AND METHODS: Next-generation targeted sequencing was used to genotype eight HLAII genes (DQA1, DQB1, DRB1, DRB3, DRB4, DRB5, DPA1, DPB1) in 1,216 participants from the Diabetes Prevention Trial-1 and Randomized Diabetes Prevention Trial with Oral Insulin sponsored by TrialNet. By the linkage disequilibrium, DQA1 and DQB1 are haplotyped to form DQ haplotypes; DP and DR haplotypes are similarly constructed. Together with available clinical covariables, we applied the Cox regression model to assess HLAII immunogenic associations with the disease progression. RESULTS: First, the current investigation updated the previously reported genetic associations of DQA1*03:01-DQB1*03:02 (hazard ratio [HR] = 1.25, P = 3.50*10-3) and DQA1*03:03-DQB1*03:01 (HR = 0.56, P = 1.16*10-3), and also uncovered a risk association with DQA1*05:01-DQB1*02:01 (HR = 1.19, P = 0.041). Second, after adjusting for DQ, DPA1*02:01-DPB1*11:01 and DPA1*01:03-DPB1*03:01 were found to have opposite associations with progression (HR = 1.98 and 0.70, P = 0.021 and 6.16*10-3, respectively). Third, DRB1*03:01-DRB3*01:01 and DRB1*03:01-DRB3*02:02, sharing the DRB1*03:01, had opposite associations (HR = 0.73 and 1.44, P = 0.04 and 0.019, respectively), indicating a role of DRB3. Meanwhile, DRB1*12:01-DRB3*02:02 and DRB1*01:03 alone were found to associate with progression (HR = 2.6 and 2.32, P = 0.018 and 0.039, respectively). Fourth, through enumerating all heterodimers, it was found that both DQ and DP could exhibit associations with disease progression. CONCLUSIONS: These results suggest that HLAII polymorphisms influence progression from islet autoimmunity to T1D among at-risk subjects with islet autoantibodies.

Type 1 diabetes-associated HLA-DQ8 transdimer accommodates a unique peptide repertoire.

HLA-DQ2 and HLA-DQ8 are strongly predisposing haplotypes for type 1 diabetes (T1D). Yet HLA-DQ2/8 heterozygous individuals have a synergistically increased risk compared with HLA-DQ2 or HLA-DQ8 homozygote subjects that may result from the presence of a transdimer formed between the α-chain of HLA-DQ2 (DQA1*05:01) and the ß-chain of HLA-DQ8 (DQB1*03:02). We generated cells exclusively expressing this transdimer (HLA-DQ8trans), characterized its peptide binding repertoire, and defined a unique transdimer-specific peptide binding motif that was found to be distinct from those of HLA-DQ2 and HLA-DQ8. This motif predicts an array of peptides of islet autoantigens as candidate T cell epitopes, many of which selectively bind to the HLA transdimer, whereas others bind to both HLA-DQ8 and transdimer with similar affinity. Our findings provide a molecular basis for the association between HLA-DQ transdimers and T1D and set the stage for rational testing of potential diabetogenic peptide epitopes.

Gluten-specific T cells cross-react between HLA-DQ8 and the HLA-DQ2α/DQ8ß transdimer.

Because susceptibility to celiac disease is associated strongly with HLA-DQ2 (DQA1*05/DQB1*02) and weakly with HLA-DQ8 (DQA1*03/DQB1*03), a subset of patients carries both HLA-DQ2 and HLA-DQ8. As a result, these patients may express two types of mixed HLA-DQ2/8 transdimers (encoded by DQA1*05/DQB1*03 and DQA1*03/DQB1*02) in addition to HLA-DQ2 and HLA-DQ8. Using T cells from a celiac disease patient expressing HLA-DQ8trans (encoded by DQA*0501/DQB*0302), but neither HLA-DQ2 nor HLA-DQ8, we demonstrate that this transdimer is expressed on the cell surface and can present multiple gluten peptides to T cell clones isolated from the duodenum of this patient. Furthermore, T cell clones derived from this patient and HLA-DQ2/8 heterozygous celiac disease patients respond to gluten peptides presented by HLA-DQ8trans, as well as HLA-DQ8, in a similar fashion. Finally, one gluten peptide is recognized better when presented by HLA-DQ8trans, which correlates with preferential binding of this peptide to HLA-DQ8trans. These results implicate HLA-DQ8trans in celiac disease pathogenesis and demonstrate extensive T cell cross-reactivity between HLA-DQ8 and HLA-DQ8trans. Because type 1 diabetes is strongly associated with the presence of HLA-DQ8trans, our findings may bear relevance to this disease as well.

Probing the hydrogen equilibrium and kinetics in zeolite imidazolate frameworks via molecular dynamics and quasi-elastic neutron scattering experiments.

The problem of simulating processes involving equilibria and dynamics of guest sorbates within zeolitic imidazolate frameworks (ZIF) by means of molecular dynamics (MD) computer experiments is of growing importance because of the promising role of ZIFs as molecular "traps" for clean energy applications. A key issue for validating such an atomistic modeling attempt is the possibility of comparing the MD results, with real experiments being able to capture analogous space and time scales to the ones pertained to the computer experiments. In the present study, this prerequisite is fulfilled through the quasi-elastic neutron scattering technique (QENS) for measuring self-diffusivity, by elaborating the incoherent scattering signal of hydrogen nuclei. QENS and MD experiments were performed in parallel to probe the hydrogen motion, for the first time in ZIF members. The predicted and measured dynamics behaviors show considerable concentration variation of the hydrogen self-diffusion coefficient in the two topologically different ZIF pore networks of this study, the ZIF-3 and ZIF-8. Modeling options such as the flexibility of the entire matrix versus a rigid framework version, the mobility of the imidazolate ligand, and the inclusion of quantum mechanical effects in the potential functions were examined in detail for the sorption thermodynamics and kinetics of hydrogen and also of deuterium, by employing MD combined with Widom averaging towards studying phase equilibria. The latter methodology ensures a rigorous and efficient way for post-processing the dynamics trajectory, thereby avoiding stochastic moves via Monte Carlo simulation, over the large number of configurational degrees of freedom a nonrigid framework encompasses.

Trans heterodimer between two non-arthritis-associated HLA alleles can predispose to arthritis in humanized mice.

OBJECTIVE: Certain HLA class II alleles are associated with susceptibility to the development of arthritis. However, the development of arthritis in some persons carrying non-rheumatoid arthritis (RA)-associated alleles remains unexplained. An individual who is heterozygous for the DQA1 and DQB1 genes can express the DQ molecule in cis or trans heterodimers. In a cis heterodimer, the α-chain interacts with the ß-chain coded by the same chromosome, while in a trans heterodimer it interacts with the ß-chain on the other chromosome. In this study, we used a humanized mouse model of arthritis in an attempt to determine whether a trans heterodimer of 2 nonassociated alleles, DQB1*0601 and DQB1*0604, can predispose to arthritis. METHODS: DQB1*0601 and *0604 occur in linkage with DQA1*0103 and *0102, respectively. To understand the role of trans heterodimers, we generated DQB1*0604/DQA1*0103-transgenic mice lacking endogenous HLA class II molecules. RESULTS: Severe arthritis developed in the DQB1*0604/A1*0103-trangenic mice, and an antigen-specific response was generated in vitro. DQB1*0604/DQA1*0103 presented type II collagen-derived peptides that were not presented by the arthritis-resistant DQB1*0601 allele, suggesting that trans heterodimer molecules between 2 DQB1 and DQA1 molecules may result in the presentation of unique antigens and susceptibility to the development of arthritis. Molecular modeling of type II collagen peptides showed that DQB1*0604/DQA1*0103 shares a p4 pocket with the arthritis-susceptible DQB1*0302 allele, suggesting a critical role of the p4 and p9 pockets in susceptibility to arthritis. CONCLUSION: These results provide a possible explanation for the parental inheritance of nonsusceptibility alleles in some patients with RA and a mechanism by which they can predispose to the development of arthritis.

A Simulation Study of the Effect of Naturally Occurring Point Mutations on the SRY-DNA Complex.

Molecular dynamics (MD) simulations were conducted in order to investigate the effect of the naturally occurring point mutations of the transcription factor (TF) sex-determining region Y (SRY) on the structure and dynamics of the SRY-DNA complex. The normal SRY, along with the two mutants I13T and G40R, comprising point mutations on the SRY chain, which have been clinically identified in patients with sex developmental disorders, were modeled as DNA complexes. Our modeling work aims at elucidating atomic-level structural determinants of the aberrant SRY-DNA complexation by means of µs-long MD. The results suggest that the observed disorders brought about by the G40R-DNA and I13T-DNA may arise predominantly from the destabilization of the complex being in accord with in vitro assays found elsewhere and from modifications of the DNA bending as revealed in this study. Comparative potential of mean force computations, over a sequence of short separation distances for the three complexes, verified a higher stability of the normal SRY-DNA. Examining the way the SRY mutations modulate the SRY-DNA complex dynamics at the microscopic level is important also toward elucidating molecular determinants of function for proteins capable of binding to DNA.

The Effect of Cold Periods on the Biological Cycle of Marchalina hellenica.

Climate change is considered a major factor affecting honeybees' behavior and productivity with major consequences in both honey and agricultural production. Many research studies have expressed serious concerns about the mass losses of bee colonies and the role of bees as pollinators, while others have underlined important issues for the impact of the increase in temperature on honeybee abundance and honey yields. In the present work, we draw our attention to Marchalina hellenica, which is the most important honeydew-producing insect in Greece. A statistically significant forecasting model for the effect of cold periods in February on the life cycle of the insect is constructed, with the aid of the Cumulative Logit Model and the theory of runs. The forecasting model may help beekeepers plan the timely exploitation of honeydew secretions of pine trees, which will be beneficial for beekeepers, the rural economy, and forest protection. The new suggested model also indicates that, in view of the climate change scenarios seen in the literature, the life cycle of M. hellenica is expected to be drastically shorter.

A Localized Enantioselective Catalytic Site on Short DNA Sequences and Their Amphiphiles.

A DNA-based artificial metalloenzyme (ArM) consisting of a copper(II) complex of 4,4'-dimethyl-2,2'-bipyridine (dmbipy-Cu) bound to double-stranded DNA (dsDNA) as short as 8 base pairs with only 2 contiguous central pairs (G for guanine and C for cytosine) catalyzes the highly enantioselective Diels-Alder reaction, Michael addition, and Friedel-Crafts alkylation in water. Molecular simulations indicate that these minimal sequences provide a single site where dmbipy-Cu is groove-bound and able to function as an enantioselective catalyst. Enantioselective preference inverts when d-DNA is replaced with l-DNA. When the DNA is conjugated to a hydrophobic tail, the obtained ArMs exhibit enantioselective performance in a methanol-water mixture superior to that of non-amphiphilic dsDNA, and dsDNA-amphiphiles with more complex G•C-rich sequences.

Association of HLA-DQ Heterodimer Residues -18ß and ß57 With Progression From Islet Autoimmunity to Diabetes in the Diabetes Prevention Trial-Type 1.

OBJECTIVE: The purpose was to test the hypothesis that the HLA-DQαß heterodimer structure is related to the progression of islet autoimmunity from asymptomatic to symptomatic type 1 diabetes (T1D). RESEARCH DESIGN AND METHODS: Next-generation targeted sequencing was used to genotype HLA-DQA1-B1 class II genes in 670 subjects in the Diabetes Prevention Trial-Type 1 (DPT-1). Coding sequences were translated into DQ α- and ß-chain amino acid residues and used in hierarchically organized haplotype (HOH) association analysis to identify motifs associated with diabetes onset. RESULTS: The opposite diabetes risks were confirmed for HLA DQA1*03:01-B1*03:02 (hazard ratio [HR] 1.36; P = 2.01 ∗ 10-3) and DQA1*03:03-B1*03:01 (HR 0.62; P = 0.037). The HOH analysis uncovered residue -18ß in the signal peptide and ß57 in the ß-chain to form six motifs. DQ*VA was associated with faster (HR 1.49; P = 6.36 ∗ 10-4) and DQ*AD with slower (HR 0.64; P = 0.020) progression to diabetes onset. VA/VA, representing DQA1*03:01-B1*03:02 (DQ8/8), had a greater HR of 1.98 (P = 2.80 ∗ 10-3). The DQ*VA motif was associated with both islet cell antibodies (P = 0.023) and insulin autoantibodies (IAAs) (P = 3.34 ∗ 10-3), while the DQ*AD motif was associated with a decreased IAA frequency (P = 0.015). Subjects with DQ*VA and DQ*AD experienced, respectively, increasing and decreasing trends of HbA1c levels throughout the follow-up. CONCLUSIONS: HLA-DQ structural motifs appear to modulate progression from islet autoimmunity to diabetes among at-risk relatives with islet autoantibodies. Residue -18ß within the signal peptide may be related to levels of protein synthesis and ß57 to stability of the peptide-DQab trimolecular complex.

HLA-DR1001 presents "altered-self" peptides derived from joint-associated proteins by accepting citrulline in three of its binding pockets.

OBJECTIVE: HLA-DRB1*1001 (DR1001) is a shared epitope allele associated with rheumatoid arthritis (RA). The present study was undertaken to assess the capacity of DR1001 to accommodate citrulline in its binding pockets and to identify citrullinated T cell epitopes derived from joint-associated proteins. METHODS: The binding of peptide derivatives containing citrulline, arginine, and other amino acid substitutions was measured. A prediction algorithm was developed to identify arginine-containing sequences from joint-associated proteins that preferentially bind to DR1001 upon citrullination. Unmodified and citrullinated versions of these sequences were synthesized and were utilized to stimulate CD4+ T cells from healthy subjects and RA patients. Responses were measured by class II major histocompatibility complex tetramer staining and confirmed by isolating CD4+ T cell clones. RESULTS: DR1001 accepted citrulline, but not arginine, in 3 of its anchoring pockets. The prediction algorithm identified sequences that preferentially bound to DR1001 with arginine replaced by citrulline. Three of these sequences elicited CD4+ T cell responses. T cell clones specific for these sequences proliferated only in response to citrullinated peptides. CONCLUSION: Conversion of arginine to citrulline generates "altered-self" peptides that can be bound and presented by DR1001. Responses to these peptides implicate the corresponding proteins (fibrinogen α, fibrinogen ß, and cartilage intermediate-layer protein) as relevant antigens. The finding of preferential responses to citrullinated sequences suggests that altered peptide binding affinity due to this posttranslational modification may be an important factor in the initiation or progression of RA. As such, measuring responsiveness to these peptides may be useful for immunologic monitoring.

The binding of antigenic peptides to HLA-DR is influenced by interactions between pocket 6 and pocket 9.

Peptide binding to class II MHC protein is commonly viewed as a combination of discrete anchor residue preferences for pockets 1, 4, 6/7, and 9. However, previous studies have suggested cooperative effects during the peptide binding process. Investigation of the DRB1*0901 binding motif demonstrated a clear interaction between peptide binding pockets 6 and 9. In agreement with prior studies, pockets 1 and 4 exhibited clear binding preferences. Previously uncharacterized pockets 6 and 7 accommodated a wide variety of residues. However, although it was previously reported that pocket 9 is completely permissive, several substitutions at this position were unable to bind. Structural modeling revealed a probable interaction between pockets 6 and 9 through beta9Lys. Additional binding studies with doubly substituted peptides confirmed that the amino acid bound within pocket 6 profoundly influences the binding preferences for pocket 9 of DRB1*0901, causing complete permissiveness of pocket 9 when a small polar residue is anchored in pocket 6 but accepting relatively few residues when a basic residue is anchored in pocket 6. The beta9Lys residue is unique to DR9 alleles. However, similar studies with doubly substituted peptides confirmed an analogous interaction effect for DRA1/B1*0301, a beta9Glu allele. Accounting for this interaction resulted in improved epitope prediction. These findings provide a structural explanation for observations that an amino acid in one pocket can influence binding elsewhere in the MHC class II peptide binding groove.

The KAG motif of HLA-DRB1 (ß71, ß74, ß86) predicts seroconversion and development of type 1 diabetes.

BACKGROUND: HLA-DR4, a common antigen of HLA-DRB1, has multiple subtypes that are strongly associated with risk of type 1 diabetes (T1D); however, some are risk neutral or resistant. The pathobiological mechanism of HLA-DR4 subtypes remains to be elucidated. METHODS: We used a population-based case-control study of T1D (962 patients and 636 controls) to decipher genetic associations of HLA-DR4 subtypes and specific residues with susceptibility to T1D. Using a birth cohort of 7865 children with periodically measured islet autoantibodies (GADA, IAA or IA-2A), we proposed to validate discovered genetic associations with a totally different study design and time-to-seroconversions prior to clinical onset of T1D. A novel analytic strategy hierarchically organized the HLA-DRB1 alleles by sequence similarity and identified critical amino acid residues by minimizing local genomic architecture and higher-order interactions. FINDINGS: Three amino acid residues of HLA-DRB1 (ß71, ß74, ß86) were found to be predictive of T1D risk in the population-based study. The "KAG" motif, corresponding to HLA-DRB1×04:01, was most strongly associated with T1D risk ([O]dds [R]atio=3.64, p = 3.19 × 10-64). Three less frequent motifs ("EAV", OR = 2.55, p = 0.025; "RAG", OR = 1.93, p = 0.043; and "RAV", OR = 1.56, p = 0.003) were associated with T1D risk, while two motifs ("REG" and "REV") were equally protective (OR = 0.11, p = 4.23 × 10-4). In an independent birth cohort of HLA-DR3 and HLA-DR4 subjects, those having the "KAG" motif had increased risk for time-to-seroconversion (Hazard Ratio = 1.74, p = 6.51 × 10-14) after adjusting potential confounders. INTERPRETATIONS: DNA sequence variation in HLA-DRB1 at positions ß71, ß74, and ß86 are non-conservative (ß74 A→E, ß71 E vs K vs R and ß86 G vs V). They result in substantial differences in peptide antigen anchor pocket preferences at p1, p4 and potentially neighboring regions such as pocket p7. Differential peptide antigen binding is likely to be affected. These sequence substitutions may account for most of the HLA-DR4 contribution to T1D risk as illustrated in two HLA-peptide model complexes of the T1D autoantigens preproinsulin and GAD65. FUNDING: National Institute of Diabetes and Digestive and Kidney Diseases and the Swedish Child Diabetes Foundation and the Swedish Research Council.