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Made available in DSpace on 2020-12-12T02:18:02Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-01-01 Aims: This study aimed to evaluate the toxicity and humoral and cellular immune response of three heterologous vaccines against Leishmania infantum, yet containing synthetic peptides from Leishmania major in the experimental model in hamsters. Methods and Results: Through bioinformatics analyses, two Leishmania major Gp63 peptides were predicted and selected for vaccine formulations. Hamsters were divided into four groups, with each group receiving doses of three vaccine formulations containing HLA-DR1 or HLA-A2 peptides plus MontanideTM or both associated with the adjuvant. The animals received three vaccine doses and were evaluated for toxicity after each dose, in addition to being analysed for the production of antibodies and lymphoproliferation on day 211 after the last vaccine dose. Peptides predicted in association with oily adjuvant induced a humoral response and strong lymphoproliferation to Leishmania infantum antigen-specific stimulation. Laboratory of Biomolecules and Vaccines Postgraduate Program in Biotechnology Agrarian Sciences and Technologic Department Federal University of Tocantins (UFT) Postgraduate Program in Biosciences Interdisciplinary Center for Life Sciences and Nature Federal University of Latin American Integration (UNILA) Bioprocess Engineering and Biotechnology Institute of Natural Resources Federal University of Itajubá Department of Biochemistry and Chemical Technology Institute of Chemistry Universidade Estadual Paulista (UNESP) Laboratory of Cell-Cell Interactions Morphology Department Institute of Biological Science Federal University of Minas Gerais (UFMG) Department of Biochemistry and Chemical Technology Institute of Chemistry Universidade Estadual Paulista (UNESP)

The nature of the H-bonds between the human protein HLA-DR1 (DRB*0101) and the hemagglutinin peptide HA306-318 has been studied using the Quantum Theory of Atoms in Molecules for the first time. We have found four H-bond groups: one conventional CO··HN bond group and three nonconventional CO··HC, π··HC involving aromatic rings and HN··HC aliphatic groups. The calculated electron density at the determined H-bond critical points suggests the follow protein pocket binding trend: P1 (2,311) >> P9 (1.109) > P4 (0.950) > P6 (0.553) > P7 (0.213) which agrees and reveal the nature of experimental findings, showing that P1 produces by a long way the strongest binding of the HLA-DR1 human protein molecule with the peptide backbone as consequence of the vast number of H-bonds in the P1 area and at the same time the largest specific binding of the peptide Tyr308 residue with aromatic residues located at the binding groove floor. The present results suggest the topological analysis of the electronic density as a valuable tool that allows a non-arbitrary partition of the pockets binding energy via the calculated electron density at the determined critical points.

Recent studies have demonstrated the impacts of 2-HG on the anti-tumor immune response in isocitrate dehydrogenase (IDH)-mutant gliomas. Our laboratory has reported that mutant IDH1 (IDH1R132H) suppresses STAT1, the master regulator of IFN-responses and IFN-inducible chemokines, thereby contributing to the “cold” tumor environment. Our data also indicated that inhibition of 2-HG production, through the use of a small molecular inhibitor of mutant IDH, can restore anti-tumor immunity. As the IDH1R132H mutation has been shown to elicit a CD4+ T-cell response in human leukocyte antigen (HLA)-DR1 hosts, to establish a clinically relevant mouse model of IDH1R132H glioma, we generated a novel IDH1R132H glioma cell line syngeneic to the HLA-A2/HLA-DR1-transgenic mice. The cell line expresses the IDH1R132H protein, produces ~65 µg/ml 2-HG in vitro, and forms 2-HG producing orthotopic glioma in vivo. Furthermore, ex vivo sorted tumor-associated myeloid cells (TAMCs) demonstrate high levels of intracellular 2-HG (60–80ng/1M cells), suggesting 2-HG may be taken up by these TAMCs in vivo. Treatment of tumor-bearing mice with vorasidenib, a pan-mutant IDH inhibitor, resulted in a 10-fold reduction of 2-HG levels in the tumor cells, correlate reduction in intracellular 2-HG levels in TAMCs, and 2-fold smaller tumors at time of sacrifice. Analysis of tumor tissues using the NanoString platform revealed enhanced pro-inflammatory IFN-related responses as a result of IDH1R132H inhibition with vorasidenib. Furthermore, vorasidenib treatment increased CD4+ tumor-infiltrating T-cells coupled with upregulation of HLA-DR on tumor-infiltrating myeloid cells, suggesting the opportunity for enhanced antigen presentation. Currently ongoing studies are evaluating the benefit of combining this enhanced immune response with additional immunomodulatory treatments, such as vaccine therapy and immune checkpoint inhibition. These data represent highly translational findings as our major histocompatibility complex (MHC)-humanized model addresses current challenges in the study of IDH1R132H-specific immunity and utilizes a clinically relevant inhibitor in phase 3 clinical development.

Copolymer 1 [Cop 1, poly (Y, E, A, K)] is a random synthetic amino acid copolymer effective in the treatment of relapsing forms of multiple sclerosis (MS), a disease that is linked to HLA-DR2 (DRB1∗1501). Another copolymer [poly (Y, A, K)] was also identified that binds to rheumatoid arthritis (RA)-associated HLA-DR1 (DRB1∗0101) or HLA-DR4 (DRB1∗0401) molecules and inhibits the response of HLA-DR1- and -DR4-restricted T cell clones to an immunodominant epitope of collagen type II (CII) 261–273 (a candidate autoantigen in RA). In the present study various peptides have been synthesized based on binding “motifs” of Cop 1 for HLA-DR1 and -DR4 molecules. Those peptides with K at P-1 or K at P8 were particularly effective as inhibitors of binding of CII 261–273, of Cop 1 and of the influenza virus hemagglutinin peptide 306–318 to these class II proteins. Moreover, several of them were also potent inhibitors of the CII 261–273-reactive T cell clones. These findings suggest that small peptides or their more stable derivatives may be able to substitute for copolymers in the treatment of RA, and by implication of MS.

SummaryHLA-DR molecules bind microbial peptides in an endosomal compartment and present them on the cell surface for CD4 T cell surveillance. HLA-DM plays a critical role in the endosomal peptide selection process. The structure of the HLA-DM–HLA-DR complex shows major rearrangements of the HLA-DR peptide-binding groove. Flipping of a tryptophan away from the HLA-DR1 P1 pocket enables major conformational changes that position hydrophobic HLA-DR residues into the P1 pocket. These conformational changes accelerate peptide dissociation and stabilize the empty HLA-DR peptide-binding groove. Initially, incoming peptides have access to only part of the HLA-DR groove and need to compete with HLA-DR residues for access to the P2 site and the hydrophobic P1 pocket. This energetic barrier creates a rapid and stringent selection process for the highest-affinity binders. Insertion of peptide residues into the P2 and P1 sites reverses the conformational changes, terminating selection through DM dissociation.

Yersinia pestis is the causative agent of plague, a rapidly fatal infectious disease that has not been eradicated worldwide. The capsular Caf1 protein of Y. pestis is a protective antigen under development as a recombinant vaccine. However, little is known about the specificity of human T-cell responses for Caf1. We characterized CD4 T-cell epitopes of Caf1 in “humanized” HLA-DR1 transgenic mice lacking endogenous major histocompatibility complex class II molecules. Mice were immunized with Caf1 or each of a complete set of overlapping synthetic peptides, and CD4 T-cell immunity was measured with respect to proliferative and gamma interferon T-cell responses and recognition by a panel of T-cell hybridomas, as well as direct determination of binding affinities of Caf1 peptides to purified HLA-DR molecules. Although a number of DR1-restricted epitopes were identified following Caf1 immunization, the response was biased toward a single immunodominant epitope near the C terminus of Caf1. In addition, potential promiscuous epitopes, including the immunodominant epitope, were identified by their ability to bind multiple common HLA alleles, with implications for the generation of multivalent vaccines against plague for use in humans.

A szerzők egy 35 éves nőbeteg kórtörténetét ismertetik, akinél 29 éves korában szeronegatív rheumatoid arthritist igazoltak a reumatológiai osztályon. A betegség remisszióját sikeres graviditás követte, majd két évvel később kizárólag axiális tüneteket mutató spondylarthritist diagnosztizáltak. A major hisztokompatibilitási komplex vizsgálata során a hagyományos szerológiai módszerekkel a HLA B27 spondylarthritis ankylopoeticára jellemző, valamint HLA DR1 rheumatoid arthritisre jellemző haplotípust mutattak ki. A HLA DRB-polimorfizmus vizsgálata során a rheumatoid arthritis létrejöttében és kórlefolyásában szerepet játszó HLA DR B1 0101 allél jelenlétét igazolták. A betegben tehát ritka kombinációként a spondylarthritis ankylopoetica és a rheumatoid arthritis létrejöttében is szerepet játszó HLA-formáció egyaránt megtalálható volt.

In an attempt to study the gene products of the HLA complex, a monoclonal antibody, named HU-30, was produced by immunizing BALB/c mice with a cultured human B lymphoblastoid cell line, Shi-C3 (Aw24, Aw31, Bw51, Bw52, DR2, DR blank, MT1, MT2, MB3). HU-30 belonged to the IgG2 subclass and was active in complement dependent cytotoxicity. When the serological specificity was evaluated with a panel of 15 cultured human lymphoblastoid cell lines, it was found that HU-30 detected a polymorphic determinant, common to HLA-DR1 and 2, with much stronger cytotoxic activity against HLA-DR2 positive B cell lines. When HU-30 was tested against a panel of B cells from 84 healthy donors at a dilution of 2(-13), it gave positive reactions only with cells typed as HLA-DR2. Furthermore, sequential coprecipitation studies indicated that the HU-30 determinant was borne on the molecules carrying the HLA-DR determinants. Thus, HU-30 appears to be of great value as a tissue typing reagent monospecific for HLA-DR2.

Evidence for a new HLA class II specificity is presented. It is recognized by LE serum, which reacts with most DR1 and/or DR4 individuals (r = 0.86). Its frequency in the French population is 0.33. Absorption-elution experiments showed that the serum reactivity was not due to a mixture of anti-DR1 and anti-DR4 antibodies, but to a single antibody population which could be absorbed on and eluted from both DR1(+) or DR4(+) cells. LE specificity seemed to be expressed on DR but not on DQ molecules since the serum reacted with and could be absorbed by DR+,DQw- cells; it did not react with a DR-,DQw+ mutant cell, but did react with the DR+,DQw+ parental cell. The relationship between LE specificity and MC1 and Te23 specificities remains to be determined.

Objective To identify the peptide anchor motif for the rheumatoid arthritis (RA)–related HLA allele, DR10, and find shared natural ligands or sequence similarities with the other disease-associated alleles, DR1 and DR4. Methods The HLA–DR10–associated peptides were purified, and a proportion of these natural ligands were de novo sequenced by mass spectrometry. Based on crystallographic structures, the complexes formed by peptide influenza virus hemagglutinin HA306–318 with DR1, DR4, and DR10 were modeled, and binding scores were obtained. Results A total of 238 peptides were sequenced, and the anchor motif of the HLA–DR10 peptide repertoire was defined. A large proportion of the DR10-associated peptides had the structural features to bind DR1 and DR4 but were theoretical nonbinders to the negatively associated alleles DR15 and DR7. Among the sequenced ligands, 10 had been reported as ligands to other RA-associated alleles. Modeling data showed that peptide HA306–318 can bind DR1, DR4, and DR10 with similar affinities. Conclusion The data show the presence of common peptides in the repertoires of RA-associated HLA alleles. The combination of the shared epitope present in DR1, DR4, and DR10 together with common putative arthritogenic peptide(s) could influence disease onset or outcome.

The immunogenicity of "novel" MART-1 and Tyrosinase class-II peptides was assessed in transgenic mice. Tyrosinase(141-161) peptide was found to be immunogenic and endogenously processed in the HLA-DRbeta1*0101 and HLA-DRbeta1*0401 transgenic mice with peptide specific production of IFNgamma or IL-5 respectively. The MART-1(29-43) peptide was only found immunogenic in HLA-DRbeta1*0101 mice.

Human papillomavirus (HPV) infection, particularly with types 16 and 18, is causally associated with the development of cervical cancer. Prophylactic vaccines against HPV have recently been licensed and have the primary aim of protecting children against future HPV infection and cervical cancer. However, these vaccines are unlikely to be effective in women with pre-existing HPV infection and disease. Previous studies have suggested that HPV16 E6-specific CD4+ T cells play a role in controlling viral infection; however, the epitopes recognized by such T-cells have not been defined. In this study, we analysed T-cell responses against HPV16 and 18 in ten healthy young women in an age group (21–31) with a high prevalence of HPV infection and clearance. Five individuals made HPV E6 responses, from which five candidate T-cell epitopes (three HPV16 E6 and two HPV18 E6) were identified. More detailed characterization of epitopes from HPV16 E6(127–141) and HPV18 E6(43–57) revealed HLA-DRB1*01 and HLA-DRB1*15 restriction, respectively. Furthermore, generation of a T-cell line against HPV16 E6(127–141) demonstrated that this epitope could be presented after endogenous processing of soluble HPV16 E6 protein. Overall we demonstrate a powerful approach for defining novel CD4+ T-cell epitopes from two oncogenic HPV types. This approach could be applied to study populations in developing countries with a high incidence of cervical cancer. Such epitopes could provide a more precise way of investigating the role of natural and vaccine-induced T-cell responses against HPV in blood and at sites of disease.

The expression of HLA-DR1 (DRB1*0101) is associated with an enhanced risk for developing rheumatoid arthritis (RA). To study its function, we have solved the three-dimensional structure of HLA-DR1 complexed with a candidate RA autoantigen, the human type II collagen peptide CII (259–273). Based on these structural data, the CII peptide is anchored by Phe263 at the P1 position and Glu266 at P4. Surprisingly, the Lys at the P2 position appears to play a dual role by participating in peptide binding via interactions with DRB1-His81 and Asn82, and TCR interaction, based on functional assays. The CII peptide is also anchored by the P4 Glu266 residue through an ionic interaction with DRB1-Arg71 and Glu28. Participation of DRB1-Arg71 is significant because it is part of the shared epitope expressed by DR alleles associated with RA susceptibility. Potential anchor residues at P6 and P9 of the CII peptide are both Gly, and the lack of side chains at these positions appears to result in both a narrower binding groove with the peptide protruding out of the groove at this end of the DR1 molecule. From the TCR perspective, the P2-Lys264, P5-Arg267, and P8-Lys270 residues are all oriented away from the binding groove and collectively represent a positive charged interface for CII-specific TCR binding. Comparison of the DR1-CII structure to a DR1-hemagglutinin peptide structure revealed that the binding of these two peptides generates significantly different interfaces for the interaction with their respective Ag-specific TCRs.

The main focus of this review has been to discuss the probable causes of the higher frequency of HLA DR1 antigen in patients with schizophrenia from Japan and Turkey, and also to see whether there was an impact of belonging to the Ural-Altaic language group. A general medline search on the terms HLA and schizophrenia was used as the method to determine HLA studies in patients with schizophrenia. Most of the findings were inconsistent regarding the increased or decreased frequencies of different Class I and II antigens. However, there were interesting results, which have been consistently repeated in several Japanese studies and in a Turkish study. HLA DR1 antigen was statistically significantly increased in Japanese and Turkish patients with schizophrenia. As Japanese and Turkish languages belong to the Ural-Altaic language group, HLA DR1 antigen might have a specific association with schizophrenia in Japanese and Turkish patients. Searching the frequency of HLA DR1 antigen in patients with schizophrenia in other members of Ural-Altaic language group is necessary to support this hypothesis. Other language groups (e.g. Indo-European) should be assessed as well.

Collagen (CII) 263-272 peptide, an autoantigen in rheumatoid arthritis, is a specific human leukocyte antigen (HLA)-DR1/4-binding peptide recognized by T-cell receptors (TCR). The affinity of influenza virus haemagglutinin (HA) 306-318 peptide for the antigen-binding groove of HLA-DR1/4 molecules is higher than that of CII263-272. The HLA-DR1/4-binding residues of HA306-318 are located in the region 308-317. Altered HA308-317 peptides with substitutions of TCR-contact residues may inhibit HLA-DR1/4-specific T-cell activation by blocking the antigen-binding site of HLA-DR1/4 molecules. To evaluate the role of altered HA308-317 peptides in HLA-DR1-restricted T-cell activation, we synthesized three altered HA308-317 peptides. The specific binding of altered HA308-317 peptides to HLA-DR1 molecules was examined using flow cytometry. Effects of altered HA308-317 peptides on HLA-DR1-specific T-cell hybridoma were studied by measuring T-cell proliferation and surface expression of CD69 or CD25. The results showed that altered HA308-317 peptides were able to bind to HLA-DR1 molecules and competed with CII263-272 or wildtype HA308-317 peptide. Compared with wildtype CII263-272 or HA308-317, altered HA308-317 peptides did not stimulate significant T-cell proliferation and CD69 or CD25 expression. Furthermore, the altered HA308-317 peptides inhibited HLA-DR1-specific T-cell activation induced by CII263-272 or wildtype HA308-317 peptide, which may suggest an effective therapeutic strategy in inhibition of HLA-DR1-specific T-cell responses in autoimmunity.

Transgenic mice expressing human HLA class II molecules provide a useful model for identifying HLA-restricted CD4+ epitopes. However, the influence of endogenous murine H-2-restricted T cell responses on HLA-restricted responses is not known. In the present study, we show that HLA-DR1 transgenic mice deficient for H-2 class II expression (HLA-DR1+/+/IAbeta0/0) exhibit an equivalent expression level of the transgene HLA-DR1 and a similar diversity in the TCR repertoire, but a slightly different number of CD4+ peripheral T cells, when compared to HLA-DR1 transgenic mice in which H-2 class II molecules were retained (HLA-DR1+/+/IAbeta+/+). More importantly, a strong antigen-specific HLA-DR1-restricted response was observed in nearly all HLA-DR1+/+/IAbeta0/0 mice immunized with HBV envelope protein (HBs) or capsid protein (HBc), whereas weak HBs- or HBc-specific HLA-DR1-restricted responses were detected in half of the immunized HLA-DR1+/+/IAbeta+/+ mice. Conversely, strong HBs- or HBc-specific H-2-restricted T cell responses were detected in HLA-DR1+/+/IAbeta+/+ mice but not in HLA-DR1+/+/IAbeta0/0 mice. Our results indicate that the coexpression of endogenous H-2 class II molecules reduces the intensity of HLA-DR1-restricted antigen-specific responses in transgenic mice, by favoring murine over human MHC recognition and education. Thus, HLA-DR1+/+/IAbeta0/0 mice represent a better model for identifying and characterizing HLA-DR1-restricted epitopes relevant for human disease.

Class II major histocompatibility complex (MHC) has tolerance for binding longer antigen peptides than those bound by class I MHC. In this paper, a normal mode analysis on HLA-DR1 class II MHC involving an antigen peptide indicated that the peptide-binding groove had some different dynamic characteristics from that of HLA-A2 class I MHC. The dynamic changes in the class I groove with removal of the bound peptide were limited primarily to the central region and the C-terminal side (corresponding to the C-terminal side of the bound peptide) of the groove, while the dynamic changes in the class II groove with removal of the bound peptide extended to the whole of the groove, and were especially remarkable around a strand located in the N-terminal side (corresponding to the N-terminal side of the bound peptide) of the groove. These results suggest that the N-terminal side of the class II groove is more flexible than the same side of the class I groove, and this flexibility may allow some N-terminal residues of the bound peptide to extend outside the class II groove. Definite anti-correlative motions with removal of the bound peptide appeared between two alpha-helical regions of class II MHC as in the case of class I MHC. These motions of the class II groove may play an important role in obtaining "a flexible dynamic fit" against diverse longer peptides both of whose terminals extend outside the groove.

Objective To identify peptides capable of altering the immune response to type II collagen (CII) in the context of HLA-DR. Methods Immunizing mice transgenic for the human HLA-DRB1*0101 immune response gene with CII elicits an arthritis (collagen-induced arthritis [CIA]) that resembles rheumatoid arthritis. We have previously identified an immunodominant determinant of CII, CII (263-270), recognized by T cells in the context of DR1. To produce synthetic peptides with the potential of disrupting the DR1-restricted immune response, synthetic analog peptides were developed that contain site-directed substitutions in critical positions. These peptides were used to treat CIA in DR1 transgenic mice. Results An analog peptide, CII (256-276, N(263), D(266)), that inhibited T cell responses in vitro, was identified. When DR1 mice were coimmunized with CII and CII (256-276, N(263), D(266)), the incidence and severity of arthritis were greatly reduced, as was the antibody response to CII. Moreover, CII (256-276, N(263), D(266)) was effective in down-regulating the immune responses to CII and arthritis, even when administered 2 weeks following immunization with CII. Spleen and lymph node cells from CII-immunized mice cultured with CII (256-276, N(263), D(266)) in vitro produced increased amounts of interleukin-4 (IL-4) compared with cells cultured with the wild-type peptide, CII (256-276). Furthermore, CII (256-276, N(263), D(266)) was incapable of preventing arthritis in DR1 IL-4(-/-) mice (genetically deficient in IL-4). Conclusion These data establish that CII (256-276, N(263), D(266)) is a potent suppressor of the DR-mediated immune response to CII. Its effect is mediated, at least in part, by IL-4. These experiments represent the first description of an analog peptide of CII recognized by T cells in the context of a human major histocompatibility complex molecule that can suppress autoimmune arthritis.

Rheumatoid arthritis is an autoimmune disease in which susceptibility is strongly associated with the expression of specific HLA-DR haplotypes, including DR1 (DRB1*0101) and DR4 (DRB1*0401). As transgenes, both of these class II molecules mediate susceptibility to an autoimmune arthritis induced by immunization with human type II collagen (hCII). The dominant T cell response of both the DR1 and DR4 transgenic mice to hCII is focused on the same determinant core, CII(263–270). Peptide binding studies revealed that the affinity of DR1 and DR4 for CII(263–270) was at least 10 times less than that of the model Ag HA(307–319), and that the affinity of DR4 for the CII peptide is 3-fold less than that of DR1. As predicted based on the crystal structures, the majority of the CII-peptide binding affinity for DR1 and DR4 is controlled by the Phe263; however, unexpectedly the adjacent Lys264 also contributed significantly to the binding affinity of the peptide. Only these two CII amino acids were found to provide binding anchors. Amino acid substitutions at the remaining positions had either no effect or significantly increased the affinity of the hCII peptide. Affinity-enhancing substitutions frequently involved replacement of a negative charge, or Gly or Pro, hallmark amino acids of CII structure. These data indicate that DR1 and DR4 bind this CII peptide in a nearly identical manner and that the primary structure of CII may dictate a different binding motif for DR1 and DR4 than has been described for other peptides that bind to these alleles.

Background Rheumatic heart disease (RHD), the only long term consequence of acute rheumatic fever, remains a leading cause of morbidity and mortality among young adults in Uganda. An inherited susceptibility to acute rheumatic fever centers around the major histocompatibility class II human leucocyte antigens. However, there is paucity of data from sub-Saharan Africa. This study compares the frequency of HLA class II DR alleles between RHD cases and normal controls in Uganda. Methods One hundred ninety-nine participants including 96 established RHD cases aged 5–60 years and 103 age and sex matched normal controls were recruited for participation. DNA was manually extracted from buffy coat samples and HLA analysis was performed. HLA-DR allelic frequency comparison between cases and controls were estimated using conditional logistic regression with 95% confidence intervals. P -values were corrected for multiple hypothesis testing. Results 199 participants (103 female, 51.8%) completed the study. The mean (SD) age in years for cases and controls were 29.6 (10.2) and 29(18), respectively. After conditional logistic regression and multiple hypothesis testing, HLA-DR1was associated with a decreased risk of RHD (OR = 0.42, CI 0.21-085, P = 0.01, Corrected P value (PC) = 0.09,) while HLA-DR11 was associated with increased risk of RHD (OR = 3.31, CI 1.57-6.97, P =

To investigate whether immunological responses to type II collagen (CII) play an important role in the patho- genesis of rheumatoid arthritis (RA), the presence of anti- CII antibodies was examined by enzyme immunoassay in 130 Japanese patients with RA, 10 systemic lupus ery- thematosus (SLE) patients, and 30 healthy subjects. In addition, the HLA-DRB1 genes of 40 RA patients were determined, and their association with positive findings of anti-CII antibodies was examined. A significantly high frequency of positive findings of anti-CII antibodies was detected in sera from RA patients (19%, P 0.05) in com- parison with that in sera from healthy subjects (3%). High frequencies of DRB1*0405 and 0101 alleles were observed in the 40 RA patients examined (40.0% and 30.0%, respec- tively). Patients with DRB1*0101 had a significantly higher rate of positive findings of anti-CII antibodies than those without DRB1*0101 (66.7% and 28.6%, respectively, P 0.05). No such association was observed for DRB1*0405. From these findings, we suggest that immunological re- sponses to CII may play an important role in the develop- ment of arthritis in some RA patients.

Frequencies of human leucocyte antigens (HLA)-A, -B and -DR were determined in 751 patients with chronic myelogenous leukaemia (CML) reported to the European Group for Blood and Marrow Transplantation after bone marrow transplantation from HLA-identical family donors and related to the occurrence of graft-versus-host disease (GVHD). HLA-A3 and DR1 were significantly associated with acute GVHD, the first with a higher risk (44% in HLA-A3(+) versus 34% in HLA-A3(-) patients) and the latter with a lower risk (28% in HLA-DR1(+) versus 38% in HLA-DR1(-) patients) for developing acute GVHD grade II--IV. Both factors were independent of known variables for GVHD as shown in a multivariate analysis. The results show that MHC alleles independently influence the incidence of GVHD in bone marrow transplantation from an HLA-identical donor for first chronic-phase CML. Possible mechanisms might include an HLA antigen-specific allele-associated effect, and/or non-specific allele-associated immune hypo- or hyper-responsiveness.

Occupational exposure to small molecules, such as metals, is frequently associated with hypersensitivity reactions. Chronic beryllium (Be) disease (CBD) is a multisystem granulomatous disease that primarily affects the lung, and occurs in ∼3% of individuals exposed to this element. Immunogenetic studies have demonstrated a strong association between CBD and possession of alleles of HLA-DP containing glutamic acid (Glu) at position 69 in the HLA-DPβ-chain. T cell clones were raised from three patients with CBD in whom exposure occurred 10 and 30 years previously. Of 25 Be-specific clones that were obtained, all were restricted by HLA-DP alleles with Glu at DPβ69. Furthermore, the proliferative responses of the clones were absolutely dependent upon DPβ Glu69 in that a single amino acid substitution at this position abolished the response. As befits a disease whose pathogenesis involves a delayed type hypersensitivity response, the large majority of Be-specific clones secreted IFN-γ (Th1) and little or no IL-4 (Th2) cytokines. This study provides insights into the molecular basis of DP2-associated susceptibility to CBD.

CTL play a pivotal role in the immune response during viral infections. In this study, the HLA class II restricted T(H) requirement for optimal in vivo induction of HLA class I restricted CTL responses has been investigated. Towards this goal, transgenic mice expressing both HLA class I (A*0201 or A2.1) and class II (DRB1*0101 or DR1) molecules have been derived. Immunization of these mice with an HLA A*0201-restricted and CMV-specific CTL epitope (pp65(495-503)), and either of three different tetanus toxin-derived MHC class II-binding T(H) epitopes, resulted in a vigorous CTL response. CTL specific for the pp65(495-503) epitope were dramatically enhanced in mice expressing both the HLA-DR1 and HLA-A*0201 transgenes. Notably, preinjection of three TT peptides (TT(639-652), TT(830-843), and TT(947-967)) increased the capability of HLA A*0201/DR1 Tg mice to respond to subsequent immunization with the T(H) + CTL peptide mixture. These results indicate that the use of HLA A*0201/DR1 Tg mice constitute a versatile model system (in lieu of immunizing humans) for the study of both HLA class I and class II restricted T-cell responses. These studies provide a rational model for the design and assessment of new minimal-epitope vaccines based on their in vivo induction of a pathogen-specific CTL response.

The human class II major histocompatibility complex protein HLA-DR1 has been shown previously to undergo a distinct conformational change from an open to a compact form upon binding peptide. To investigate the role of peptide in triggering the conformational change, the minimal requirements for inducing the compact conformation were determined. Peptides as short as two and four residues, which occupy only a small fraction of the peptide-binding cleft, were able to induce the conformational change. A mutant HLA-DR1 protein with a substitution in the beta subunit designed to fill the P1 pocket from within the protein (Gly(86) to Tyr) adopted to a large extent the compact, peptide-bound conformation. Interactions important in stabilizing the compact conformation are shown to be distinct from those responsible for high affinity binding or for stabilization of the complex against thermal denaturation. The results suggest that occupancy of the P1 pocket is responsible for partial conversion to the compact form but that both side chain and main chain interactions contribute to the full conformational change. The implications of the conformational change to intracellular antigen loading and presentation are discussed.

The design and synthesis of two pyrrolinone−peptide hybrid ligands (3 and 20) for the rheumatoid arthritis-associated class II MHC HLA-DR1 protein are described. The hybrids incorporate bispyrrolinones 4 and 21 as tetrapeptide mimics for amino acids VKQN (residues 309−312) of the virus hemagglutinin peptide HA 306−318 (PKYVKQNTLKLAT). Ligand construction employed our polypyrrolinone synthetic protocol, in conjunction with Fmoc-based solid-phase peptide synthesis. Bioaffinity studies reveal that hybrid ligand 3 bound the HLA-DR1 protein with affinity (IC50 = 137 nM) comparable to those of both the native HA 306−318 peptide (IC50 = 89 nM) and a control peptide (IC50 = 176 nM). This result demonstrates that the polypyrrolinone scaffold can be employed in the construction of bioactive peptide hybrid ligands, thus considerably expanding the scope and utility of the pyrrolinone scaffold.

The first hydrophobic pocket, P1, of class II MHC has been shown to be an important site of peptide anchoring. Two polymorphisms occur in this pocket in the human class II MHC beta chain at position 85 and 86. beta 85 is usually Val, occasionally Ala, whereas beta 86 can be Gly or Val. However, Ala85 is found only in conjunction with Val86. The independent effect of the polymorphism at these two positions on the binding of normal and substituted antigenic peptides has never been examined. To do so, three soluble HLA-DR1 variants that contain the naturally occurring combinations of these side chains at these two positions were generated and tested with a panel of influenza matrix peptides varying at anchor P1. DR1 alleles differing only at position 86 are very similar in the binding of a panel of antigenic peptide, indicating that beta 86 does not substantially influence the peptide binding of DR1. In contrast, DR1 varying only at position beta 85 differ in their binding of substituted peptides containing Ala, Tyr or Trp at the P1 anchor position. Thus, beta 85 shows the predominant effect on the P1 anchor side chain preference of the P1 pocket in DR1. This is in contrast to other HLA-DR alleles where beta 86 has been shown to control the nature of the P1 anchor. These previous data together with our own imply that the role of polymorphism in P1 may be influenced by the contextual framework of the remaining allelic polymorphism.

Lassueur-Graham-Little-Piccardi syndrome (LGLPS) is a rare lichenoid dermatosis characterized by progressive cicatricial alopecia of the scalp, loss of pubic and axillary hairs and keratosis pilaris. The syndrome is considered a form of follicular lichen planus (LP). Although the familial occurrence of LP is a well-described phenomenon, no familial case of LGLPS has ever been reported. We describe the occurrence of LGLPS in a mother and her daughter. HLA typing revealed HLA-DR1 in both patients. Topical tacrolimus was of partial benefit in the daughter. Copyright © 2004 S. Karger AG, Basel.

The predominant peptides bound to major histocompatibility complex class II molecules expressed on human B cells are derived from a relatively limited number of self proteins. To determine whether any of the prebound self peptides might be released in endosomes during recycling, water-soluble HLA-DR1 molecules were incubated with a high affinity synthetic peptide at pH 4.0 and 7.0 at 37 degrees C. The resulting bound peptide repertoire was then acid extracted, and separated by reversed-phase high performance liquid chromatography. Using a combination of mass spectrometry and ultraviolet spectroscopy, prebound self peptides and newly bound synthetic peptide were characterized. Most self peptides bound to HLA-DR1 were not appreciably released during extended exposure to pH 4.0. However, some invariant chain-derived peptides were uniquely released at this pH.

Molecules of the major histocompatibility complex (MHC) present antigenic peptides for surveillance by T cells and, thereby, are central for the initiation of an adaptive immune response. The structure of MHC class II (MHC II) has been known for almost 20 years and all structures solved so far show a single overall conformation. However, there is ample evidence for variations of this uniform picture. In this thesis I wanted to lay the structural basis for these conformers. Empty MHC II molecules quickly lose their ability to rebind peptides, what is thought to be accompanied by a conformational change, but direct structural proof is missing. Here, a molecular dynamics simulations-derived model for the transition of empty MHC II from a peptide- receptive to a non-receptive state is presented. It predicts a closure of the binding site by straightening and inwards movement of a flanking helix, stabilized by the formation of a hydrogen bond between highly conserved residues, functioning as lock. This model was verified by in vitro mutagenesis studies. First, an intramolecular disulfide bridge was designed that proved the conformational flexibility predicted by the model. More importantly, disabling the locking mechanism by mutagenesis led to highly peptide receptive MHC II species. Thus, the data presented here strongly supports the postulated model for the non-receptive state of MHC II. In addition, also ligand- dependent conformers of MHC II have been described. For the human leukocyte antigen (HLA)-DR1 it has been reported that terminal extension of peptide ligands beyond the core binding motif influences the MHC II conformation. Here, the structural basis for this effect was probed by X-ray crystallography. Structures of four HLA-DR1 molecules in complex with length variants of a viral antigen were solved, but no conformational difference was identified. In contrast, X-ray crystallography of HLA-DR1 in complex with two length variants of the class II-associated invariant chain derived peptide (CLIP), a natural intermediate in antigen-processing, surprisingly revealed a new conformation. The short version of CLIP was found in an inversed orientation as compared to the canonical binding mode. Remarkably, no rearrangements in the binding groove were necessary to establish the same conserved hydrogen bonding network for both directions. Accompanying NMR experiments proved that thermodynamic stability of the complex drives CLIP to reorient within the binding groove from the canonical to the reversed state. It was possible to trap the antigen in only one orientation by introduction of favorable anchors. Finally, after screening databases for other potentially reversed antigens, five new HLA-DR1 structures with different peptides were solved. However, none of these showed the non-canonical binding mode. Instead, the structures illustrated the high adaptability of the MHC to different peptide sequences, e.g. by a before unseen opening of the binding pocket 7. In conclusion, the structures of several pMHC II suggest a novel peptide binding mode and underscore the various strategies of MHC II to form highly stable peptide complexes., Moleküle des Haupthistokompatibilitätskomplexes (MHC) präsentieren Antigenpeptide für die Überwachung durch T-Zellen und sind somit ein zentraler Bestandteil der adaptiven Immunantwort. Die Struktur von MHC Klasse II (MHC II) Komplexen ist seit fast 20 Jahren bekannt und alle Strukturen zeigen eine einheitliche Konformation. Es gibt jedoch reichhaltige Beweise für weitere Konformationen. Mit der vorliegenden Arbeit möchte ich die strukturelle Grundlage hierfür legen. Leere MHC II Moleküle verlieren schnell die Fähigkeit, erneut Peptide zu binden. Es wird angenommen, dass dies mit einer Änderung ihrer Konformation einhergeht, aber bislang fehlt ein direkter struktureller Beweis. In dieser Arbeit wird ein Modell für den Übergang von einem peptidrezeptiven zu einem nicht rezeptiven Zustand vorgestellt, das auf Simulation der Molekulardynamik des leeren MHC basiert. Dabei bewirkt die Streckung und Einwärtsbewegung einer flankierenden Helix das Verschließen der Antigenbindungsstelle, was durch die Ausbildung einer als Verschluss fungierenden Wasserstoffbrückenbindung zwischen hoch konservierten Aminosäuren stabilisiert wird. Dieses Modell konnte durch in vitro Mutagenesestudien verifiziert werden. Dafür wurde zunächst eine intramolekulare Schwefelbrücke eingebaut, die die konformationelle Flexibilität des Modells bewies. Von größerer Bedeutung war jedoch die Erkenntis, dass das Ausschalten des Verschlussmechanismus zu einer hoch peptidrezeptiven MHC II Art führte. Die vorgelegten Daten unterstützen somit das postulierte Modell des nicht- rezeptiven MHC II. Weiterhin sind auch ligandabhängige MHC II Konformationen beschrieben worden. Die Konformation des humanen Leukozytenantigen (HLA)-DR1 wird beispielsweise durch Verlängerungen der Peptidligandentermini über das Kernbindungsmotiv hinaus beeinflusst. Die strukturelle Basis hierfür wurde mittels Röntgenstrukturanalyse untersucht. Dabei wurden vier Strukturen von HLA-DR1 im Komplex mit Längenvarianten eines viralen Antigens gelöst, wobei jedoch keine Änderung in der Konformation zu beobachten war. Im Gegensatz dazu zeigte die Röntgenstrukturanalyse von HLA-DR1 im Komplex mit zwei Längenvarianten des „class II-associated invariant chain derived peptide“ (CLIP), ein natürliches Intermediat bei der Antigenprozessierung, überraschenderweise eine neuartige Konformation. Die kürzere CLIP Version lag, im Vergleich zur kanonischen Bindungsweise, umgedreht in der Antigenbindungstasche. Dabei waren bemerkenswerterweise keine Änderungen innerhalb der Bindungsfurche nötig, um dasselbe konservierte Wasserstoffbrückennetzwerk für beide Richtungen auszubilden. Begleitende NMR- Experimente bewiesen, dass die Neuorientierung des Peptides durch die thermodynamische Stabilität des Komplexes angetrieben wurde. Es war möglich, diese Neuorientierung durch den Einbau von Peptidankern zu beeinflussen. Zusätzlich wurden Datenbanken nach weiteren potentiell umgedrehten Peptiden durchsucht und fünf neue Strukturen von HLA-DR1 im Komplex mit unterschiedlichen Peptiden gelöst. Dabei zeigte jedoch keine die gewünschte Orientierung. Stattdessen betonten sie die hohe Anpassungsfähigkeit des MHC an verschiedene Peptidsequenzen, z.B. durch eine zuvor unbeobachtete Öffnung der Bindungstasche 7. Zusammenfassend deuten die verschiedenen Strukturen von Peptid-MHC II Komplexen einen neuartigen Peptidbindungsmodus an und unterstreichen die verschiedenen Möglichkeiten des MHC II, hochstabile Peptidkomplexe auszubilden.

A monoclonal antibody 137BL7 raised against purified DR1 protein was shown to bind specifically to 5/5 DR1, 18/18 DR2 cells and 0/23 non-DR1,2 cells by cell-EIA. Further analysis by FACS using HLA-transfectants revealed that 137BL7 also bound specifically to the DRB5 transfectants in addition to the expected DR1 transfectants. However, it did not bind to the DR2 (DR15) transfectants, showing that cross-reactivity with DR2 cells lies with the DRB5 (DR51) rather than the DRB1 gene product. A comparison of the HLA-DR amino acid sequences of DR1 and DR51 antigens revealed a common glutamic acid residue at position 96, which may form the putative binding epitope of this mAb.

The DRB1/3/4/5 loci from 97 patients with MS and 100 normal control subjects were analyzed. DRB1*15 increased the risk of MS (OR = 4.2, p0.0001), whereas DR1 decreased the risk (OR = 0.30, p(c )= 0.005). In analyses of the DR1 risk in relation to DR51, DR52, and DR53 alleles, DR1 in combination with DR53 was found to have the strongest protective effect against MS in all subjects (OR = 0.05, p0.0001) and in DRB1*15-negative subjects (OR = 0.09, p(c )= 0.026). DR53 may be an important allele or haplotype, acting together with DR1 to protect against MS.

HLA DR1 molecules are coded by a single polymorphic DRB1 gene. We have observed rare DR1 cells in one Caucasoid family and three unrelated individuals that also reacted with some anti-DR2 sera. Since the second DR antigen was normally expressed, these cells appeared as triplets. Contrary to serology, the cells were not typed by HTCs defining Dw2, Dw12, and Dw21. Further investigations on these unusual DR1 + 2 ∗ haplotypes were conducted by DNA oligotyping and by sequencing of the DRB first-domain exon. The results showed that these DR1 haplotypes, besides their DRB1 ∗ 0101 allel, carried also a DRB5 ∗ 0101 allele.

In Eastern Hungary, vitiligo is found to be associated with HLA-DR1. When other autoimmune disorders are also present, DR3 is also increased.

HLAMatchmaker is a matching algorithm that can be used to characterize antibodies specific for structurally defined epitopes. Under the auspices of the 15(th) International Histocompatibility Workshop, we are conducting a multilaboratory collaborative project to characterize these epitopes and also to determine how often they induce specific antibodies in patients with rejected kidney transplants. This report addresses the reactivity of post-allograft nephrectomy sera tested for DRB antibodies with Luminex assays using single alleles. This analysis was performed for 19 informative kidney transplant cases contributed by 13 laboratories worldwide. There were 11 cases with a single DR2 mismatch (DR15 or DR16), and nine of them (82%) showed antibodies with both DR2 and DR1. Although these antigens might share an epitope recognized by these antibodies, this interpretation is incorrect. The HLAMatchmaker analysis offers a clearly different explanation that involves antibodies induced by DR51, which commonly associates with DR2. DR51 has an epitope defined by the 96EV eplet, which is also present on DR1 but no other DR antigen. This means that the reactivity with DR51 and DR1 reflects the presence of 96EV-specific antibodies. Conversely, we analyzed eight patients sensitized by a single DR1 mismatch that has no associated DR51. All of these patients reacted also with DR51, and this could be explained only by antibodies against the shared 96EV eplet. These findings demonstrate that 96EV represents a highly immunogenic epitope that can induce cross-sensitization between antigens encoded by the different DRB loci, and also that DR51 is important in determining DRB mismatch acceptability of potential donors. This analysis has also demonstrated that antibody responses are restricted to a few epitopes on these immunizing DR antigens. For DR2 they are 142M3 (unique for DR2), 71QAA (shared with DB5*02), and 96QV (shared with DR10). DR51 mismatches appear to have three immunogenic eplets: 96EV (shared with DR1), 108T3 (unique for DR51), and 40HFD (shared with DR9). Immunogenic eplets on DR1 are 12LKF2 (unique for DR1), 14FEH (shared with DR9 and DR10), and 25HRL (shared with DR10).

This study of 110 seropositive rheumatoid arthritis (RA) patients confirms the significant association of susceptibility to RA with HLA-DR4 specificity (P less than 0.001). The DR1 frequency is elevated in the entire seropositive patient group, reaching marginal significance (P less than 0.025). The DR4-negative patients, however, have a much higher prevalence of DR1 (P less than 0.001). Surprisingly, the DRw6 specificity is significantly increased in the remaining DR4- and DR1-negative patients (P less than 0.01). These results demonstrate that RA is not associated with a single HLA-specificity, but to various degrees with DR4, DR1, and DRw6. These findings, and particularly the newly recognized association with DRw6, support the hypothesis that functionally equivalent shared epitopes or conformations on otherwise distinct MHC molecules may confer risk for developing RA.