CD28 costimulation is critical for experimental allergic asthma in HLA-DQ8 transgenic mice

The objective of this study was to investigate the contribution of the CD28 costimulatory molecules to allergen-induced primary and chronic inflammatory responses. To this end, we have developed and characterized a short ragweed allergen-induced asthma model involving sensitization of HLA-DQ transgenic mice followed by intranasal challenge with allergen. Forty-eight hours after primary challenge, sensitized DQ8 mice developed pulmonary eosinophilic inflammation, airway hyperreactivity, Th2 cytokines, and IgE/IgG1 Ab. This allergic inflammatory response was absent in H-2Abeta(0) and DQ8/CD28(0) mice. Secondary rechallenge with allergen 4 weeks later induced even greater inflammatory changes in the airways of DQ8 mice with eosinophils being the predominant inflammatory cells while only pulmonary lymphocytosis was observed in DQ8/CD28(0) mice. No inflammation was detected in H-2Abeta(0) mice. Proliferation and cytokine profile studies demonstrated that CD28 regulates T-cell activation and effector function. Therefore, CD28 is essential for the extrinsic asthma and can be a target for immunotherapy.     

To B or not to B: role of B cells in pathogenesis of arthritis in HLA transgenic mice

Population studies have shown that amongst all the genetic factors linked with autoimmune disease development, MHC class II genes are the most significant. Experimental autoimmune arthritis resembling human rheumatoid arthritis (RA) can be induced in susceptible strains of mice following immunization with type II collagen (CIA). We generated transgenic mice lacking endogenous class II molecules and expressing various HLA genes including RA-associated, HLA-DRB1*0401 and HLA-DQ8, and RA-resistant, DRB1*0402, genes. The HLA molecules in these mice are expressed on the cell surface and can positively select CD4+ T cells expressing various Vβ T cell receptors. Endogenous class II invariant chain is required for proper functioning of the class II transgene. Arthritis development in transgenic mice is CD4+ and B cells dependent. Studies in humanized mice showed that B cells are required as antigen presenting cells in addition to antibody producing cells for the development of CIA. The transgenic mice expressing *0401 and *0401/DQ8 genes developed sex-biased arthritis with predominantly females being affected, similar to that of human RA. Further, the transgenic mice produced autoantibodies like rheumatoid factor and anti-cyclic antibodies. Antigen presentation by B cells leads to a sex-specific immune response in DRB1*0401 mice suggesting a role of B cells and HLA-DR in rendering susceptibility to develop arthritis in females.     

H2-O influence on antigen presentation in H2-E-expressing mice

MHC class II molecules sample peptides generated in the endosomal/lysosomal system for cell surface presentation to CD4+ T cells. Peptide loading requires the endosomal/lysosomal resident HLA-DM (DM; H2-DM, mouse), but in B cells, DM is tightly associated with HLA-DO (DO; H2-O, mouse). We have previously shown that H2-O differentially modulates the processing and presentation of different antigenic epitopes on H2-Ab molecules. Using H2-Ead-transgenic mice, we here show that presentation of different epitopes by H2-Ed/b molecules is similarly influenced by H2-O after membrane immunoglobulin-mediated uptake of antigen. In addition, B cells from H2-Ead-transgenic mice (which co-express H2-Ab and H2-Ed/b molecules) show an altered pattern of presentation of H2-Ab-restricted epitopes. In H2-Ead-transgenic, H2-O-deficient mice, further changes in the peptide repertoire were observed. Thus, H2-Ed/b expression influences the epitopes presented by H2-Ab, and this effect is further altered by expression of H2-O.     

PRBAM: a new tool to analyze the MHC class I and HLA-DR anchor motifs

Major histocompatibility complex (MHC) genes are highly polymorphic, which makes each MHC molecule different regarding their peptide repertoire, so they can bind and present to T lymphocytes. The increasing importance of immunopeptidomics and its use in personalized medicine in different fields such as oncology or autoimmunity demand the correct analysis of the peptide repertoires bound to human leukocyte antigen type 1 (HLA-I) and HLA-II molecules. Purification of the peptide pool by affinity chromatography and individual peptide sequencing using mass spectrometry techniques is the standard protocol to define the binding motifs of the different MHC-I and MHC-II molecules. The identification of MHC-I binding motifs is relatively simple, but it is more complicated for MHC-II. There are some programs that identify the anchor motifs of MHC-II molecules. However, these programs do not identify the anchor motif correctly for some HLA-II molecules and some anchor motifs have been deduced using subjective interpretation of the data. Here, we present a new software, called PRBAM (Peptide Repertoire-Based Anchor Motif) that uses a new algorithm based on the peptide–MHC interactions and, using peptide lists obtained by mass spectrometry sequencing, identifies the binding motif of MHC-I and HLA-DR molecules. PRBAM has an easy-to-use interface, and the results are presented in graphics, tables and peptide lists. Finally, the fact that PRBAM uses a new algorithm makes it complementary to other existing programs.     

Modulation of insulitis and type 1 diabetes by transgenic HLA-DR3 and DQ8 in NOD mice lacking endogenous MHC class II

To evaluate the contributions of DR3 and DQ8 to the etiopathogenesis of type 1 diabetes in a diabetes-predisposing milieu, we developed human leukocyte antigen (HLA) transgenic mice on the nonobese diabetic (NOD) background in the absence of the endogenous class II molecule, I-A(g7) and studied the incidence of both spontaneous and experimental (induced) autoimmune diabetes. Transgenic expression of HLA-DR3 and -DQ8 (either alone or in combination) did not confer susceptibility to spontaneous or cyclophosphamide-induced type 1 diabetes. Expression of I-A(g7) was mandatory for development of spontaneous or cyclophosphamide-induced diabetes. However, multiple low doses of streptozotocin could induce diabetes in all groups of mice independent of the class II molecules expressed. In unmanipulated mice, only islets from I-A(g7+/+) mice revealed significant intra-islet infiltration. Although a characteristic peri-insulitis/peri-ductulitis was present in Abeta(0)/NOD mice, islets from DR3, DQ8 and DR3 x DQ8 double transgenic mice demonstrated significantly less infiltration. In conclusion, transgenic expression of HLA-DR3 and -DQ8 associated with predisposition to type 1 diabetes alone is not sufficient to induce spontaneous diabetes in NOD mice lacking endogenous class II molecules.     

Quality control of MHC class II associated peptides by HLA-DM/H2-M

For many years the crucial components involved in MHC class II mediated antigen presentation have been thought to be known: polymorphic MHC class II molecules, the monomorphic invariant chain (li) and a set of conventional proteases that cleave antigenic proteins thereby generating ligands able to associate with MHC class II molecules. However, in 1994 it was found that without an additional molecule, HLA-DM (DM), efficient presentation of protein antigens cannot be achieved. Biochemical studies showed that DM acts as a molecular chaperone protecting empty MHC class II molecules from functional inactivation. In addition, it serves as a peptide editor: DM catalyzes not only the release of the invariant chain remnant CLIP, but of all sorts of low-stability peptides, and simultaneously favors binding of high-stability peptides. Through this quality control of peptide loading, DM enables APCs to optimize MHC restriction and to display their antigenic peptide cargo on the surface for prolonged periods of time to be scrutinized by T cells.     

Ubiquitination of HLA‐DO by MARCH family E3 ligases

HLA‐DO (DO) is a nonclassical MHC class II (MHCII) molecule that negatively regulates the ability of HLA‐DM to catalyse the removal of invariant chain‐derived CLIP peptides from classical MHCII molecules. Here, we show that DO is posttranslationally modified by ubiquitination. The location of the modified lysine residue is shared with all classical MHCII beta chains, suggesting a conserved function. Three membrane‐associated RING‐CH (MARCH1, 8 and 9) family E3 ligases that polyubiquitinate MHCII induce similar profiles of polyubiquitination on DOβ. All three MARCH proteins also influenced trafficking of DO indirectly by a mechanism that required the DOβ encoded di‐leucine and tyrosine‐based endocytosis motifs. This may be the result of MARCH‐induced ubiquitination of components of the endocytic machinery. MARCH9 was by far the most efficient at inducing intracellular redistribution of DO but did not target molecules for lysosomal degradation. The specificity of MARCH9 for HLA‐DQ and HLA‐DO suggests a need for common regulation of these two MHC‐encoded molecules.     

Dendritic cell function in mice lacking Plexin C1

Semaphorins are secreted or transmembrane proteins that provide essential repulsive guidance cues to growing axons or endothelial cells through their receptors of the Plexin and Neuropilin family. Semaphorins and Plexins are also expressed in the immune system where their function remains elusive. In particular, Plexin C1 is expressed by mouse dendritic cells (DCs) and is the receptor for the poxvirus semaphorin homolog A39R. We previously found that Plexin C1 engagement by A39R inhibits integrin-mediated DC adhesion and chemokine-induced migration. Here, we show that a cellular ligand for Plexin C1 is expressed both by activated T cells and DCs, suggesting that Plexin C1 might be engaged on DCs both in cis and in trans. We used Plexin C1(-/-) mice to explore the role of Plexin C1 in DC function. DC development is unaffected in these mice. In two different in vivo assays, Plexin C1(-/-) DC migration to lymph nodes (LNs) was lower than that of wild-type (WT) DC but this difference was not statistically significant. Plexin C1(-/-) bone marrow-derived DCs induced normal in vitro T cell responses but reduced in vivo T cell responses when injected subcutaneously to WT mice. Finally, in vivo T cell responses to ovalbumin peptide and contact hypersensitivity to dinitrofluorobenzene were slightly decreased in Plexin C1(-/-) mice. These results suggest a role for Plexin C1 in DC migration or mobility within the LNs.     

MHC class I dimer formation by alteration of the cellular redox environment and induction of apoptosis

Many MHC class I molecules contain unpaired cysteine residues in their cytoplasmic tail domains, the function of which remains relatively uncharacterized. Recently, it has been shown that in the small secretory vesicles known as exosomes, fully folded MHC class I dimers can form through a disulphide bond between the cytoplasmic tail domain cysteines, induced by the low levels of glutathione in these extracellular vesicles. Here we address whether similar MHC class I dimers form in whole cells by alteration of the redox environment. Treatment of the HLA-B27-expressing Epstein-Barr virus-transformed B-cell line Jesthom, and the leukaemic T-cell line CEM transfected with HLA-B27 with the strong oxidant diamide, and the apoptosis-inducing and glutathione-depleting agents hydrogen peroxide and thimerosal, induced MHC class I dimers. Furthermore, induction of apoptosis by cross-linking FasR/CD95 on CEM cells with monoclonal antibody CH-11 also induced MHC class I dimers. As with exosomal MHC class I dimers, the formation of these structures on cells is controlled by the cysteine at position 325 in the cytoplasmic tail domain of HLA-B27. Therefore, the redox environment of cells intimately controls induction of MHC class I dimers, the formation of which may provide novel structures for recognition by the immune system.     

Peptidomic analysis of type 1 diabetes associated HLA‐DQ molecules and the impact of HLA‐DM on peptide repertoire editing

HLA‐DM and class II associated invariant chain (Ii) are key cofactors in the MHC class II (MHCII) antigen processing pathway. We used tandem mass spectrometry sequencing to directly interrogate the global impact of DM and Ii on the repertoire of MHCII‐bound peptides in human embryonic kidney 293T cells expressing HLA‐DQ molecules in the absence or presence of these cofactors. We found that Ii and DM have a major impact on the repertoire of peptides presented by DQ1 and DQ6, with the caveat that this technology is not quantitative. The peptide repertoires of type 1 diabetes (T1D) associated DQ8, DQ2, and DQ8/2 are altered to a lesser degree by DM expression, and these molecules share overlapping features in their peptide binding motifs that are distinct from control DQ1 and DQ6 molecules. Peptides were categorized into DM‐resistant, DM‐dependent, or DM‐sensitive groups based on the mass spectrometry data, and representative peptides were tested in competitive binding assays and peptide dissociation rate experiments with soluble DQ6. Our data support the conclusion that high intrinsic stability of DQ‐peptide complexes is necessary but not sufficient to confer resistance to DM editing, and provide candidate parameters that may be useful in predicting the sensitivity of T‐cell epitopes to DM editing.     

Humanized transgenic mice expressing HLA DR4-DQ3 haplotype: reconstitution of phenotype and HLA-restricted T-cell responses

Many autoimmune conditions have close genetic linkages to particular human histocompatibility leukocyte antigen (HLA) class II genes. With the aim of establishing a murine model of autoimmune disease, we have generated an HLA DR4-DQ3 haplotype transgenic (Tg) mouse that expresses a 440-kb yeast artificial chromosome harbouring DRA, DRB1*040101, DRB4*010301, DQA1*030101, DQB1*0302 and all the internal regulatory segments. This Tg mouse line was crossed to human CD4 (hCD4) Tg mice and endogenous class II knockout mice (I-A(o/o) and I-E(o/o)) lines to generate a DR4-DQ3.hCD4.IAE(o/o) Tg line. The Tg DR and DQ molecules are expressed on the physiological cell types in these animals, i.e. on most B cells (>85%), dendritic cells (DCs) and macrophages but not on T cells, with levels of expression comparable with those of human B cells (where DR > DQ expression). The DR4/DQ3 transgenes fully reconstituted the CD4 T-cell compartment, in both the thymus and the periphery, and the analysis of the T-cell receptor repertoire in the Tg mice confirmed that these class II molecules were able to mediate thymic selection of a broad range of Vbeta families. HLA DR4- and DQ3-restricted T-cell responses were elicited following immunization with known T-cell determinants presented by these molecules. Furthermore, the DR4-DQ3-restricted CD4(+) T cells conferred protective antibody-mediated immunity against an otherwise lethal infection with Salmonella enterica var. typhimurium. These new DR4-DQ3 Tg mice should prove to be valuable tools for dissecting the importance of this class II haplotype in autoimmune disorders like rheumatoid arthritis.     

B cells as effectors and regulators of sex-biased arthritis

B cells have been implicated both with pathogenic as well as protective capabilities in induction and regulation of autoimmune diseases. Rheumatoid arthritis (RA) is an autoimmune disease that occurs more often in women than men. A significant role of B cells as antibody producing and antigen-presenting cells has been demonstrated in RA. Predisposition to RA is associated with the presence of certain HLA class II alleles that share sequences with DRB1*0401. To determine the role of HLA genes and B cells in vivo, we have generated transgenic mice carrying HLA genes, DRB1*0401 and DQ8, known to be associated with susceptibility to RA. Humanized mice can be induced to develop arthritis that mimics human disease in clinical, histopathological and sex bias. Effect of hormones on immune cells and their function has been described in humans and mice and has been suggested to be the major reason for female bias of autoimmune diseases. An immune response to an antigen requires presentation by HLA molecules thus suggesting a critical role of MHC in combination with sex hormones in susceptibility to develop rheumatoid arthritis. Based on our observations, we hypothesize that modulation of B cells by estrogen, presentation of modified antigens by DR4 and production of antigen-specific B cell modulating cytokines leads to autoreactivity in females. These data suggest that considering patient's sex may be crucial in selecting the optimal treatment strategy. Humanized mice expressing RA susceptible and resistant haplotype provide a means to investigate mechanism sex-bias of arthritis and future strategies for therapy.     

HLA-DR and HLA-DQ polymorphism in human thyroglobulin-induced autoimmune thyroiditis: DR3 and DQ8 transgenic mice are susceptible

In contrast to H2-based susceptibility to experimental autoimmune thyroiditis (EAT) induced with thyroglobulin (Tg), human leukocyte antigen (HLA) association with Hashimoto's thyroiditis, the human counterpart, is less clear, and determining association is further complicated by DR/DQ linkage disequilibrium. Previously, we addressed the controversial implication of HLA-DR genes by introducing HLA-DRA/DRB1*0301 (DR3) transgene into endogenous class II negative H2Ab(0) mice. EAT induction with either human (h) or mouse (m) Tg demonstrated the permissiveness of DR3 molecules for shared Tg epitopes. Here, we examined the participation of HLA-DQ genes by introducing DQA1*0301/DQB1*0302 (DQ8) transgene into class II negative Ab(0) or class I and II negative beta(2)m((-/-)) Ab(0) mice. About 50% and 80% of HLA-DQ8(+) Ab(0) and beta(2)m(-) Ab(0) mice, respectively, developed moderate EAT after hTg immunization, but only minimal response to mTg. The hTg presentation to hTg-primed cells was blocked by anti-DQ mAb in vitro. By contrast, HLA-DRB1*1502 (DR2) and *0401 (DR4) transgenes contributed little to hTg induction. Similarly, DQA1*0103/DQB1*0601 or DQA1*0103/DQB1*0602 (DQ6) transgenic Ab(0) mice were unresponsive to hTg induction and carried no detectable influence in DQ8/DQ6 double transgenic mice. Thus, both HLA-DR and -DQ polymorphism exists for hTg in autoimmune thyroiditis. The use of defined single or double transgenic mice obviates the complications seen in polygenic human studies.     

Allergy to antibiotics: T-cell recognition of amoxicillin is HLA-DR restricted and does not require antigen processing

Allergic immune responses are initiated and maintained by T cells that recognize peptidic fragments of allergens in the context of major histocompatibility complex (MHC) class II molecules. An anomaly of this model exists in the T-cell response to haptens. Haptens are nonpeptide antigens that alone are too small to provoke an immune response. Nevertheless. T-cell responses to haptenic allergens clearly occur and are critically involved in allergic immune responses to drugs such as penicillin. Although the mechanisms that generate T-cell epitopes from protein antigens are well understood, haptens create T-cell epitopes by alternative mechanisms. These may include binding of haptens directly to preformed MHC-peptide complexes on the cell surface, or indirect association with MHC molecules after conjugation with self cell surface or serum proteins that are then processed and presented as haptenated peptide antigens. Which of these unorthodox mechanisms of epitope generation is dominant in allergy to penicillin is unknown. This study aims to determine the nature of the epitopes recognized by amoxicillin-specific T cells from allergic donors, and to clarify whether T-cell responses to penicillin antibiotics are MHC restricted and require haptenated self proteins to be processed before recognition. Human T-cell lines specific for amoxicillin were raised and used in assays with processing-disabled and MHC-class Il-typed antigenpresenting cells to determine the MHC restriction and processing requirements of T cells recognizing amoxicillin. Fixation of antigenpresenting cells with paraformaldehyde. before or after pulsing with amoxicillin. established that T cells can recognize amoxicillin-containing epitopes with a similar ezfficiency irrespective of whether the antigenic conjugate has been internalized and processed. These results suggest that amoxicillin can bind directly to preformed MHC-peptide complexes and need not necessarily involve the processing of haptenated self carrier proteins before recognition of the conjugate by amoxicillinpecific T cells.     

Thermal-exchange HLA-E multimers reveal specificity in HLA-E and NKG2A/CD94 complex interactions

There is growing interest in HLA-E-restricted T-cell responses as a possible novel, highly conserved, vaccination targets in the context of infectious and malignant diseases. The developing field of HLA multimers for the detection and study of peptide-specific T cells has allowed the in-depth study of TCR repertoires and molecular requirements for efficient antigen presentation and T-cell activation. In this study, we developed a method for efficient peptide thermal exchange on HLA-E monomers and multimers allowing the high-throughput production of HLA-E multimers. We optimized the thermal-mediated peptide exchange, and flow cytometry staining conditions for the detection of TCR and NKG2A/CD94 receptors, showing that this novel approach can be used for high-throughput identification and analysis of HLA-E-binding peptides which could be involved in T-cell and NK cell-mediated immune responses. Importantly, our analysis of NKG2A/CD94 interaction in the presence of modified peptides led to new molecular insights governing the interaction of HLA-E with this receptor. In particular, our results reveal that interactions of HLA-E with NKG2A/CD94 and the TCR involve different residues. Altogether, we present a novel HLA-E multimer technology based on thermal-mediated peptide exchange allowing us to investigate the molecular requirements for HLA-E/peptide interaction with its receptors.     

Differential up-regulation of HLA-DM, invariant chain, and CD83 on myeloid and plasmacytoid dendritic cells from peripheral blood

Two main dendritic cell (DC) subsets have been described in peripheral blood, the myeloid subset or DC1 that is characterized by the presence of CD11c and the plasmacytoid subset or DC2 negative for this marker. The two subsets may perform different functions and have been defined as immunogenic (the myeloid subset) or tolerogenic (the plasmacytoid subset). The expression of human leukocyte antigen (HLA)-DM molecules, which act as peptide editors in the antigen presentation process, was studied in freshly isolated plasmacytoid and myeloid DCs from peripheral blood. The expression of the invariant chain (Ii), the major histocompatibility complex class II (MHC-II) : class II-associated Ii peptide (CLIP) complex, and CD83 was also investigated. The results showed that intracellular expression of HLA-DM and the Ii was significantly higher in the plasmacytoid than in the myeloid DC subset. In contrast, a higher fraction of cell expressing MHC-II : CLIP complex was found in the myeloid than in the plasmacytoid DC subpopulation. CD83 was not detected in any of these two subsets. Following culture of these cells with interleukin-3 (IL-3), tumor necrosis factor-alpha (TNFalpha) and/or heat shock protein-70 (HSP-70), the expression of intracellular HLA-DM was up-regulated in the myeloid DCs to levels similar to those found in the plasmacytoid DCs, whilst the Ii was down-regulated in the plasmacytoid subset to similar levels to those expressed in the myeloid DCs. In addition, CD83 was up-regulated in the myeloid (CD11c+) but not in the plasmacytoid (CD11c-) DCs. The expression pattern of these antigen-processing molecules could be related to the immaturity and function attributed to these DC subsets.     

Antigen processing and immune regulation in the response to tumours

The MHC class I and II antigen processing and presentation pathways display peptides to circulating CD8⁺ cytotoxic and CD4⁺ helper T cells respectively to enable pathogens and transformed cells to be identified. Once detected, T cells become activated and either directly kill the infected / transformed cells (CD8⁺ cytotoxic T lymphocytes) or orchestrate the activation of the adaptive immune response (CD4⁺ T cells). The immune surveillance of transformed/tumour cells drives alteration of the antigen processing and presentation pathways to evade detection and hence the immune response. Evasion of the immune response is a significant event tumour development and considered one of the hallmarks of cancer. To avoid immune recognition, tumours employ a multitude of strategies with most resulting in a down‐regulation of the MHC class I expression at the cell surface, significantly impairing the ability of CD8⁺ cytotoxic T lymphocytes to recognize the tumour. Alteration of the expression of key players in antigen processing not only affects MHC class I expression but also significantly alters the repertoire of peptides being presented. These modified peptide repertoires may serve to further reduce the presentation of tumour‐specific/associated antigenic epitopes to aid immune evasion and tumour progression. Here we review the modifications to the antigen processing and presentation pathway in tumours and how it affects the anti‐tumour immune response, considering the role of tumour‐infiltrating cell populations and highlighting possible future therapeutic targets.     

Characterization of HLA DR3/DQ2 transgenic mice: a potential humanized animal model for autoimmune disease studies

Linkage studies indicate close associations of certain HLA alleles with autoimmune diseases. To better understand how specific HLA alleles are related to disease pathogenesis, we have generated an HLA DR3/DQ2 transgenic mouse utilizing a 550-kb yeast artificial chromosome (YAC) construct containing the complete DRalpha, DRbeta1, DRbeta3, DQalpha, and DQbeta regions. The transgenic mouse (4D1/C2D) in an I-Abeta(o) background appears healthy with no signs of autoimmune diseases. Lymphoid tissues as well as CD4(+) T cells develop normally. Characterization of the transgene expression demonstrates that approximately 90% of B cells express high levels of DR3 and 50-70% of B cells express DQ2. CD11c(+) dendritic cells express high levels of DR and DQ. Approximately 12-18% of resting T cells are positive for DR expression, and further up-regulation to 40-50% expression is seen upon activation with anti-CD3/anti-CD28 mAb. These results suggest that the transgenic construct confers a high fidelity to the normal human temporal and spatial expression profile. Analysis of T cell receptor repertoire in transgenic mice confirms that DR3/DQ2 are able to mediate thymic selection. Furthermore, transgenic mice respond to a DR3-restricted antigen, demonstrating antigen processing and presentation by antigen-presenting cells (APC). Purified T cells from ovalbumin (OVA)-immunized 4D1 mice respond to human APC co-cultured with OVA, suggesting appropriate antigen/DR3 or DQ2 recognition by murine T cells. Immunoglobulin isotype switching is also observed, indicating functional T-B cognate interactions. Thus, the DR3/DQ2 transgenic mouse has normal lymphoid development and functionality that are mediated by HLA transgenes and can be used to investigate HLA-associated immunological questions.