Murine Major Histocompatibility Complex Research Articles

Generation of Antibodies of Distinct Subclasses and Specificity Is Linked to H2s in an Active Mouse Model of Epidermolysis Bullosa Acquisita

Epidermolysis bullosa acquisita (EBA) is an autoimmune blistering disease, characterized by antibodies to type VII collagen (COL7). EBA can be induced in mice by immunization with a fragment of the non-collagenous 1 domain of murine COL7. Contrary to other autoimmune diseases, e.g., rheumatoid arthritis, little is known about the genetic susceptibility for EBA. We therefore used the EBA mouse model to address the hypothesis that disease induction depends on the major histocompatibility complex (MHC) haplotype. Mice from different inbred strains were immunized with recombinant murine COL7. Five distinct responses were observed: induction of (i) severe disease in SJL/J (H2s) and female MRL/MpJ (H2k), (ii) mild and transient disease in C57Bl/10.s (H2s), (iii) microscopic blistering in DBA/1J (H2q), (iv) only presence of non-pathogenic autoantibodies in C57Bl/6J (H2b), NZM2410/J (H2z), BXD2 (H2b), and male MRL/MpJ, and (v) complete resistance to EBA in NOD/ShiLtJ (H2g7) and C57Bl/10.q (H2q) mice. Overall, susceptibility to EBA was strongly associated with H2s. In addition, the diseased phenotype was associated with autoantibodies to specific regions of COL7. Our findings show that induction of antibodies with a distinct specificity is linked to the MHC haplotype in experimental EBA. Furthermore, our data are the basis for future studies with the goal of identifying non-MHC EBA susceptibility genes.

The Signal Peptide of the IgE Receptor α-Chain Prevents Surface Expression of an Immunoreceptor Tyrosine-based Activation Motif-free Receptor Pool

The high affinity receptor for IgE, Fc epsilon receptor I (FcepsilonRI), is an activating immune receptor and key regulator of allergy. Antigen-mediated cross-linking of IgE-loaded FcepsilonRI alpha-chains induces cell activation via immunoreceptor tyrosine-based activation motifs in associated signaling subunits, such as FcepsilonRI gamma-chains. Here we show that the human FcepsilonRI alpha-chain can efficiently reach the cell surface by itself as an IgE-binding receptor in the absence of associated signaling subunits when the endogenous signal peptide is swapped for that of murine major histocompatibility complex class-I H2-K(b). This single-chain isoform of FcepsilonRI exited the endoplasmic reticulum (ER), trafficked to the Golgi and, subsequently, trafficked to the cell surface. Mutational analysis showed that the signal peptide regulates surface expression in concert with other described ER retention signals of FcepsilonRI-alpha. Once the FcepsilonRI alpha-chain reached the cell surface by itself, it formed a ligand-binding receptor that stabilized upon IgE contact. Independently of the FcepsilonRI gamma-chain, this single-chain FcepsilonRI was internalized after receptor cross-linking and trafficked into a LAMP-1-positive lysosomal compartment like multimeric FcepsilonRI. These data suggest that the single-chain isoform is capable of shuttling IgE-antigen complexes into antigen loading compartments, which plays an important physiologic role in the initiation of immune responses toward allergens. We propose that, in addition to cytosolic and transmembrane ER retention signals, the FcepsilonRI alpha-chain signal peptide contains a negative regulatory signal that prevents expression of an immunoreceptor tyrosine-based activation motif-free IgE receptor pool, which would fail to induce cell activation.

Helper T Cell Epitope-Mapping Reveals MHC-Peptide Binding Affinities That Correlate with T Helper Cell Responses to Pneumococcal Surface Protein A

Understanding the requirements for protection against pneumococcal carriage and pneumonia will greatly benefit efforts in controlling these diseases. Several proteins and polysaccharide capsule have recently been implicated in the virulence of and protective immunity against Streptococcus pneumonia. Pneumococcal surface protein A (PspA) is highly conserved among S. pneumonia strains, inhibits complement activation, binds lactoferrin, elicits protective systemic immunity against pneumococcal infection, and is necessary for full pneumococcal virulence. Identification of PspA peptides that optimally bind human leukocyte antigen (HLA) would greatly contribute to global vaccine efforts, but this is hindered by the multitude of HLA polymorphisms. Here, we have used an experimental data set of 54 PspA peptides and in silico methods to predict peptide binding to HLA and murine major histocompatibility complex (MHC) class II. We also characterized spleen- and cervical lymph node (CLN)-derived helper T lymphocyte (HTL) cytokine responses to these peptides after S. pneumonia strain EF3030-challenge in mice. Individual, yet overlapping peptides, 15 amino acids in length revealed residues 199 to 246 of PspA (PspA199–246) consistently caused the greatest IFN-γ, IL-2, IL-5 and proliferation as well as moderate IL-10 and IL-4 responses by ex vivo stimulated splenic and CLN CD4+ T cells isolated from S. pneumonia strain EF3030-challeged F1 (B6×BALB/c) mice. IEDB, RANKPEP, SVMHC, MHCPred, and SYFPEITHI in silico analysis tools revealed peptides in PspA199–246 also interact with a broad range of HLA-DR, -DQ, and -DP allelles. These data suggest that predicted MHC class II-peptide binding affinities do not always correlate with T helper (Th) cytokine or proliferative responses to PspA peptides, but when used together with in vivo validation can be a useful tool to choose candidate pneumococcal HTL epitopes.

Mechanism for Amyloid Precursor-like Protein 2 Enhancement of Major Histocompatibility Complex Class I Molecule Degradation

Earlier studies have demonstrated interaction of the murine major histocompatibility complex (MHC) class I molecule K(d) with amyloid precursor-like protein 2 (APLP2), a ubiquitously expressed member of the amyloid precursor protein family. Our current findings indicate that APLP2 is internalized in a clathrin-dependent manner, as shown by utilization of inhibitors of the clathrin pathway. Furthermore, we demonstrated that APLP2 and K(d) bind at the cell surface and are internalized together. The APLP2 cytoplasmic tail contains two overlapping consensus motifs for binding to the adaptor protein-2 complex, and mutation of a tyrosine shared by both motifs severely impaired APLP2 internalization and ability to promote K(d) endocytosis. Upon increased expression of wild type APLP2, K(d) molecules were predominantly directed to the lysosomes rather than recycled to the plasma membrane. These findings suggest a model in which APLP2 binds K(d) at the plasma membrane, facilitates uptake of K(d) in a clathrin-dependent manner, and routes the endocytosed K(d) to the lysosomal degradation pathway. Thus, APLP2 has a multistep trafficking function that influences the expression of major histocompatibility complex class I molecules at the plasma membrane.

Comparative analysis of the impact of a free cysteine in tapasin on the maturation and surface expression of murine MHC class I allotypes

Tapasin is a key molecule in the major histocompatibility complex (MHC) class I peptide-loading complex, interacting with several other proteins in the complex. An amino acid substitution at a free cysteine position in tapasin has been shown to disrupt the covalent association of tapasin with ERp57. In this study, we mutated the free cysteine in mouse tapasin, and analysed the effects on the cell surface expression of the mouse MHC class I molecules K(d) and K(b). The C95S substitution in mouse tapasin increased the proportion of open forms relative to folded forms for both types of MHC class I molecules at the cell surface. Furthermore, the C95S substitution resulted in increased association of tapasin with folded K(d). Overall, our studies with these mouse MHC class I allotypes have revealed that the free cysteine 95 in mouse tapasin influences stable expression at the plasma membrane for both MHC class I allotypes, and have shown that tapasin's interaction with folded K(d) is elevated by the C95S substitution in tapasin.

Naturally occurring regulatory dendritic cells regulate murine cutaneous chronic graft-versus-host disease

AbstractChronic graft-versus-host disease (cGVHD) is a limiting factor in allogeneic hematopoietic stem cell transplantation (alloHSCT) for the treatment of leukemia and other malignancies. Relative to the process that initiates and promotes cGVHD, the regulation is poorly understood. In this study, we examined the role of naturally occurring regulatory dendritic cells (DCregs) in murine major histocompatibility complex (MHC)-compatible and multiple minor histocompatibility antigen (miHAg)–incompatible model of cGVHD in alloHSCT. DCregs generated from bone marrow in vitro (BM-DCregs) exclusively expressed CD200 receptor 3 (CD200R3), which exerted a suppressive function in the Ag-specific CD4+ T-cell response. CD49+CD200R3+ cells showed similarities in phenotype and function to BM-DCregs, which formally distinguishes them from other leukocytes, suggesting that they are the natural counterpart of BM-DCregs. Treatment of the recipient mice after alloHSCT with the recipient-type CD49+CD200R3+ cells as well as BM-DCregs protected against cGVHD, and the protection was associated with the generation of Ag-specific anergic CD4+ T cells as well as CD4+CD25+Foxp3+ regulatory T cells (Tregs) from donor-derived alloreactive CD4+CD25−Foxp3− T cells. In addition, the depletion of CD49+CD200R3+ cells before alloHSCT enhanced the progression of cGVHD. In conclusion, CD49+CD200R3+ cells act as naturally occurring DCregs to regulate the pathogenesis of cGVHD in alloHSCT mediated through the control of the transplanted alloreactive CD4+ T cells.

In Vitro Differentiation of Adult Bone Marrow Progenitors into Antigen-Specific CD4 Helper T Cells Using Engineered Stromal Cells Expressing a Notch Ligand and a Major Histocompatibility Complex Class II Protein

A murine stromal cell line (OP9-DL1) expressing a notch ligand, Delta-like-1, has been shown to be able to drive the differentiation of both murine and human hematopoietic progenitors into T cells in vitro. Further studies showed that hematopoietic progenitors transduced by a retroviral vector to express a human CD8 T-cell receptor (TCR) followed by an OP9-DL1 monolayer coculture could generate antigen-specific cytotoxic T lymphocytes in vitro. It remains unknown if a similar method could be applied to produce CD4 helper T cells. In this report, we show that murine adult bone marrow (BM) cells transduced with an OT2 CD4 TCR and cocultured with OP9 stromal cells expressing Delta-like-1 can differentiate into antigen-specific CD4 T cells in vitro. These cells are capable of inducing the expression of T-cell activation markers and producing cytokines upon stimulation. We have also constructed a new stromal cell line (OP9-DL1-IA(b)) ectopically expressing a murine major histocompatibility complex class II protein, I-A(b), in OP9-DL1 cells. This new line could accelerate the development of TCR-transduced BM cells into CD4 T cells, resulting in cells with an improved capacity to respond to T-cell stimulation to secrete cytokines. Taken together, we demonstrate a general and potentially useful method to generate autologous antigen-specific CD4 helper T cells in vitro from easily accessible BM cells.

Influence of the tapasin C terminus on the assembly of MHC class I allotypes

Several endoplasmic reticulum proteins, including tapasin, play an important role in major histocompatibility complex (MHC) class I assembly. In this study, we assessed the influence of the tapasin cytoplasmic tail on three mouse MHC class I allotypes (H2-K(b), -K(d), and -L(d)) and demonstrated that the expression of truncated mouse tapasin in mouse cells resulted in very low K(b), K(d), and L(d) surface expression. The surface expression of K(d) also could not be rescued by human soluble tapasin, suggesting that the surface expression phenotype of the mouse MHC class I molecules in the presence of soluble tapasin was not due to mouse/human differences in tapasin. Notably, soluble mouse tapasin was able to partially rescue HLA-B8 surface expression on human 721.220 cells. Thus, the cytoplasmic tail of tapasin (either mouse or human) has a stronger impact on the surface expression of murine MHC class I molecules on mouse cells than on the expression of HLA-B8 on human cells. A K408W mutation in the mouse tapasin transmembrane/cytoplasmic domain disrupted K(d) folding and release from tapasin, but not interaction with transporter associated with antigen processing (TAP), indicating that the mechanism whereby the tapasin transmembrane/cytoplasmic domain facilitates MHC class I assembly is not limited to TAP stabilization. Our findings indicate that the C terminus of mouse tapasin plays a vital role in enabling murine MHC class I molecules to be expressed at the surface of mouse cells.

Relevance of viral context and diversity of antigen-processing routes for respiratory syncytial virus cytotoxic T-lymphocyte epitopes

Antigen processing of respiratory syncytial virus (RSV) fusion (F) protein epitopes F85-93 and F249-258 presented to cytotoxic T-lymphocytes (CTLs) by the murine major histocompatibility complex (MHC) class I molecule Kd was studied in different viral contexts. Epitope F85-93 was presented through a classical endogenous pathway dependent on the transporters associated with antigen processing (TAP) when the F protein was expressed from either RSV or recombinant vaccinia virus (rVACV). At least in cells infected with rVACV encoding either natural or cytosolic F protein, the proteasome was required for epitope processing. In cells infected with rVACV encoding the natural F protein, an additional endogenous TAP-independent presentation pathway was found for F85-93. In contrast, epitope F249-258 was presented only through TAP-independent pathways, but presentation was brefeldin A sensitive when the F protein was expressed from RSV, or mostly resistant when expressed from rVACV. Therefore, antigen-processing pathways with different mechanisms and subcellular localizations are accessible to individual epitopes presented by the same MHC class I molecule and processed from the same protein but in different viral contexts. This underscores both the diversity of pathways available and the influence of virus infection on presentation of epitopes to CTLs.

Structure and expression of MHC class Ib genes of the central M region in rat and mouse: M4, M5, and M6

The M region at the telomeric end of the murine major histocompatibility complex (MHC) contains class I genes that are highly conserved in rat and mouse. We have sequenced a cosmid clone of the LEW rat strain (RT1 haplotype) containing three class I genes, RT1.M6-1, RT1.M4, and RT1.M5. The sequences of allelic genes of the BN strain (RT1n haplotype) were obtained either from cDNAs or genomic clones. For the coding parts of the genes few differences were found between the two RT1 haplotypes. In LEW, however, only RT1.M5 and RT1.M6 have open reading frames; whereas in BN all three genes were intact. In line with the findings in BN, transcription was found for all three rat genes in several tissues from strain Sprague Dawley. Protein expression in transfectants could be demonstrated for RT1.M6-1 using the monoclonal antibody OX18. By sequencing of transcripts obtained by RT-PCR, a second, transcribed M6 gene, RT1.M6-2, was discovered, which maps next to RT1.M6-1 outside of the region covered by the cosmid. In addition, alternatively spliced forms for RT1.M5 and RT1.M6 were detected. Of the orthologous mouse genes, H2-M4, H2-M5, and H2-M6, only H2-M5 has an open reading frame. Other important differences between the corresponding parts of the M region of the two species are insertion of long LINE repeats, duplication of RT1.M6, and the inversion of RT1.M5 in the rat. This demonstrates substantial evolutionary dynamics in this region despite conservation of the class I gene sequences themselves.

CD137 Is Expressed in Human Atherosclerosis and Promotes Development of Plaque Inflammation in Hypercholesterolemic Mice

Atherosclerosis is a multifactorial disease in which inflammatory processes play an important role. Inflammation underlies lesion evolution at all stages, from establishment to plaque rupture and thrombosis. Costimulatory molecules of the tumor necrosis factor superfamily such as CD40/CD40L and OX40/OX40L have been implicated in atherosclerosis. This study shows that the tumor necrosis factor superfamily members CD137 and CD137 ligand (CD137L), which play a major role in several autoimmune diseases, may constitute a pathogenic pair in atherogenesis. We detected CD137 protein in human atherosclerotic lesions not only on T cells but also on endothelial cells and showed that CD137 in cultured endothelial cells and smooth muscle cells was induced by proinflammatory cytokines implicated in atherosclerosis. Activation of CD137 by CD137L induced adhesion molecule expression on endothelial cells and reduced smooth muscle cell proliferation. In addition, treatment of atherosclerosis-prone apolipoprotein E-deficient mice with a CD137 agonist caused increased inflammation. T-cell infiltration, mainly of CD8(+) cells, and expression of the murine major histocompatibility complex class II molecule I-A(b) increased significantly in atherosclerotic lesions, as did the aortic expression of proinflammatory cytokines. Taken together, these observations suggest that CD137-CD137L interactions in the vasculature may contribute to the progression of atherosclerosis via augmented leukocyte recruitment, increased inflammation, and development of a more disease-prone phenotype.

Enhancement of experimental Graves' disease by intranasal administration of a T cell epitope of the thyrotropin receptor

We previously showed that immunization of mice with murine fibroblasts transfected with the thyrotropin receptor (TSHR) and a murine major histocompatibility complex (MHC) class II molecule induces immune thyroid disease with the humoral and histological features of human Graves' disease in about 20% of mice. In this model, based on the proliferative response of T cells from hyperthyroid mice to a panel of overlapping TSHR peptides, we now demonstrate that TSHR 121–140 peptide contains an immunodominant T cell epitope. Supporting this conclusion, spleen cells from mice immunized with TSHR 121–140 peptide showed a strong proliferative response to fibroblasts transfected with the TSHR and a murine I-A k molecule, but not either alone. Also, intranasal administration of 100 μg of TSHR 121–140 peptide led to suppressed proliferative response of lymph node cells to the peptide. Interestingly, however, administration of this peptide enhanced, rather than suppressed, the frequency and severity of Graves' disease induced by the immunization of the fibroblasts transfected with the TSHR and a murine I-A k molecule. Spleen cells from hyperthyroid mice that were pretreated with intranasal peptide tended to produce lesser amounts of IL-4, IL-10 and IFN-gamma than those from normothyroid control mice. Although precise mechanisms of this enhancement remain to be determined, the results suggest that attempts to treat Graves' disease by intranasal administration of an immunodominant TSHR T cell epitope may aggravate, not prevent, the disease.

Cellular Expression and Crystal Structure of the Murine Cytomegalovirus Major Histocompatibility Complex Class I-like Glycoprotein, m153

Mouse cytomegalovirus (MCMV), a beta-herpesvirus that establishes latent and persistent infections in mice, is a valuable model for studying complex virus-host interactions. MCMV encodes the m145 family of putative immunoevasins with predicted major histocompatibility complex, class I (MHC-I) structure. Functions attributed to some family members include down-regulation of host MHC-I (m152) and NKG2D ligands (m145, m152, and m155) and interaction with inhibitory or activating NK receptors (m157). We present the cellular, biochemical, and structural characterization of m153, which is a heavily glycosylated homodimer, that does not require beta2m or peptide and is expressed at the surface of MCMV-infected cells. Its 2.4-A crystal structure confirms that this compact molecule preserves an MHC-I-like fold and reveals a novel mode of dimerization, confirmed by site-directed mutagenesis, and a distinctive disulfide-stabilized extended N terminus. The structure provides a useful framework for comparative analysis of the divergent members of the m145 family.

Similarities and differences in tapasin interaction with disparate murine MHC class I allotypes (93.11)

Abstract Tapasin is a key molecule in the major histocompatibility complex (MHC) class I peptide-loading complex. In this study, we first assessed the influence of the tapasin C-terminus on H2-Kb, -Kd, and -Ld in mouse cells. Truncated mouse tapasin (lacking the transmembrane and C-terminal region) was unable to associate with TAP or any of three murine MHC class I allotypes, and did not assist murine MHC class I folding and surface expression. A tapasin mutant with a single substitution in the C-terminus (K408W) increased the amount of Kd (but not Ld) in the open, peptide-free form, relative to wild type tapasin. We investigated whether Kd was also particularly responsive to other tapasin mutations, and found that a C95S substitution in tapasin also caused an elevation in open Kd (but not open Ld or Kb). Folded Kd was found to be amply present in association with wild type tapasin, and even more so with tapasin K408W and C95S. In summary, our results suggest that the C-terminus of mouse tapasin plays a vital role in the surface expression of murine MHC class I molecules, and that mouse MHC allotypes vary in their sensitivity to specific alterations in tapasin sequence and in their release from tapasin after folding. Overall, our results reveal new complexity in tapasin’s interactions with polymorphic MHC class I molecules. (NIH Grant GM57428)

Hepatitis B Virus Splice-Generated Protein Induces T-Cell Responses in HLA-Transgenic Mice and Hepatitis B Virus-Infected Patients

Hepatitis B virus splice-generated protein (HBSP), encoded by a spliced hepatitis B virus RNA, was recently identified in liver biopsy specimens from patients with chronic active hepatitis B. We investigated the possible generation of immunogenic peptides by the processing of this protein in vivo. We identified a panel of potential epitopes in HBSP by using predictive computational algorithms for peptide binding to HLA molecules. We used transgenic mice devoid of murine major histocompatibility complex (MHC) class I molecules and positive for human MHC class I molecules to characterize immune responses specific for HBSP. Two HLA-A2-restricted peptides and one immunodominant HLA-B7-restricted epitope were identified following the immunization of mice with DNA vectors encoding HBSP. Most importantly, a set of overlapping peptides covering the HBSP sequence induced significant HBSP-specific T-cell responses in peripheral blood mononuclear cells from patients with chronic hepatitis B. The response was multispecific, as several epitopes were recognized by CD8(+) and CD4(+) human T cells. This study provides the first evidence that this protein generated in vivo from an alternative reading frame of the hepatitis B virus genome activates T-cell responses in hepatitis B virus-infected patients. Given that hepatitis B is an immune response-mediated disease, the detection of T-cell responses directed against HBSP in patients with chronic hepatitis B suggests a potential role for this protein in liver disease progression.

Constitutive internalization of murine MHC class I molecules

The total number of cell surface glycoprotein molecules at the plasma membrane results from a balance between their constitutive internalization and their egress to the cell surface from intracellular pools and/or biosynthetic pathway. Constitutive internalization is net result of constitutive endocytosis and endocytic recycling. In this study we have compared spontaneous internalization of murine major histocompatibility complex (MHC) class I molecules (K(d), D(d), full L(d), and empty L(d)) after depletion of their egress to the cell surface (Cycloheximide [CHX], brefeldin A [BFA]) and internalization after external binding of monoclonal antibody (mAb). MHC class I alleles differ regarding their cell surface stability, kinetics, and in the way of internalization and degradation. K(d) and D(d) molecules are more stable at the cell surface than L(d) molecules and, thus, constitutively internalized more slowly. Although the binding of mAbs to cell surface MHC class I molecules results in faster internalization than depletion of their egress, it is still slow and, thereby, can serve as a model for tracking of MHC class I endocytosis. Internalization of fully conformed MHC class I molecules (K(d), D(d), and L(d)) was neither inhibited by chlorpromazine (CP) (inhibitor of clathrin endocytosis), nor with filipin (inhibitor of lipid raft dependent endocytosis), indicating that fully conformed MHC class I molecules are internalized via the bulk pathway. In contrast, internalization of empty L(d) molecules was inhibited by filipin, indicating that non-conformed MHC class I molecules require intact cholesterol-rich membrane microdomains for their constitutive internalization. Thus, conformed and non-conformed MHC class I molecules use different endocytic pathways for constitutive internalization.

Bovine polymorphonuclear cells passively acquire membrane lipids and integral membrane proteins from apoptotic and necrotic cells

Immune cells can acquire membrane fragments and integral membrane proteins from dead and dying cells or in the case of immature dendritic cells, from live cells. While investigating the possibility that bovine polymorphonuclear cells (PMNs) might present antigen, coculture assays confirmed that integral membrane proteins were transferred rapidly and efficiently to bovine PMNs from a variety of apoptotic and necrotic cells. Specifically, we observed that PMNs rapidly acquired proteins such as major histocompatibility complex (MHC) class II and CD3 from a variety of syngeneic, allogeneic, and xenogeneic cell types. Such acquisition occurred within 40 min of PMN coculture with isolated peripheral blood mononuclear cells, and this acquisition occurred with equal efficiency at 4 degrees C and 37 degrees C. The transfer of murine MHC class II to bovine PMNs precluded the possibility of endogenous protein expression. We also demonstrated the transfer of fluorescently labeled plasma membrane lipids and biotinylated integral membrane proteins. Collectively, these observations support the hypothesis that membrane protein transfer was mediated by the fusion of membrane fragments or microvesicles with the PMN plasma membrane and not by phagocytosis of cell fragments. These observations indicate that phenotypic studies of PMNs must consider circumstances whereby PMNs may passively acquire membrane lipids and a variety of integral membrane proteins from dead or dying cells.

Intranasal Exposure to Bacterial Superantigens Induces Airway Inflammation in HLA Class II Transgenic Mice

Staphylococcus aureus is widely prevalent in the nasopharynges of healthy individuals (carriers) but can also cause serious infections. S. aureus can elaborate a variety of superantigen exotoxins in "carrier" or "pathogenic" states. Streptococcus pyogenes can also colonize the nasopharynx and elaborate superantigens. Unlike the acute effects of superantigen exotoxins absorbed through the gut or vaginal mucosa, little is known regarding the pathogenesis of superantigens entering through the intranasal route. In the current study, we evaluated the local and systemic effects of staphylococcal enterotoxin B (SEB) and streptococcal pyrogenic exotoxin A (SPEA) delivered through the intranasal route. Superantigens were administered intranasally on multiple occasions, and experimental animals were sacrificed on day 8 for experimental analyses. SEB-induced airway inflammation was more pronounced for HLA-DR3 transgenic mice than for BALB/c mice, consistent with bacterial superantigens binding more efficiently to human than murine major histocompatibility complex class II. The nature of the airway inflammation in HLA-DR3 mice was determined by the concentration of SEB applied intranasally. Low concentrations (20 ng) induced eosinophilic airway inflammation as well as eosinophil degranulation, whereas intranasal exposure to higher concentrations (2,000 ng) resulted in neutrophilic airway inflammation, permanent airway destruction, toxic shock, and mortality. SEB-induced eosinophilic inflammatory response was enhanced in signal transducer and activator of transcription (STAT)-4-deficient HLA-DQ8 transgenic mice with defective interleukin-12 signaling. Intranasal administration of SPEA induced airway inflammation and systemic immune activation in HLA-DQ8 transgenic mice. In conclusion, repeated chronic intranasal exposure to bacterial superantigens causes airway inflammation and systemic immune activation.

Structural Basis of the Differential Stability and Receptor Specificity of H-2D b in Complex with Murine versus Human β 2-Microglobulin

beta(2)-Microglobulin (beta(2)m) is non-covalently linked to the major histocompatibility complex (MHC) class I heavy chain and interacts with CD8 and Ly49 receptors. Murine MHC class I heavy chains can bind human beta(2)m (hbeta(2)m) and peptide, and such hybrid molecules are often used in structural and functional studies. The replacement of mouse beta(2)m (mbeta(2)m) with hbeta(2)m has several functional consequences for MHC class I complex stability and specificity, but the structural basis for this is presently unknown. To investigate the impact of species-specific beta(2)m subunits on MHC class I conformation, we provide a crystallographic comparison of H-2D(b) in complex with LCMV-derived gp33 peptide and either hbeta(2)m or mbeta(2)m. The conformation of the gp33 peptide is not affected by the beta(2)m species. Comparison of the interface between beta(2)m and the alpha(1)alpha(2) domains of the heavy chain in these two crystal structures reveals a marked increase in both polarity and number of hydrogen bonds between hbeta(2)m and the alpha(1)alpha(2) domains of H-2D(b). We propose that the positioning of two hydrogen bond rich regions at the hbeta(2)m/alpha(1)alpha(2) interface plays a central role in the increased overall stability and peptide exchange capacity in the H-2D(b)/hbeta(2)m complex. These two regions act as bridges, holding and stabilizing the underside of the alpha(1) and alpha(2) helices, enabling a prolonged peptide-receptive conformation of the peptide binding cleft. Furthermore, analysis of H-2D(b) in complex with either mbeta(2)m or hbeta(2)m provides a structural explanation for the differential binding of H-2D(b)/hbeta(2)m to both Ly49A and Ly49C. Our comparative structural study emphasizes the importance of beta(2)m residues at positions 3, 6 and 29 for binding to Ly49A and suggests that sterical hindrance by residue K6 on hbeta(2)m impairs the recognition of Ly49C by H-2D(b)/gp33/hbeta(2)m. Finally, comparison of the two H-2D(b) crystal structures implies that the beta(2)m species may affect the strength of TCR recognition by affecting CD8 binding.

Reconstitution of expression of H-2K region-encoded murine MHC class I glycoproteins in MHC class I-deficient B16BL6 melanoma cells affects the expression and function of antigen-processing machinery

We have recently reported that reconstitution of expression of major histocompatibility complex (MHC) class I glycoproteins in MHC-deficient and highly metastatic B16BL6 melanoma cells attenuates their malignant capacities by modulation of compartmentalization and functions of cell membrane receptors for growth factors [Assa-Kunik E, et al. J Immunol 2003;171:2945–52]. Our present study provides evidence that re-expression of an H-2K MHC class I-encoding gene in these cells also augments the expression of the Tap-2 peptide transporter and the inducible proteasome subunits, i.e. Lmp-2, Lmp-7 and Lmp-10. Up-regulation of inducible proteasome subunits was also followed by a significant changed in the proteolytic activity of the proteasome complex. We suggest that, in addition to providing a framework for proper presentation of antigenic peptides, MHC class I glycoproteins may regulate the immune response by modulating the expression and function of other genes, whose products are essential for proper antigen processing and presentation.