A Conformationally-Constrained MHC Class II I-A-Derived Peptide Protects NOD Mice from the Development of Diabetes

Allele-specific peptide vaccination against disease-associated MHC class II molecules is a promising new strategy for modulating self-antigen presentation to autoreactive T cells in autoimmune diseases. To evaluate the potential of this approach for treatment of insulin-dependent diabetes mellitus (IDDM), we have designed a cyclic peptide vaccine, DiavaX, from the third hypervariable region of the β-chain of the NOD mouse MHC class II I-A^g7. NOD mice were treated at 5 and 9 weeks of age with 100 μg DiavaX emulsified in alum, a control peptide in alum, or alum alone. At the end of the study, 87% of alum treated mice had developed diabetes, compared with only 28% of DiavaX-treated mice. None of the control peptides, including a linear I-A^g7, a scrambled cyclic I-A^g7, or an analogous cyclic I-A^speptide, reduced the incidence of diabetes, demonstrating that the protective effect of DiavaX is conformationally dependent and both allele- and sequence-specific. DiavaX treatment did not cause any general immune suppression, but did induce peptide-specific antibodies and memory T cells. DiavaX-induced protection from diabetes was associated with the maintenance of a non-destructive islet-associated autoimmune response. These data indicate that a conformationally constrained peptide from the disease-associated MHC represents a potential vaccine candidate for the prevention of clinical IDDM.     

Naturally processed peptides from HLA-DQ7 (α1*0501-β1*0301): influence of both α and β chain polymorphism in the HLA-DQ peptide binding specificity

Self peptides bound to HLA-DQ7 (α1*0501-β1*0301), one of the HLA molecules associated with protection against insulin-dependent diabetes mellitus, were characterized after their acid elution from immunoaffinity-purified HLA-DQ7 (α1*0501-β1*0301) molecules. The majority of these self peptides derived from membrane-associated proteins including HLA class I, class II, class II-associated invariant chain peptide and the transferrin-receptor (TfR). By in vitro binding assays, the specificity of these endogenous peptides for HLA-DQ7 (α1*0501-β1*0301) molecules was confirmed. Among these peptides, the binding specificity of the TfR 215 – 230 self peptide was further examined on a variety of HLA-DQ and DR dimers. Several findings emerged from this analysis: (1) this peptide displayed HLA-DQ allelic specificity, binding only to HLA-DQ7 (α1*0501-β1*0301); (2) when either the DQα or DQβ chain was exchanged, little or no binding was observed, indicating that specificity of HLA-DQ peptide binding was determined by polymorphic residues of both the α and β chains. (3) Unexpectedly, the TfR 215 – 230 self peptide, eluted from DQ, was promiscuous with regard to HLA-DR binding. This distinct DR and DQ binding pattern could reflect the structure of these two molecules as recently evidenced by crystallography.     

Carboxy-terminal residues of major histocompatibility complex class II-associated peptides control the presentation of the bacterial superantigen toxic shock syndrome toxin-1 to T cells

Previous studies have shown that the presentation of some bacterial superantigens by major histocompatibility complex (MHC) class II molecules is strongly influenced by class II-associated peptides. For example, presentation of the toxic shock syndrome toxin-1 (TSST-1) superantigen by antigen-processing-defective T2-I-A^b cells (which expresses I-A^b that is either empty or associated with invariant chain-derived peptides) can be strongly enhanced by some, but not other, I-A^b-binding peptides. Here we investigate the contribution of I-A^b-associated peptides in the presentation of TSST-1 to T cells. The data show that overlapping peptides expressing the same core I-A^b-restricted epitope, but with various N and C termini, can differ profoundly in their ability to promote TSST-1 presentation to T cells. Analysis of altered and truncated peptides indicates that residues at the C-terminal end of the peptide have a dramatic effect on TSST-1 presentation. This effect does not involve a cognate interaction between the peptide and the TSST-1 molecule, but appears to depend on the length of the C-terminal region. These data are consistent with crystallographic studies suggesting that TSST-1 may interact with the C-terminal residues of MHC class II-associated peptides. We also examined the capacity of naturally processed peptides to promote TSST-1 binding using a superantigen blocking assay. The data demonstrated that a naturally processed epitope is dominated by peptides that do not promote strong TSST-1 binding to I-A^b. Taken together, these data suggest that TSST-1 binding to MHC class II molecules is controlled by the C-terminal residues of the associated peptide, and that many naturally processed peptide/class II complexes do not present TSST-1 to T cells. Thus, the peptide dependence of TSST-1 binding to class II molecules may significantly reduce the capacity of TSST-1 to stimulate T cells.     

Differential expression of CLIP: MHC class II and conventional endogenous peptide: MHC class II complexes by thymic epithelial cells and peripheral antigen-presenting cells

Major histocompatibility complex (MHC) class II molecules expressed by thymic epithelial cells are involved in positive selection of CD4 T cells, whereas the high-avidity interaction of T cell receptors with the endogenous peptide : MHC class II complexes expressed on bone marrow (BM)-derived antigen-presenting cells (APC) and, to a lesser extent, on thymic epithelial cells mediate negative selection. To understand better the generation of the CD4 T cell repertoire both in the thymus and in the periphery we analyzed relative levels of expression of specific endogenous peptide: MHC class II complexes in thymic epithelial cells (TEC) and peripheral APC. Expression of Eα52–68: I-A^b and class II-associated invariant chain peptide (CLIP): I-A^b complexes in thymic epithelial cells and in the bone-marrow derived splenic APC, i.e. B cells, was studied using YAe and 30-2 monoclonal antibodies which are specific for the corresponding complexes. To distinguish between expression of both complexes in radioresistant thymic epithelial elements and radiation sensitive BM-derived APC, radiation BM chimeras were constructed. Using immunohistochemical and immunochemical approaches we demonstrated that the level of expression of Eα52–68:I-A^b complexes in thymic epithelial cells is approximately 5–10 % of that seen in splenic cells whereas total class II levels were comparable. In contrast, CLIP: I-A^b complexes are expressed at substantially higher levels in TEC vs. splenic APC. This result demonstrates quantitative differences in expression of distinct peptide: MHC class II complexes in thymic epithelial cells and peripheral splenic APC.     

Substitution of aspartic acid at beta57 with alanine alters MHC class II peptide binding activity but not protein stability: HLA-DQ (alpha1*0201, beta1*0302) and (alpha1*0201, beta1*0303)

In class II major histocompatibility complex (MHC) proteins, residue beta57 is usually aspartic acid. Alleles carrying serine, valine, or alanine at this position are strongly correlated with the development of insulin-dependent diabetes mellitus (IDDM). Asp(beta)57 participates in a conserved salt bridge that bridges the alpha and beta subunits in the peptide-binding site. It has been proposed that the correlation between IDDM and MHC alleles lacking Asp(beta)57 may be due to an instability of the protein caused by loss of this salt bridge. Using a pair of HLA-DQ proteins (alpha1*0201, beta1*0302) and (alpha1*0201, beta1*0303) differing only in having aspartic acid or alanine at position beta57, we show that the polymorphism does not have a significant effect on protein stability for either the empty or peptide-loaded forms. However, the circular dichroism spectra indicate that empty and peptide-loaded Alabeta57 proteins display slightly different secondary structures relative to their Aspbeta57 counterparts. A set of three peptides shows different binding affinities for DQ(alpha1*0201, beta1*0302) relative to DQ(alpha1*0201, beta1*0303). We propose that substitution of Asp(beta)57 residue causes a local rearrangement within the DQ peptide-binding site that alters the peptide-binding specificity. This rearrangement may help to explain the previously observed differences in SDS stability between Asp and non-Asp(beta)57 DQ proteins.     

Poor loading of major histocompatibility complex class II molecules with endogenously synthesized short peptides in the absence of invariant chain

In normal antigen-presenting cells, newly synthesized major histocompatibility complex (MHC) class II molecules associate with the invariant chain (Ii) glycoprotein in the endoplasmic reticulum (ER). They are loaded with peptides only after proteolytic removal of the Ii in post-Golgi endocytic vesicles. Since the Ii inhibits peptide binding to MHC class II molecules, this association could protect MHC class II molecules from being loaded with endogenous peptides early after biosynthesis. If this were an important function of the Ii in vivo, MHC class II molecules synthesized in cells lacking the Ii should be loaded efficiently with short endogenous peptides in the ER; such peptides are known to be present there due to TAP-mediated import from the cytosol. To examine this possibility, we have studied peptide loading in HeLa transfectants expressing murine H-2A^k MHC class II molecules either alone or together with an excess of Ii. Endogenous peptides could readily be extracted from conformationally intact A^k αβ dimers of biosynthetically labeled Ii⁺ cells, whereas peptide loading was greatly (> 95%) diminished in the absence of Ii. Significant amounts of sodium dodecyl sulfate-(SDS) stable 55-kDa peptide: A^k complexes were only found in the Ii⁺ transfectants. In the absence of Ii, the MHC class II molecules instead formed stable complexes with long (20 and 50 kDa) polypeptides. Known A^k-binding peptides bound stably to A^k molecules on Ii^? cells, could be co-purified with them, and were resistant to release in SDS, suggesting that poor recovery of endogenous peptides was not due to decreased stability of A^k: peptide complexes in the absence of Ii. We conclude that protection of MHC class II molecules from endogenous short peptides does not appear to be a quantitatively important function of the Ii molecule, because peptide loading is inefficient in its absence.     

A retro-inverso analog mimicks the cognate peptide epitope of a CD4+ T cell clone

Synthetic analogs of peptide epitopes may activate specific T helper cells, antagonize their antigen receptors, or block recognition by competing for major histocompatibility complex (MHC) class II binding sites. Rationally designed peptides may therefore prove useful as vaccines and for treatment of autoimmune diseases and allergies mediated by CD4⁺ T cells. However, their susceptibility to proteolytic degradation limits the applicability of conventional peptides in vivo. By contrast, retro-inverso analogs, in which a native sequence is substituted with D -amino acids linked with a reversed backbone, resist proteolysis and still maintain the side chain topology of the corresponding natural peptide. We report here that an end group-modified retro-inverso analog of the IgG2a^b heavy chain allopeptide determinant γ2a^b 435–447 was recognized by an I-A^d-restricted, γ2a^b 435–447-reactive T cell clone. The pseudopeptide elicited near-maximal interleukin-2 responses, although 300-fold higher concentrations were needed than the native determinant. The weaker antigenicity of the retro-inverso analog could be fully accounted for by an impaired I-A^d binding capacity, which might reflect reduced ability of the distorted main chain to form hydrogen bonds with I-A^d. Glycine substitution at the residue corresponding to the first primary anchor (P1) of the native peptide abrogated I-A^d binding and antigenicity of the retro-inverso analog. Thus, the pseudopeptide resembled the native determinant with respect to orientation in the class II binding site, configuration of the epitopic side chains, and the constraints that governed the interactions between a major anchoring side chain and I-A^d. In conclusion, proteolytically resistant compounds with predefined capacity to interact with MHC class II allelic products and T cell antigen receptors may be designed by retro-inverso modification of native determinants.     

Masking of a cathepsin G cleavage site in vivo contributes to the proteolytic resistance of major histocompatibility complex class II molecules

The expression of major histocompatibility complex class II (MHC II) molecules is post‐translationally regulated by endocytic protein turnover. Here, we identified the serine protease cathepsin G (CatG) as an MHC II‐degrading protease by in vitro screening and examined its role in MHC II turnover in vivo. CatG, uniquely among endocytic proteases tested, initiated cleavage of detergent‐solubilized native and recombinant soluble MHC II molecules. CatG cleaved human leukocyte antigen (HLA)‐DR isolated from both HLA‐DM‐expressing and DM‐null cells. Even following CatG cleavage, peptide binding was retained by pre‐loaded, soluble recombinant HLA‐DR. MHC II cleavage occurred on the loop between fx1 and fx2 of the membrane‐proximal β2 domain. All allelic variants of HLA‐DR tested and murine I‐A^g7 class II molecules were susceptible, whereas murine I‐E^k and HLA‐DM were not, consistent with their altered sequence at the P1’ position of the CatG cleavage site. CatG effects were reduced on HLA‐DR molecules with DRB mutations in the region implicated in interaction with HLA‐DM. In contrast, addition of CatG to intact B‐lymphoblastoid cell lines (B‐LCLs) did not cause degradation of membrane‐bound MHC II. Moreover, inhibition or genetic ablation of CatG in primary antigen‐presenting cells did not cause accumulation of MHC II molecules. Thus, in vivo, the CatG cleavage site is sterically inaccessible or masked by associated molecules. A combination of intrinsic and context‐dependent proteolytic resistance may allow peptide capture by MHC II molecules in harshly proteolytic endocytic compartments, as well as persistent antigen presentation in acute inflammatory settings with extracellular proteolysis.     

A Peptide-Binding Assay for the Disease-Associated HLA-DQ8 Molecule

The study of peptide binding to HLA class II molecules has mostly concentrated on DR molecules. Since many autoimmune diseases show a primary association to particular DQ molecules rather than DR molecules, it is also important to study the peptide-binding properties of DQ molecules. Here we report a biochemical peptide-binding assay for the type I diabetes-associated DQ8, i.e. DQ (α1*0301, β1*0302), molecule. Affinity-purified DQ8 molecules were tested in peptide-binding assays using a radiolabelled influenza haemagglutinin (Ha) peptide encompassing positions 255–271(Y) as an indicator peptide. The Ha 255–271(Y) peptide bound to DQ8 in a pH-dependent fashion showing optimal binding around pH 5. The association kinetics were relatively slow and the resulting complexes were heat labile. The specificity of peptide binding to DQ8 was investigated in competitive inhibition experiments with a panel of 43 peptides of different lengths and sequences. The DQ8 molecules showed a different pattern of peptide binding compared to a previously studied DQ2 molecule. Peptides derived from thyroid peroxidase, HLA-DQ(α1*0301), HLA-DQ(α1*0302), retinol receptor and p21ras were among the high-affinity binders, whereas peptides derived from myelin basic protein were among the low-affinity binders. The sequence of the high-affinity peptides conformed with a previously published peptide-binding motif of DQ8.     

An allo-specific peptide derived from HLA-A2 is presented by HLA-DQ7

The peptide motifs of two HLA class II molecules, DR11 and DQ7, were determined from natural peptides. The EBV transformed B cells JVM (HLA-A2-DRB1^*1102-DQA1^*0501-DQB1^*0301) were cultured to a final yield of 2 10¹⁰ cells. DR11 and DQ7 molecules were immunopurified and peptides were extracted after acid elution and separated by reverse-phase HPLC. Five peptides from DR11 and five from DQ7 were sequenced using Edman degradation and other peptides were analysed by pool sequencing. Peptides were from 11 to 15 amino acids in length and P often occurred at the second residue for both DR5 and DQ7. The peptide motif for DR11 was I at position i and K or R at position i + 4. For DQ7 the most significant signal was A in the middle of the peptide as also described by Falk et al. The source of one peptide eluted from DQ7 was a polymorphic part of the HLA-A2 heavy chain (from 56th to 69th amino-acid). It was also exactly the same peptide than the synthetic peptide used by Krensky et al in the 10th international workshop to modulate lysis by HLA-A2-specific cytotoxic T lymphocytes. The biochemical characterisation of this peptide from HLA-DQ7 strongly supports functional tests showing an indirect presentation of alloantigens by MHC molecules.     

Substitution of aspartic acid at β57 with alanine alters MHC class II peptide binding activity but not protein stability: HLA-DQ (α1*0201, β1*0302) and (α1*0201, β1*0303)

In class II major histocompatibility complex (MHC) proteins, residue β57 is usually aspartic acid. Alleles carrying serine, valine, or alanine at this position are strongly correlated with the development of insulin-dependent diabetes mellitus (IDDM). Aspβ57 participates in a conserved salt bridge that bridges the α and β subunits in the peptide-binding site. It has been proposed that the correlation between IDDM and MHC alleles lacking Aspβ57 may be due to an instability of the protein caused by loss of this salt bridge. Using a pair of HLA-DQ proteins (α1*0201, β1*0302) and (α1*0201, β1*0303) differing only in having aspartic acid or alanine at position β57, we show that the polymorphism does not have a significant effect on protein stability for either the empty or peptide-loaded forms. However, the circular dichroism spectra indicate that empty and peptide-loaded Alaβ57 proteins display slightly different secondary structures relative to their Aspβ57 counterparts. A set of three peptides shows different binding affinities for DQ(α1*0201, β1*0302) relative to DQ(α1*0201, β1*0303). We propose that substitution of Aspβ57 residue causes a local rearrangement within the DQ peptide-binding site that alters the peptide-binding specificity. This rearrangement may help to explain the previously observed differences in SDS stability between Asp and non-Aspβ57 DQ proteins.     

Binding of conserved islet peptides by human and murine MHC class II molecules associated with susceptibility to type I diabetes

The major histocompatibility complex (MHC) is the most important susceptibility locus for type I diabetes in humans and NOD mice. NOD mice express a single MHC class II molecule (I-Ag7) which carries a unique beta chain sequence. In humans, DQ alleles that encode DQ8 and DQ2 confer the highest risk for the disease. Soluble DQ8 and I-Ag7 were used to directly compare the binding specificity of these MHC molecules. Peptides from three islet antigens--insulin, GAD 65 and HSP 60--bound to both CQ8 and I-Ag7. These peptides included epitopes that are immunodominant in NOD mice, namely insulin (9-23), GAD (206-220) and HSP 60 (441-460). All of these peptide sequences are highly conserved between the human and murine antigens. The binding specificity of DQ8 and I-Ag7 was similar, but not identical, since two peptides eluted from splenocytes of NOD mice did not bind to DQ8. DQ8 formed long-lived complexes with the majority of these peptides, indicating that DQ8 is not a poor peptide binder. These results demonstrate functional similarities between human and murine MHC class II molecules that confer susceptibility to type I diabetes.     

Extensive major histocompatibility complex class I binding promiscuity for Mycobacterium tuberculosis TB10.4 peptides and immune dominance of human leucocyte antigen (HLA)‐B*0702 and HLA‐B*0801 alleles in TB10.4 CD8+ T‐cell responses

The molecular definition of major histocompatibility complex (MHC) class I‐presented CD8⁺ T‐cell epitopes from clinically relevant Mycobacterium tuberculosis (Mtb) target proteins will aid in the rational design of T‐cell‐based diagnostics of tuberculosis (TB) and the measurement of TB vaccine‐take. We used an epitope discovery system, based on recombinant MHC class I molecules that cover the most frequent Caucasian alleles [human leucocyte antigen (HLA)‐A*0101, A*0201, A*0301, A*1101, A*2402, B*0702, B*0801 and B*1501], to identify MHC class I‐binding peptides from overlapping 9‐mer peptides representing the Mtb protein TB10.4. A total of 33 MHC class I‐binding epitopes were identified, spread across the entire amino acid sequence, with some clustering at the N‐ and C‐termini of the protein. Binding of individual peptides or closely related peptide species to different MHC class I alleles was frequently observed. For instance, the common motif of xIMYNYPAMx bound to six of eight alleles. Affinity (50% effective dose) and off‐rate (half life) analysis of candidate Mtb peptides will help to define the conditions for CD8⁺ T‐cell interaction with their nominal MHC class I‐peptide ligands. Subsequent construction of tetramers allowed us to confirm the recognition of some of the epitopes by CD8⁺ T cells from patients with active pulmonary TB. HLA‐B alleles served as the dominant MHC class I restricting molecules for anti‐Mtb TB10.4‐specific CD8⁺ T‐cell responses measured in CD8⁺ T cells from patients with pulmonary TB.     

Type 1 diabetes associated HLA‐DQ2 and DQ8 molecules are relatively resistant to HLA‐DM mediated release of invariant chain‐derived CLIP peptides

HLA‐DM is essential for editing peptides bound to MHC class II, thus influencing the repertoire of peptides mediating selection and activation of CD4⁺ T cells. Individuals expressing HLA‐DQ2 or DQ8, and DQ2/8 trans‐dimers, have elevated risk for type 1 diabetes (T1D). Cells coexpressing DM with these DQ molecules were observed to express elevated levels of CLIP (Class II associated invariant chain peptide). Relative resistance to DM‐mediated editing of CLIP was further confirmed by HPLC‐MS/MS analysis of eluted peptides, which also demonstrated peptides from known T1D‐associated autoantigens, including a shared epitope from ZnT8 that is presented by all four major T1D‐susceptible DQ molecules. Assays with purified recombinant soluble proteins confirmed that DQ2‐CLIP complexes are highly resistant to DM editing, whereas DQ8‐CLIP is partially sensitive to DM, but with an apparent reduction in catalytic potency. DM sensitivity was enhanced in mutant DQ8 molecules with disruption of hydrogen bonds that stabilize DQ8 near the DM‐binding region. Our findings show that T1D‐susceptible DQ2 and DQ8 share significant resistance to DM editing, compared with control DQ molecules. The relative resistance of the T1D‐susceptible DQ molecules to DM editing and preferential presentation of T1D‐associated autoantigenic peptides may contribute to the pathogenesis of T1D.     

The phenotype of H-2M-deficient mice is dependent on the MHC class II molecules expressed

For a broader view of the role of H-2M as an accessory molecule in antigen presentation, we investigated the degree to which different MHC class II isotypes and alleles depend on H-2M to function in vivo. We generated H-2M-deficient animals expressing E^{k / b} or A^k molecules in addition to the A^b molecules already present in the mutant strain, and compared the ability of the different MHC class II molecules to present antigen at the cell surface for recognition by T cells, and contribute to positive selection of CD4⁺ T cells in the thymus. Biochemical analyses were performed to assess MHC class II maturation, and to determine the peptide content of the molecules. In the absence of H-2M, E^{k / b} molecules containd a more heterogeneous set of class II-associated invariant chain peptides (CLIP) than A^b did, which, unlike A^b -CLIP complexes, were not SDS-stable. Unlike A^b molecules, both E^{k / b} and A^k efficiently presented exogenously added peptides to T cells in the absence of H-2M. In addition, epitopes from some proteins, especially those known to be invariant chain independent, were presented by A^k molecules in the mutant animals. To our surprise, expression of E^{k / b} overcame the positive selection defect observed in H-2M-deficient mice expressing A^b alone. In contrast, A^k expression did not augment positive selection of CD4⁺ T cells in the mutant animals. Some of these findings in vivo contrast significantly with findings from in vitro studies on murine MHC class II molecules in human DM-deficient cell lines.     

CDw78 — a determinant on a major histocompatibility complex class II subpopulation that can be induced to associate with the cytoskeleton

In the present study we demonstrate that CDw78 monoclonal antibody (mAb) recognizes a distinct subpopulation of major histocompatibility complex (MHC) class II molecules. We show that the CDw78 epitope is present on less than 10 % of the total number of MHC class II molecules expressed on different cells, is not linked to a single isotype, and exhibits a characteristic expression pattern in tonsils. While mAb against MHC class II (DR, DP and DQ) stained the majority of cells both in the mantle zone and in germinal centers, the CDw78 staining was more heterogeneous with the strongest reactivity and the highest number of positive cells in the mantle zone and in the light centrocyte-rich part of the germinal centers. Antibodies to this MHC class II subpopulation (e.g. FN1) induced association with the cytoskeleton and a subsequent capping in more than 90 % of peripheral blood B cells. In contrast, mAb against MHC class II (DR, DP and DQ) did not induce association with the cytoskeleton and only 10–20 % of B cells were induced to cap, suggesting that CDw78 defines a population of MHC class II molecules functionally different from the majority of these antigens. Scatchard plot analysis indicates that FN1 mAb is of relatively low affinity (K_a = 1.5 × 10⁸M^?1) and monovalent Fab fragments fail to bind to the cell surface with measurable affinity. Our data seen in the context of the ability of FN1 to co-stimulate B cells with a suboptimal dose of anti-μ suggest that CDw78 mAb might recognize a functional important subpopulation of MHC class II molecules so far not described. It seems likely that this subpopulation represents dimerized or aggregated MHC class II molecules that can selectively bind this low-affinity mAb.     

Multiple genes/multiple autoantigens role in type 1 diabetes

Conclusion We hypothesize that Type 1 diabetes of humans and NOD mouse results from non-MHC genes, which determine susceptibility to T-cell autoimmunity. MHC class II alleles (and perhaps class I) determines the tissue targeted by these T-cells (DQ8/DQ2 Type 1 diabetes and Addison's disease, DQ2 or DQ8 for celiac disease). The high propensity for islet beta cells to be the target of autoimmunity we believe is the result of a peculiarity of the insulin B-chain peptide, such that it is a dominant beta cell-specific autoantigen. In the NOD mouse, autoreactive T-cells show a restricted Vα repertoire. The ability of selected class II molecules to prevent disease may relate to effects of these molecules (I-E, DQB1^*0602, and so forth) on the T-cell repertoire. With improving assays for autoantibodies and T-cell autoimmunity and the availability of transgenic mouse, we believe that the aforementioned hypotheses can be thoroughly tested.     

Processing of exogenous hepatitis B surface antigen particles for Ld-restricted epitope presentation depends on exogenous β2- microglobulin

Processing of exogenous hepatitis B surface antigen (HBsAg) particles in an endolysosomal compartment generates peptides that bind to the major histocompatibility complex (MHC) class I molecule L^d and are presented to CD8⁺ cytotoxic T lymphocytes. Surface-associated ‘empty’ MHC class I molecules associated neither with peptide, nor with β2-microglobulin (β2m) are involved in this alternative processing pathway of exogenous antigen for MHC class I-restricted peptide presentation. Here, we demonstrate that internalization of exogenous β2m is required for endolysosomal generation of presentation-competent, trimeric L^d molecules in cells pulsed with exogenous HBsAg. These data point to a role of endocytosed exogenous β2m in the endolysosomal assembly of MHC class I molecules that present peptides from endosomally processed, exogenous antigen.     

PREDNOD,a prediction server for peptide binding to the H-2g7 haplotype of the non-obese diabetic mouse

The non-obese diabetic (NOD) mouse is a widely used animal model for study of autoimmune diseases, in particular human type 1 diabetes mellitus (T1DM). Identification of the subset of peptides that bind MHC molecules comprising the H-2^g7 haplotype of NOD mouse and thereby representing potential NOD T-cell epitopes is important for research into the pathogenesis and immunotherapy of T1DM. The H-2^g7 haplotype comprises the MHC class-I molecules K^d and D^b and a single class-II molecule I-A^g7. We have developed a prediction system, PRED^NOD, for accurate identification of peptides that bind the MHC molecules constituting the H-2^g7 haplotype. PRED^NOD is accessible at http://antigen.i2r.a-star.edu.sg/Ag7.