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Background: The crossreactivity of Epstein-Barr virus (EBV Epstein-Barr virus nuclear antigen 3A [EBNA3A])-specific CD8 T cells against allogeneic human leukocyte antigen (HLA)-B*44:02 has been shown to be dependent on presentation of self-peptide EEYLQAFTY by the target antigen. In this study, we report that allogeneic HLA-B*44:02+ proximal tubular epithelial cells (PTECs) and human umbilical vein endothelial cells (HUVECs) are poor targets for EBV EBNA3A-specific T cells. Methods: The EEY peptide was exogenously loaded onto HLA-B*44:02 and HLA-B*44:03-expressing PTECs and HUVECs. EEY-peptide-loaded, and unloaded, PTECs and HUVECs were then incubated with serial dilutions of our EBNA3A T-cell clone, in a cytotoxicity assay. Results: Although HLA-B*44:02-expressing PTECs were specifically lysed in proportion to the effector/target ratio by the EBNA3A T-cell clone, without peptide loading, lysis was greatly increased by exogenous EEY peptide loading (15% vs. 75%; P

Type I natural killer T cells (NKT cells) are characterized by an invariant variable region 14-joining region 18 (V(α)14-J(α)18) T cell antigen receptor (TCR) α-chain and recognition of the glycolipid α-galactosylceramide (α-GalCer) restricted to the antigen-presenting molecule CD1d. Here we describe a population of α-GalCer-reactive NKT cells that expressed a canonical V(α)10-J(α)50 TCR α-chain, which showed a preference for α-glucosylceramide (α-GlcCer) and bacterial α-glucuronic acid-containing glycolipid antigens. Structurally, despite very limited TCRα sequence identity, the V(α)10 TCR-CD1d-α-GlcCer complex had a docking mode similar to that of type I TCR-CD1d-α-GalCer complexes, although differences at the antigen-binding interface accounted for the altered antigen specificity. Our findings provide new insight into the structural basis and evolution of glycolipid antigen recognition and have notable implications for the scope and immunological role of glycolipid-specific T cell responses.

Human anti-La/SS-B autoantibodies are known to react with highly conserved epitopes suggested to be functional or active sites on the La/SS-B polypeptide. This study was designed to determine whether the autoantibodies also react with poorly conserved regions of La/SS-B as predicted by an antigen-driven autoimmune response. Binding of human autoantibodies to purified human, mouse, and bovine recombinant fragments representing immunodominant regions of the La/SS-B polypeptide was compared using Western blotting and ELISA. A cross-reactive epitope was located in the highly conserved NH2-terminal region of La/SS-B. Significantly, human-specific epitopes were identified in both the conserved RNA-recognition motif and a poorly conserved COOH-terminal fragment, providing direct evidence for an autoantigen-driven response. The lack of autoantibody cross-reactivity with a conserved domain of mouse and bovine La/SS-B implies that a small number of residues in human autoepitopes may be critical for autoimmunogenicity.

Class I major histocompatibility complex (MHC)-restricted cytotoxic T lymphocyte responses to ovalbumin (OVA) were evaluated following delivery of soluble antigen mixed with commercial liposomes or by electroporation of soluble protein into target cells. Splenic antigen presenting cells (APC) and transfected L cell lines were sensitised for recognition by OVA-specific, class I-restricted T hybridomas when antigen was introduced by either method into live cells. Delivery of soluble OVA by both electroporation and commercial liposomes proved more efficient than osmotic loading in sensitising for class I presentation. OVA-specific cytotoxic T lymphocytes (CTL) were effectively primed in naive mice following reinjection of spleen cells pulsed with soluble OVA encapsulated by liposomes or electroporated in vitro. These CTL recognised the well defined OVA257-264 determinant in association with H-2Kb and were derived under conditions where CTL activity obtained from cross priming by soluble OVA alone was undetectable. In addition, using electroporation and commercial liposomes, the loading of APC for OVA recognition required intact MHC-linked antigen presentation genes deleted in the T2-Kb cell line. Antigen delivery to APC by electroporation and commercial liposomes provides a simple and efficient way of studying class I-restricted T cell recognition of soluble protein antigens.

Celiac disease is a genetic condition that results in a debilitating immune reaction in the gut to antigens in grain. The antigenic peptides recognized by the T cells that cause this disease are incompletely defined. Our understanding of the epitopes of pathogenic CD4(+ )T cells is based primarily on responses shown by intestinal T-cells in vitro to hydrolysates or polypeptides of gluten, the causative antigen. A protease-resistant 33-amino acid peptide from wheat alpha-gliadin is the immunodominant antigen, but little is known about the spectrum of T cell epitopes in rye and barley or the hierarchy of immunodominance and consistency of recognition of T-cell epitopes in vivo. We induced polyclonal gluten-specific T cells in the peripheral blood of celiac patients by feeding them cereal and performed a comprehensive, unbiased analysis of responses to all celiac toxic prolamins, a class of plant storage protein. The peptides that stimulated T cells were the same among patients who ate the same cereal, but were different after wheat, barley and rye ingestion. Unexpectedly, a sequence from omega-gliadin (wheat) and C-hordein (barley) but not alpha-gliadin was immunodominant regardless of the grain consumed. Furthermore, T cells specific for just three peptides accounted for the majority of gluten-specific T cells, and their recognition of gluten peptides was highly redundant. Our findings show that pathogenic T cells in celiac disease show limited diversity, and therefore suggest that peptide-based therapeutics for this disease and potentially other strongly HLA-restricted immune diseases should be possible.

An SV40-based expression vector was used to generate CD4-negative murine L cell lines which stably expressed the human immunodeficiency virus envelope glycoprotein (env). Despite the presence of abundant intracellular envelope glycoprotein, the expression of env gp120/41 was not detected on the cell surface. Pulse-chase studies showed that the majority of the gp120 detected at the end of a 20-h chase was in the culture medium. Therefore gp120 was shed and/or secreted from these cells. Transfected L cells (H-2k) served as targets for specific lysis by CTL raised against vaccinia virus-encoded env gp160. The discrepancy in relative levels of intracellular versus surface expression of env was probably due to the highly inefficient processing of newly synthesized gp160, as well as the apparent instability of the gp120/41 complex in the transfected cell lines. Digestion of immunoprecipitated gp120 and gp160 with endoglycosidase H and peptide N-glycosidase F revealed that the envelope glycoprotein in transfected L cells possessed both high mannose and complex N-glycans, analogous to the posttranslational modification of the mature envelope glycoprotein in infected T cells. These studies indicate that the relatively inefficient processing of env gp160 occurs in the absence of CD4, and that the stable surface expression of envelope gp120/41 complex may require additional factors not present in transfected cells.

The semi-invariant natural killer (NK) T-cell receptor (NKTcr) recognises structurally diverse glycolipid antigens presented by the monomorphic CD1d molecule. While the alpha-chain of the NKTcr is invariant, the beta-chain is more diverse, but how this diversity enables the NKTcr to recognise diverse antigens, such as an alpha-linked monosaccharide (alpha-galactosylceramide and alpha-galactosyldiacylglycerol) and the beta-linked trisaccharide (isoglobotriaosylceramide), is unclear. We demonstrate here that NKTcrs, which varied in their beta-chain usage, recognised diverse glycolipid antigens with a similar binding mode on CD1d. Nevertheless, the NKTcrs recognised distinct epitopic sites within these antigens, including alpha-galactosylceramide, the structurally similar alpha-galactosyldiacylglycerol and the very distinct isoglobotriaosylceramide. We also show that the relative roles of the CDR loops within the NKTcr beta-chain varied as a function of the antigen. Thus, while NKTcrs characteristically use a conserved docking mode, the NKTcr beta-chain allows these cells to recognise unique aspects of structurally diverse CD1d-restricted ligands.

Hereditary haemochromatosis (HH) is an autosomal recessive disease involving mutations in the recently characterised HFE gene linked to HLA-A in the major histocompatibility complex. The known HFE polymorphisms include the wild-type allele, a G-->A substitution at base 845 (845A) and a C-->G substitution at position 187 (187G). Although most cases of HH are accountable by homozygosity of the 845A allele the exact risk of other HFE genotypes, especially those involving the 187G allele has not been determined. We have compiled estimates of disease risk for all known HFE genotypes by re-analyzing published studies. The data show a hierarchical risk calculated as odds ratio (OR) for each genotype 845A/ 845A (OR=2101); 845A/187G (OR=24); 187G/187G (OR=9); 845A/Wt (OR=5); 187G/Wt (OR=2). Interestingly, the disease risk of 187G-genotypes suggests that subtle functional changes in the HFE product can interact with other genetic factors (e.g. trans allele, gender) and environmental factors (e.g. diet) to manifest either as clinical disease, altered iron stores or a normal phenotype. This paradigm is potentially useful in understanding the contribution of HLA alleles to risk of various disorders especially autoimmunity.

CD1d presents lipid-based antigens (Ag) that are recognised by the semi-invariant T cell receptor (TCR) expressed on Natural Killer T (NKT) cells. While the TCR α-chain is typically invariant, the TCR β-chain expression is more diverse, particularly in mice where at least three different Vβ chains are commonly expressed. We report the structures of Vα14-Vβ8.2 and Vα14-Vβ7 NKT TCRs in complex with CD1d-α-galactosylceramide (α-GalCer), as well as a 2.5 Å structure of the human NKT TCR-CD1d-α-GalCer complex. Both Vβ8.2 and Vβ7 NKT TCRs, as well as the human NKT TCR, ligated CD1d-α-GalCer in a broadly similar manner, thereby highlighting the evolutionarily-conserved nature of this interaction. However, differences within the Vβ domains of the Vβ8.2 and Vβ7 NKT TCR-CD1d complexes not only resulted in altered TCR-β-CD1d-mediated contacts, but also surprisingly modulated recognition mediated by the invariant α-chain. Mutagenesis studies revealed the differing contributions of Vβ8.2 and Vβ7 residues within the CDR2β loop in mediating contacts with CD1d. Collectively we provide a structural basis for the differential NKT TCR Vβ usage in NKT cells.

The major ligands presented by MHC class I molecules after natural antigen processing are peptides of eight to ten residues in length, and it is widely accepted that the binding preferences of MHC class I molecules play a dominant role in dictating this classic feature of antigen presentation. In this report, we have reassessed the peptide size specificity of class I human leukocyte antigens (HLAs). By lengthening previously defined T cell epitopes by central amino acid insertion, we demonstrate that the peptide length specificity of some common HLA class I alleles (HLA-B*3501, B*0702 and A*2402) is very broad, and includes peptides of up to 25 residues. These data suggest that the length limitation of naturally processed MHC class I-associated peptides is primarily controlled by peptide availability after antigen processing rather than the binding specificity of MHC class I molecules. Furthermore, the findings provide an explanation for recent reports highlighting that epitopes of >10 amino acids play a minor but significant role in virus-specific immune surveillance by CD8(+) T cells.

Malathion is an organophosphate pesticide commonly used on field crops, fruit trees, livestock, agriculture, and for mosquito and medfly control. Aerial applications can result in solubilized malathion in swimming pools and other recreational waters that may come into contact with human skin. To evaluate the human skin absorption of malathion for the assessment of risk associated with human exposures to aqueous solutions, human volunteers were selected and exposed to aqueous solutions of malathion. Participants submerged their arms and hands in twenty liters of dilute malathion solution in either a stagnant or stirred state. The "disappearance method" was applied by measuring malathion concentrations in the water before and after human exposure for various periods of time. No measurable skin absorption was detected in 42% of the participants; the remaining 58% of participants measured minimal absorbed doses of malathion. Analyzing these results through the Hazard Index model for recreational swimmer and bather exposure levels typically measured in contaminated swimming pools and surface waters after bait application indicated that these exposures are an order of magnitude less than a minimal dose known to result in a measurable change in acetylcholinesterase activity. It is concluded that exposure to aqueous malathion in recreational waters following aerial bait applications is not appreciably absorbed, does not result in an effective dose, and therefore is not a public health hazard.

Hepatitis C virus (HCV)-specific cytotoxic T lymphocytes (CTLs) play an important role in HCV clearance. The frequency of HCV-specific T(CD8) in peripheral blood of HCV-infected donors is very low and HCV cannot be cultivated for reinfection of antigen presenting cells, making it difficult to detect T(CD8) of broad HCV specificities from peripheral blood mononuclear cells (PBMCs). We have developed a recombinant adenoviral system that efficiently reactivates and expands HCV-specific CTLs from PBMCs of HCV-infected donors. Replication-incompetent adenoviruses expressing individual HCV proteins (core and NS3) were produced and PBMCs from HCV-infected donors were transduced with these recombinant adeno-HCV constructs to stimulate HCV-specific CTL populations. T cells expanded from adeno-HCV stimulated cultures were potent producers of HCV-specific IFN-gamma and TNF-alpha and efficiently lysed target cells pulsed with HCV peptides. These constructs could stimulate T(CD8) directed towards multiple HCV peptides while preserving the determinant hierarchy. This approach therefore overcomes some of the shortcomings of the selective expansion of CTLs with peptide-based vaccine strategies. These findings provide an effective approach for the expansion of HCV-specific CTLs from PBMCs of HCV-infected patients and have potential for immunotherapeutic/vaccine development.

The expression of human immunodeficiency virus 1 (HIV-1) envelope glycoprotein products was studied in cells transfected with env gene constructs transcribed from an SV40 promoter. Gene constructs possessing the complete tat, rev (tat+ rev+) and env genes were transiently expressed in COS-1 cells as precursor SU-TM (gp160), SU.TM (gp120 x 41), and nucleolar rev protein. In addition, envelope glycoprotein was detected on the surface of those transfected COS-1 cells expressing abundant levels of env protein. Transfected constructs possessing a mutated tat translational initiation codon (tat-rev+) were expressed in COS-1 cells with at least a 10-fold increase in the level of envelope glycoprotein expression compared to the analogous constructs with an intact tat AUG codon (tat+ rev+). Mutation of the rev initiation codon (tat+ rev-) and (tat-rev-) resulted in no detectable expression of env products but expression of these proteins could be rescued by co-transfection of a cDNA encoding the rev gene. Subgenomic tat/rev transcripts were detected following transfection of all of the gene constructs indicating splicing of the env mRNAs transcribed from a heterologous promoter. Unspliced env transcripts were only detected in the cytoplasm of cells transfected with (rev+) constructs or with the (tat- rev-) construct in the presence of the rev cDNA supplied in trans. In contrast, unspliced transcripts were detected in the nuclear and total cellular RNA of all transfected cells. Expression of rev protein was localized to the nucleolus of transfected COS-1 cells. These results indicate that the export of unspliced env mRNA to the cytoplasm is facilitated by the expression of rev. Following env synthesis, the conversion of SU-TM (gp160) to SU.TM (gp120 x 41) was not quantitative. After a 20-h pulse-chase, only 40% of the SU-TM (gp160) present at the start of the chase period was subsequently accountable as mature SU (gp120), and approximately 30% of the detectable SU (gp120) was found in the culture medium of transfected COS-1 cells. The findings indicate that the surface expression of SU.TM (gp120 x 41) derived from heterologous gene transcripts is modulated (i) the co-expression of rev, (ii) the efficiency of proteolytic processing of SU-TM (gp160), and (iii) the degree of SU (gp120) shedding and/or secretion from the cell.

The use of peptides as therapeutics is experiencing renewed enthusiasm owing to advances in delivery, stability and design. Moreover, there is a growing emphasis on the use of peptides in vaccine design as insights into tissue-specific processing of the immunogenic epitopes of proteins and the discovery of unusually long cytotoxic T-lymphocyte epitopes broaden the range of targets and give clues to enhancing peptide immunogenicity. Peptides can also be synthesized with known post-translational modifications and/or deliberately introduced protease-resistant peptide bonds to regulate their processing independent of tissue-specific proteolysis and to stabilize these compounds in vivo. We discuss the potential of peptide-based vaccines for the treatment of chronic viral diseases and cancer, and review recent developments in the field of peptide-based vaccines.

CD1d is a non-polymorphic antigen-presenting molecule that is structurally related to the MHC class I molecule. CD1d molecules are heterodimers that are composed of a heavy chain that is non-covalently associated with β2-microglobulin (β2m). The heavy chain is composed of three extracellular domains designated α1, α2 and α3 as well as a transmembrane and an intracytoplasmic domain. The antigen-binding cavity is formed from the α1 and α2 domains of the heavy chain. These domains comprise two α-helices which flank the opening of the cleft and a β-sheet floor (see Fig. 1a and b). and Jamie Rossjohn

Mucosal associated invariant T (MAIT) cells have a semi-invariant TCRVα chain, and depend on riboflavin metabolites (ribityllumazines, RLAg) produced by commensal flora and presented by the nonpolymorphic class Ib molecule MR1 for optimal development. We used MR1/RLAg tetramers and TCR Vα19 transgenic mice to study MR1-dependency, subset heterogeneity and protective effector functions important for tuberculosis (TB) immunity. We found that Vβ6/8+NK1.1+ MAIT cell subsets maximally expand peripherally in mice expressing MR1. In addition, we show that MR1/RLAg tetramer+ MAIT cells expressing CD4, CD8 or neither contain populations variably co-expressing Vβ6/8 and/or NK1.1, indicating that MAIT cells are more heterogeneous than previously thought. Furthermore, NK1.1 (NKR-P1C) is minimally expressed on mature Vβ6/8+ tetramer+ MAIT cells in the thymus but progressively increases with peripheral activation. Importantly, after virulent mycobacterial pulmonary infection, heterogeneous subsets of tetramer+ MAIT cells (CD4-CD8->CD4-CD8+>CD4+CD8-) co-expressing Vβ6/8 and NK1.1 and the homing molecules α4β1 and CXCR3, were recruited into the lungs and afforded maximal early protection in MR1-sufficient mice. Surprisingly, MAIT cells developing in the absence of MR1 were partially protective. Overall, we demonstrate diversity of MAIT cells, evidence that NK1.1 is an activation rather than developmental MAIT cell marker, and also that MAIT cells are important for TB protective immunity.

Antigen-specific T-cell responses induced by infection, transplantation, autoimmunity or hypersensitivity are characterized by cells expressing biased profiles of T-cell receptors (TCRs) that are selected from a diverse, naive repertoire. Here, we review the evidence for these TCR biases, focusing on crystallographic analysis of the structural constraints that determine the binding of a TCR to its ligand and the persistence of certain TCRs in an immune repertoire. We discuss the ways in which diversity in a selected TCR repertoire can contribute to protective immunity and the implications of this for vaccine design and immunotherapy.

Our understanding of the molecular mechanisms of T cell alloreactivity remains limited by the lack of systems for which both the T cell receptor allo- and cognate ligand are known. Here we provide evidence that a single alloreactive T cell receptor interacts with analogous structural regions of its cognate ligand, HLA-B*0801(FLRGRAYGL), as its allogeneic ligand, HLA-B*3501(KPIVVLHGY). The crystal structures of the binary peptide-major histocompatibility complexes show marked differences in the conformation of the heavy chains as well as the bound peptides. Nevertheless, both epitopes possess a prominent solvent-exposed aromatic residue at position 7 flanked by a small glycine at position 8 of the peptide determinant. Moreover, regions of close structural homology between the heavy chains of HLA B8 and HLA B35 coincided with regions that have previously been implicated in "hot spots" of T cell receptor recognition. The avidity of this human T cell receptor was also comparable for the allo- and cognate ligand, consistent with the modes of T cell receptor binding being broadly similar for these complexes. Collectively, it appears that highly focused structural mimicry against a diverse structural background provides a basis for the observed alloreactivity in this system. This cross-reactivity underpins the T cell degeneracy inherent in the limited mature T cell repertoire that must respond to a vast diversity of microbial antigens.

The interaction between the alphabeta T cell receptor (TCR) and the peptide bound to the major histocompatibility complex class I molecule (pMHC-I) constitutes a central interaction in adaptive immunity. How these receptors interact with such low affinity while maintaining exquisite specificity for peptide antigen and host MHC (MHC-I restriction) remains a challenge to be explained by structural immunologists. Moreover, how this extracellular interaction is transmitted as an intracellular signal via the CD3 complex remains unresolved. Nevertheless, several structures of TCRs, non-liganded and ligated to a defined pMHC-I, combined with detailed biophysical analyses, have provided insight of the structural basis of MHC-I restriction. In addition, structures of isolated CD3 components have enabled T cell signalling mechanisms to be postulated. Recent findings in this area, which include seven distinct TCR/pMHC-I complexes, have fundamental implications in adaptive immunity as well as therapeutic applications to modulate the adaptive immune response.

The focusing of cellular immunity toward one, or just a few, antigenic determinant, even during immune responses to complex microorganisms or antigens, is known as immunodominance. Although described in many systems, the mechanisms of determinant immunodominance are only just beginning to be appreciated, especially in relation to the interplay between T cells of differing specificities and the interactions between T cells and the antigen-presenting cells (APCs). The outcome of these cellular interactions can lead to a form of immune suppression of one specificity by another-described as "immunodomination". The specific and detailed mechanisms involved in this process are now partly defined. A full understanding of all the factors that control immunodominance and influence immunodomination will help us to develop better viral and cancer vaccines.

Human leukocyte antigen-G (HLA-G) is a class Ib major histocompatibility complex (MHC) molecule that is specifically expressed in immune-privileged tissues. The overall structure of HLA-G resembles other class I MHC molecules, in which a heavy chain comprised of three domains is noncovalently associated with beta(2)microglobulin (beta(2)m). A nine-residue self-peptide is bound within a cleft formed by two alpha-helices and a beta-sheet floor. An extensive network of contacts is formed between the peptide and the binding cleft, leading to a constrained mode of binding reminiscent of that observed in HLA-E. The alpha3 domain of HLA-G, the putative binding site for leukocyte immunoglobulinlike receptor-1 (LIR-1) and -2, is structurally distinct from class Ia MHC molecules, providing a basis for the observed differences in affinity for these ligands. In addition, a disulfide-bonded dimer adopts an oblique conformation, providing the possibility of a 1:2 (HLA-G dimer:receptor) complex stoichiometry. The relative orientation of the HLA-G protomers in the dimer structure suggests that it is unlikely that dimerization is involved in killer immunoglobulinlike receptor 2DL4 (KIR2DL4) binding.

Methotrexate (MTX), used as a graft-versus-host disease (GvHD) prophylactic agent in hematopoietic stem cell transplantation (HSCT), exerts its effect via folate cycle inhibition. A critical enzyme involved in folate metabolism is 5,10-methylenetetrahydrofolate reductase (MTHFR). We examined the association of a single nucleotide polymorphism (SNP) at position 677 in the MTHFR gene on GvHD outcomes in allogeneic HSCT patients administered MTX. MTHFR genotyping was performed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) on 193 HSCT patients and donors. A total of 140 patients were transplanted with an HLA-matched related donor and 53 with an unrelated donor. GvHD outcomes were compared between genotypes by univariate and multivariate analysis. The combined donor 677CT and TT genotypes were associated with a decreased incidence of GvHD (acute and chronic combined) in HSCT recipients with an HLA-matched related donor (75% at 1 year in the CT and TT group compared with 91% in the wild type CC group, P=0.01), increased time to onset of first GvHD (P=0.001) and time to first GvHD treated with systemic therapy (P=0.022). Unrelated donor MTHFR genotype was not associated with outcome parameters and no associations of recipient genotype in either related or unrelated donor cohorts were observed.

The focusing of cellular immunity toward one, or just a few, antigenic determinant, even during immune responses to complex microorganisms or antigens, is known as immunodominance. Although described in many systems, the mechanisms of determinant immunodominance are only just beginning to be appreciated, especially in relation to the interplay between T cells of differing specificities and the interactions between T cells and the antigen‐presenting cells (APCs). The outcome of these cellular interactions can lead to a form of immune suppression of one specificity by another—described as “immunodomination”. The specific and detailed mechanisms involved in this process are now partly defined. A full understanding of all the factors that control immunodominance and influence immunodomination will help us to develop better viral and cancer vaccines.

Alphabeta T-cell receptor (TcR) recognition of antigenic peptides bound to the major histocompatibility complex (pMHC), is integral to the cellular immune system. Crystallographic studies over the last decade have provided significant insight into this unique trimolecular recognition event. The TcR-pMHC structural information has been paralleled by biophysical studies that have further explored the emerging binding models in an attempt to answer fundamental immunological questions regarding MHC restriction, T-cell immunodominance and TcR cross-reactivity. However, despite the important data that has been generated regarding TcR-pMHC interactions, the scope of this information is still incomplete due to the limited range of TcRs that have been studied. These limitations are primarily due to difficulties in obtaining high yields of recombinant alphabeta TcR for crystallographic and biophysical analysis; here we will discuss some of the protein engineering strategies that have been employed to expand the pool of recombinant TcRs suitable for crystallographic studies and the subsequent studies that have utilized these proteins.

Tapasin (Tpn) is a chaperone of the endoplasmic reticulum involved in peptide loading to MHC class I proteins. The influence of mouse Tpn (mTpn) on the HLA-B*2705-bound peptide repertoire was analyzed to characterize the species specificity of this chaperone. B*2705 was expressed on Tpn-deficient human 721.220 cells cotransfected with human (hTpn) or mTpn. The heterodimer to β2-microglobulin-free H chain ratio on the cell surface was reduced with mTpn, suggesting lower B*2705 stability. The B*2705-bound peptide repertoires loaded with hTpn or mTpn shared 94–97% identity, although significant differences in peptide amount were observed in 16–17% of the shared ligands. About 3–6% of peptides were bound only with either hTpn or mTpn. Nonamers differentially bound with mTpn had less suitable anchor residues and bound B*2705 less efficiently in vitro than those loaded only with hTpn or shared nonamers. Decamers showed a different pattern: those found only with mTpn had similarly suitable residues as shared decamers and bound B*2705 with high efficiency. Peptides differentially presented by B*2705 on human or mouse cells showed an analogous pattern of residue suitability, suggesting that the effect of mTpn on B*2705 loading is comparable in both cell types. Thus, mTpn has quantitative and qualitative effects on the B*2705-bound peptide repertoire, impairing presentation of some suitable ligands and allowing others with suboptimal anchor residues and lower affinity to be presented. Our results favor a size-dependent peptide editing role of Tpn for HLA-B*2705 that is species-dependent and suboptimally performed, at least for nonamers, by mTpn.

Antiretroviral drug-resistant human immunodeficiency virus type 1 (HIV-1) is a major, growing, public health problem. Immune responses targeting epitopes spanning drug resistance sites could ameliorate development of drug resistance. We studied 25 individuals harboring multidrug-resistant HIV-1 for T-cell immunity to HIV-1 proteins and peptides spanning all common drug resistance mutations. CD8 T cells targeting epitopes spanning drug-induced mutations were detected but only in the 3 individuals with robust HIV-specific T-cell activity. Novel CD8 T-cell responses were detected against the common L63P and L10I protease inhibitor fitness mutations. Induction of T-cell immunity to drug-resistant variants was demonstrated in simian human immunodeficiency virus-infected macaques, where both CD8 and CD4 T-cell immune responses to reverse transcriptase and protease antiretroviral mutations were elicited using a novel peptide-based immunotherapy. T-cell responses to antiretroviral resistance mutations were strongest in the most immunocompetent animals. This study suggests feasible strategies to further evaluate the potential of limiting antiretroviral drug resistance through induction of T-cell immunity.

Although HLA class I alleles can bind epitopes up to 14 amino acids in length, little is known about the immunogenicity or the responding T-cell repertoire against such determinants. Here, we describe an HLA-B*3508-restricted cytotoxic T lymphocyte response to a 13-mer viral epitope (LPEPLPQGQLTAY). The rigid, centrally bulged epitope generated a biased T-cell response. Only the N-terminal face of the peptide bulge was critical for recognition by the dominant clonotype SB27. The SB27 public T-cell receptor (TcR) associated slowly onto the complex between the bulged peptide and the major histocompatibility complex, suggesting significant remodeling upon engagement. The broad antigen-binding cleft of HLA-B*3508 represents a critical feature for engagement of the public TcR, as the narrower binding cleft of HLA-B*3501(LPEPLPQGQLTAY), which differs from HLA-B*3508 by a single amino acid polymorphism (Arg156 --Leu), interacted poorly with the dominant TcR. Biased TcR usage in this cytotoxic T lymphocyte response appears to reflect a dominant role of the prominent peptide x major histocompatibility complex class I surface.

Using both 'reverse genetics' and structural analysis, we have examined the in vivo relationship between antigenicity and T cell receptor (TCR) repertoire diversity. Influenza A virus infection of C57BL/6 mice induces profoundly different TCR repertoires specific for the nucleoprotein peptide of amino acids 366-374 (NP366) and the acid polymerase peptide of amino acids 224-233 (PA224) presented by H-2D(b). Here we show the H-2D(b)-NP366 complex with a 'featureless' structure selected a limited TCR repertoire characterized by 'public' TCR usage. In contrast, the prominent H-2D(b)-PA224 complex selected diverse, individually 'private' TCR repertoires. Substitution of the arginine at position 7 of PA224 with an alanine reduced the accessible side chains of the epitope. Infection with an engineered virus containing a mutation at the site encoding the exposed arginine at position 7 of PA224 selected a restricted TCR repertoire similar in diversity to that of the H-2D(b)-NP366-specific response. Thus, the lack of prominent features in an antigenic complex of peptide and major histocompatibility complex class I is associated with a diminished spectrum of TCR usage.

The transporter associated with antigen processing (TAP) is a member of the ATP-binding cassette transporter family that specializes in delivering cytosolic peptides to class I molecules in the endoplasmic reticulum. The TAP is a major target of genetic alteration in tumours and disruption by viral inhibitors. In some species, TAP genes have co-evolved with MHC class I molecules to deliver peptides that are customised for particular alleles. In humans, MHC class I polymorphism determines the level of tapasin-mediated association with TAP and subsequent peptide optimisation within the peptide-loading complex (PLC). MHC class I molecules that still load peptides without complexing to the TAP might be more resistant to viral interference of the PLC and less sensitive to competition for TAP by other class I allotypes.

The light activated position sensing array will bring great independence to many persons with disabilities. Enabling those that have limited to no use of their limbs, this product will allow its user to perform many day to day activities that may have not otherwise been possible without outside assistance. The system is a hands-free electric wheelchair control that will also function as a powerful environmental control tool. Guided by a light emitter worn on the user's head, it will replace orally activated (sip and puff) switches, as well as any other switches that may be mounted on or near the user's head.

The application of the light-guided environmental control device will greatly increase the quality of life of certain persons with disabilities. This hands-free mechanism will allow the user to perform many daily tasks without the need of an aid. It is an environmental control and a wheelchair control that is driven by a light emitting head switch. One of the main foci of the project is to remove many of the cumbersome control switches being implemented today, and replacing them with one compact model. This product is to perform all operations currently performed by head arrays and switches, as well as standard joysticks. It is being designed to be used primarily by those people without the use of their limbs, especially those looking to gain a great deal of independence.

Human CD3 is an essential multisubunit complex that plays a fundamental role in T-cell signalling, T-cell development and surface expression of the alphabeta T-cell receptor. The CD3 complex comprises the CD3epsilongamma and CD3epsilondelta heterodimers and the CD3zetazeta homodimer. Here, the expression of the human CD3epsilongamma and CD3epsilondelta heterodimers, both of which were expressed as single-chain polypeptides, is reported. Following refolding, functional heterodimers were immunoaffinity purified from improperly folded heterodimers using OKT3, a therapeutic monoclonal antibody specific for the CD3epsilon chain. Subsequently, the Fab fragment of OKT3 was used to complex individually with the CD3epsilongamma and CD3epsilondelta heterodimers. Crystals of scCD3epsilongamma-FabOKT3 were grown using 15%(w/v) PEG 3350, 200 mM potassium fluoride, 100 mM Tris-HCl pH 8.0. Crystals of scCD3epsilondelta-FabOKT3 were grown using 20%(w/v) PEG 3350, 200 mM potassium formate, 100 mM Tris-HCl pH 8.0, 2%(v/v) MPD. Crystals of both complexes diffract to beyond 3 A resolution. scCD3epsilongamma-FabOKT3 crystals belonged to space group P2(1), with unit-cell parameters a = 67.70, b = 55.77, c = 96.05 A, beta = 100.85 degrees and one complex per asymmetric unit. scCD3epsilondelta-FabOKT3 crystals belong to space group P2(1), with unit-cell parameters a = 101.67, b = 50.36, c = 138.7 A, beta = 108.84 degrees, suggesting two complexes per asymmetric unit.

We aimed to generate T-cell clones specific for human pre-proinsulin. An HLA DQ8, CD4+ T-cell clone that recognised a 10mer (C65-A9) peptide from pre-proinsulin was isolated. Further analysis revealed that the clone responded neither to recombinant proinsulin nor to re-synthesised C65-A9 peptide. Analysis of the original peptide revealed minor contamination (

This overview presents nomenclature and serology information on human leucocyte antigens, or HLA molecules, which are encoded by a cluster of genes linked on the short arm of chromosome 6. This region is known as the major histocompatibility coclass II molecules based upon their structure, tissue distribution, and source of peptide antigen, as well as upon their interactions with T cell subsets.

We have examined the basis for immunodominant or "public" TCR usage in an antiviral CTL response. Residues encoded by each of the highly selected genetic elements of an immunodominant clonotype recognizing Epstein-Barr virus were critical to the antigen specificity of the receptor. Upon recognizing antigen, the immunodominant TCR undergoes extensive conformational changes in the complementarity determining regions (CDRs), including the disruption of the canonical structures of the germline-encoded CDR1alpha and CDR2alpha loops to produce an enhanced fit with the HLA-peptide complex. TCR ligation induces conformational changes in the TCRalpha constant domain thought to form part of the docking site for CD3epsilon. These findings indicate that TCR immunodominance is associated with structural properties conferring receptor specificity and suggest a novel structural link between TCR ligation and intracellular signaling.

Tapasin is critical for efficient loading and surface expression of most HLA class I molecules. The high level surface expression of HLA-B*2705 on tapasin-deficient 721.220 cells allowed the influence of this chaperone on peptide repertoire to be examined. Comparison of peptides bound to HLA-B*2705 expressed on tapasin-deficient and -proficient cells by mass spectrometry revealed an overall reduction in the recovery of B*2705-bound peptides isolated from tapasin-deficient cells despite similar yields of B27 heavy chain and β2-microglobulin. This indicated that a proportion of suboptimal ligands were associated with B27, and they were lost during the purification process. Notwithstanding this failure to recover these suboptimal peptides, there was substantial overlap in the repertoire and biochemical properties of peptides recovered from B27 complexes derived from tapasin-positive and -negative cells. Although many peptides were preferentially or uniquely isolated from B*2705 in tapasin-positive cells, a number of species were preferentially recovered in the absence of tapasin, and some of these peptide ligands have been sequenced. In general, these ligands did not exhibit exceptional binding affinity, and we invoke an argument based on lumenal availability and affinity to explain their tapasin independence. The differential display of peptides in tapasin-negative and -positive cells was also apparent in the reactivity of peptide-sensitive alloreactive CTL raised against tapasin-positive and -negative targets, demonstrating the functional relevance of the biochemical observation of changes in peptide repertoire in the tapasin-deficient APC. Overall, the data reveal that tapasin quantitatively and qualitatively influences ligand selection by class I molecules.

Rheumatoid arthritis (RA) is a chronic inflammatory disease for which immunogenetic susceptibility factors have been defined. In a recent case control study, it was shown that a prior intimate relationship with pet cats or budgerigars confers risk for subsequent development of RA after a period of latency. Pets are a potential reservoir for putative microbial agents that could be a stimulus for chronic inflammation subject to the influence of immunogenetic factors. Therefore, a study was undertaken to determine whether the presence of HLA-DRB1 alleles bearing the RA susceptibility motif influenced risk for RA associated with prior exposure to pets.Blood samples were obtained from available RA patients and their case controls who had participated in the prior epidemiologic study. DR and DQ genotypes were determined by sequence analysis of oligonucleotides amplified from the DRB1 and DQB1 genes by polymerase chain reactions (PCR). Subjects were segregated according to pet exposure (as determined previously) and genotype for statistical analyses.The odds ratio (OR) for prepubertal exposure to cats and RA in available subjects irrespective of DRB1 genotype was 4.2 (CI, 2.1 to 8.5; P.00002). The OR between prior exposure to cats and RA in subjects with the RA susceptibility genotype DRB1 *0401 and *0404 was 5.8 (CI, 1.4 to 26; P.02) and24 (CI, 1.6 to 813; P.01), respectively. In subjects with the genotype DRB1 *1501, the association between RA and prior cat exposure was OR 8.4 (CI, 1.7 to 45; P.01). No significant association between RA and pet exposure was found in patients selected according to other genotypes. The association between RA and the recognized HLA-DR susceptibility motif was slightly stronger in subjects with a history of intimate cat exposure (OR 4.7 [CI, 1.5 to 14.8], P.005) than subjects without prior intimate exposure (OR 3.3 [CI; 1.2 to 9.3], P.02). In the small number of subjects who had reported an intimate association with pet birds, no influence of DR genotype on risk for RA was discerned.Risk for RA associated with prior intimate exposure to cats is concentrated in subjects with the RA-susceptibility conferring genotypes DRB1 *0401 and *0404. The findings suggest an interaction between an environmen-tal agent associated with pet cats and certain RA susceptibility-conferring DR genotypes. The risk for RA associated with intimate cat exposure also was significant in subjects with DRB1*1501, a genotype not otherwise associated with RA, but which shares with known RA susceptibility-bearing alleles the presence of an electropositive pocket (Pocket 4) in the DR peptide binding groove.

Mannose-binding lectin (MBL) is an important complement-activating protein of the human innate immune system. Deficiency of MBL is associated with an increased risk of various infections and arises from three structural gene mutations in exon 1 (variants B, C and D) and/or the presence of a low efficiency promoter. The C allele is found in sub-Saharan Africa whereas the B allele is found elsewhere, suggesting that these mutations occurred after the suggested hominid migration out of Africa [100-150 000 years before present (BP)]. Paradoxically, these alleles may have a selective advantage in protection against intracellular pathogens and occur at particularly high frequencies in sub-Saharan Africa (C variant) and South America (B variant). Since hominids reached Australia at least 50 000 years ago, a study of MBL polymorphisms in the indigenous population was of interest. Using heteroduplex technology we found a paucity of MBL structural gene mutations in two population groups from geographically distinct regions. Of 293 individuals tested, 289 were wild-type and four were heterozygous for either the B or D allele. In each individual with an MBL mutation the HLA haplotype profile suggested some Caucasian admixture. We also found a restricted range of MBL promoter haplotypes and the serum MBL levels were higher than those of any other ethnic group studied to date (median 3.07 microg/ml). Our data suggest that the B mutation probably arose between 50 000 and 20 000 BP. Its absence from the founder gene pool of indigenous Australians may also partly explain their vulnerability to intracellular infections such as tuberculosis.

Calreticulin (CR) is widely recognized as a new human autoantigen but there are conflicting data concerning its relationship with the Ro(SS-A) ribonucleoprotein (RNP). Recent evidence suggests that CR binds to 52 kDaRo (Ro52) by a protein/protein interaction and binds to hY RNA and rubella virus RNA. Other studies have shown that initiation of immunity to either Ro52 or 60 kDaRo (Ro60) can lead to reciprocal spreading of autoimmunity to Ro60 or Ro52, respectively, and induce anti-La autoantibodies in some strains of mice. These findings support a physical association of these polypeptides in Ro/La complexes. To test the hypothesis that CR is physically associated with Ro52 and/or Ro60 we examined the sera of Ro52-, Ro60 and La-immunized mice for intermolecular spreading to CR. Immune sera from BALB/c and C3H/HeJ mice immunized with recombinant 6xHis-mouse Ro52, 6xHis-human Ro60 or 6xHis-human La were tested for reactivity by ELISA and immunoblotting with a full-length human CR protein expressed as a soluble maltose binding protein fusion protein (CR-MBP). Five of the six Ro52-immunized C3H/HeJ mice sera and all six Ro60-immunized C3H/HeJ mice sera reacted with the CR-MBP (but not a MBP control) on ELISA. In the BALB/c group, the responder rate was lower with one in six of the Ro52-immunized and one in five of the Ro60-immunized mice spreading to CR. In contrast, none of the BALB/corC3H/HeJ mice which was immunized with La showed evidence of a recruited anti-CR antibody response. Immunoblotting of the different recombinant proteins with immune sera from the C3H/HeJ mice confirmed the specificity of the initial and recruited antibody responses. The spreading of immunity from Ro52 and Ro60 to CR in Ro-immunized mice suggests that a subpopulation of CR or CR-like molecules must associate under certain circumstances with Ro52 and Ro60 polypeptides in vivo, possibly as Ro/CR complexes concentrated in surface membrane blebs of apoptotic cells. The lack of spreading to CR in Laimmunized mice suggests that CR may be associated with a subpopulation of Ro particles from which La has already dissociated.

In the present report, cytotoxic T lymphocyte (CTL) clones are described that display dual specificity for one of two common human leukocyte antigens (HLA B14 or B35) as alloantigens, and an immunodominant epitope (FLRGRAYGL) from Epstein-Barr virus (EBV) that binds to HLA B8. These T cell clonotypes were isolated from several unrelated HLA B8+, EBV-exposed individuals, and each distinct cross-reactivity pattern was associated with a common, public T cell receptor (TCR). In some individuals, CTL cross-reactive with these alloantigens completely dominated the memory response to this EBV epitope. Moreover, these memory T cells to EBV could be reactivated as a significant component of the repertoire of CTL responding to allogeneic stimulator cells expressing either HLA B14 or B35. These data illustrate how a history of infection with an immunogenic virus such as EBV can augment responsiveness to particular alloantigens; such influences may underlie the observed clinical association between herpesvirus infection and both allograft rejection and graft-versus-host disease. We have also explored the molecular basis for T cell cross-reactivity with alloantigens using the HLA B35 allo-reactive CTL clonotype. To elucidate the structural features of peptides that may be cross-recognized by these T cells, mono-substituted analogs of the viral epitope were screened for recognition, revealing broad specificity for major histocompatibility complex (MHC)-bound peptide. Based on the particular amino acid changes tolerated by the CTL at each peptide position, the human protein sequence database was searched for possible sequences that were recognized in association with HLA B35. Four peptides were identified (MPEATVYGL, IPIAPVYGM, KPSPPYFGL, and KPIVVLHGY) that were powerful activating ligands for the CTL when presented on HLA B35 but not B8. Thus, equivalent epitopes, capable of fully activating a single TCR, were formed by peptides with minimal obvious sequence homology bound to either HLA B8 or B35. These data indicate that degenerate peptide recognition by TCR may play an important role in the vigorous response of self-MHC-restricted T cells to alloantigens.