Spliced HLA-bound peptides: a Black Swan event in immunology.

Peptides that bind to and are presented on the cell surface by human leucocyte antigen (HLA) molecules play a critical role in adaptive immunity. For a long time it was believed that all the HLA-bound peptides were generated through simple proteolysis of linear sequences of cellular proteins, and therefore are templated in the genome and proteome. However, evidence for untemplated peptide ligands of HLA molecules has accumulated during the last two decades, with a recent global analysis of HLA-bound peptides suggesting that a considerable proportion of HLA-bound peptides are potentially generated through splicing/fusion of discontinuous peptide segments from one or two distinct proteins. In this review, we will evaluate recent discoveries and debates on the contribution of spliced peptides to the HLA class I immunopeptidome, consider biochemical rules for splicing and the potential role of these spliced peptides in immune recognition.

A comprehensive analysis of peptides presented by HLA-A1.

Human leukocyte antigen (HLA)-A1 is one of the most common Caucasian HLA-A alleles. Here, we describe the comprehensive analysis of the HLA-A*01:01 ligand repertoire with the identification of 4735 naturally processed and presented peptides derived from 2477 source proteins. We found HLA-A*01:01 bound an equivalent number of ligands of 9 or 10 amino acids in length as well as being remarkably tolerant of even longer peptides. Indeed close to half of the HLA-A1 bound peptides identified ranged between 11 and 13 amino acids in length. These longer peptides contained the strong canonical motif of and acidic E/D residue at position 3 (P3) and Y at the C-terminus (CΩ), a motif that was still apparent in peptides of up to 18 amino acids in length. The identification of this large database of natural ligands will facilitate the refinement of predictive algorithms particularly with respect to longer peptide ligands.

A comprehensive analysis of constitutive naturally processed and presented HLA-C*04:01 (Cw4)-specific peptides.

The human B lymphoblastoid cell line C1R is widely regarded as human leukocyte antigen-A (HLA-A)/HLA-B negative and is therefore frequently exploited as a recipient cell line to study HLA class I functions. However, the normal levels of HLA-C*04:01 often hamper the investigation of introduced HLA class I allomorphs, which is particularly evident in sensitive applications such as mass spectrometry. Here we describe the comprehensive analysis of endogenous HLA-C*04:01 ligands expressed on the surface of C1R cells to (i) define a large sequence dataset of HLA-C*04:01 ligands, to (ii) refine the HLA-C*04:01 peptide-binding motif and (iii) to provide a resource that allows discrimination between peptides bound to introduced HLA class I subtypes and to the endogenous HLA-C*04:01 molecules.

Serum phosphorylated neurofilament-heavy chain levels in multiple sclerosis patients.

OBJECTIVES: We evaluated whether the measurement of serum phosphorylated neurofilament heavy chain (pNF-H) titre is likely to be a valid biomarker of axonal injury in multiple sclerosis (MS). METHODS: Serum pNF-H concentrations were measured by ELISA in cases with relapsing-remitting (RR)-MS (n=81), secondary progressive (SP) MS (n=13) and primary progressive (PP)-MS; n=6) MS; first demyelinating event (FDE; n=82); and unaffected controls (n=135). A subset of MS cases (n=45) were re-sampled on one or multiple occasions. The Multiple Sclerosis Severity Score (MSSS) and MRI measures were used to evaluate associations between serum pNF-H status, disease severity and cerebral lesion load and activity. RESULTS: We confirmed the presence of pNF-H peptides in serum by ELISA. We showed that a high serum pNF-H titre was detectable in 9% of RR-MS and FDE cases, and 38.5% of SP-MS cases. Patients with a high serum pNF-H titre had higher average MSSS scores and T2 lesion volumes than patients with a low serum pNF-H titre. Repeated sampling of a subset of MS cases showed that pNF-H levels can fluctuate over time, likely reflecting temporal dynamics of axonal injury in MS. CONCLUSIONS: A subset of FDE/MS cases was found to have a high serum pNF-H titre, and this was associated with changes in clinical outcome measures. We propose that routine measurement of serum pNF-H should be further investigated for monitoring axonal injury in MS.

Hierarchical self-tolerance to T cell determinants within the ubiquitous nuclear self-antigen La (SS-B) permits induction of systemic autoimmunity in normal mice.

Systemic autoimmune diseases are frequently associated with clustering of high titer autoantibody responses towards nuclear self-antigens. Little is known, however, about the extent of immune tolerance to the target nuclear antigens or the events leading to the complex autoantibody responses that are characteristic of systemic autoimmunity. To address these issues, we have examined the mouse immune response to La autoantigen (mLa) and the homologous human La antigen (hLa), which are components of the La(SS-B)/Ro(SS-A) ribonucleoprotein (RNP) complex targeted in systemic lupus erythematosus and primary Sjögren's syndrome. The findings reveal the presence of hierarchical T cell tolerance involving multiple autodeterminants within the La autoantigen expressed by normal H-2k and H-2a mice. At one end of this spectrum, there was no detectable T or B cell autoimmunity observed in mice that were immunized with the immunodominant mLa287-301 determinant, which differed by a single residue in its core sequence from the homologous but highly immunogenic human La288-302 determinant. Interestingly, the mLa287-301 peptide acted as an altered peptide ligand that specifically antagonized the activation of an hLa288-302-specific T cell hybridoma. In contrast to the tolerogenic mLa287-301 determinant, a range of autoimmune potential was identified among poorly tolerizing, subdominant self-peptides present within mouse La autoantigen. Notably, immunization of normal mice with the autologous subdominant La25-44 and La106-129 determinants resulted in limited or no detectable autoantibody response. In contrast, immunization with the subdominant mouse La13-30 determinant induced a proliferative T cell response associated with the appearance of specific autoantibodies recognizing multiple intrastructural (La) and intermolecular components (Ro) of the murine La/Ro RNP. The findings suggest how diversified autoimmunity might follow initiation of immunity to simple peptide mimics of poorly tolerogenic determinants that are present within ubiquitous self-antigens.

The A-chain of insulin is a hot-spot for CD4+ T cell epitopes in human type 1 diabetes.

Type 1 diabetes (T1D) is caused by T cell-mediated destruction of the pancreatic insulin-producing beta cells. While the role of CD4(+) T cells in the pathogenesis of T1D is accepted widely, the epitopes recognized by pathogenic human CD4(+) T cells remain poorly defined. None the less, responses to the N-terminal region of the insulin A-chain have been described. Human CD4(+) T cells from the pancreatic lymph nodes of subjects with T1D respond to the first 15 amino acids of the insulin A-chain. We identified a human leucocyte antigen-DR4-restricted epitope comprising the first 13 amino acids of the insulin A-chain (A1-13), dependent upon generation of a vicinal disulphide bond between adjacent cysteines (A6-A7). Here we describe the analysis of a CD4(+) T cell clone, isolated from a subject with T1D, which recognizes a new HLR-DR4-restricted epitope (KRGIVEQCCTSICS) that overlaps the insulin A1-13 epitope. This is a novel epitope, because the clone responds to proinsulin but not to insulin, T cell recognition requires the last two residues of the C-peptide (Lys, Arg) and recognition does not depend upon a vicinal disulphide bond between the A6 and A7 cysteines. The finding of a further CD4(+) T cell epitope in the N-terminal A-chain region of human insulin underscores the importance of this region as a target of CD4(+) T cell responses in human T1D.

Immunodominance hierarchies and gender bias in direct T(CD8)-cell alloreactivity.

Allogeneic solid organ transplantation often occurs across multiple donor-recipient HLA mismatches with consequent risk of allograft rejection. However, there is growing evidence that not all HLA mismatches are equivalent in their stimulation of allogeneic T cells making it important to determine which of these might be more significant as predictors of allograft rejection. To this end, we used defined antigen-presenting cell (APC) transfectants expressing single MHC-I allotypes as target cells that could discriminate the relative contribution of individual mismatched MHC-I allotypes to direct T-cell alloreactivity. We demonstrate remarkably reproducible patterns of immunodominance in reactivity across mismatched MHC-I allotypes. These patterns are HLA context-dependent, partly reflecting alloantigenic competition in responder cell responses. In strong alloresponses, we also observed an increased percentage of alloreactive T(CD8) cells in female responders, regardless of the stimulator gender, highlighting HLA-independent factors in the potency of the alloresponse. This approach provides a potential measure of specific alloreactive T cells that could be used in clinical practice for selection of donors, assessment of posttransplant outcomes, modulation of immunosuppression and detection of rejection episodes.

The molecular basis of cross-reactivity in the Australian Snake Venom Detection Kit (SVDK).

The Snake Venom Detection Kit (SVDK) is of major medical importance in Australia, yet it has never been rigorously characterised in terms of its sensitivity and specificity, especially when it comes to reports of false-negative and false-positive results. This study investigates reactions and cross-reactions of five venoms the SVDK is directed against and a number of purified toxins. Snakes showing the closest evolutionary relationships demonstrated the lowest level of cross-reactivity between groups. This was, instead, far more evident between snakes that are extraordinarily evolutionary separated. These snakes: Pseudechis australis, Acanthophis antarcticus and Notechis scutatus, in fact displayed more false-positive results. Examination of individual toxin groups showed that phospholipase A(2)s (PLA(2)s) tends to react strongly and display considerable cross-reactivity across groups while the three-finger toxins (3FTx) reacted poorly in all but the Acanthophis well. The hook effect was evident for all venoms, particularly Oxyuranus scutellatus. The results of this study show considerable variation in toxin detection, with implications in further development of venom detection, both in Australia and other countries.

The peptide-loading complex and ligand selection during the assembly of HLA class I molecules.

The identification and characterisation of the class I peptide loading complex has resulted in an appreciation of the co-ordinated and multifaceted nature of HLA class I assembly in the lumen of the endoplasmic reticulum. This loading complex consists of the assembling class I heterodimer in association with a number of molecular chaperones. These chaperones can be classified as generic to the folding of most glycoproteins in the endoplasmic reticulum or specific to the class I loading pathway. The functions of the various components of the loading complex in class I molecule assembly are reviewed. A critical component of the class I loading complex is the specialised chaperone tapasin. The role of tapasin in the stabilisation and retention of empty or suboptimally loaded class I molecules and the facilitation of the loading of these molecules with more appropriate ligands is discussed. As such, it is proposed that tapasin is a major determinant of peptide repertoire selection for class I-restricted presentation in normal antigen presenting cells. The potential implications in vaccine design and autoimmunity are discussed.

The use of post-source decay in matrix-assisted laser desorption/ionisation mass spectrometry to delineate T cell determinants.

The identification of naturally processed peptides presented by molecules of the major histocompatibility complex (MHC) has progressed significantly over the past decade. The elution of peptides from immunoaffinity purified complexes of MHC class I or class II molecules has provided highly specific biochemical information regarding the nature of endogenous peptides capable of binding to and being presented by particular MHC alleles. Whilst Edman chemistry is sufficient for the identification of abundant or homogeneous immunodominant peptides contained in samples of fractionated peptides, mass spectrometry has proved more powerful for sequencing less abundant species present in the typically heterogeneous fractions of eluted peptides. This review focuses on the characterisation of T cell determinants by matrix-assisted laser desorption/ionisation (MALDI)-time-of-flight (TOF) mass spectrometry (MS). We demonstrate, with specific examples, the utility of post-source decay in MALDI-TOF MS for the characterisation of the amino acid sequences of both native and modified T cell determinants. The potential advantages and pitfalls of this technique relative to the more commonly used forms of tandem mass spectrometry in electrospray and ion spray modes of ionisation as well as hybrid quadrupole-quadrupole-TOF instruments are discussed. We highlight the complementarity between these techniques and discuss the advantages in the combined use of both MALDI- and electrospray-based instrumentation in epitope identification strategies.

The central role played by peptides in the immune response and the design of peptide-based vaccines against infectious diseases and cancer.

Vaccines are one of the most cost effective methods of improving public health thereby increasing the quality of life. Prophylactic and therapeutic treatment by vaccines can prevent infectious diseases and some cancers and could also be used in the treatment of autoimmune disorders. An appreciation of this potential has resulted in a burgeoning literature which not only describes the scientific efforts being made into designing new and improved vaccines but also drives the efforts being made by public health organizations world-wide in delivering vaccines to the community. At the forefront of technologies being applied to the design of vaccines is the use of synthetic peptides; the chemical technologies used to assemble peptides have made great strides over the last decade and assembly of hi-fidelity peptides which can be of high molecular weight, multimeric or even branched is now almost routine. Together with the advances in peptide technology our understanding of the molecular events that are necessary to induce immune responses has also made great strides. The central role that peptides play in immune recognition is now recognised and rules are emerging that are being applied to the construction of peptide-based vaccines that, in the right context, can induce humoral (antibody) and cellular (cytotoxic and helper T cell) immune responses. Synthetic peptides are exquisitely placed to answer questions about immune recognition and along the way to provide us with new and improved vaccines.

Endogenous and exogenous factors contributing to the surface expression of HLA B27 on mutant APC.

We have examined the expression of HLA B*2705 in the mutant cell line 721.220, which lacks endogenous HLA A and B alleles and expresses a defective tapasin molecule. Several peptide sensitive mAbs distinguish between HLA B*2705 expressed on the surface of 721.220 cells (B27.220) and 721.220 cells co-transfected with human tapasin (B27.220.hTsn). This differential staining defines subtle differences in the conformation of HLA B27, which most likely reflect changes in the repertoire of antigenic peptides bound to B27 in the presence and absence of wild type tapasin. HLA B27 molecules expressed on the surface of 721.220 display increased levels of "free" B27 heavy chain (HC-10 staining), an epitope that is dependent on TAP-translocated peptides. The conformation and stability of B27 molecules was examined by investigating the integrity of mAb epitopes and the half-lives of these complexes on cells cultured with and without serum. The decay of surface B27 epitopes occurred more rapidly in B27.220 and this effect was exaggerated in serum free media. Importantly, the decay of surface B27 molecules in B27.220.hTsn cells was characterized by an early increase in HC-10 staining when the cells were grown in serum free media. This decay of B27 molecules via HC-10 reactive intermediates was not observed in B27.220 cells, implying molecules on these cells may already have passed through this stage prior to surface expression. Taken together these observations indicate that tapasin has a significant contribution to the composition and stability of the B27-bound peptide repertoire.

Conformational effects in reversed-phase high-performance liquid chromatography of polypeptides. I. Resolution of insulin variants.

In order to further characterise the role of conformation in the retention behaviour of polypeptides and proteins in reversed-phase high-performance liquid chromatography (RP-HPLC), the chromatographic properties of four different insulins have been studied as a function of temperature (over the range 5-85 degrees C) and column residence time (over the range 10-60 min). The role of the ligand structure was also investigated by comparing results obtained with a n-octadecyl (C18) and a n-butyl (C4) ligand immobilised to the same porous silica. Comparative structure-retention-stability relationships were determined from an examination of the influence of temperature on a number of chromatographic parameters including the chromatographic contact area, the affinity constant and the experimental band width. The results demonstrated that variations in temperature can be used to affect significant changes in selectivity between the different insulins despite their very high degree of sequence homology. These observations have permitted specific amino acid residues, and in particular those residues encompassing the region A8-A10, to be proposed to be directly involved in the chromatographic contact area of the insulin molecules. Overall, the analysis of the changes in various chromatographic parameters in response to variation of the amino acid sequence, temperature and other experimental parameters provides a powerful tool to elucidate the structural basis for the interfacial stability and the role of conformation on the retention behaviour of polypeptides and proteins in RP-HPLC.

Conformational effects in reversed-phase high-performance liquid chromatography of polypeptides. II. The role of insulin A and B chains in the chromatographic behaviour of insulin.

The contribution of the insulin A- and B-chain to the retention and bandwidth behaviour of bovine insulin has been investigated. The influence of temperature and residence time on the logarithmic capacity factor (log k) versus the mole fraction of organic modifier psi, i.e. the effect of temperature and ligand residency on the S and log k0 values of the individual peptide chains, were assessed at temperatures between 5 and 85 degrees C and elution times between 30 to 90 min with an n-octadecyl (C18) and an n-butyl (C4) sorbent. Analysis of these log k versus psi dependencies revealed that the insulin A-chain exhibits retention behaviour significantly different to the intact insulin molecule whilst the B-chain exhibits retention behaviour which is remarkably similar to the parent protein. However, in terms of kinetic processes, the A-chain exhibited a peak-splitting phenomenon at higher temperatures which was similar to the behaviour of the intact insulin molecule, whilst only bandbroadening with no peak splitting was apparent for the B-chain. Overall, the similarity of the retention behaviour of the insulin B-chain and the intact insulin molecule with regard to their temperature and residency dependencies suggests that the insulin B-chain makes a significant contribution to the chromatographic contact region of the insulin molecule when this polypeptide is exposed to hydrocarbonaceous ligands at low to intermediate temperatures due to the progressive unfolding of the molecule and greater accessibility of the previously buried B-chain residues.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparison between the isocratic and gradient retention behaviour of polypeptides in reversed-phase liquid chromatographic environments.

The isocratic and gradient elution behaviour of beta-endorphin and glucagon, two polypeptides known to exist in amphipathic alpha-helical conformations in lipophilic environments, have been examined under reversed-phase high-performance liquid chromatographic (RP-HPLC) conditions with low pH, aquo-acetonitrile mobile phases. The effects of changes in the volume fraction, psi, of the organic solvent modifier and temperature, T, on the magnitudes of the S and log k(o) values of these two polypeptides, obtained from the plots of logarithmic capacity factor (log k') vs. psi using isocratic elution conditions have been determined. These data have then been compared to the corresponding S and log k(o) values, obtained from the plots of logarithmic median capacity factor (log k) versus the median volume fraction of the organic solvent modifier (psi) derived from the linear gradient elution data, using the same n-butyl silica sorbent and related aquo-acetonitrile mobile phase conditions. As apparent from these studies, substantial differences occur in the temperature-dependent trends and magnitudes of the corresponding S and S values, or the log k(o) and log k(o) values, when these parameters are derived from experimental data acquired by these two different elution methods. Moreover, when gradient elution data for beta-endorphin and glucagon are utilised, the extrapolated values of the intercept and slope of the plots of log k vs. 1/T (corresponding to an apparent change in the median enthalpy of association, deltaH(o)assoc, or an apparent change in the median entropy of association, deltaS(o)assoc) substantially deviated from the values obtained for the thermodynamic parameters, deltaH(o)assoc and deltaS(o)assoc, derived from the log k' vs. 1/T plots using the corresponding isocratic data. These findings thus have important implications for biophysical and thermodynamic investigations when gradient elution data are employed to assess the molecular basis of the interaction of polypeptides with non-polar ligates.

Immunoproteomics: Mass spectrometry-based methods to study the targets of the immune response.

The mammalian immune system has evolved to display fragments of protein antigens derived from microbial pathogens to immune effector cells. These fragments are typically peptides liberated from the intact antigens through distinct proteolytic mechanisms that are subsequently transported to the cell surface bound to chaperone-like receptors known as major histocompatibility complex (MHC) molecules. These complexes are then scrutinized by effector T cells that express clonally distributed T cell receptors with specificity for specific MHC-peptide complexes. In normal uninfected cells, this process of antigen processing and presentation occurs continuously, with the resultant array of self-antigen-derived peptides displayed on the surface of these cells. Changes in this peptide landscape of cells act to alert immune effector cells to changes in the intracellular environment that may be associated with infection, malignant transformation, or other abnormal cellular processes, resulting in a cascade of events that result in their elimination. Because peptides play such a crucial role in informing the immune system of infection with viral or microbial pathogens and the transformation of cells in malignancy, the tools of proteomics, in particular mass spectrometry, are ideally suited to study these immune responses at a molecular level. Here we review recent advances in the studies of immune responses that have utilized mass spectrometry and associated technologies, with specific examples from collaboration between our laboratories.

High-performance liquid chromatography of amino acids, peptides and proteins. CXV. Thermodynamic behaviour of peptides in reversed-phase chromatography.

The thermodynamic behaviour of three peptides, bombesin, beta-endorphin and glucagon, was studied under reversed-phase high-performance liquid chromatographic conditions. Experimental data related to the interactive surface contact area (S values) and solute affinity (log k0) were derived over a range of temperatures between 5 and 85 degrees C. These experimental conditions allowed changes in the secondary structure of the solute to be monitored. The influence of the nature of the stationary phase ligand on the relative conformational stability of the three peptides was analysed by acquiring data with n-octadecyl silica (C18) and n-butyl silica (C4) sorbents. Values for the relative changes in entropy and enthalpy associated with the interactive process were also determined. The results provide further insight into the factors involved with the stabilization of secondary structure and the mechanism of the interaction of peptides with hydrophobic surfaces.

High-performance liquid chromatography of amino acids, peptides and proteins. XC. Investigations into the relationship between structure and reversed-phase high-performance liquid chromatography retention behaviour of peptides related to human growth hormone.

The gradient elution behaviour of eight synthetic peptides encompassing residues [6-13] of human growth hormone, i.e. Leu1-Ser-Arg-Leu-Phe-Asp-Asn-Ala8, has been investigated, by using an octadecylsilica, a butylsilica, and a polymeric fluorocarbon as stationary phases. Quantitative expressions, derived from the linear-solvent-strength theory and the general plate-height theory, were used to assess the influence of gradient time on the relative retention and bandwidths of these peptides. It was demonstrated that the chromatographic properties of the cyclised imide form involving Asp6 are consistent with the formation of a highly stabilised amphipathic helix, while the open-chain alpha- and beta-rearranged forms eluted as less rigid structures. The putative hydrophobic contact region consists of two leucine residues and one phenylalanine residue. From an analysis of the retention and bandwidth data obtained at pH 9, a surface-induced molecular reorientation of the beta-linked peptides was observed, in which the repulsion of the aspartyl carboxyl group from the hydrophobic stationary phase directs the C-terminal moiety away from the sorbent surface. Furthermore, the fluorocarbon sorbent exhibited characteristics favourable for use in preparative purification of these peptides. The present results demonstrate the sensitivity of reversed-phase high-performance liquid chromatography (RP-HPLC) to monitor small changes in the interactive behaviour of peptides with hydrocarbonaceous ligands and aquo-organic solvent combinations in reversed-phase systems. These observations further illustrate the general utility of HPLC for investigating the conformational behaviour of peptides at solid-liquid interfaces.

High-performance liquid chromatography of amino acids, peptides and proteins. XCI. The influence of temperature on the chromatographic behaviour of peptides related to human growth hormone.

The influence of temperature on the gradient elution properties of synthetic peptides related to residues [6-13] of human growth hormone, e.g., Leu1-Ser-Arg-Leu-Phe-Asp-Asn-Ala8, has been studied by using both an octadecylsilica and a polymeric fluorocarbon stationary phase. Correlation of changes in the solute hydrophobic contact area and affinity for the stationary phase, as given by S and log k0 values respectively, revealed that the alpha- and imide forms are more conformationally stable than the beta-linked peptide. In addition, negative values of the standard entropy change, delta S0 assoc, for the transfer of the solute to the stationary phase, were observed for both alpha- and beta-linked peptides. These results are indicative of an increased ordering of the system upon solute adsorption and implies that the open-chain peptides exist in solution in more flexible conformations, while the helical structure of the cyclised imide is more rigid and constrained. The implications of the relative conformational stability of these peptides in their role as insulin-potentiating agents is also discussed.

High-performance liquid chromatography of amino acids, peptides, and proteins. 123. Dynamics of peptides in reversed-phase high-performance liquid chromatography.

The dynamics of several peptides in reversed-phase high-performance liquid chromatography (RP-HPLC) have been investigated on both n-octadecyl (C18) silica and n-butyl (C4) silica sorbents. In particular, the conformational interconversions and the relative rates of chromatographic relaxation of bombesin, glucagon, and beta-endorphin on both C18 and C4 n-alkylsilicas were monitored by examining changes in the experimental bandwidths of these peptides as a function of temperature and column residence time under linear gradient elution RP-HPLC conditions. The observed band-broadening trends were correlated with previously derived retention parameters and thermodynamic descriptors of the association process determined for bombesin, beta-endorphin, glucagon, and a control peptide, penta-L-phenylalanine. This study confirms that bandwidth measurements can be used as an integral experimental component to study the effect of the secondary structure of peptidic solutes on their RP-HPLC retention behavior. Further, the data demonstrate the utility of RP-HPLC as a tool to examine peptide conformational dynamics at hydrophobic surfaces. The relevance of these results to the general phenomenon of peptide-lipid interactions is discussed in terms of the associated evidence for lipid-induced changes in the conformation of these three bioactive peptides.