Protective Allele for Multiple Sclerosis HLA-DRB1*01:01 Provides Kinetic Discrimination of Myelin and Exogenous Antigenic Peptides.

Risk of the development of multiple sclerosis (MS) is known to be increased in individuals bearing distinct class II human leukocyte antigen (HLA) variants, whereas some of them may have a protective effect. Here we analyzed distribution of a highly polymorphous HLA-DRB1 locus in more than one thousand relapsing-remitting MS patients and healthy individuals of Russian ethnicity. Carriage of HLA-DRB1*15 and HLA-DRB1*03 alleles was associated with MS risk, whereas carriage of HLA-DRB1*01 and HLA-DRB1*11 was found to be protective. Analysis of genotypes revealed the compensatory effect of risk and resistance alleles in trans. We have identified previously unknown MBP153−161 peptide located at the C-terminus of MBP protein and MBP90−98 peptide that bound to recombinant HLA-DRB1*01:01 protein with affinity comparable to that of classical antigenic peptide 306-318 from the hemagglutinin (HA) of the influenza virus demonstrating the ability of HLA-DRB1*01:01 to present newly identified MBP153−161 and MBP90−98 peptides. Measurements of kinetic parameters of MBP and HA peptides binding to HLA-DRB1*01:01 catalyzed by HLA-DM revealed a significantly lower rate of CLIP exchange for MBP153−161 and MBP90−98 peptides as opposed to HA peptide. Analysis of the binding of chimeric MBP-HA peptides demonstrated that the observed difference between MBP153−161, MBP90−98, and HA peptide epitopes is caused by the lack of anchor residues in the C-terminal part of the MBP peptides resulting in a moderate occupation of P6/7 and P9 pockets of HLA-DRB1*01:01 by MBP153−161 and MBP90−98 peptides in contrast to HA308−316 peptide. This leads to the P1 and P4 docking failure and rapid peptide dissociation and release of empty HLA-DM–HLA-DR complex. We would like to propose that protective properties of the HLA-DRB1*01 allele could be directly linked to the ability of HLA-DRB1*01:01 to kinetically discriminate between antigenic exogenous peptides and endogenous MBP derived peptides

Conformational Plasticity of HLA-B27 Molecules Correlates Inversely With Efficiency of Negative T Cell Selection

The development of autoimmune disorders is incompletely understood. Inefficient thymic T cell selection against self-peptides presented by major histocompatibility antigens (HLA in humans) may contribute to the emergence of auto-reactive effector cells, and molecular mimicry between foreign and self-peptides could promote T cell cross-reactivity. A pair of class I subtypes, HLA-B2705 and HLA-B2709, have previously been intensely studied, because they are distinguished from each other only by a single amino acid exchange at the floor of the peptide-binding groove, yet are differentially associated with the autoinflammatory disorder ankylosing spondylitis. Using X-ray crystallography in combination with ensemble refinement, we find that the non-disease-associated subtype HLA-B2709, when presenting the self-peptide pGR (RRRWHRWRL), exhibits elevated conformational dynamics, and the complex can also be recognized by T cells. Both features are not observed in case of the sequence-related self-peptide pVIPR (RRKWRRWHL) in complex with this subtype, and T cell cross-reactivity between pGR, pVIPR, and the viral peptide pLMP2 (RRRWRRLTV) is only rarely observed. The disease-associated subtype HLA-B2705, however, exhibits extensive conformational flexibility in case of the three complexes, all of which are also recognized by frequently occurring cross-reactive T cells. A comparison of the structural and dynamic properties of the six HLA-B27 complexes, together with their individual ability to interact with T cells, permits us to correlate the flexibility of HLA-B27 complexes with effector cell reactivity. The results suggest the existence of an inverse relationship between conformational plasticity of peptide-HLA-B27 complexes and the efficiency of negative selection of self-reactive cells within the thymus

Identification of Class I HLA T Cell Control Epitopes for West Nile Virus

The recent West Nile virus (WNV) outbreak in the United States underscores the importance of understanding human immune responses to this pathogen. Via the presentation of viral peptide ligands at the cell surface, class I HLA mediate the T cell recognition and killing of WNV infected cells. At this time, there are two key unknowns in regards to understanding protective T cell immunity: 1) the number of viral ligands presented by the HLA of infected cells, and 2) the distribution of T cell responses to these available HLA/viral complexes. Here, comparative mass spectroscopy was applied to determine the number of WNV peptides presented by the HLA-A*11:01 of infected cells after which T cell responses to these HLA/WNV complexes were assessed. Six viral peptides derived from capsid, NS3, NS4b, and NS5 were presented. When T cells from infected individuals were tested for reactivity to these six viral ligands, polyfunctional T cells were focused on the GTL9 WNV capsid peptide, ligands from NS3, NS4b, and NS5 were less immunogenic, and two ligands were largely inert, demonstrating that class I HLA reduce the WNV polyprotein to a handful of immune targets and that polyfunctional T cells recognize infections by zeroing in on particular HLA/WNV epitopes. Such dominant HLA/peptide epitopes are poised to drive the development of WNV vaccines that elicit protective T cells as well as providing key antigens for immunoassays that establish correlates of viral immunity. © 2013 Kaabinejadian et al

Promiscuous Binding of Invariant Chain-Derived CLIP Peptide to Distinct HLA-I Molecules Revealed in Leukemic Cells

Antigen presentation by HLA class I (HLA-I) and HLA class II (HLA-II) complexes is achieved by proteins that are specific for their respective processing pathway. The invariant chain (Ii)-derived peptide CLIP is required for HLA-II-mediated antigen presentation by stabilizing HLA-II molecules before antigen loading through transient and promiscuous binding to different HLA-II peptide grooves. Here, we demonstrate alternative binding of CLIP to surface HLA-I molecules on leukemic cells. In HLA-II-negative AML cells, we found plasma membrane display of the CLIP peptide. Silencing Ii in AML cells resulted in reduced HLA-I cell surface display, which indicated a direct role of CLIP in the HLA-I antigen presentation pathway. In HLA-I-specific peptide eluates from B-LCLs, five Ii-derived peptides were identified, of which two were from the CLIP region. In vitro peptide binding assays strikingly revealed that the eluted CLIP peptide RMATPLLMQALPM efficiently bound to four distinct HLA-I supertypes (-A2, -B7, -A3, -B40). Furthermore, shorter length variants of this CLIP peptide also bound to these four supertypes, although in silico algorithms only predicted binding to HLA-A2 or -B7. Immunization of HLA-A2 transgenic mice with these peptides did not induce CTL responses. Together these data show a remarkable promiscuity of CLIP for binding to a wide variety of HLA-I molecules. The found participation of CLIP in the HLA-I antigen presentation pathway could reflect an aberrant mechanism in leukemic cells, but might also lead to elucidation of novel processing pathways or immune escape mechanisms

HLA class I allele promiscuity revisited

The peptide repertoire presented on human leukocyte antigen (HLA) class I molecules is largely determined by the structure of the peptide binding groove. It is expected that the molecules having similar grooves (i.e., belonging to the same supertype) might present similar/overlapping peptides. However, the extent of promiscuity among HLA class I ligands remains controversial: while in many studies T cell responses are detected against epitopes presented by alternative molecules across HLA class I supertypes and loci, peptide elution studies report minute overlaps between the peptide repertoires of even related HLA molecules. To get more insight into the promiscuous peptide binding by HLA molecules, we analyzed the HLA peptide binding data from the large epitope repository, Immune Epitope Database (IEDB), and further performed in silico analysis to estimate the promiscuity at the population level. Both analyses suggest that an unexpectedly large fraction of HLA ligands (>50%) bind two or more HLA molecules, often across supertype or even loci. These results suggest that different HLA class I molecules can nevertheless present largely overlapping peptide sets, and that “functional” HLA polymorphism on individual and population level is probably much lower than previously anticipated

A study of KIR binding to HLA-C

NK cell cytotoxic functions are under the control of a large array of inhibitory and activating cell surface receptors. One class of inhibitory receptors are the MHC class I specific Killer Immunoglobulin-like Receptors (KIRs). Different KIRs recognise different groups of HLA molecules. KIR2DL2 and KIR2DL3 segregate as alleles of the same locus and recognise the same HLA-C group 1 allotypes (HLA-C1). Loss of an inhibitory signal is one mechanism by which NK cells might recognise an infected cell. KIR-Ig proteins were generated to study the direct binding of KIR2DL2 and KIR2DL3 to HLA-C1: peptide complexes. The TAP-deficient T2 cell line expresses HLA-Cw*0102 (HLA-C1) and can be loaded with exogenous peptide to generate stable peptide: MHC class I complexes. Using an endogenous peptide, VAPWNSLSL as a template, known to bind to HLA-Cw*0102, a panel of seven peptides with mutations at residues 7 and 8 was studied to assess KIR binding. A peptide hierarchy was defined for KIR2DL2 and KIR2DL3 binding; peptides that mediated strong, intermediate or weak recognition of KIR to HLA-Cw*0102 were identified. A similar peptide hierarchy was observed to inhibit NK cells in CD107a assays. We showed that KIR2DL3 binds more strongly to HLA-Cw*0102: peptide than KIR2DL2, although in CD107a assays, KIR2DL2+ NK cells were not more inhibited than KIR2DL3+ NK cells. The peptide hierarchy was used to generate in vitro peptide repertoires containing strong and weak KIR-binding peptides. We observed that the weak KIR-binding peptide VAPWNSDAL can abrogate the inhibition of NK cells by the strong KIR-binding peptide VAPWNSFAL, despite levels of HLA-C remaining constant. This loss of NK cell inhibition in the presence of VAPWNSDAL occurred at more than 1 log higher concentration of VAPWNSFAL than in the absence of VAPWNSDAL. These data indicate that changes in peptide repertoire are significantly more efficient than class I down-regulation at releasing NK cells from inhibition

Disruption of Hydrogen Bonds between Major Histocompatibility Complex Class II and the Peptide N-Terminus Is Not Sufficient to Form a Human Leukocyte Antigen-DM Receptive State of Major Histocompatibility Complex Class II

Peptide presentation by MHC class II is of critical importance to the function of CD4+ T cells. HLA-DM resides in the endosomal pathway and edits the peptide repertoire of newly synthesized MHC class II molecules before they are exported to the cell surface. HLA-DM ensures MHC class II molecules bind high affinity peptides by targeting unstable MHC class II:peptide complexes for peptide exchange. Research over the past decade has implicated the peptide N-terminus in modulating the ability of HLA-DM to target a given MHC class II:peptide combination. In particular, attention has been focused on both the hydrogen bonds between MHC class II and peptide, and the occupancy of the P1 anchor pocket. We sought to solve the crystal structure of a HLA-DR1 molecule containing a truncated hemagglutinin peptide missing three N-terminal residues compared to the full-length sequence (residues 306–318) to determine the nature of the MHC class II:peptide species that binds HLA-DM. Here we present structural evidence that HLA-DR1 that is loaded with a peptide truncated to the P1 anchor residue such that it cannot make select hydrogen bonds with the peptide N-terminus, adopts the same conformation as molecules loaded with full-length peptide. HLA-DR1:peptide combinations that were unable to engage up to four key hydrogen bonds were also unable to bind HLA-DM, while those truncated to the P2 residue bound well. These results indicate that the conformational changes in MHC class II molecules that are recognized by HLA-DM occur after disengagement of the P1 anchor residue

Immunogenicity of HLA Class i and II double restricted influenza a-derived peptides

The aim of the present study was to identify influenza A-derived peptides which bind to both HLA class I and-II molecules and by immunization lead to both HLA class I and class II restricted immune responses. Eight influenza A-derived 9-11mer peptides with simultaneous binding to both HLA-A02:01 and HLA-DRB101:01 molecules were identified by bioinformatics and biochemical technology. Immunization of transgenic HLA-A02:01/HLADRB101:01 mice with four of these double binding peptides gave rise to both HLA class I and class II restricted responses by CD8 and CD4 T cells, respectively, whereas four of the double binding peptides did result in HLA-A02:01 restricted responses only. According to their cytokine profile, the CD4 T cell responses were of the Th2 type. In influenza infected mice, we were unable to detect natural processing in vivo of the double restricted peptides and in line with this, peptide vaccination did not decrease virus titres in the lungs of intranasally influenza challenged mice. Our data show that HLA class I and class II double binding peptides can be identified by bioinformatics and biochemical technology. By immunization, double binding peptides can give rise to both HLA class I and class I restricted responses, a quality which might be of potential interest for peptide-based vaccine development.Fil: Pedersen, Sara Ram. Universidad de Copenhagen; DinamarcaFil: Christensen, Jan Pravsgaard. Universidad de Copenhagen; DinamarcaFil: Buus, Søren. Universidad de Copenhagen; DinamarcaFil: Rasmussen, Michael. Universidad de Copenhagen; DinamarcaFil: Korsholm, Karen Smith. Statens Serum Institute; DinamarcaFil: Nielsen, Morten. Technical University of Denmark; Dinamarca. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Claesson, Mogens Helweg. Universidad de Copenhagen; Dinamarc

HLA class I maturation - in the presence and absence of tapasin

Human leukocyte antigen class I (HLA-I) molecules are present on all nucleated cells and present the cell content to cytotoxic T lymphocytes in the form of peptides. Maturation of HLA-I occurs in the endoplasmic reticulum and results in stable peptide-HLA-I complexes, in the presence of proper quality control. For proper peptide binding most HLA-I molecules interact with the peptide-loading complex (PLC), which is a multi-protein complex consisting of the transporter associated with antigen presentation, calreticulin, ERp57 and tapasin. Tapasin integrates HLA-I into the PLC and mediates quality control of the HLA-I maturation. When an optimal peptide is bound, tapasin releases the peptide-loaded HLA-I molecule that is next transported to the cell surface. The mechanisms for tapasin quality control of HLA-I maturation and the criteria defining optimal peptides are not completely known. Here, a recombinant part of tapasin, the first 87 N-terminal amino acids (Tpn1-87), was produced and shown to facilitate folding of different HLA-I molecules, i.e. allomorphs, to different degree in a peptide dependent manner. Folding of HLA-A*02:01 molecules with natural ligands, i.e. with peptides purified mainly from HLA-I expressed on the cell surface, was not facilitated by Tpn1-87, while folding of non-natural ligands, i.e. not presented at the cell surface, was facilitated. The folding facilitation exerted by Tpn1-87, tapasin-facilitation, inversely correlated with the stability of the peptide-HLA-I complex to some extent. The inverse correlation of these two parameters, tapasin-facilitation and stability, was studied in detail in the third paper. An increased stability was shown to not necessarily be associated with a decreased tapasin-facilitation. In the last paper of this thesis the tapasin-facilitation was studied in a large set of allomorphs folded with peptides of 7 to 13 amino acids in length. The influence of peptide-length for the different allomorphs increased with their tapasin dependence. In conclusion, Tpn1-87 facilitates folding of HLA-I in a peptide- and allomorph-dependent manner. Based on the above studies and data showing tapasin retention of immature HLA-I molecules, and studies of a suggested peptide-editing role of tapasin, we propose that tapasin keeps HLA-I molecules in a peptide-receptive conformation, which reduces the risk degradation of HLA-I molecules and increases the possibility for binding of optimal peptides