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1.  Conformational variation in structures of classical and nonclassical MHCII proteins and functional implications 
Immunological reviews  2012;250(1):144-157.
Summary
Recent structural characterizations of classical and nonclassical major histocompatibility complex class II (MHCII) proteins have provided a view into the dynamic nature of the MHCII peptide binding groove and the role that structural changes play in peptide loading processes. Although there have been numerous reports of crystal structures for MHCII-peptide complexes, a detailed analysis comparing all the structures has not been reported, and subtle conformational variations present in these structures may not have been fully appreciated. We compared the 91 MHCII crystal structures reported in the PDB to date, including a HLA-DR mutant particularly susceptible to DM-mediated peptide exchange, and reviewed experimental and computational studies of the effect of peptide binding on MHCII structure. These studies provide evidence for conformational lability in and around the α subunit 310 helix at residues α48-51, a region known to be critical for HLA-DM-mediated peptide exchange. A biophysical study of MHC-peptide hydrogen bond strengths and a recent structure of the nonclassical MHCII protein HLA-DO reveal changes in the same region. Conformational variability was observed also in the vicinity of a kink in the β subunit helical region near residue β66 and in the orientation and loop conformation in the β2 Ig domain. Here, we provide an overview of the regions within classical and nonclassical MHCII proteins that display conformational changes and the potential role that these changes may have in the peptide loading/exchange process.
doi:10.1111/imr.12003
PMCID: PMC3471379  PMID: 23046127
major histocompatibility complex; human lymphocyte antigen; antigen presentation; peptide binding; protein structure; conformational change
2.  HLA-DM constrains epitope selection in the human CD4 T cell response to vaccinia virus by favoring the presentation of peptides with longer HLA-DM-mediated half-lives1 
HLA-DM (DM) is a non-classical major histocompatibility complex II (MHC II) protein that acts as a peptide editor to mediate the exchange of peptides loaded onto MHC II during antigen presentation. Although the ability of DM to promote peptide exchange in vitro and in vivo is well established, the role of DM in epitope selection is still unclear, especially in human response to infectious disease. In this study, we addressed this question in the context of the human CD4 T cell response to vaccinia virus. We measured the IC50, intrinsic dissociation half-life, and DM-mediated dissociation half-life for a large set of peptides derived from the major core protein A10L and other known vaccinia epitopes bound to HLA-DR1, and compared these properties to the presence and magnitude of peptide-specific CD4+ T cell responses. We found that MHC II-peptide complex kinetic stability in the presence of DM distinguishes T cell epitopes from non-recognized peptides in A10L peptides and also in a set of predicted tight binders from the entire vaccinia genome. Taken together, these analyses demonstrate that DM-mediated dissociation half-life is a strong and independent factor governing peptide immunogenicity by favoring the presentation of peptides with greater kinetic stability in the presence of DM.
doi:10.4049/jimmunol.1200626
PMCID: PMC3466378  PMID: 22966084
3.  HLA-DM Focuses on Conformational Flexibility Around P1 Pocket to Catalyze Peptide Exchange 
Peptides presented by major histocompatibility complex class II (MHCII) molecules to CD4+ T cells play a central role in the initiation of adaptive immunity. This antigen presentation process is characterized by the proteolytic cleavage of foreign and self proteins, and loading of the resultant peptides onto MHCII molecules. Loading and exchange of antigenic peptides is catalyzed by a non-classical MHCII molecule, HLA-DM. The impact of HLA-DM on epitope selection has been appreciated for a long time. However, the molecular mechanism by which HLA-DM mediates peptide exchange remains elusive. Here, we review recent efforts in elucidating how HLA-DM works, highlighted by two recently solved co-structures of HLA-DM bound to HLA-DO (a natural inhibitor of HLA-DM), or to HLA-DR1 (a common MHCII). In light of these efforts, a model for HLA-DM action in which HLA-DM utilizes conformational flexibility around the P1 pocket of the MHCII-peptide complex to catalyze peptide exchange is proposed.
doi:10.3389/fimmu.2013.00336
PMCID: PMC3797982  PMID: 24146666
antigen presentation; epitope selection; MHCII-peptide complex; HLA-DM susceptibility; hydrogen bonds; conformational heterogeneity
4.  Human CD4+ T Cell Response to Human Herpesvirus 6 
Journal of Virology  2012;86(9):4776-4792.
Following primary infection, human herpesvirus 6 (HHV-6) establishes a persistent infection for life. HHV-6 reactivation has been associated with transplant rejection, delayed engraftment, encephalitis, muscular dystrophy, and drug-induced hypersensitivity syndrome. The poor understanding of the targets and outcome of the cellular immune response to HHV-6 makes it difficult to outline the role of HHV-6 in human disease. To fill in this gap, we characterized CD4 T cell responses to HHV-6 using peripheral blood mononuclear cell (PBMC) and T cell lines generated from healthy donors. CD4+ T cells responding to HHV-6 in peripheral blood were observed at frequencies below 0.1% of total T cells but could be expanded easily in vitro. Analysis of cytokines in supernatants of PBMC and T cell cultures challenged with HHV-6 preparations indicated that gamma interferon (IFN-γ) and interleukin-10 (IL-10) were appropriate markers of the HHV-6 cellular response. Eleven CD4+ T cell epitopes, all but one derived from abundant virion components, were identified. The response was highly cross-reactive between HHV-6A and HHV-6B variants. Seven of the CD4+ T cell epitopes do not share significant homologies with other known human pathogens, including the closely related human viruses human herpesvirus 7 (HHV-7) and human cytomegalovirus (HCMV). Major histocompatibility complex (MHC) tetramers generated with these epitopes were able to detect HHV-6-specific T cell populations. These findings provide a window into the immune response to HHV-6 and provide a basis for tracking HHV-6 cellular immune responses.
doi:10.1128/JVI.06573-11
PMCID: PMC3347333  PMID: 22357271
5.  Tetherin/BST-2 Antagonism by Nef Depends on a Direct Physical Interaction between Nef and Tetherin, and on Clathrin-mediated Endocytosis 
PLoS Pathogens  2013;9(7):e1003487.
Nef is the viral gene product employed by the majority of primate lentiviruses to overcome restriction by tetherin (BST-2 or CD317), an interferon-inducible transmembrane protein that inhibits the detachment of enveloped viruses from infected cells. Although the mechanisms of tetherin antagonism by HIV-1 Vpu and HIV-2 Env have been investigated in detail, comparatively little is known about tetherin antagonism by SIV Nef. Here we demonstrate a direct physical interaction between SIV Nef and rhesus macaque tetherin, define the residues in Nef required for tetherin antagonism, and show that the anti-tetherin activity of Nef is dependent on clathrin-mediated endocytosis. SIV Nef co-immunoprecipitated with rhesus macaque tetherin and the Nef core domain bound directly to a peptide corresponding to the cytoplasmic domain of rhesus tetherin by surface plasmon resonance. An analysis of alanine-scanning substitutions identified residues throughout the N-terminal, globular core and flexible loop regions of Nef that were required for tetherin antagonism. Although there was significant overlap with sequences required for CD4 downregulation, tetherin antagonism was genetically separable from this activity, as well as from other Nef functions, including MHC class I-downregulation and infectivity enhancement. Consistent with a role for clathrin and dynamin 2 in the endocytosis of tetherin, dominant-negative mutants of AP180 and dynamin 2 impaired the ability of Nef to downmodulate tetherin and to counteract restriction. Taken together, these results reveal that the mechanism of tetherin antagonism by Nef depends on a physical interaction between Nef and tetherin, requires sequences throughout Nef, but is genetically separable from other Nef functions, and leads to the removal of tetherin from sites of virus release at the plasma membrane by clathrin-mediated endocytosis.
Author Summary
Tetherin (BST-2, CD317 or HM1.24) is an interferon-inducible cellular restriction factor that prevents the release of enveloped viruses from infected cells. Human and simian immunodeficiency viruses have evolved to use different viral proteins to overcome the anti-viral effects of tetherin. Whereas HIV-1 Vpu and HIV-2 Env counteract human tetherin, most SIVs use the accessory protein Nef to counteract tetherin in their non-human primate hosts. Here we show that the mechanism of tetherin antagonism by SIV Nef involves a direct physical interaction between the core domain of Nef and the cytoplasmic domain of tetherin, which results in the removal of tetherin from sites of virus assembly and release on the cell surface by a mechanism that depends on clathrin and dynamin 2. The Nef-mediated internalization of tetherin leads to the accumulation of tetherin within lysosomal compartments, suggesting that, similar to CD4− and MHC I-downregulation, Nef promotes the lysosomal degradation of tetherin.
doi:10.1371/journal.ppat.1003487
PMCID: PMC3708871  PMID: 23853598
6.  HLA-DO acts as a substrate mimic to inhibit HLA-DM by a competitive mechanism 
MHCII proteins bind peptide antigens in endosomal compartments of antigen-presenting cells. The non-classical MHCII protein HLA-DM chaperones peptide-free MHCII against inactivation and catalyzes peptide exchange on loaded MHCII. Another non-classical MHCII protein, HLA-DO, binds HLA-DM and influences the repertoire of peptides presented by MHCII proteins. However, the mechanism by which HLA-DO functions is unclear. Here we use x-ray crystallography, enzyme kinetics and mutagenesis approaches to investigate human HLA-DO structure and function. In complex with HLA-DM, HLA-DO adopts a classical MHCII structure, with alterations near the alpha subunit 310 helix. HLA-DO binds to HLA-DM at the same sites implicated in MHCII interaction, and kinetic analysis demonstrates that HLA-DO acts as a competitive inhibitor. These results show that HLA-DO inhibits HLA-DM function by acting as a substrate mimic and place constraints on possible functional roles for HLA-DO in antigen presentation.
doi:10.1038/nsmb.2460
PMCID: PMC3537886  PMID: 23222639
7.  HLA-DR: Molecular insights and vaccine design 
Current pharmaceutical design  2009;15(28):3249-3261.
Vaccines are one of the most cost effective methods to control infectious diseases and at the same time one of the most complex products of the Pharmaceutical industry. Contrary to other drugs, vaccines are used mainly in healthy individuals, many cases children. For this reason, very high standards are set for their production.
Subunit vaccines, especially peptide vaccines, provide a cost effective alternative. Structural studies of peptide-MHC complexes and biochemical characterizations of the peptide-MHC interaction have provided conceptual foundation for the rational design of subunit vaccines and in the other side of the spectrum, development of “immunological silent” therapeutic proteins. In this review, we explain the manner in which the analysis of crystal structures of peptide-class II MHC complexes opened the door to the understanding of the major rules that govern this interaction. We also describe biochemical studies that allowed the development of “virtual matrices” of the peptide-class II MHC interaction and MHC-peptide binding algorithms that incorporate in one or other manner structural and/or biochemical information of the interaction. Finally, and using malaria as a model, we describe the development of a minimal subunit vaccine for the human malaria parasite Plasmodium falciparum.
PMCID: PMC3615543  PMID: 19860674
8.  Antigen-specific T cell phenotyping microarrays using Grating Coupled Surface Plasmon Resonance Imaging and Surface Plasmon Coupled Emission 
Biosensors & bioelectronics  2011;31(1):264-269.
The circulating population of peripheral T lymphocytes obtained from a blood sample can provide a large amount of information about an individual's medical status and history. Recent evidence indicates that the detection and functional characterization of antigen-specific T cell subsets within the circulating population may provide a diagnostic indicator of disease and has the potential to predict an individual's response to therapy. In this report, a microarray detection platform that combines grating-coupled surface plasmon resonance imaging (GCSPRI) and grating-coupled surface plasmon coupled emission (SPCE) fluorescence detection modalities was used to detect and characterize CD4+ T cells. The microspot regions of interest (ROIs) printed on the array consisted of immobilized antibodies or peptide loaded MHC monomers (p/MHC) as T cell capture ligands mixed with additional antibodies as cytokine capture ligands covalently bound to the surface of a corrugated gold sensor chip. Using optimized parameters, an unlabelled influenza peptide reactive T cell clone could be detected at a frequency of 0.1% in a mixed T cell sample using GCSPRI. Additionally, after cell binding was quantified, differential TH1 cytokine secretion patterns from a T cell clone cultured under TH1 or TH2 inducing conditions was detected using an SPCE fluorescence based assay. Differences in the secretion patterns of 3 cytokines, characteristic of the inducing conditions, indicated that differences were a consequence of the functional status of the captured cells. A dual mode GCSPRI/SPCE assay can provide a rapid, high content T cell screening/characterization tool that is useful for diagnosing disease, evaluating vaccination efficacy, or assessing responses to immunotherapeutics.
doi:10.1016/j.bios.2011.10.029
PMCID: PMC3249003  PMID: 22104646
T cell microarray; SPR; SPCE
9.  A role for differential variable gene pairing in creating T cell receptors specific for unique major histocompatibility ligands 
Immunity  2011;35(5):694-704.
SUMMARY
A limited set of T cell receptor (TCR) variable (V) gene segments are used to create a repertoire of TCRs that recognize all major histocompatibility complex (MHC) ligands within a species. How individual αβTCRs are constructed to specifically recognize a limited set of MHC ligands is unclear. Here we have identified a role for the differential paring of particular V gene segments in creating TCRs that recognized MHC class II ligands exclusively, or cross-reacted with classical and non-classical MHC class I ligands. Biophysical and structural experiments indicated TCR specificity for MHC ligands is not driven by germline encoded pairwise interactions. Rather, identical TCRβ chains can have altered peptide-MHC (pMHC) binding modes when paired with different TCRα chains. The ability of TCR chain pairing to modify how V region residues interact with pMHC helps to explain how the same V genes are used to create TCRs specific for unique MHC ligands.
doi:10.1016/j.immuni.2011.10.012
PMCID: PMC3253227  PMID: 22101158
10.  Fluorogenic probes for monitoring peptide binding to class II MHC proteins in living cells 
Nature chemical biology  2007;3(4):222-228.
A crucial step in the immune response is the binding of antigenic peptides to MHC proteins. Class II MHC proteins present their bound peptides to CD4+T cells, helping to activate both the humoral and the cellular arms of the adaptive immune response. Peptide loading onto class II MHC proteins is regulated temporally, spatially, and developmentally in antigen-presenting cells1. To help visualize these processes, we have developed a series of novel fluorogenic probes that incorporate the environment-sensitive amino acid analogs 6-N,N-dimethylamino-2-3-naphthalimido-alanine and 4-N,N-dimethylaminophthalimido-alanine. Upon binding to class II MHC proteins these fluorophores exhibit large changes in emission spectra, quantum yield, and fluorescence lifetime. Peptides incorporating these fluorophores bind specifically to class II MHC proteins on antigen presenting cells, and can be used to follow peptide binding in vivo. Using these probes we have tracked a developmentally-regulated cell surface peptide binding activity in primary human monocyte-derived dendritic cells.
doi:10.1038/nchembio868
PMCID: PMC3444530  PMID: 17351628
11.  Minimal conformational plasticity enables TCR cross-reactivity to different MHC class II heterodimers 
Scientific Reports  2012;2:629.
Successful immunity requires that a limited pool of αβ T-cell receptors (TCRs) provide cover for a vast number of potential foreign peptide antigens presented by ‘self’ major histocompatibility complex (pMHC) molecules. Structures of unligated and ligated MHC class-I-restricted TCRs with different ligands, supplemented with biophysical analyses, have revealed a number of important mechanisms that govern TCR mediated antigen recognition. HA1.7 TCR binding to the influenza hemagglutinin antigen (HA306–318) presented by HLA-DR1 or HLA-DR4 represents an ideal system for interrogating pMHC-II antigen recognition. Accordingly, we solved the structure of the unligated HA1.7 TCR and compared it to both complex structures. Despite a relatively rigid binding mode, HA1.7 T-cells could tolerate mutations in key contact residues within the peptide epitope. Thermodynamic analysis revealed that limited plasticity and extreme favorable entropy underpinned the ability of the HA1.7 T-cell clone to cross-react with HA306–318 presented by multiple MHC-II alleles.
doi:10.1038/srep00629
PMCID: PMC3432979  PMID: 22953050
12.  Intrinsically Disordered Cytoplasmic Domain of T Cell Receptor Zeta Chain Binds to the Nef Protein of Simian Immunodeficiency Virus Without a Disorder-to-Order Transition† 
Biochemistry  2008;47(49):12942-12944.
Intrinsically disordered proteins are thought to undergo coupled binding and folding upon interaction with their folded partners. In this study, we investigate whether binding of the intrinsically disordered T cell receptor zeta cytoplasmic tail to the well-folded simian immunodeficiency virus Nef core domain is accompanied by a disorder-to-order transition. We show that zeta forms a 1:1 complex with Nef and remains unfolded in the complex. Thus, our findings oppose the generally accepted view on the behavior of intrinsically disordered proteins and provide new evidence for the existence of specific interactions for unfolded protein molecules.
doi:10.1021/bi801602p
PMCID: PMC3226742  PMID: 19012413
13.  Structural Basis For Antigenic Peptide Precursor Processing by the Endoplasmic Reticulum Aminopeptidase ERAP1 
ERAP1 trims antigen precursors to fit into MHC class I proteins. To perform this function, ERAP1 has unique substrate preferences, trimming long peptides while sparing shorter ones. To identify the structural basis for ERAP1's unusual properties, we determined the X-ray crystal structure of human ERAP1 bound to bestatin. The structure reveals an open conformation with a large interior compartment. An extended groove originating from the enzyme's catalytic center can accommodate long peptides and has features that explain ERAP1's broad specificity for antigenic peptide precursors. Structural and biochemical analysis suggest a mechanism for ERAP1's length-dependent trimming activity, whereby binding of long but not short substrates induces a conformational change with reorientation of a key catalytic residue towards the active site. ERAP1's unique structural elements suggest how a generic aminopeptidase structure has been adapted for the specialized function of trimming antigenic precursors.
doi:10.1038/nsmb.2021
PMCID: PMC3087843  PMID: 21478864
14.  Peptide-MHC Cellular Microarray with Innovative Data Analysis System for Simultaneously Detecting Multiple CD4 T-Cell Responses 
PLoS ONE  2010;5(6):e11355.
Background
Peptide:MHC cellular microarrays have been proposed to simultaneously characterize multiple Ag-specific populations of T cells. The practice of studying immune responses to complicated pathogens with this tool demands extensive knowledge of T cell epitopes and the availability of peptide:MHC complexes for array fabrication as well as a specialized data analysis approach for result interpretation.
Methodology/Principal Findings
We co-immobilized peptide:DR0401 complexes, anti-CD28, anti-CD11a and cytokine capture antibodies on the surface of chamber slides to generate a functional array that was able to detect rare Ag-specific T cell populations from previously primed in vitro T cell cultures. A novel statistical methodology was also developed to facilitate batch processing of raw array-like data into standardized endpoint scores, which linearly correlated with total Ag-specific T cell inputs. Applying these methods to analyze Influenza A viral antigen-specific T cell responses, we not only revealed the most prominent viral epitopes, but also demonstrated the heterogeneity of anti-viral cellular responses in healthy individuals. Applying these methods to examine the insulin producing beta-cell autoantigen specific T cell responses, we observed little difference between autoimmune diabetic patients and healthy individuals, suggesting a more subtle association between diabetes status and peripheral autoreactive T cells.
Conclusions/Significance
The data analysis system is reliable for T cell specificity and functional testing. Peptide:MHC cellular microarrays can be used to obtain multi-parametric results using limited blood samples in a variety of translational settings.
doi:10.1371/journal.pone.0011355
PMCID: PMC2902358  PMID: 20634998
15.  An Expanded Self-Antigen Peptidome Is Carried by the Human Lymph As Compared to the Plasma 
PLoS ONE  2010;5(3):e9863.
Background
The pre-nodal afferent lymph is the fluid which directly derives from the extracellular milieu from every parenchymal organ and, as it continues to circulate between the cells, it collects products deriving from the organ metabolism/catabolism. A comprehensive qualitative and quantitative investigation of the self-antigenic repertoire transported by the human lymph is still missing.
Methodology/Principal Findings
A major difference between lymph and plasma could be visualized by FPLC and 2D gel in the amount of low molecular weight products corresponding to peptide fragments. Naturally processed peptides in normal pre-nodal human lymph were then fractionated by HPLC and characterized by multidimensional mass spectrometry. Analysis of more then 300 sequences identified self-peptides derived from both intracellular and extracellular proteins revealing the variety of catabolic products transported by human lymph. Quantitative analysis established that at least some of these peptides are present in the circulating lymph in nanomolar concentration.
Conclusions/Significance
The peptidome, generated by physiological tissue catabolism and transported by the pre-nodal lymph, is in addition to the self-peptidome generated in endosomal compartment. Unlike self antigen processed by local or nodal APC, which mostly produce epitopes constrained by the endosomal processing activity, self antigens present in the lymph could derived from a wider variety of processing pathways; including caspases, involved in cellular apoptosis, and ADAM and other metalloproteinases involved in surface receptor editing, cytokines processing and matrix remodeling. Altogether, expanding the tissue-specific self-repertoire available for the maintenance of immunological tolerance.
doi:10.1371/journal.pone.0009863
PMCID: PMC2845622  PMID: 20360855
16.  Pseudo-merohedral twinning and noncrystallographic symmetry in orthorhombic crystals of SIVmac239 Nef core domain bound to different-length TCRζ fragments 
P212121 crystals of SIV Nef core domain bound to a peptide fragment of the T-cell receptor ζ subunit exhibited noncrystallographic symmetry and nearly perfect pseudo-merohedral twinning simulating tetragonal symmetry. For a different peptide fragment, nontwinned tetragonal crystals were observed but diffracted to lower resolution. The structure was determined after assignment of the top molecular-replacement solutions to various twin or NCS domains followed by refinement under the appropriate twin law.
HIV/SIV Nef mediates many cellular processes through interactions with various cytoplasmic and membrane-associated host proteins, including the signalling ζ subunit of the T-­cell receptor (TCRζ). Here, the crystallization strategy, methods and refinement procedures used to solve the structures of the core domain of the SIVmac239 isolate of Nef (Nefcore) in complex with two different TCRζ fragments are described. The structure of SIVmac239 Nefcore bound to the longer TCRζ polypeptide (Leu51–Asp93) was determined to 3.7 Å resolution (R work = 28.7%) in the tetragonal space group P43212. The structure of SIVmac239 Nefcore in complex with the shorter TCRζ polypeptide (Ala63–Arg80) was determined to 2.05 Å resolution (R work = 17.0%), but only after the detection of nearly perfect pseudo-merohedral crystal twinning and proper assignment of the orthorhombic space group P212121. The reduction in crystal space-group symmetry induced by the truncated TCRζ polypeptide appears to be caused by the rearrangement of crystal-contact hydrogen-bonding networks and the substitution of crystallographic symmetry operations by similar noncrystallographic symmetry (NCS) operations. The combination of NCS rotations that were nearly parallel to the twin operation (k, h, −l) and a and b unit-cell parameters that were nearly identical predisposed the P212121 crystal form to pseudo-merohedral twinning.
doi:10.1107/S090744490904880X
PMCID: PMC2815668  PMID: 20124696
pseudo-merohedral twinning; noncrystallographic symmetry; pseudosymmetry; human immunodeficiency virus; Nef; T-cell receptor
17.  Peptide immunotherapy in allergic asthma generates IL-10–dependent immunological tolerance associated with linked epitope suppression 
The Journal of Experimental Medicine  2009;206(7):1535-1547.
Treatment of patients with allergic asthma using low doses of peptides containing T cell epitopes from Fel d 1, the major cat allergen, reduces allergic sensitization and improves surrogate markers of disease. Here, we demonstrate a key immunological mechanism, linked epitope suppression, associated with this therapeutic effect. Treatment with selected epitopes from a single allergen resulted in suppression of responses to other (“linked”) epitopes within the same molecule. This phenomenon was induced after peptide immunotherapy in human asthmatic subjects and in a novel HLA-DR1 transgenic mouse model of asthma. Tracking of allergen-specific T cells using DR1 tetramers determined that suppression was associated with the induction of interleukin (IL)-10+ T cells that were more abundant than T cells specific for the single-treatment peptide and was reversed by anti–IL-10 receptor administration. Resolution of airway pathophysiology in this model was associated with reduced recruitment, proliferation, and effector function of allergen-specific Th2 cells. Our results provide, for the first time, in vivo evidence of linked epitope suppression and IL-10 induction in both human allergic disease and a mouse model designed to closely mimic peptide therapy in humans.
doi:10.1084/jem.20082901
PMCID: PMC2715096  PMID: 19528258
18.  Vaccinia peptides eluted from HLA-DR1 isolated from virus-infected cells are recognized by CD4+ T cells from a vaccinated donor 
Journal of proteome research  2008;7(7):2703-2711.
Class II MHC proteins bind peptides and present them to CD4+ T cells as part of the immune system's surveillance of bodily tissues for foreign and pathogenic material. Antigen processing and presentation pathways have been characterized in detail in normal cells, but there is little known about the actual viral peptides that are presented to CD4+ T cells that signal infection. In this study, two-dimensional LC-MS/MS was used to identify vaccinia virus-derived peptides among the hundreds to thousands of peptide antigens bound to the human class II MHC protein HLA-DR1 on the surface of vaccinia virus-infected cells. The peptides, derived from the I6L, D6R, and A10L viral proteins, were 15 residues in length, bound efficiently to HLA-DR1 as synthetic peptides, and were recognized by vaccinia-specific CD4+ T cells obtained from an immunized donor.
doi:10.1021/pr700780x
PMCID: PMC2515592  PMID: 18507432
tandem mass spectrometry; vaccinia virus; epitope; antigen processing; antigen presentation; peptide binding; T cell response; CD4+ T-cell; immune response; vaccine
19.  Antagonism of HIV-specific CD4+ T cells by C-terminal truncation of a minimum epitope 
Molecular immunology  2005;43(9):1349-1357.
Antagonism of T cell responses by variants of the cognate peptide is a potential mechanism of viral escape from immune responses and may play a role in the ability of HIV to evade immune control. We show here a rarely described mechanism of antagonism by a peptide shorter than the minimum length epitope for an HIV p24-specific CD4+ T cell clone. The shorter antagonist peptide-MHC complex bound the T cell receptor (TCR), albeit with lower affinity than the full-length agonist peptide. Prior work showing the crystal structure of the peptide-MHC complex revealed a unique glycine hinge near the C-terminus of the agonist peptide, allowing the generation of full-length antagonist peptide lacking the hinge. These results confirm the dependence of productive TCR engagement on residues spilling out from the C-terminus of the MHC binding groove and show that partial engagement of the TCR with a truncated, low-affinity ligand can result in T cell antagonism.
doi:10.1016/j.molimm.2005.09.004
PMCID: PMC2561961  PMID: 16216327
20.  Lipid-binding activity of intrinsically unstructured cytoplasmic domains of multichain immune recognition receptor signaling subunits† 
Biochemistry  2006;45(51):15731-15739.
Multichain immune recognition receptors (MIRRs) found on the surface of T cells, B cells, mast cells, natural killer cells, basophils, and other immune cells, are formed by the association of several single-pass transmembrane proteins, with immunoglobulin-like ligand recognition domains and signal-transducing domains present on separate subunits. The MIRR signaling subunits all have cytoplasmic domains containing one or more copies of an immunoreceptor tyrosine-based activation motif (ITAM), tyrosine residues of which are phosphorylated upon receptor engagement in an early and obligatory event in the signaling cascade. Despite the proximity to the cell membrane and crucial role in transmembrane signal transduction, little is known about the structure and lipid-binding activity of the ITAM-containing cytoplasmic domains. Here we investigate the conformation and lipid-binding activity of several MIRR cytoplasmic domains, namely T cell receptor ζcyt, CD3εcyt, CD3δcyt, and CD3γcyt, B cell receptor Igαcyt and Igβcyt, and Fc receptor FcεRIγcyt, using purified recombinant proteins. Secondary structure prediction analysis and experimental circular dichroism spectra identify each of these cytoplasmic domains as natively unfolded proteins. We also report that ζcyt, CD3εcyt, and FcεRIγcyt bind to acidic and mixed phospholipid vesicles and that the binding strength correlates with the protein net charge and the presence of clustered basic amino acid residues. Circular dichroism analysis reveals the lack of secondary structure for these domains in lipid-bound form. Phosphorylation of ζcyt and FcεRIγcyt does not alter their random-coil conformation but weakens binding to membranes. The implications of these results for transmembrane signal transduction by immune receptors are discussed.
doi:10.1021/bi061108f
PMCID: PMC2528957  PMID: 17176095
21.  Model for the Peptide-Free Conformation of Class II MHC Proteins 
PLoS ONE  2008;3(6):e2403.
Background
Major histocompatibility complex proteins are believed to undergo significant conformational changes concomitant with peptide binding, but structural characterization of these changes has remained elusive.
Methodology/Principal Findings
Here we use molecular dynamics simulations and experimental probes of protein conformation to investigate the peptide-free state of class II MHC proteins. Upon computational removal of the bound peptide from HLA-DR1-peptide complex, the α50-59 region folded into the P1-P4 region of the peptide binding site, adopting the same conformation as a bound peptide. Strikingly, the structure of the hydrophobic P1 pocket is maintained by engagement of the side chain of Phe α54. In addition, conserved hydrogen bonds observed in crystal structures between the peptide backbone and numerous MHC side chains are maintained between the α51-55 region and the rest of the molecule. The model for the peptide-free conformation was evaluated using conformationally-sensitive antibody and superantigen probes predicted to show no change, moderate change, or dramatic changes in their interaction with peptide-free DR1 and peptide-loaded DR1. The binding observed for these probes is in agreement with the movements predicted by the model.
Conclusion/Significance
This work presents a molecular model for peptide-free class II MHC proteins that can help to interpret the conformational changes known to occur within the protein during peptide binding and release, and can provide insight into possible mechanisms for DM action.
doi:10.1371/journal.pone.0002403
PMCID: PMC2408972  PMID: 18545669
22.  Human CD4+ T Cell Epitopes from Vaccinia Virus Induced by Vaccination or Infection 
PLoS Pathogens  2007;3(10):e144.
Despite the importance of vaccinia virus in basic and applied immunology, our knowledge of the human immune response directed against this virus is very limited. CD4+ T cell responses are an important component of immunity induced by current vaccinia-based vaccines, and likely will be required for new subunit vaccine approaches, but to date vaccinia-specific CD4+ T cell responses have been poorly characterized, and CD4+ T cell epitopes have been reported only recently. Classical approaches used to identify T cell epitopes are not practical for large genomes like vaccinia. We developed and validated a highly efficient computational approach that combines prediction of class II MHC-peptide binding activity with prediction of antigen processing and presentation. Using this approach and screening only 36 peptides, we identified 25 epitopes recognized by T cells from vaccinia-immune individuals. Although the predictions were made for HLA-DR1, eight of the peptides were recognized by donors of multiple haplotypes. T cell responses were observed in samples of peripheral blood obtained many years after primary vaccination, and were amplified after booster immunization. Peptides recognized by multiple donors are highly conserved across the poxvirus family, including variola, the causative agent of smallpox, and may be useful in development of a new generation of smallpox vaccines and in the analysis of the immune response elicited to vaccinia virus. Moreover, the epitope identification approach developed here should find application to other large-genome pathogens.
Author Summary
Although the routine use of vaccinia virus for vaccination against smallpox was stopped after eradication of this disease, there is a possibility for an accidental or intentional release of this virus. In response to this challenge, vaccination of at least emergency personnel has been suggested. However, adverse reactions induced by the smallpox vaccine have had a negative impact in the success of this program. For these reasons development of new smallpox vaccines is a public health priority. Identification of strong helper T cell epitopes is central to these efforts. However, identification of T cell epitopes in large genomes like vaccinia is difficult using current screening methods. In this work, we develop a new computational approach for prediction of T cell epitopes, validate it using epitopes already identified by classical methods, and apply it to the prediction of vaccinia epitopes. Twenty-five of 36 peptides containing predicted sequences were recognized by T cells from individuals exposed to vaccinia virus. These peptides are highly conserved across the orthopox virus family and may be useful in development of a new generation of smallpox vaccines and in the analysis of the immune response against vaccinia virus.
doi:10.1371/journal.ppat.0030144
PMCID: PMC2014795  PMID: 17937498

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