Peptide antagonism and T cell receptor interactions with peptide-MHC complexes

We describe antagonist peptides that specifically inhibit cytolytic activity of T cell clones and lines that express the antigen-specific receptor of CD8+ T lymphocyte clone 2C, which recognizes peptides in association with syngeneic (Kb) and allogeneic (Ld) MHC proteins. Addition of an antagonist peptide that can bind to Kb on 2C cells decreased the tyrosine phosphorylation of CD3 zeta chains elicited by prior exposure of the cells to an agonist peptide-Kb complex. Contrary to previous agonist-antagonist comparisons, the 2C T cell receptor had higher affinity for an antagonist peptide-Kb complex than for a weak agonist peptide-Kb complex. This difference is considered in light of evidence that antigen-specific receptor affinity values can be substantially higher when determined with the receptor on live cells than with the receptor in cell-free systems.     

Structural basis of plasticity in T cell receptor recognition of a self peptide-MHC antigen

The T cell receptor (TCR) inherently has dual specificity. T cells must recognize self-antigens in the thymus during maturation and then discriminate between foreign pathogens in the periphery. A molecular basis for this cross-reactivity is elucidated by the crystal structure of the alloreactive 2C TCR bound to self peptide-major histocompatibility complex (pMHC) antigen H-2Kb-dEV8 refined against anisotropic 3.0 angstrom resolution x-ray data. The interface between peptide and TCR exhibits extremely poor shape complementarity, and the TCR beta chain complementarity-determining region 3 (CDR3) has minimal interaction with the dEV8 peptide. Large conformational changes in three of the TCR CDR loops are induced upon binding, providing a mechanism of structural plasticity to accommodate a variety of different peptide antigens. Extensive TCR interaction with the pMHC alpha helices suggests a generalized orientation that is mediated by the Valpha domain of the TCR and rationalizes how TCRs can effectively "scan" different peptides bound within a large, low-affinity MHC structural framework for those that provide the slight additional kinetic stabilization required for signaling.     

The negative dominant effects of T340M mutation on mammalian pyruvate kinase

A fundamental issue in allosteric regulatory enzymes is the identification of pathways of signal transmission. Rabbit muscle and kidney pyruvate kinase isozymes are ideal to address this issue because these isozymes exhibit different enzymatic regulatory patterns, and the sequence differences between these isozymes have identified the amino acid residues that alter their kinetic behavior. In an earlier study, Cheng et al. (Cheng, X., Friesen, R. H. E., and Lee, J. C. (1996) J. Biol. Chem. 271, 6313-6321), reported the effects of a threonine to methionine mutation at residue 340 in the muscle isozyme. In this study, the same mutation was effected in the kidney isozyme. Qualitatively, the same negative effects are observed in both isozymes, namely a significant decrease in catalytic efficiency and decrease in apparent affinity for phosphoenolpyruvate but no change in affinity for ADP, and a decrease in responsiveness to the presence of effectors, be it activator or inhibitor. Because the diversity in the primary sequence between these two isozymes does not alter the negative impact of the T340M mutation, it can be concluded that this mutation exerts a dominant, negative effect. The negative effects of T340M mutation on the kinetic properties imply that there is communication between residue 340 and the active site. Residue 340 is located at the 1,4 subunit interface; however, a T340M mutation enhances the dimerization affinity along the 1,2 subunit interface. Thus, this study has identified a communication network among the active site, residue 340, and the 1,2 subunit interface.     

On defining the rules for interactions between the T cell receptor and its ligand: a critical role for a specific amino acid residue of the T cell receptor beta chain

The specificity of T cell-mediated immune responses is primarily determined by the interaction between the T cell receptor (TCR) and the antigenic peptide presented by the major histocompatibility complex (MHC) molecules. To refine our understanding of interactions between the TCR and the antigenic peptide of vesicular stomatitis virus (VSV) presented by the class I MHC molecule H-2Kb, we constructed a TCR alpha chain transgenic mouse in a TCR alpha-deficient background to define specific structural features in the TCR beta chain that are important for the recognition of the VSV/H-2Kb complex. We found that for a given peptide, a peptide-specific, highly conserved amino acid could always be identified at position 98 of the complementarity-determining region 3 (CDR3) loop of TCR beta chains. Further, we demonstrated that substitutions at position 6, but not position 1, of the VSV peptide induced compensatory changes in the TCR in both the amino acid residue at position 98 and the length of the CDR3beta loop. We conclude that the amino acid residue at position 98 of the CDR3beta loop is a key residue that plays a critical role in determining the specificity of TCR-VSV/H-2Kb interactions and that a specific length of the CDR3beta loop is required to facilitate such interactions. Further, these findings suggest that the alpha and beta chains of TCRs interact with amino acid residue(s) toward the N and C termini of the VSV peptide, respectively, providing functional evidence for the orientation of a TCR with its peptide/MHC ligand as observed in the crystal structures of TCR/peptide/MHC complexes.     

The effect of amino acids and amino acid derivatives on cell proliferation

The effect of certain amino acids and amino acid derivatives on cell proliferation have been studied in the author's Institute for more than 25 years. The optically active forms of arginine, lysine, aspartic acid and glutamic acid influence the growth of transplantable rat tumors. L-arginine, D-lysine, L-aspartic acid and D-glutamic acid promoted; D-aspartic acid, L-glutamic acid, D-arginine and L-lysine inhibited tumour growth. E-amino trimethyl-lysine (TML) stimulated cell proliferation in various cell systems (bone marrow, small intestine, cultured lymphocytes). When administered simultaneously with high doses of Cyclophosphamide, Vincristin or Doxorubicin to tumour-bearing mice, TML decreased the toxicity of the antitumour drugs, resulting in a higher rate of survivors. L-leucine methyl ester caused cell death of mouse peritoneal macrophages by inducing disruption of macrophages.     

Tolerance to a dominant T cell epitope in the acetylcholine receptor molecule induces epitope spread and suppresses murine myasthenia gravis

Myasthenia gravis (MG) is a T cell-dependent, Ab-mediated autoimmune disease. T cells reactive to a dominant peptide alpha 146-162 of acetylcholine receptor (AChR) alpha subunit participate in murine MG pathogenesis. To suppress the autoimmune response to AChR, a high dose of alpha146-162 peptide in IFA was administered parenterally as a tolerogen, after the development of a primary T cell immune response to AChR. This form of AChR T cell peptide tolerance suppressed the in vitro T cell proliferative response to AChR and its dominant alpha146-162 and subdominant alpha182-198 peptides through epitope spread. Administration of alpha146-162 peptide in IFA after the primary immune response to AChR also significantly suppressed the serum anti-AChR Ab of the IgG2b isotype and clinical incidence of MG in C57BL/6 mice. Furthermore, the production of IFN-gamma, IL-2, and IL-10 cytokines by AChR, alpha146-162, and alpha182-198 peptide-reactive cells was suppressed by alpha146-162 peptide tolerance, and the epitope spread observed could be attributed to the reduction in the above cytokine production. Therefore, AChR T cell-dominant peptide tolerance could be adapted in the Ag-specific therapy of MG.     

Separately expressed T cell receptor alpha and beta chain transgenes exert opposite effects on T cell differentiation and neoplastic transformation

Two aspects of T cell differentiation in T cell receptor (TCR)-transgenic mice, the generation of an unusual population of CD4-CD8-TCR+ thymocytes and the absence of gamma delta cells, have been the focus of extensive investigation. To examine the basis for these phenomena, we investigated the effects of separate expression of a transgenic TCR alpha chain and a transgenic TCR beta chain on thymocyte differentiation. Our data indicate that expression of a transgenic TCR alpha chain causes thymocytes to differentiate into a CD4-CD8-TCR+ lineage at an early developmental stage, depleting the number of thymocytes that differentiate into the alpha beta lineage. Surprisingly, expression of the TCR alpha chain transgene is also associated with the development of T cell lymphosarcoma. In contrast, expression of the transgenic TCR beta chain causes immature T cells to accelerate differentiation into the alpha beta lineage and thus inhibits the generation of gamma delta cells. Our observations provide a model for understanding T cell differentiation in TCR-transgenic mice.     

The dominant T cell clone is present in multiple regressing skin lesions and associated T cell lymphomas of patients with lymphomatoid papulosis

This study was undertaken to determine the clonality of lymphomatoid papulosis (LyP), its clonal relationship to lymphomas, which occur at high frequency in LyP patients, and to define the cell lineage of Reed-Sternberg-like cells in type A lesions of LyP. Punch biopsies of skin of 11 adult patients with LyP were analyzed for morphologic subtype of LyP, surface antigens, and clonal T-cell receptor (TCR) gene rearrangements. Clonal rearrangements were identified by semiquantitative polymerase chain reaction amplification and sequencing of TCR-beta chain genes in nine patients and TCR-gamma chain genes in two patients. A single dominant clone was detected in multiple separate LyP lesions, often of different histologies, in nine patients. The same clone was detected in LyP lesions and the anaplastic large cell lymphoma (ALCL) of 2 patients and the mycosis fungoides (MF) of 2 other patients. No dominant clone could be detected in one patient with LyP uncomplicated by lymphoma or in a second patient with LyP and MF. A T-cell lineage was evident for RS-like cells in cell culture and in type A lesions. These results show that multiple regressing skin lesions and associated T cell lymphomas (MF and ALCL) are clonally related in most LyP patients, which suggest that the disease in these patients was initiated by a non-random genetic event.     

The role of I-Ag7 beta chain in peptide binding and antigen recognition by T cells

We examine here how the beta chain of the class II MHC molecule I-Ag7 influences T cell recognition. Three sets of T cell clones were identified. The first set recognizes peptides bound to I-Ag7, I-Ad and I-Ag7 mutant in which the allele-specific residues His and Ser at position 56 and 57 were changed to the Pro at residue 56 and to non-polymorphic Asp at residue 57. The second set responds to the antigen presented only by I-Ag7 and does not recognize the peptides bound to the other class II molecules. The third set is also specific for I-Ag7 as a result of the poor binding of the peptide to I-Ad and the mutant I-Ag7. These results indicate that positions 56 and 57 of the I-Ag7 class II MHC beta chain play a role in both T cell recognition of the MHC-peptide complex and peptide binding to MHC. These two different functions may be involved in I-Ag7-restricted beta cell antigen recognition by diabetogenic T cell clones.     

T cell recognition and tolerance of antibody diversity

The capacity of B cells to self-present their Ab variable regions in the context of class II MHC structures suggests a potential regulatory problem. If T cells were able to recognize self-presented Ab, then T cell help might be delivered to B cells independently of a foreign carrier epitope, resulting in a chronic state of unregulated Ab synthesis. For this reason, we have proposed that T cells normally attain a state of tolerance to Ab V region diversity. Here, we tested this idea by performing direct immunizations with unmutated isologous mAb. We also identified and analyzed epitopes recognized by class II MHC-restricted T cell hybridomas that were originally generated against two physiologically mutated isologous mAb. Our results indicate that the class II MHC-restricted T cell repertoire is tolerant of germ-line-encoded Ab diversity and that the physiologic somatic hypermutation process creates immunogenic epitopes in Ab V regions, in some cases by producing class II MHC-binding peptides. In agreement with these findings, we found that germ-line-encoded Ab V regions are presented by endogenous splenic APC at a level that is physiologically significant.     

The avidity spectrum of T cell receptor interactions accounts for T cell anergy in a double transgenic model

The mechanism of self-tolerance in the CD4(+) T cell compartment was examined in a double transgenic (Tg) model in which T cell receptor (TCR)-alpha/beta Tg mice with specificity for the COOH-terminal peptide of moth cytochrome c in association with I-Ek were crossed with antigen Tg mice. Partial deletion of cytochrome-reactive T cells in the thymus allowed some self-specific CD4(+) T cells to be selected into the peripheral T cell pool. Upon restimulation with peptide in vitro, these cells upregulated interleukin (IL)-2 receptor but showed substantially lower cytokine production and proliferation than cells from TCR Tg controls. Proliferation and cytokine production were restored to control levels by addition of saturating concentrations of IL-2, consistent with the original in vitro definition of T cell anergy. However, the response of double Tg cells to superantigen stimulation in the absence of exogenous IL-2 was indistinguishable from that of TCR Tg controls, indicating that these self-reactive cells were not intrinsically hyporesponsive. Measurement of surface expression of Tg-encoded TCR alpha and beta chains revealed that cells from double Tg mice expressed the same amount of TCR-beta as cells from TCR Tg controls, but only 50% of TCR-alpha, implying expression of more than one alpha chain. Naive CD4(+) T cells expressing both Tg-encoded and endogenous alpha chains also manifested an anergic phenotype upon primary stimulation with cytochrome c in vitro, suggesting that low avidity for antigen can produce an anergic phenotype in naive cells. The carboxyfluorescein diacetate succinimidyl ester cell division profiles in response to titered peptide +/- IL-2 indicated that expression of IL-2 receptor correlated with peptide concentration but not TCR level, whereas IL-2 production was profoundly affected by the twofold decrease in specific TCR expression. Addition of exogenous IL-2 recruited double Tg cells into division, resulting in a pattern of cell division indistinguishable from that of controls. Thus, in this experimental model, cells expressing more than one alpha chain escaped negative selection to a soluble self-protein in the thymus and had an anergic phenotype indistinguishable from that of low avidity naive cells. The data are consistent with the notion that avidity-mediated selection for self-reactivity in the thymus may lead to the appearance of anergy within the peripheral, self-reactive T cell repertoire, without invoking the induction of hyporesponsiveness to TCR-mediated signals.     

The recognition of the nonclassical major histocompatibility complex (MHC) class I molecule, T10, by the gammadelta T cell, G8

Recent studies have shown that many nonclassical major histocompatibility complex (MHC) (class 1b) molecules have distinct antigen-binding capabilities, including the binding of nonpeptide moieties and the binding of peptides that are different from those bound to classical MHC molecules. Here, we show that one of the H-2T region-encoded molecules, T10, when produced in Escherichia coli, can be folded in vitro with beta2-microglobulin (beta2m) to form a stable heterodimer in the absence of peptide or nonpeptide moieties. This heterodimer can be recognized by specific antibodies and is stimulatory to the gammadelta T cell clone, G8. Circular dichroism analysis indicates that T10/beta2m has structural features distinct from those of classical MHC class I molecules. These results suggest a new way for MHC-like molecules to adopt a peptide-free structure and to function in the immune system.     

The Plasmodium falciparum-CD36 interaction is modified by a single amino acid substitution in CD36

CD36 is an 88-kD glycoprotein involved in the cytoadherence of Plasmodium falciparum-parasitized erythrocytes (PE) to endothelial cells. The molecular mechanisms involved in CD36-dependent cytoadherence were examined by expressing three CD36 homologues (human, murine, and rat) in COS-7 cells and observing their PE-binding characteristics over a pH range of 6.0 to 7.4 and following iodination of these receptors. PE binding to human CD36 was pH dependent, with peak binding at pH 6.8 to 7.0, and binding was unaffected by iodination. In contrast, PE adherence to murine and rat CD36 was insensitive to changes in pH, and iodination significantly reduced binding. We further show that the differences observed in the binding phenotype of human and rodent CD36 can be attributed to a single residue. Site-directed mutagenesis of the histidine at position 242 of human CD36 to tyrosine (found in rodent CD36) conferred the binding phenotype of rodent CD36 onto human CD36. Furthermore, substitution of the tyrosine at position 242 of rat CD36 for histidine conferred the binding phenotype of human CD36 onto rat CD36. These findings suggest that residue 242 is part of, or important to the conformation of, the PE-binding domain of CD36.     

Antigen mimicry in autoimmune disease sharing of amino acid residues critical for pathogenic T cell activation

A nonamer peptide from murine nicotinic acetylcholine receptor delta chain (ACR delta), which shared four amino acid residues with a nonamer peptide of murine ovarian zona pellucida glycoprotein ZP3, induced murine autoimmune oophoritis and IgG autoantibody to the zona pellucida. Crossreaction between the ACR delta and ZP3 peptides was established by the response of a ZP3 peptide-specific, oophoritogenic T cell clone to both peptides in association with IA (alpha k beta b). By substituting the ZP3 peptides with a single alanine, four amino acids within the ZP3 peptide were found to be important for ovarian autoimmune disease, autoantibody response, and stimulation of the ZP3-specific T cell clone. Substitution with conservative amino acids of three residues also ablated activity, whereas the fourth, a phenylalanine, was replaceable by tyrosine without loss of activity. Of the four critical amino acids, three were shared between the ZP3 peptide and the ACR delta peptide. Moreover, polyalanine peptides with the four critical ZP3 amino acids or the four amino acids common to the ZP3 and ACR delta peptides induced immune response to ZP3 and elicited severe ovarian autoimmune disease. Thus, organ-specific autoimmune disease can occur through immune response against unrelated self (or foreign) peptides that share with a self-peptide sufficient common amino acid residues critical for activation of pathogenic, autoreactive T cells.     

The primacy of perceiving: Emotion recognition buffers negative effects of emotional labor.

There is ample empirical evidence for negative effects of emotional labor (surface acting and deep acting) on workers' well-being. This study analyzed to what extent workers' ability to recognize others' emotions may buffer these effects. In a 4-week study with 85 nurses and police officers, emotion recognition moderated the relationship between emotional labor and work engagement: Workers with high emotion recognition engaging in emotional labor did not report lower work engagement after 4 weeks, whereas those with low emotion recognition did. These effects pertained to both surface and deep acting. The results suggest that emotional labor be not necessarily detrimental to workers' engagement. Instead, the impact of emotional labor hinges upon workers' ability to correctly identify interaction partners' emotions. (PsycINFO Database Record (c) 2011 APA, all rights reserved)     

Modulation of T cell cytokine profiles and peptide-MHC complex availability in vivo by delivery to scavenger receptors via antigen maleylation

We have previously shown that conversion of proteins to scavenger receptor (SR) ligands by maleylation increases their immunogenicity. We now show that maleyl-Ag-immune spleen cells make relatively more IFN-gamma and less IL-4 or IL-10 than native Ag-immune cells. This is also reflected in the IgG1:IgG2a ratios in Abs generated in vivo. SR engagement on macrophages does not alter their surface levels of the adhesive/costimulatory molecules CD11a/CD18, CD11b/CD18, CD24, CD54, or CD40, nor does it enhance their ability to support anti-CD3-driven proliferation of naive T cells in vitro. Costimulatory molecules implicated in differential Th1/Th2 commitment--CD80, CD86, and IL-12--are not inducible by SR ligation. In addition to macrophages and dendritic cells, B cells also show receptor-mediated uptake and enhanced presentation of maleyl-Ags. Using a monoclonal T cell line to detect peptide-MHC complexes expressed on spleen cells in Ag-injected mice, we find that higher levels of these complexes are generated in vivo from maleyl-proteins and they persist longer than those generated from the native protein. Together, these data suggest that in certain situations, the levels of cognate ligand available and/or the time course of their availability may play a major role in determining the cytokine profiles of the responding T cells in addition to the costimulatory signals implicated so far.     

Polysialic acid and the regulation of cell interactions

Polysialic acid, a unique glycosylation of the neural cell adhesion molecule, is highly regulated in its expression. Its function is manifested in the modulation of cell interactions, probably through its unusual physical properties. Recent advances have clarified the enzymatic mechanism of polysialic acid biosynthesis, expanded its role in cell migration and axon guidance, and suggested that it promotes plasticity in the adult nervous system.     

Critical interactions in binding antibody NC41 to influenza N9 neuraminidase: amino acid contacts on the antibody heavy chain

Antibody NC41 binds to the subtype N9 neuraminidase (NA) of influenza virus A/tern/Australia/ G70c/75 and inhibits its enzyme activity. To address the molecular mechanisms by which antibodies interact with neuraminidase and the requirements for successful escape from antibody inhibition, we made amino acid substitutions in heavy chain CDRs of NC41. Antibody proteins expressed as a single-chain Fv (scFv) fused with maltose-binding protein were assayed for binding to NA by ELISA. Association constants (Ka) for wild-type and mutant scFvs are as follows: wild type, 2 x 10(7) M-1; Asn31-->Gln, 2 x 10(7) M-1; Glu96-->Asp, 1 x 10(7) M-1; Asp97-->Lys, 6 x 10(6) M-1; and Asn98-->Gln, 8 x 10(6) M-1. The Ka for intact NC41 antibody was 4 x 10(8) M-1 in the same assay, reflecting increased stability compared to that of the scFv. Mutations in the scFv antibody had less of an effect on binding than mutations in their partners on the NA, and modeling studies suggest that interactions involving the mutant antibody side chains occur, even without taking increased flexibility into account. Asp97 forms a salt link with NA critical contact Lys434; of the four mutants, D97K shows the largest reduction in binding to NA. Mutant N98Q also shows reduced binding, most likely through the loss of interaction with NA residue Thr401. Substitution N31Q had no effect on Ka. NC41 residue Glu96 interacts with NA critical contact Ser368, yet E96D showed only a 2-fold reduction in binding to NA, apparently because the H bond can still form. Asp97 and Asn98 provide the most important interactions, but some binding is maintained when they are mutated, in contrast to their partners on the NA. The results are consistent with maturation of the immune response, when the protein epitope is fixed while variation in the antibody paratope allows increasing affinity. Influenza viruses may exploit this general mechanism since single amino acid changes in the epitope allow the virus to escape from the antibody.