Yu Zhen Chen

Single amino acid substitutions on a Japanese cedar pollen allergen (Cry j 1)-derived peptide induced alterations in human T cell responses and T cell receptor antagonism

We generated T cell clones specific to a Japanese cedar pollen allergen (Cry j 1) and investigated effects of altered T cell receptor (TCR) ligand on changes of T cell responses. One of these Cry j 1-specific T cell clones established from patients with Japanese cedar pollinosis, ST1.9, recognized an antigenic peptide Cry j 1 p335-346 in the context of HLA-DRA+DRB3*0301 molecules and secreted interleukin-4 dominantly, with a smaller amount of interferon-gamma. ST1.9 represented one of the major T cell clones specific to Cry j 1 in the donor, because a short-term cultured polyclonal T cell line specific to Cry j 1 exhibited the same character as the ST1.9. We synthesized various analog peptides derived from Cry j 1 p335-346 with single amino acid substitutions and determined key residues for interactions between TCR of ST1.9 and HLA-DR molecules. We also analyzed changes in the responses of ST1.9 to Cry j 1 p335-346-derived analog peptides. Of interest was that the substitution of 339threonine to valine resulted in a significant increase in interferon-gamma production, with no remarkable changes either in proliferative response or interleukin-4 production. Analog peptides carrying the substitutions of 339threonine to glycine or glutamine revealed TCR antagonism, without changes in their binding affinities to the DR molecule. Therefore single amino acid substitutions on an allergen peptide carrying the T cell epitope may suppress helper-T-dependent class switch pressure to IgE in B cells either by inducing increased interferon-gamma production or by inhibiting proliferative responses in helper-T cells.

The CLIP-substituted invariant chain efficiently targets an antigenic peptide to HLA class II pathway in L cells

The presentation of antigenic peptides by major histocompatibility complex (MHC) class II to CD4+ T cells is crucial to initiate immune responses. We developed a new system for delivery of an antigenic peptide to the MHC class II pathway, using the invariant chain (Ii). We designed a mutated human p33-form Ii, CLIP-substituted Ii, in which streptococcal M12p55-68 (RDLEQAYNELSGEA) was substituted for CLIP (class II associated invariant chain peptide). We examined the peptide presenting function of this construct, in comparison with the previously reported C-terminal fused Ii, in which a cathepsin cleavage site and M12p54-68 was ligated to the C-terminus of Ii. Mouse L cell transfectants expressing either of these two mutated Ii along with HLA-DR4 could process and present M12p55-68 to the peptide specific and DR4-restricted CD4+ T cell clone. CLIP-substituted Ii was much more efficient in antigen presentation than was the C-terminal fused Ii. Similar to the wild-type Ii, the CLIP-substituted Ii was associated intracellularly with DR4 molecules. These results indicate that the peptide substituted for CLIP of Ii p33 bound to the groove of DR molecules in the same manner as CLIP and it was preferentially presented to the CD4+ T cell clone in the absence of HLA-DM molecules. This system may prove useful for immunotherapy with DNA vaccines or for construction of an antigen presenting cell library with diverse peptides.

An Amiloride-Sensitive and Voltage-Dependent Na+ Channel in an HLA-DR-Restricted Human T Cell Clone

We investigated changes in voltage-gated Na+ currents and effects of extracellular Na+ on proliferation in HLA-DR-restricted human CD4+ αβ T cells after stimulation with a non-self antigenic peptide, M12p54–68. In the absence of antigenic peptide, neither single (n = 80) nor APC-contacted (n = 71) T cells showed voltage-gated inward currents recording with whole-cell patch-clamp techniques, even with Ca2+ and Na+ ions present in the perfusion solution. However, with the same recording conditions, 31% (26 of 84) of APC-contacted T cells stimulated with the antigenic peptide showed voltage-dependent inward currents that were elicited from −60 mV. The inward currents were not inhibited in extracellular Ca2+-free conditions or in the presence of 1 mM NiCl2. However, they were completely inhibited in extracellular Na+-free conditions, which were made by replacing Na+ with iso-osmotic N-methyl-d-glucamine or choline. The Na+ currents were insensitive to tetrodotoxin, a classical blocker of Na+ channels, but were dose-dependently inhibited by amiloride, a potassium-sparing pyrazine diuretic. Furthermore, the Ag-specific proliferative response of T cells was completely inhibited in Na+-free Tyrode’s solution and was suppressed by amiloride in a dose-dependent manner. Our findings suggest that activation of amiloride-sensitive and voltage-gated Na+ channels would be an important step to allow an adequate influx of Na+ and maintain a sustained high Ca2+ level during T cell activation.

Systematic Analysis of the Combinatorial Nature of Epitopes Recognized by TCR Leads to Identification of Mimicry Epitopes for Glutamic Acid Decarboxylase 65-Specific TCRs

Accumulating evidence indicates that recognition by TCRs is far more degenerate than formerly presumed. Cross-recognition of microbial Ags by autoreactive T cells is implicated in the development of autoimmunity, and elucidating the recognition nature of TCRs has great significance for revelation of the disease process. A major drawback of currently used means, including positional scanning synthetic combinatorial peptide libraries, to analyze diversity of epitopes recognized by certain TCRs is that the systematic detection of cross-recognized epitopes considering the combinatorial effect of amino acids within the epitope is difficult. We devised a novel method to resolve this issue and used it to analyze cross-recognition profiles of two glutamic acid decarboxylase 65-autoreactive CD4+ T cell clones, established from type I diabetes patients. We generated a DNA-based randomized epitope library based on the original glutamic acid decarboxylase epitope using class II-associated invariant chain peptide-substituted invariant chains. The epitope library was composed of seven sublibraries, in which three successive residues within the epitope were randomized simultaneously. Analysis of agonistic epitopes indicates that recognition by both TCRs was significantly affected by combinations of amino acids in the antigenic peptide, although the degree of combinatorial effect differed between the two TCRs. Protein database searching based on the TCR recognition profile proved successful in identifying several microbial and self-protein-derived mimicry epitopes. Some of the identified mimicry epitopes were actually produced from recombinant microbial proteins by APCs to stimulate T cell clones. Our data demonstrate the importance of the combinatorial nature of amino acid residues of epitopes in molecular mimicry.

Modulation of calcium responses by altered peptide ligands in a human T cell clone

To determine whether altered peptide ligands (APL) affect calcium signaling events, we investigated changes in intracellular calcium concentration ([Ca2+]i) in human T cell clone stimulated with either the fully agonistic peptide M12p54 – 68, the partially agonistic analogue E63V or the simple antagonistic analogue E58M. Both E63V and E58M stimulated a Ca2+ response in ∼ 40 % of T cells, whereas M12p54 – 68 did so in ∼ 70 % of T cells. The most predominant pattern of a Ca2+ increase induced by M12p54 – 68 was a small sinusoidal peak followed by a sustained high response. The most frequent pattern of calcium response induced by E63V was a continuous high response without a preceding sinusoidal peak, whereas that induced by E58M was large with frequent oscillations. Genistein, an inhibitor of the protein tyrosine kinases (PTK), markedly inhibited the wild‐type peptide‐induced increase in [Ca2+]i, whereas it marginally inhibited the response induced by E63V or E58M. In contrast, GF109203X, a protein kinase C (PKC)‐specific inhibitor, markedly inhibited the E63V‐ or E58M‐induced Ca2+ response, whereas it marginally affected the wild peptide‐induced Ca2+ response. Furthermore, in nominal Ca2+‐free medium, the E58M‐induced Ca2+ response was almost completely blocked, while the M12p54 – 68‐ or E63V‐induced responses were only partially inhibited. Our results suggest that the Ca2+ response induced by the fully agonistic peptide depends on activation of the genistein‐sensitive signaling pathway, including PTK, whereas the Ca2+ response to a simple antagonistic APL completely depends on extracellular Ca2+ and activation of the GF109203X‐sensitive signaling pathway, including PKC. These differences in the CA2+i response in recognition of different APL may parallel the unique T cell activation patterns induced by APL in human T cells.

TCR ligand avidity determines the mode of B-Raf/Raf-1/ERK activation leading to the activation of human CD4+ T cell clone.

The interactions between peptide/MHC complexes and their cognate TCR are essential for various T cell responses. However, the relationship between the avidity of TCR ligand and the subsequent intracellular signaling through the TCR is still unclear. To investigate the effects of TCR ligand avidity on TCR‐mediated signaling, we established L cells expressing HLA‐DR4 molecules covalently linked with agonistic peptide (high‐affinity ligand) or altered peptide ligand (APL; low‐affinity ligand) at various densities as APC for a cognate human CD4+ T cell clone. Using this system, we demonstrated that the T cell clone stimulated with APL/HLA‐DR4 complexes presented at an excessive density provoked the up‐regulation of CD69, IL‐2 production and proliferation, but no detectable phosphorylation of ZAP‐70/LAT/SLP‐76. Furthermore, in contrast to the high‐affinity stimulation, the low‐affinity stimulation evoked delayed and sustained activation of the B‐Raf/extracellular signal‐regulated kinase (ERK) pathway without Raf‐1 activation. The strength and duration of B‐Raf/ERK activations closely correlated with the density of the TCR ligand. A knockdown approach confirmed that B‐Raf activation was indispensable for the APL‐induced T cell responses. These observations suggest that the differences in TCR‐peptide/MHC interactions reflect the strength and duration of B‐Raf/Raf‐1/ERK activation in the human CD4+ T cells.

Peptide-Based Molecular Analyses of HLA class II-Associated Susceptibility to Autoimmune Diseases

Recent advances in knowledge of crystal structures of MHC class II molecules has advanced understanding of the molecular basis for interactions between peptides and HLA class II molecules. Polymorphism of HLA class II molecules influences structures of peptides bound to HLA class II molecules. To better understand mechanisms related to particular HLA class II alleles and autoimmune diseases, it is important to identify self-peptides presented by disease-susceptible HLA class II molecules and triggering disease-causative autoreactive T cells. Autoimmune diseases occur in Caucasians, Blacks and Asians, albeit with a different incidence. In some autoimmune diseases, disease-susceptible HLA class II alleles are closely related but different, and clinical manifestations of diseases differ among ethnic groups. These phenomena strongly suggest that difference in autoimmune self-peptide(s) in the context of disease-susceptible HLA class II molecules may explain the different clinical manifestations of diseases. Therefore, a comparison among disease-susceptible HLA class II alleles, autoimmune self-peptides and clinical manifestations of autoimmune diseases in different ethnic groups would be instructive. We directed efforts to determining: (1) HLA-class II alleles specific to Asian populations and which are associated with susceptibility to autoimmune diseases, (2) binding-peptide motifs for these HLA class II molecules, and (3) self-peptides presented by susceptible HLA class II molecules to stimulate autoreactive T cells related to the development of autoimmune diseases in Asians. In this review, our related recent investigations are described and the uniqueness of HLA class II-associated autoimmune diseases in Asians is given emphasis.

A Single Residue Polymorphism at DRβ37 Affects Recognition of Peptides by T Cells

Single amino acid polymorphism at residue 37 of the HLA-DR beta chain (DR beta 37) between DRB1*0406 and 0403 markedly influences susceptibility to the insulin autoimmune syndrome. We investigated the effects of DR beta 37 polymorphism regarding recognition of nonself peptides by a T-cell clone, YN5-32, specific to a streptococcal peptide (M12p54-68) presented by the DRB1*0406 molecule. YN5-32 responded better to M12p54-68 presented by allogeneic DRB1*0403 with a single Tyr-substitution at DR beta 37-Ser of the DRB1*0406 molecule. One hundred and fifty-four peptides carrying single residue substitutions at each of the core residues 57-65 of M12p54-68, were tested for full agonistic and TCR antagonistic activities. Forty-six peptides showed full agonism, 34 analogues exhibited TCR antagonism, and 45 analogues exhibited neither full agonism nor TCR antagonism, irrespective of the presenting molecules (DRB1*0406 or 0403). On the other hand, 29 analogue peptides substituted at each of residues 57-63 of M12p54-68 were recognized differently by YN5-32, depending on the presenting molecules. These observations indicate that 1) single amino acid polymorphism (Ser-Tyr) at the DR beta 37 residue induced a conformational change distinguished by TCR in some but not all peptides; and 2) these conformational changes were observed even in analogue peptides carrying single residue substitutions at residues far from a putative DR beta 37 contact site. These findings provide further evidence for altered human T-cell responses induced by TCR ligands with minor modifications.

Modulation of intracellular Cl- homeostasis by lectin-stimulation in Jurkat T lymphocytes.

We investigated changes in intracellular Cl(-) concentration ([Cl(-)](i)) during lectin-induced activation and proliferation in human Jurkat T lymphocytes. [Cl(-)](i) was measured using Cl(-) fluorescence dye (N-(6-methoyquinolyl) acetoxy-acetyl-ester, MQAE) methods. Lectins, phytohemagglutinin and concanavalin A, dose-dependently increased [Cl(-)](i) and triggered intracellular Cl(-) oscillation in human Jurkat T lymphocytes. However, some mitochondria metabolism inhibitors, such as m-chlorocarbonylcyanide phenylhydrazone (CCP) and 2,4-dinitrophenol, increased [Cl(-)](i) without triggering any Cl(-) oscillation. Furthermore, both lectins and metabolism inhibitors-induced elevation in [Cl(-)](i) were blocked by removal of extracellular Cl(-) from perfusion solution or by application of anthracene-9-carboxylate, a blocker of Cl(-) channels. Since an extracellular Cl(-)-free condition and application of 9-AC also inhibited PHA-induced proliferation, we suggested that elevation of [Cl(-)](i) via activation of Cl(-) channels and increase in incidence of Cl(-) oscillation would play an important role in modulation of Jurkat T cell activation and proliferation.

Unique T cell proliferation associated with PKCμ activation and impaired ZAP-70 phosphorylation in recognition of overexpressed HLA/partially agonistic peptide complexes

Altered peptide ligands (APL) induce T cell responses different from those induced by the original agonistic peptide. As shown for CD4+ T cells, partial agonists induce partial T cellactivation without proliferation because of lower affinities and higher off rates to TCR than those of agonists. To determine whether overexpression of partially agonistic TCR ligands on antigen‐presenting cells provides high‐avidity TCR ligands, we generated L cell transfectants expressing various numbers of HLA‐DR4 covalently linked with APL derived from a streptococcal peptide and observed responses of the cognate T cells. Some overexpressed HLA‐DR4/partially agonistic APL complexes induced T cell proliferation in a density‐dependent manner. However, tyrosine phosphorylation of zeta‐associated protein‐70 (ZAP‐70) and linker for activation of T cells and kinase activity of ZAP‐70 were not detectable. T cell proliferation stimulated with L cell transfectants was sensitive to thePKC inhibitor Gö6976, but to a lesser extent to Gö6983, suggesting the involvement of μ isotype of PKC (PKCμ). In vitro kinase assays revealed that PKCμ activity was up‐regulated only in T cells stimulated with L cell transfectants that induced T cell proliferation. Our data suggest the presence of a unique signaling pathway coupling TCR ligation with T cell proliferation associated with PKCμ activation and impaired ZAP‐70 activation.