Cross-reactive antigen recognition by an encephalitogenic T cell receptor. Implications for T cell biology and autoimmunity.

The dominant immune response to rat myelin basic protein in H-2u mice is directed against the acetylated, N-terminal peptide Ac1-11 (AcASQKR-PSQRHG). This peptide causes encephalomyelitis on injection into mice of the H-2u haplotype. Only two residues of the peptide are required for ligation of the TCR from an Ac1-11-specific T cell hybridoma. Proline at position 6 could not be substituted by any other L-amino acid, whereas glutamine at position 3 could be replaced by phenylalanine, histidine, methionine, or tyrosine. Cross-reactive recognition of these residues appears to be specific, because increasing the affinity of each analogue for its MHC restriction element, by replacing lysine with tyrosine at position 4, did not alter the pattern of cross-reactivity. For the majority of substitutions at this position, a lack of stimulation could not be explained by failure to bind to I-Au. However, competition binding studies showed that introduction of proline at position 3 reduced the efficacy of binding to I-Au. Cross-reactive analogues of Ac1-11 were injected into H-2u mice to test the extent to which cross-reactive T cell activation might lead to autoimmune disease in this model. An analogue containing methionine at position 3 caused clinical experimental autoimmune encephalomyelitis in a small percentage of H-2u mice.     

Identification of a naturally processed cytotoxic CD8 T-cell epitope of coxsackievirus B4, presented by HLA-A2.1 and located in the PEVKEK region of the P2C nonstructural protein

The adaptive immune system generates CD8 cytotoxic T lymphocytes (CTLs) as a major component of the protective response against viruses. Knowledge regarding the nature of the peptide sequences presented by HLA class I molecules and recognized by CTLs is thus important for understanding host-pathogen interactions. In this study, we focused on identification of a CTL epitope generated from coxsackievirus B4 (CVB4), a member of the enterovirus group responsible for several inflammatory diseases in humans and often implicated in the triggering and/or acceleration of the autoimmune disease type 1 diabetes. We identified a 9-mer peptide epitope that can be generated from the P2C nonstructural protein of CVB4 (P2C(1137-1145)) and from whole virus by antigen-presenting cells and presented by HLA-A2.1. This epitope is recognized by effector memory (gamma interferon [IFN-gamma]-producing) CD8 T cells in the peripheral blood at a frequency of responders that suggests that it is a major focus of the anti-CVB4 response. Short-term CD8 T-cell lines generated against P2C(1137-1145) are cytotoxic against peptide-loaded target cells. Of particular interest, the epitope lies within a region of viral homology with the diabetes-related autoantigen, glutamic acid decarboxylase-65 (GAD(65)). However, P2C(1137-1145)-specific cytotoxic T lymphocyte (CTL) lines were not activated to produce IFN-gamma by the GAD(65) peptide homologue and did not show cytotoxic activity in the presence of appropriately labeled targets. These results describe the first CD8 T-cell epitope of CVB4 that will prove useful in the study of CVB4-associated disease.     

Structure of a rice β-glucanase gene regulated by ethylene,cytokinin, wounding,salicylic acid and fungal elicitors

A rice -glucanase gene was sequenced and its expression analyzed at the level of mRNA accumulation. This gene (Gns1) is expressed at relatively low levels in germinating seeds, shoots, leaves, panicles and callus, but it is expressed at higher levels in roots. Expression in the roots appears to be constitutive. Shoots expressGns1 at much higher levels when treated with ethylene, cytokinin, salicylic acid, and fungal elicitors derived from the pathogenSclerotium oryzae or from the non-pathogenSaccharomyces cereviseae. Shoots also expressGns1 at higher levels in response to wounding. Expression in the shoots is not significantly affected by auxin, gibberellic acid or abscisic acid. The -glucanase shows 82% amino acid similarity to the barley 1,3;1,4--D-glucanases, and from hybridization studies it is the -glucanase gene in the rice genome closest to the barley 1,3;1,4--glucanase EI gene. The mature peptide has a calculated molecular mass of 32 kDa. The gene has a large 3145 bp intron in the codon for the 25th amino acid of the signal peptide. The gene exhibits a very strong codon bias of 99% G+C in the third position of the codon in the mature peptide coding region, but only 61% G+C in the signal peptide region.Abbreviations ABA abscisic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - EtBr ethidium bromide - GA gibberellic acid - n.p. nucleotide position in gene sequence - PCR polymerase chain reaction - 1×SSPE 150 mM NaCl, 10 mM NaH₂PO₄, 1 mM EDTA pH 7.4     

Structural features of an antigen required for cellular interactions and for T cell activation in a MHC-restricted response

The protein Ag, tobacco mosaic virus protein, (TMVP) and its tryptic peptide number 8 (residues 93-112 of the protein) exhibit cross-reactivity on the T cell level in some strains of mice (e.g., C3H.SW, C57BL/10); these strains are termed cross-reactive (CR). In other strains such as A/J or B10.BR, no cross-reactivity is exhibited; these strains are termed non-cross-reactive (NCR). Genetic experiments indicated that the cross-reactivity is dominant and that it is mapped to the I-A or I-E region of the MHC, with cross-reactivity exhibited by the I-Ab haplotype but not by I-Ak or I-Ek. Cell reconstitution experiments have indicated that the non-cross-reactivity is associated with the inability of the NCR APC to present Ag. Analysis of the area(s) on peptide 8 which serve(s) as epitope revealed that both strains recognize an overlapping area consisting of 11 amino acid residues in the middle of peptide 8 (residues 97-107), which by itself is nonstimulatory to TMVP- or peptide 8-immune T cells of the CR or the NCR strains. However, the addition of a few amino acid residues of the sequence of peptide 8 to this area converts it to a complete stimulatory epitope. Additivity experiments revealed that the CR strain contains two major T cell populations each recognizing this middle region of peptide 8 when elongated by a few amino acids N-terminally and C-terminally, respectively. In contrast, the NCR strain contains one major T cell population recognizing elongation only N-terminally. Because TMVP (but not peptide 8) requires processing before presentation to T cells, it is postulated that, during processing of TMVP, there occur alterations in the area of the proximal three or four N-terminal amino acids of the region consisting of peptide 8, destroying the only region containing the T cell epitope recognized by the NCR strain, hence TMVP and peptide 8 do not exhibit cross-reactivity in this strain. The same alterations of TMVP still leave intact an epitope consisting of amino acid residues C-terminal to the altered area which is recognized by the CR strain, hence the cross-reactivity exhibited by this strain. The results suggest that the difference in cross-reactivity on the T cell level between TMVP and peptide 8 exhibited by the strains may be due to differences in the orientation of presentation and the subsequent cell recognition of an epitope contained within peptide 8.(ABSTRACT TRUNCATED AT 400 WORDS)     

Complete dissection of the Hb(64-76) determinant using T helper 1, T helper 2 clones, and T cell hybridomas.

We have generated cloned Th1 cells, Th2 cells, and T cell hybridomas specific for the single immunogenic peptide from the beta-chain of murine hemoglobin (Hb(64-76)). The availability of these various types of T cells provided us an unique opportunity to examine and dissect the T cell response to an immunogenic peptide. A panel of altered Hb peptides was made by replacing each amino acid in the Hb peptide (positions 64-76) with a conservative amino acid substitution or an alanine. Although none of the eleven T cell clones and hybridomas tested exhibited the same pattern of reactivity to the substituted Hb peptides, some general features were identified for all T cell responses. The primary T cell contact residue of Hb(64-76) was shown to be asparagine 72. For every Hb(64-76) specific T cell, no activation was observed using a peptide containing the conservative substitution of a glutamine for the asparagine at position 72. The flanking glutamic acid at position 73 was also required for a proliferative response for all of the Th1 and Th2 clones. The Th subtypes were not grossly unique in their responses to the substituted Hb peptides, but exhibited minor differences in fine specificity with the Th1 cells identifying more critical amino acids then did the Th2 cells. For the Th1 cells and also the T cell hybridomas, the phenylalanine at position 71 was critical for a T cell response. Analysis of peptide affinity for IEk molecules indicated that position 71 played a role in peptide binding to MHC. Secondary T cell contact residues, which were important for many but not all of the T cells, were identified at positions 69, 70, and 76. Overall T cell responses were minimally affected by changes in the amino acid residues at positions 64-68, 74, and 75. We have also demonstrated that cloned Th1 cells, Th2 cells and T hybridomas can be generated against the same Hb(64-76) determinant.     

CD8 independence and specificity of cytotoxic T lymphocytes restricted by HLA-Aw68.1.

The crystal structure of the HLA-Aw68.1 antigen binding site revealed a negatively charged pocket centred on aspartic acid 74 (Garrett et al. 1989). Access to this '74 pocket' is blocked in HLA-Aw68.2 and HLA-Aw69 by two substitutions at positions 97 and 116. This key feature suggests that the Aw68.1-peptide-specific interactions may involve salt bridges between oppositely charged residues. In this paper, the influenza epitope recognized by virus-specific HLA-Aw68.1-restricted cytotoxic T lymphocytes (CTL) has been defined in vitro with a synthetic peptide corresponding to residues 89-101 of the nucleoprotein (NP). Amino acid substitutions of the peptide NP 89-101 showed that the arginine at position 99 is an anchor point of the peptide within the Aw68.1 antigen binding site. Consistent with this we find that neither HLA-Aw68.2 nor HLA-Aw69 positive cells can present peptide NP 89-101 to Aw68.1-restricted CTL. Our results therefore suggest a model in which presentation of NP 89-101 by HLA-Aw68.1 is dependent upon interaction of the positively charged arginine residue at position 99 of the peptide, with the negatively charged aspartic acid in the '74 pocket' of HLA-Aw68.1. We also show that influenza-virus-specific HLA-Aw68.1-restricted CTL are CD8 independent. This result is consistent with the low affinity of HLA-Aw68.1 for CD8 (Salter et al. 1989) and reveals a unique example of CD8-independent priming of CTL by natural infection with a common pathogen in humans.     

Characterization of a Mycobacterium tuberculosis peptide that is recognized by human CD4+ and CD8+ T cells in the context of multiple HLA alleles

The secreted Mycobacterium tuberculosis 10-kDa culture filtrate protein (CFP)10 is a potent T cell Ag that is recognized by a high percentage of persons infected with M. tuberculosis. We determined the molecular basis for this widespread recognition by identifying and characterizing a 15-mer peptide, CFP10(71-85), that elicited IFN-gamma production and CTL activity by both CD4(+) and CD8(+) T cells from persons expressing multiple MHC class II and class I molecules, respectively. CFP10(71-85) contained at least two epitopes, one of 10 aa (peptide T1) and another of 9 aa (peptide T6). T1 was recognized by CD4(+) cells in the context of DRB1*04, DR5*0101, and DQB1*03, and by CD8(+) cells of A2(+) donors. T6 elicited responses by CD4(+) cells in the context of DRB1*04 and DQB1*03, and by CD8(+) cells of B35(+) donors. Deleting a single amino acid from the amino or carboxy terminus of either peptide markedly reduced IFN-gamma production, suggesting that they are minimal epitopes for both CD4(+) and CD8(+) cells. As far as we are aware, these are the shortest microbial peptides that have been found to elicit responses by both T cell subpopulations. The capacity of CFP10(71-85) to stimulate IFN-gamma production and CTL activity by CD4(+) and CD8(+) cells from persons expressing a spectrum of MHC molecules suggests that this peptide is an excellent candidate for inclusion in a subunit antituberculosis vaccine.     

Trypanosoma cruzi: cellular and antibody response against the parasite in mice immunized with a 19-amino acid synthetic peptide

Several monoclonal antibodies were prepared against the flagellar fraction of Trypanosoma cruzi epimastigotes (Tulahuén strain, stock Tul 2). One of them, FCH-F8-4, has previously shown biologic activity against the parasite (complement-mediated lysis and neutralization of the trypomastigote infectivity). Immunopurified antigens using this monoclonal antibody elicited a protective immune response in mice. Two recombinant cDNA clones were detected with this anti-flagellar fraction monoclonal antibody on a lambda gt11 expression library prepared from T. cruzi epimastigote mRNA. The insert of one of these cDNA clones, lambda(FCH-F8-4)1 (150 bp) coded for a 19-amino acid peptide (PAFLGCSSRFSGSFSGVEP). This insert hybridized with a 5.0-kb mRNA from epimastigotes. The beta-galactosidase fusion protein was produced in lysogenic bacteria. The monoclonal antibody recognized the epitope present in the fusion protein after western blotting of the crude lysate. A synthetic peptide (SP4) containing the complete sequence of lambda(FCH-F8-4)1 was constructed on solid phase. This peptide was able to inhibit the ELISA reactivity (in a range from 13 to 52%) of flagellar fraction immunized mouse sera and when administered (coupled to KLH or alone) to BALB/c mice with Bordetella pertussis as adjuvant, it induced a humoral and cellular immune response which was detected by ELISA, immunofluorescence, blotting, and DTH reactions against T. cruzi antigens. The immune response obtained indicates that this synthetic peptide resembles the parasite antigen conformation and could be useful for diagnosis purposes or be able to elicit immunoprotection against T. cruzi infection.     

Correlation between low-temperature immunosuppression and the absence of unsaturated fatty acid synthesis in murine T cells.

1. Studies were performed to determine if the inability of murine T cells to provide primary helper function at low temperature (27 degrees C) could be correlated with their inability to synthesize unsaturated fatty acids (UFAs). 2. In the absence of exogenous oleic acid (18:1), splenocytes responded to a T-dependent (TD) Ag (trinitrophenyl-keyhole limpet hemocyanin, TNP-KLH) at 37 degrees C but not at 27 degrees C. The addition of 150 microM 18:1 almost completely restored plaque-forming cell (PFC) responses to TNP-KLH at 27 degrees C but markedly suppressed PFC responses to the TD Ag at 37 degrees C. 3. During incubation at 27 degrees C, B cells converted 3- to 5-fold more stearic acid (18:0) to 18:1 and showed a greater accumulation of monounsaturated phospholipid molecular species than did T cells. 4. Following incubation in the presence of a rescuing dose of 18:1 (150 microM), both B and T cells accumulated large amounts of dioleoyl PC. 5. It is proposed that the absence of 18:1 synthesis in T cells is responsible for the unique low temperature susceptibility of this lymphocyte population.     

Complete amino acid sequence of the A chain of human complement-classical-pathway enzyme C1r.

The amino acid sequence of human C1r A chain was determined, from sequence analysis performed on fragments obtained from C1r autolytic cleavage, cleavage of methionyl bonds, tryptic cleavages at arginine and lysine residues, and cleavages by staphylococcal proteinase. The polypeptide chain has an N-terminal serine residue and contains 446 amino acid residues (Mr 51,200). The sequence data allow chemical characterization of fragments alpha (positions 1-211), beta (positions 212-279) and gamma (positions 280-446) yielded from C1r autolytic cleavage, and identification of the two major cleavage sites generating these fragments. Position 150 of C1r A chain is occupied by a modified amino acid residue that, upon acid hydrolysis, yields erythro-beta-hydroxyaspartic acid, and that is located in a sequence homologous to the beta-hydroxyaspartic acid-containing regions of Factor IX, Factor X, protein C and protein Z. Sequence comparison reveals internal homology between two segments (positions 10-78 and 186-257). Two carbohydrate moieties are attached to the polypeptide chain, both via asparagine residues at positions 108 and 204. Combined with the previously determined sequence of C1r B chain [Arlaud & Gagnon (1983) Biochemistry 22, 1758-1764], these data give the complete sequence of human C1r.     

Structure-antiviral activity relationships of cecropin A-magainin 2 hybrid peptide and its analogues.

In order to elucidate the structure-antiviral activity relationship of cecropin A (1-8)-magainin 2 (1-12) (termed CA-MA) hybrid peptide, several analogues with amino acid substitutions were synthesized. In a previous study, it was shown that serine at position 16 in CA-MA hybrid peptide was very important for antimicrobial activity. Analogues were designed to increase the hydrophobic property by substituting a hydrophobic amino acid residue (S --> A, V, F or W, position 16) in the CA-MA hybrid peptide. In this study, the structure-antiviral activity relationships of CA-MA and its analogues were investigated. In particular, substitution of Ser with a hydrophobic amino acid, Val, Phe or Trp at position 16 caused a dramatic increase in the virus-cell fusion inhibitory activity. These results suggested that the hydrophobicity at position 16 in the hydrophobic region of CA-MA is important for potent antiviral activity.     

Post-translational amino acid isomerization: a functionally important D-amino acid in an excitatory peptide

The post-translational modification of an L- to a D-amino acid has been documented in relatively few gene products, mostly in small peptides under 10 amino acids in length. In this report, we demonstrate that a 46-amino acid polypeptide toxin has one D-phenylalanine at position 44, and that the epimerization from an L-Phe to a D-Phe has a dramatic effect on the excitatory effects of the peptide. In one electrophysiological assay carried out, the D-Phe-containing peptide was extremely potent, whereas the unmodified polypeptide had no biological activity, demonstrating that the chirality of the post-translationally modified amino acid is functionally significant. The peptide toxin analyzed, r11a, belongs to the I-gene superfamily of conotoxins that has four disulfide cross-links. The D-Phe in r11a is at the third amino acid from the C terminus, the same relative position from the C-terminal end as the d-amino acid in omega-agatoxin TK from a spider, an unrelated peptide. Thus, although post-translational amino acid isomerization appears to have no strong specificity for the chemical nature of the amino acid side chain, the few peptides where this modification has been established suggest that there may be favored positions near the N or C terminus that are preferential sites for isomerization to a D-amino acid.     

Single amino acid replacements in an antigenic peptide are sufficient to alter the TCR V beta repertoire of the responding CD8+ cytotoxic lymphocyte population.

Cytotoxic CD8+ T lymphocytes are activated upon the engagement of their Ag-specific receptors by MHC class I molecules loaded with peptides 8-11 amino acids long. T cell responses triggered by certain antigenic peptides are restricted to a limited number of TCR V beta elements. The precise role of the peptide in causing this restricted TCR V beta expansion in vivo remains unclear. To address this issue, we immunized C57BL/6 mice with the immunodominant peptide of the vesicular stomatitis virus (VSV) and several peptide variants carrying single substitutions at TCR-contact residues. We observed the expansion of a limited set of TCR V beta elements responding to each peptide variant. To focus our analysis solely on the TCR beta-chain, we created a transgenic mouse expressing exclusively the TCR alpha-chain from a VSV peptide-specific CD8+ T cell clone. These mice showed an even more restricted TCR V beta usage consequent to peptide immunization. However, in both C57BL/6 and TCR alpha transgenic mice, single amino acid replacements in TCR-contact residues of the VSV peptide could alter the TCR V beta usage of the responding CD8+ T lymphocytes. These results provide in vivo evidence for an interaction between the antigenic peptide and the germline-encoded complementarity-determining region-beta loops that can influence the selection of the responding TCR repertoire. Furthermore, only replacements at residues near the C terminus of the peptide were able to alter the TCR V beta usage, which is consistent with the notion that the TCR beta-chain interacts in vivo preferentially with this region of the MHC/peptide complex.     

Epitope Mapping and Identification of Amino Acids Critical for Rabbits IgG-Binding to Linear Epitopes on Buffalo beta-lactoglobulin

Buffalo milk safety was highlighted with the increase in dietary consumption, and a little information is available on buffalo milk allergy except for cross-reactivity between buffalo and cow milk. In this work, linear epitopes and critical amino acids of buffalo β-lactoglobulin were defined by 4 rabbit's sera using SPOTTM peptide arrays approach based on the defined mimotopes. The eight epitopes on buffalo β-lactoglobulin were located in the position of A6(21-30), A7(25-34), A8 (29-38), B4 (73-82), B5(77-86), C(87-96), F4(134-143) and F8(150-159), respectively. Among them, four epitopes (A7, A8, F4 and F8) were described as the most major epitopes and peptide (A6, B4, B5 and C) as the second major epitopes. Following single AA substitutions (Alanine or Glycine) at each position of the major epitopes, 2,3,2,3,5 and 3 of critical amino acids were identified on epitopes of A6, A8, B5, C , F4 and F8, respectively, which vary in distribution among the epitopes, such as in C terminal or N terminal and in continuous or discontinuous forms, characteristics including hydrophobicity, polar and charge, and existed frequency.     

Synthesis and hybridization analysis of a small library of peptide-oligonucleotide conjugates.

A small library of 49 peptide-oligonucleotide conjugates were synthesized to explore the influence of various peptide side chains on the hybridization properties of the DNA. An invariant 8mer oligonucleotide was coupled to a peptide portion that contained a five residue variable region composed of the cationic amino acids lysine, ornithine, histidine and arginine, the hydrophobic amino acid tryptophan, and alanine as a spacer. Melting temperature analysis indicated that T m depended principally on the number of cationic residues. The free energies of binding for polycationic peptide-oligonucleotides were enhanced compared with the unmodified 8mer. The origin of this stabilizing effect was found to be derived from a more exothermic enthalpic term. Improvement in Delta G vH was found to depend on the presence of positive charge and also the exact identity of the cationic amino acid, with the polyarginine peptide giving the most favourable Delta G vH value and the most exothermic Delta H vH. Further exploration suggested that the cationic peptide fragments interacted mainly with single-stranded rather than duplex DNA. A study of pH dependence showed that the polyhistidine conjugate was particularly sensitive to pH changes near neutrality, as indicated by a significant rise in T m from 19.5 degrees C at pH 8.0 to 28.5 degrees C at pH 6.0.     

Altered -3 substrate specificity of Escherichia coli signal peptidase 1 mutants as revealed by screening a combinatorial peptide library

Signal peptidase functions to cleave signal peptides from preproteins at the cell membrane. It has a substrate specificity for small uncharged residues at -1 (P1) and aliphatic residues at the -3 (P3) position. Previously, we have reported that certain alterations of the Ile-144 and Ile-86 residues in Escherichia coli signal peptidase I (SPase) can change the specificity such that signal peptidase is able to cleave pro-OmpA nuclease A in vitro after phenylalanine or asparagine residues at the -1 position (Karla, A., Lively, M. O., Paetzel, M. and Dalbey, R. (2005) J. Biol. Chem. 280, 6731-6741). In this study, screening of a fluorescence resonance energy transfer-based peptide library revealed that the I144A, I144C, and I144C/I86T SPase mutants have a more relaxed substrate specificity at the -3 position, in comparison to the wild-type SPase. The double mutant tolerated arginine, glutamine, and tyrosine residues at the -3 position of the substrate. The altered specificity of the I144C/I86T mutant was confirmed by in vivo processing of pre-beta-lactamase containing non-canonical arginine and glutamine residues at the -3 position. This work establishes Ile-144 and Ile-86 as key P3 substrate specificity determinants for signal peptidase I and demonstrates the power of the fluorescence resonance energy transfer-based peptide library approach in defining the substrate specificity of proteases.     

Peptide-specific CD8 T regulatory cells use IFN-gamma to elaborate TGF-beta-based suppression

We identified a murine peptide-specific CD8 T regulatory cell population able to suppress responding CD4 T cells. Immunization with OVA, poly(I:C), and anti-4-1BB generated a population of SIINFEKL-specific CD8 T regulatory cells that profoundly inhibited peptide-responding CD4 T cells from cellular division. The mechanism of suppression required IFN-gamma, but IFN-gamma alone was not sufficient to suppress the responding CD4 T cells. The data show that CD8 T regulatory cells were unable to suppress unless they engaged IFN-gamma. Furthermore, even in the absence of recall with peptide, the CD8 T regulatory cells suppressed CD4 responses as long as IFN-gamma was present. To examine the effector mechanism of suppression, we showed that neutralizing TGF-beta inhibited suppression because inclusion of anti-TGF-beta rescued the proliferative capacity of the responding cells. TGF-beta-based suppression was dependent completely upon the CD8 T regulatory cells being capable of binding IFN-gamma. This was the case, although peptide recall of primed IFN-gamma (-/-) or IFN-gammaR(-/-) CD8 T cells up-regulated pro-TGF-beta protein as measured by surface latency-associated peptide expression but yet were unable to suppress. Finally, we asked whether the CD8 T regulatory cells were exposed to active TGF-beta in vivo and showed that only wild-type CD8 T regulatory cells expressed the TGF-beta-dependent biomarker CD103, suggesting that latency-associated peptide expression is not always congruent with elaboration of active TGF-beta. These data define a novel mechanism whereby IFN-gamma directly stimulates CD8 T regulatory cells to elaborate TGF-beta-based suppression. Ultimately, this mechanism may permit regulation of pathogenic Th1 responses by CD8 T regulatory cells.     

Autoactivation of Mouse Trypsinogens Is Regulated by Chymotrypsin C via Cleavage of the Autolysis Loop

Chymotrypsin C (CTRC) is a proteolytic regulator of trypsinogen autoactivation in humans. CTRC cleavage of the trypsinogen activation peptide stimulates autoactivation, whereas cleavage of the calcium binding loop promotes trypsinogen degradation. Trypsinogen mutations that alter these regulatory cleavages lead to increased intrapancreatic trypsinogen activation and cause hereditary pancreatitis. The aim of this study was to characterize the regulation of autoactivation of mouse trypsinogens by mouse Ctrc. We found that the mouse pancreas expresses four trypsinogen isoforms to high levels, T7, T8, T9, and T20. Only the T7 activation peptide was cleaved by mouse Ctrc, causing negligible stimulation of autoactivation. Surprisingly, mouse Ctrc poorly cleaved the calcium binding loop in all mouse trypsinogens. In contrast, mouse Ctrc readily cleaved the Phe-150–Gly-151 peptide bond in the autolysis loop of T8 and T9 and inhibited autoactivation. Mouse chymotrypsin B also cleaved the same peptide bond but was 7-fold slower. T7 was less sensitive to chymotryptic regulation, which involved slow cleavage of the Leu-149–Ser-150 peptide bond in the autolysis loop. Modeling indicated steric proximity of the autolysis loop and the activation peptide in trypsinogen, suggesting the cleaved autolysis loop may directly interfere with activation. We conclude that autoactivation of mouse trypsinogens is under the control of mouse Ctrc with some notable differences from the human situation. Thus, cleavage of the trypsinogen activation peptide or the calcium binding loop by Ctrc is unimportant. Instead, inhibition of autoactivation via cleavage of the autolysis loop is the dominant mechanism that can mitigate intrapancreatic trypsinogen activation.     

Fine specificity of CD4+ T cell responses to the dominant encephalitogenic PLP 139-151 peptide in SJL/J mice

PLP 139-151(S) is the major encephalitogenic epitope of PLP in the SJL/J mouse. CD4⁺ T cells specific for PLP 139-151(S) induce a relapsing-remitting form of EAE which is similar to the human demyelinating disease MS in both clinical course and histopathology. We are interested in events involved in activation of autoreactive T cells and how to specifically regulate these immune response to both prevent and treat ongoing demyelinating disease. In the current study, we examined the effect of both amino acid substitutions and deletions in the native PLP 139-151(S) peptide to identify which residues are critical for immunogenicity and encephalitogenicity. Conservative and nonconservative substitutions at position 145 diminished or completely destroyed the encephalitogenic potential of the peptide without affecting the ability to recall a proliferative response in lymph node T cells primed with the native PLP 139-151(S) peptide indicating an interesting dichotomy between ability to induce T cell proliferation and ability to induce active clinical disease. In addition, tryptophan at position 144 was identified as a critical TCR contact site as a peptide containing an alanine for tryptophan at this position (A144) primed a unique population of T cells which did not cross react with the native PLP 139-151(S). In addition, A144 was unable to stimulate PLP 139-151(S)-specific T cells in vitro or to induce active relapsing EAE in vivo. The significance of these results to the potential development of new strategies for preventing and treating T cell-mediated autoimmune diseases is discussed.Special issue dedicated to Dr. Majorie B. Lees.