Hen Egg Lysozyme Epitope Research Articles

Epitope-dependent thermodynamic signature of single-domain antibodies against hen egg lysozyme.

A substantial body of work has been carried out describing the structural features of the complex between single-domain antibodies (VHHs) and antigens, and the preeminence for epitopes located at concave surfaces of the antigen. However, the thermodynamic basis of binding is far less clear. Here, we have analysed the energetic profiles of five VHHs binding to the catalytic cleft or to a noncleft epitope of hen egg lysozyme. Various binding energetic profiles with distinctive enthalpic/entropic contributions and structural distribution of critical residues were found in the five antibodies analysed. Collectively, we suggest that from an energetic point of view the binding mechanism is influenced by the shape of the epitope. This information may be beneficial for the design of tailored epitopes for VHHs and their practical use.

Functional Requirements for the Lysosomal Thiol Reductase GILT in MHC Class II-Restricted Antigen Processing

Ag processing and presentation via MHC class II is essential for activation of CD4(+) T lymphocytes. gamma-IFN-inducible lysosomal thiol reductase (GILT) is present in the MHC class II loading compartment and has been shown to facilitate class II Ag processing and recall responses to Ags containing disulfide bonds such as hen egg lysozyme (HEL). Reduction of proteins within the MHC class II loading compartment is hypothesized to expose residues for class II binding and protease trimming. In vitro analysis has shown that the active site of GILT involves Cys(46) and Cys(49), present in a CXXC motif that shares similarity with the thioredoxin family. To define the functional requirements for GILT in MHC class II Ag processing, a GILT-deficient murine B cell lymphoma line was generated and stably transduced with wild-type and cysteine mutants of GILT. Intracellular flow cytometric, immunoblotting, and immunofluorescence analyses demonstrated that wild-type and mutant GILT were expressed and maintained lysosomal localization. Transduction with wild-type GILT reconstituted MHC class II processing of a GILT-dependent HEL epitope. Mutation of either Cys(46) or Cys(49) abrogated MHC class II processing of a GILT-dependent HEL epitope. In addition, biochemical analysis of these mutants suggested that the active site facilitates processing of precursor GILT to the mature form. Precursor forms of GILT-bearing mutations in Cys(200) or Cys(211), previously found to display thiol reductase activity in vitro, could not mediate Ag processing. These studies demonstrate that the thiol reductase activity of GILT is its essential function in MHC class II-restricted Ag processing.

SENSITIVITY TO BLEOMYCIN-INDUCED LUNG INJURY IS NOT MODERATED BY AN ANTIGEN-LIMITED T-CELL REPERTOIRE

Pulmonary fibrosis is a progressive scarring disease of the lung. It has been suggested that fibrosis is an inflammatory process, and cytokines such as tumor necrosis factor (TNF)-α and transforming growth factor (TGF)-β have been shown to play key roles in the pathogenesis of fibrotic lung disease. However, the source of these cytokines remains in question and there is controversy over the role that infiltrating inflammatory cells play in fibrosis. T cells could play a key role by releasing cytokines upon engaging autoantigens revealed as a result of necrosis or apoptosis following epithelial injury. Some studies have shown that disrupting T-cell function leads to more severe disease, whereas others have shown that T-cell deficiency protects against fibrotic injury. To investigate whether specific antigen engagement by T cells is required for the development of fibrosis, bleomycin was instilled into the lungs of mice expressing a transgenic T-cell receptor β (TCRβ) gene. Expression of the TCRβ transgene prevents effective recognition of antigens other than a single epitope of hen egg lysozyme. These mice therefore have defective antigen-specific responses but a normal representation of mature T-cell subsets. If antigen-specific T-cell engagement is required for the development of lung fibrosis, bleomycin-induced fibrosis should be reduced in the TCRβ transgenic mice. In fact, there is no difference in the inflammatory or fibrotic response to bleomycin between TCRβ transgenic and control mice. Thus, if T cells are required for fibrogenesis, it must involve an antigen-independent mechanism.

B-cell repertoire specific for an unfolded self-determinant of mouse lysozyme escape tolerance and dominantly participate in the autoantibody response.

We previously found that autoantibodies against mouse lysozyme (ML) were strongly induced in normal BALB/c mice when immunized with mutant ML that has triple mutations rendering the dominant T-cell epitope of hen egg lysozyme (HEL), HEL 107-116. As T cells specific for HEL 107-116 were primed in these mice, the anti-ML immunoglobulin G (IgG) responses would be the result of collaborations between autoreactive B cells specific for ML and T cells specific for HEL 107-116. Serum IgG responses against ML were dominantly focused on the ML 14-69 region, indicating that B cells responding to the epitope escape tolerance. In the present study, we prepared several monoclonal antibodies (mAbs) specific for ML 14-69 and examined their antigen specificities in detail, to characterize the nature of the remaining B-cell repertoire specific for ML. mAbs specific for ML 14-69 interacted weakly with soluble, native ML, but the interactions were strengthened by denaturation of ML. The apparent affinity constants between these mAbs and ML showed an increase, ranging from six- to 80-fold, by denaturation of ML. Therefore, these mAbs were more specific for the denatured determinant than for the determinant in the native structure. These results indicate that a substantial number of autoreactive B cells, specific for the unfolded conformation of ML, escape tolerance and are dominantly involved in the autoantibody response to ML. Our finding provides important information to understand the naturally occurring autoreactive B-cell repertoire in normal mice.

Proteolytic Sensitivity and Helper T-cell Epitope Immunodominance Associated with the Mobile Loop in Hsp10s

Antigen three-dimensional structure potentially limits antigen processing and presentation to helper T-cell epitopes. The association of helper T-cell epitopes with the mobile loop in Hsp10s from mycobacteria and bacteriophage T4 suggests that the mobile loop facilitates proteolytic processing and presentation of adjacent sequences. Sites of initial proteolytic cleavage were mapped in divergent Hsp10s after treatment with a variety of proteases including cathepsin S. Each protease preferentially cleaved the Hsp10s in the mobile loop. Flexibility in the 22-residue mobile loop most probably allows it to conform to protease active sites. Three variants of the bacteriophage T4 Hsp10 were constructed with deletions in the mobile loop to test the hypothesis that shorter loops would be less sensitive to proteolysis. The two largest deletions effectively inhibited proteolysis by several proteases. Circular dichroism spectra and chemical cross-linking of the deletion variants indicate that the secondary and quaternary structures of the variants are native-like, and all three variants were more thermostable than the wild-type Hsp10. Local structural flexibility appears to be a general requirement for proteolytic sensitivity, and thus, it could be an important factor in antigen processing and helper T-cell epitope immunogenicity.

C-terminal extension of the MHC class II-associated invariant chain by an antigenic sequence triggers activation of naive T cells.

In vitro and in vivo activation of T cells was investigated with invariant chain-antigen fusion protein. The CD4 T cell epitope amino acid 52-61 of hen egg lysozyme (HEL) was attached to the C-terminal end of invariant chain (Ii). Expression of this recombinant Ii HEL directs the T cell epitope to the class II processing pathway. Class II molecules of transfected antigen presenting cells (APC) are charged with this HEL epitope. The endogenously provided epitope competes with processing and presentation of exogenously added antigen. APC expressing recombinant Ii HEL stimulate a maximal IL-2 response of HEL-specific T hybridoma cells. Nonprofessional APC expressing recombinant Ii HEL and H2-Ak are also able to activate naive T cells from 3A9 TCR transgenic mice, a result not achieved with peptide pulsed APC. To elicit an in vivo immune response dendritic cells (DC) were transfected with rIi HEL cDNA: following immunization of CBA mice with transfected DC, a primary T cell response against the HEL epitope was induced. Thus the procedure described here could be used to introduce antigens into the class II processing pathway and to elicit T cell activation both in vitro and in vivo.

T Cell Receptor Recognition of MHC Class II–Bound Peptide Flanking Residues Enhances Immunogenicity and Results in Altered TCR V Region Usage

Naturally processed MHC class II–bound peptides possess ragged NH 2 and COOH termini. It is not known whether these peptide flanking residues (PFRs), which lie outside the MHC anchor residues, are recognized by the TCR or influence immunogenicity. Here we analyzed T cell responses to the COOH-terminal PFR of the H-2A k immunodominant epitope of hen egg lysozyme (HEL) 52–61. Surprisingly, the majority of T cells were completely dependent on, and specific for, the COOH-terminal PFR of the immunogen. In addition, there were striking correlations between TCR Vβ usage and PFR dependence. We hypothesize that the Vα CDR1 region recognizes NH 2-terminal PFRs, while the Vβ CDR1 region recognizes COOH-terminal PFRs. Last, peptides containing PFRs were considerably more immunogenic and mediated a greater recall response to the HEL protein. These results demonstrate that PFRs, which are a unique characteristic of peptides bound to MHC class II molecules, can have a profound effect on TCR recognition and T cell function. These data may have important implications for peptide-based immunotherapy and vaccine development.

In a vaccine model, selected substitution of a highly stimulatory T cell epitope of hen's egg lysozyme into a Salmonella flagellin does not result in a homologous, specific, cellular immune response and may alter the way in which the total antigen is processed

A 13 amino acid peptide corresponding to a potent BALB c mouse T cell epitope of hen's egg lysozyme (HEL) was substituted singly at five sites in the d flagellin of Salmonella muenchen. The resulting chimeric proteins were unable to expand T cells capable of being stimulated by the HEL epitope and induced T cell populations which either failed to respond or responded at a low level to a normally highly stimulatory flagellin T cell epitope that was present in all chimeras. The results suggested that substitution of a T cell epitope in flagellin may alter the processing of the resulting immunogen.

Distinct effects of recombinant cholera toxin B subunit and holotoxin on different stages of class II MHC antigen processing and presentation by macrophages.

Cholera toxin (CT) is a potent mucosal adjuvant with enhancing effects on Ag presentation, although the mechanisms of its adjuvanticity remain poorly understood. Using an in vitro Ag presentation assay, we found CT and recombinant B subunit (rCTB) to have distinct effects on different stages of processing and class II MHC (MHC-II)-restricted presentation of hen egg lysozyme (HEL). CT treatment of macrophages resulted in enhanced presentation of soluble HEL(48-61) peptide to3A9 hybridoma cells. However, CT had inhibitory effects on intracellular processing of soluble native Ag. Thus, CT inhibited presentation when added prior to HEL, whereas presentation was enhanced when CT was added after HEL exposure and the generation of peptide-MHC-II complexes. Pretreatment of macrophages with CT also markedly inhibited phagocytic processing of a Crl-HEL fusion protein (containing the HEL(48-61) epitope) expressed in intact bacteria (Escherichia coli HB101.Crl-HEL or Salmonella typhimurium 14028s.Crl-HEL), whereas addition of CT to macrophages after a 2-h incubation with the bacteria again enhanced presentation. CT produced little effect on overall uptake and catabolism of radiolabeled HEL or HB101.Crl-HEL. In contrast to the holotoxin, purified rCTB subunit did not inhibit intracellular processing of soluble or bacterial Ag, although it similarly enhanced the presentation of surface HEL-(48-61)-I-Ak complexes to 3A9 cells. These data suggest that the inhibitory effects of CT on Ag processing are mediated by the A subunit.

T cell epitope recognition involved in the low-responsiveness to a region of hen egg lysozyme (46–61) in C57BL/6 mice

The predominant T cell epitope of hen egg lysozyme (HEL) in high-responder C3H mice has been previously identified as the HEL 46–61 region. In contrast, this region is poorly recognized by T cells from low-responder C57BL/6 mice upon immunization with HEL. In previous studies, we have demonstrated that several C57BL/6 derived T cell hybridomas reactive to this epitope and other HEL epitopes preferentially recognize phosphorylcholine (PC)-conjugated HEL over unconjugated HEL. To understand the mechanisms involved in this difference of T cell recognition, we have further analysed the reactivity of T cells and T cell hybridomas from low-responder C57BL/6 mice. T cells from HEL-immunized mice were preferentially reactive to HEL 47–60. These results suggest a potential deficiency in generating an appropriate T cell epitope from the 46–61 region of native HEL in low-responder C57BL/6 mice. The minimal T cell epitope of this region was defined as HEL 51–60 using the PCH4.1 T hybridoma clone. This minimal epitope represents a single amino acid shift from the minimal epitope of HEL high-responder C3H mice (HEL 52–61). Various peptides representing this region were synthesized with single alanine substitutions at each position. The residues at positions 51, 52, 53 and 57 of HEL appear to be involved in Ia binding and the residues at 55 and 56 in contacting the TCR. T cell reactivity to HEL 51–61 peptides with various substitutios at position 61 strongly suggest that primarily the size of the C-terminal residue interferes with binding to the Ia molecules of low-responder mice. In addition, substitutions of the TCR contacting residues at positions 55 and 56 with similar residues (isoleucine → leucine or leucine → isoleucine) significantly increased the T cell reactivity, suggesting a low reactivity with the native residues. Therefore, the requirement of many residues in the T cell epitope for interaction with Ia, the necessity for additional Ag processing to facilitate Ia binding, and the low affinity of the TCR contacting residues may together render C57BL/6 mice unresponsive to the HEL 46–61 region.

Threshold detection of self-antigen/MHC class II complexes formed in vivo: constitutive presentation of an immunodominant epitope of hen egg lysozyme (HEL) in HEL-transgenic mice.

Hen egg lysozyme (HEL)-specific T hybridomas were used to study the presentation of HEL as a self component in tolerant, HEL-transgenic mice expressing different serum levels of HEL (AL3, 3-7 nM: ML5, 1-3 nM). Constitutive presentation of the HEL46-61 determinant was detectable in antigen presenting cell (APC)-enriched preparations of spleen, thymus, and lymph node from AL3 transgenic mice. The most likely cell responsible for constitutive presentation of HEL antigen was a non-B cell. In addition, constitutive presentation of HEL46-61 was clearly evident in thymic rosettes (complexes of macrophages or dendritic cells and lymphoid cells) derived from AL3 HEL-transgenic mice, but not from ML5 HEL-transgenic mice. In contrast to the HEL46-61 determinant, constitutive presentation of the HEL25-43 and HEL112-129 determinants was not detectable on APC-enriched preparations from either of the HEL-transgenic lines tested. Thus, although T cell tolerance was evident in transgenic mice expressing a range of serum HEL concentrations, constitutive presentation of processed HEL antigen was not detectable on APCs from mice expressing less than 3-7 nM. This threshold of detectable antigen presentation is consistent with the notion that the concentration of nominal antigen needed for tolerizing T cells is less than that required for activation of mature T cells.

Recombinant Escherichia coli express a defined, cytoplasmic epitope that is efficiently processed in macrophage phagolysosomes for class II MHC presentation to T lymphocytes.

Although the processing of soluble Ag for presentation to T cells has been extensively studied in vitro, similar studies of phagocytic Ag processing have been limited. We have developed an in vitro model system to study the ability of macrophages to process recombinant Escherichia coli strain HB101 with cytoplasmic or surface expression of the well characterized T cell epitope of hen egg lysozyme (HEL) 52-61. This epitope was expressed within full length HEL or within a fusion protein containing the HEL epitope. Phagocytosis of E. coli with cytoplasmic expression of HEL or the HEL fusion protein resulted in strong presentation of HEL(52-61) to T cells. Surface-conjugated HEL was processed with even greater efficiency. Processing required viable macrophages, was inhibited by cytochalasin D, and was achieved within 20 min of bacterial contact with the macrophages. Within this time span, phagosomes containing bacteria fused with lysosomes, and the bacteria were extensively degraded. Uptake of as few as four bacteria per macrophage produced an Ag-specific T cell response. We conclude that bacterial compartmentalization of the antigenic epitope (cytoplasmic vs surface) had some effect on its processing, but that phagocytic Ag processing organelles contain extensive capacity to degrade internalized bacteria and liberate intracellular Ag epitopes for recycling and presentation, consistent with a central role for phagolysosomes. Thus, future recombinant bacterial vaccines may be effectively designed with T cell epitopes expressed either on the surface or within the bacterial cytoplasm.

Constraints in antigen processing result in unresponsiveness to a T cell epitope of hen egg lysozyme in C57BL/6 mice.

T cell hybridoma clones were derived after fusion of BW-5147 parent cells with lymphocytes from C57BL/6 mice injected with phosphorylcholine (PC)-hen egg lysozyme (HEL) conjugates. Several T cell hybridomas were preferentially reactive with PC-HEL over unconjugated HEL, and a particular clone (PC-H4.1) was further analyzed. This T cell hybridoma clone could respond to its maximal level toward unconjugated HEL only when the dose of HEL was increased to 5-10-fold of the PC-HEL concentration. Interestingly, this clone was not stimulated by unfolded HEL (by S-carboxymethylation) to the level of PC-HEL. A synthetic peptide representing the amino acid position 47-61 of HEL, which is known to be non-immunogenic upon HEL injection in C57BL/6 mice, was able to stimulate the hybridoma only to a level comparable to that induced by unconjugated HEL. The T cell response to this synthetic peptide required an additional antigen-processing step, based on its inability to stimulate T cells after treatment of antigen-presenting cells with leupeptin, chloroquine or paraformaldehyde. Deletion of a single C-terminal amino acid residue of HEL 47-61 (arginine) significantly enhanced (10-100-fold of HEL 47-61) the T cell reactivity and abrogated the necessity of further antigen processing. These results suggest that the lack of a T cell response to a certain epitope may not be due to the lack of a T cell repertoire reactive to the epitope. In some cases, the unresponsiveness may be due to the difficulty in generating the particular epitopes. Taken together, modification of the lysozyme molecule with PC conjugation may facilitate further antigen processing of HEL to generate an optimal epitope for the nonresponder mice.

Immunization with antigen bound to bispecific antibody induces antibody that is restricted in epitope specificity and contains antiidiotype.

Mice can be efficiently immunized in the absence of adjuvant using chemically cross-linked bispecific antibody (biAb) that bind to both class II MHC molecules and a protein Ag of interest. In our experiments, mice were immunized with the protein Ag hen egg lysozyme (HEL) bound to several different biAb, each of which contained a different mAb specific for a distinct (nonoverlapping) epitope of HEL. Primary and secondary serum antibody responses of the immunized mice were analyzed for their specificity for different epitopes of HEL. The results show that immunization with each HEL-biAb complex produced a bias in the epitope specificity of the primary antibody response. This bias was determined by the individual specificity of the anti-HEL mAb used in each biAb. The primary response was dominated by antibody reacting with epitopes distinct from that bound by the mAb in the immunizing complex, and was deficient in antibody that recognized the epitope bound by the biAb during immunization. This bias in antibody specificity was maintained during the secondary antibody response that followed a single challenge with soluble HEL alone. However, an additional challenge with HEL induced a switch in the specificity pattern, with increased amounts of antibody against the epitope that was previously ignored. In addition, immunization with Ag bound to biAb resulted in a substantial primary anti-Id response, detected by serum antibody specific only for the Fab'2 fragment of the mAb used in the biAb. These studies illustrate two unique features of immunization using biAb that allow for fine manipulation of the epitope specificity and anti-Id repertoires of the antibody response to whole protein Ag.

Class II-restricted presentation of an endogenously derived immunodominant T-cell determinant of hen egg lysozyme.

An in vitro model was used to investigate the potential for different structural forms of endogenous antigen to be processed and presented by major histocompatibility complex class II molecules. For this purpose the class II-restricted presentation of an immunodominant epitope of hen egg lysozyme [HEL-(46-61)] was studied in class II-positive B-lymphoma cells (M12.C3) transfected with genes encoding HEL molecules either (i) secreted in high (hi) or low (lo) amounts as soluble antigen [sHEL(hi/lo)], (ii) localized within the endoplasmic reticulum (ER)/salvage compartment (ER-HEL), or (iii) anchored on the cell surface as an integral membrane protein (mHEL). The corresponding sHEL, ER-HEL, and mHEL gene products were expressed as predicted except that HEL determinants accumulated in the culture supernatant as well as on the cell membrane of mHEL-transfected cells. Class II-positive cells endogenously expressing all three forms of HEL antigen constitutively presented the immunodominant HEL-(46-61) determinant with differential efficiency (mHEL, sHEL greater than ERHEL) to a class II-restricted T hybridoma. A second T hybridoma recognized endogenous HEL-(46-61) determinants constitutively presented on sHEL(hi) and mHEL transfectants but not on sHEL(lo) or ERHEL transfectants. The formation of HEL-(46-61)/I-Ak complexes in the ERHEL and sHEL(lo) transfectants was therefore limiting. Mixing experiments with different antigen-presenting cells indicated that the HEL-(46-61) determinant was derived from endogenous antigen rather than by reuptake of shed or secreted HEL determinants. We conclude that MHC class II molecules can present some antigenic determinants derived from endogenous proteins that are sequestered in the ER/salvage compartment as well as distally transported in the form of secretory or membrane antigens.