A single amino acid substitution in a self protein is sufficient to trigger autoantibody response.

We determined if a single amino acid substitution in a self protein causes autoantibody responses. Mouse lysozyme (ML) was used as a model self protein, and a mutant ML (F57L ML) was prepared by replacing 57Phe of ML to Leu, an approach which resulted in introducing into ML the immunogenic sequence of peptide 50-61 of hen egg lysozyme (HEL) restricted to I-A(k) MHC class II molecule. We found that F57L ML but not native ML primed HEL specific T cells and triggered ML specific autoantibody responses in B10.A and C3H mice (I-A(k), I-E(k)). Peptide regions, ML 14-69 and ML 98-130, were major epitopes of autoantibodies in both strains of mice. These findings indicate that a single amino acid substitution in self proteins can cause an autoantibody response when the mutated region is presented by MHC class II molecules and recognized by T cells.     

Autoreactive B-cell elimination by pathogenic IgG specific for the same antigen: implications for peripheral tolerance

Harmful pathogenic IgG auto-antibodies are produced against desmoglein 3 (Dsg3) in pemphigus vulgaris, an autoimmune blistering disease. Dsg3 is a cadherin-type cell adhesion molecule expressed in desmosomes of the skin and mucous membranes. In AK7-transgenic mice expressing non-pathogenic AK7 IgM against Dsg3, autoreactive transgenic B cells escape from the deletion or inactivation and exist in the periphery. However, when a pathogenic anti-Dsg3 IgG1 mAb (AK23) capable of inducing blisters was injected into AK7-transgenic mice, AK7 B cells were eliminated from the bone marrow (BM) and spleen only when Dsg3 was expressed in the periphery. In contrast, non-pathogenic IgG mAbs (AK7, AK9) failed to eliminate AK7 B cells. Interestingly, the AK23-mediated elimination of mature AK7 B cells in the spleen was significantly diminished in AK7-transgenic mice on a Rag2(-/-) background while BM B cells were still eliminated, suggesting the presence of T-cell-dependent and -independent mechanisms. T cell transfer studies into AK7-Rag2(-/-) mice revealed that autoreactive B-cell elimination in the periphery requires CD4(+) T cells from wild-type mice but not from gld (FasL mutant) mice. The B-cell elimination was impaired in both BM and periphery when Bcl2 was over-expressed in AK7 B cells. These findings suggest that autoreactive B cells exist unless they are harmful, but once harmful or dangerous events such as tissue destruction are sensed, the mature autoreactive B cells in the periphery are eliminated via a Fas-mediated process in a CD4(+) T cell-dependent manner.     

Induction of autoimmunity by immunization with hapten-modified hen egg lysozyme in hen egg lysozyme-transgenic mice

To understand the mechanism of autoimmunity induction, hen egg lysozyme (HEL)-transgenic (Tg) C57BL/6 (B6) mice were immunized with HEL or phosphorylcholine-conjugated HEL (PC-HEL). Repeated immunization of HEL-Tg mice with native HEL failed to induce the antibody response against HEL. However, immunization with PC-HEL generated a significant anti-HEL antibody response. Immunization of the Tg mice with dominant (HEL(74-88)) or cryptic (HEL(47-61)) T-cell epitope peptide stimulated the corresponding T-cell response and similarly yielded the anti-HEL antibody response. Predominance of immunoglobulin G1 (IgG1) anti-HEL antibody response in the HEL-Tg mice and preferential IL-4 production by HEL-specific T cells suggested the dependency of the antibody response to the presence of T helper 2. HEL-Tg mice received HEL-primed B6 T cells, but not HEL-primed Tg T cells, were able to generate anti-HEL antibody response following PC-HEL immunization. The pattern and the level of epitope peptides generated by splenic antigen-presenting cells indicated that PC-HEL results in much more efficient processing as compared to HEL. These results strongly suggest that the enhancement of antigen processing by hapten (PC) conjugation to the antigen facilitates more efficient stimulation of T cells reactive to self antigen, HEL in HEL-Tg mice resulting in the production of anti-self HEL antibody.     

Anergic B cells constitutively present self antigen: Enhanced immunoglobulin receptor-mediated presentation of antigenic determinants by B cells is hierarchical

Presentation of hen egg lysozyme (HEL) by HEL-specific B cells was studied in transgenic mice expressing anti-HEL immunoglobulin (Ig-transgenic). In T hybridoma assays, presentation of the HEL_46–61 determinant by B cells from Ig-transgenic mice required 10³?10⁴-fold lower concentrations of HEL than were required for presentation by B cells from non-transgenic mice. In contrast, presentation of the HEL determinants 112–129 and 25–43 by HEL-specific B cells was either not significantly enhanced, or enhanced only 10-fold compared with B cells from non-transgenic mice. Enhanced presentation of HEL determinants by B cells from Ig-transgenic donors was specific for HEL, since keyhole limpet hemocyanin or synthetic HEL_46–61 peptide were presented comparably by B cells from Ig-transgenic mice and non-transgenic littermates. A minimum of 1–4% Ig-transgenic B cells was required to detect enhanced presentation of HEL_46–61 in vitro. Constitutive presentation of the HEL_46–61 determinant, but not the HEL_25–43 or HEL_112–129 determinants, was detectable on anergic HEL-specific B cells from double (HEL/Ig)-transgenic mice. In the presence of exogenously added HEL, anergic B cells presented all three HEL determinants. Constitutively presented HEL_46–61 was not due to endogenous synthesis of HEL antigen by anergic B cells from double-transgenic mice, as comparable levels of the HEL_46–61 determinant were constitutively presented by B cells from Ig-Tg → HEL-Tg irradiation bone marrow chimeric mice. Firstly, these results indicate that the enhanced antigen presentation mediated by Ig receptors on B cells is not equivalent for all antigenic determinants. Secondly, the data demonstrate that anergic, autoreactive B cells efficiently process and present nominal antigens in addition to constitutively presenting specific self antigen in vivo.     

Modification of the inhibitory amino acid for epitope peptide binding onto major histocompatibility complex class II molecules enhances immunogenicity of the antigen

Previously, the arginine at hen egg-white lysozyme 61 (HEL 61) was characterized as inhibiting T-lymphocyte stimulation due to the inefficient binding of the arginine-containing epitope peptide to the corresponding major histocompatibility complex class II molecules in C57BL/6 mice. In this study, we produced recombinant HEL, with arginine or alanine at HEL 61, and compared its ability to induce immune responses in mice to see whether modification of an inhibitory amino acid could enhance the immunogenicity of an inefficient antigen. Immunization of the mice with modified HEL induced strong antibody and T-cell immune responses against the native antigen. The enhanced T-cell immune response was due to a more specific elevation of the T-cell responses to the HEL 46-61 epitope region than to other epitope regions, although recognition of the other epitope peptides of HEL was generally increased. Mass spectrometric analyses of the epitope peptides generated by splenic antigen-presenting cells indicated that production of the epitope peptides encompassing HEL 46-61 was efficient using the modified antigen. These results suggest that modification of the critical amino acid residue(s) involved in hampering induction of an efficient immune response is an effective method to improve the immunogenicity of an inefficient antigen.     

Increasing the frequency of T-cell precursors specific for a cryptic epitope of hen-egg lysozyme converts it to an immunodominant epitope

Efforts to understand the mechanisms that govern how immunodominant T-cell epitopes are selected from protein antigens have focused mostly on differences in the efficiency of processing and presentation of peptide/major histocompatibility complex (MHC) complexes by antigen-presenting cells, while little attention has been directed at the role of the T-cell repertoire. In this report, the influence of the T-cell repertoire on immunodominance was investigated using transgenic mice that express the beta chain from a T-cell receptor specific for a cryptic Ek restricted epitope of hen-egg lysozyme, HEL85-96. In these mice, the frequency of HEL85-96-specific T-cell precursors is increased 10-20-fold over non-transgenic mice. Transgenic mice respond as well as non-transgenic controls to intact HEL, even though they respond poorly or not at all to a variety of other antigens, including the dominant H-2k restricted epitopes of HEL. Following immunization with native HEL, the only HEL peptide that could recall a response in vitro in the transgenic mice was HEL85-96. Therefore, this normally cryptic epitope is the sole immunodominant epitope in the transgenic mice, and this alteration in immune response is due solely to an increase in the frequency of specific T-cell precursors. An analysis of four additional H-2k restricted cryptic epitopes of HEL suggests that three are similarly limited by T-cell frequency, and that only one is consistent with a defect in efficient antigen presentation. This indicates that there are at least two different types of cryptic epitopes, one in which crypticity is caused by inefficient processing or presentation, and another in which the frequency of specific T-cell progenitors is limiting.     

mAb against hen egg-white lysozyme regulate its presentation to CD4(+) T cells.

Specific antibodies increase antigen uptake and presentation by antigen-presenting cells via the B cell receptor in B cells or FcgammaR in dendritic cells. To determine whether the interaction between antibody and antigen could influence the set of peptides presented by MHC II molecules, we analyzed the presentation of different CD4(+) T cell epitopes of hen egg-white lysozyme (HEL) after the capture of immune complexes formed between HEL and seven different specific mAb. The 103-117 T cell epitope (I-E(d)) was specifically and selectively up-regulated by the D1.3 and F9.13.7 mAb that binds to proximal loops in the native structure of HEL. Furthermore, Ii-independent T cell epitopes exposed on the HEL surface (116-129 and 34-45, I-A(k) restricted) which require a mild processing involving the recycling of MHC II molecules were selectively up-regulated by mAb that overlap those T cell epitopes (D1.3 and D44.1). However, F10.6.6, somatically derived from the same germ line genes as D44.1 and exhibiting an higher affinity for HEL, was without effect on the presentation of the 34-45 epitope. An Ii-dependent T cell epitope buried into the tertiary structure of HEL (45-61, I-A(k) restricted) and requiring the neosynthesis of MHC II was up-regulated by high-affinity mAb recognizing epitopes located at the N- or C-terminus of the T cell epitope. These results strongly suggest that (i) the spatial relationship linking the T cell epitope and the B cell epitope recognized by the mAb, (ii) the intrinsic processing requirements of the T cell epitope, and (iii) the antibody affinity influences the presentation of a given T cell epitope.     

Cell type-specific processing of the I-Ed-restricted hen egg lysozyme determinant 107–116

Class II major histocompatibility complex heterodimers present to T cells determinants as sets of antigen fragments with ragged N and C termini. It is not yet elucidated whether different types of antigen-presenting cells generate identical sets of peptides containing the same determinant. Taking advantage of recombinant I-E^d molecules produced by insect cells as empty heterodimers, a sensitive T cell stimulation assay was developed to analyze naturally processed hen egg lysozyme (HEL) peptides. I-E^d preparations were isolated from antigen-presenting cells cultured with HEL. Following acid treatment, peptides eluted from I-E^d were chromatographed and the fractions incubated at acidic pH with purified recombinant I-E^d molecules, conditions which favor peptide binding. The stimulatory capacity of the reconstituted peptide-I-E^d complexes adsorbed on the well surface of cell culture plates was then evaluated by measuring interleukin-2 secreted by an HEL 107–116-specific, I-E^d-restricted T cell hybridoma. We found that the B lymphoma A20 and an I-E^d-transfected fibroblast cell line generated distinct sets of peptides containing the HEL sequence 107–116. Our results suggest the possibility that presentation of one determinant by different types of antigen-presenting cells stimulates populations of T cells with distinct fine antigen specificities.     

Structural relationships between carrier epitopes and antigenic epitopes on hen egg-white lysozyme.

The spatial relationship for T-B cell cooperation between antigenic epitopes and carrier epitopes on the antigen molecule was studied. Two mono-DNP substituted derivatives of hen egg-white lysozyme (HEL), DNP1-33HEL and DNP1-96HEL were used as antigens; the former is dinitrophenylated only at lysine-33 and the latter only at lysine-96. Fragment peptides of HEL were used to induce specific T cells to the respective sites of the antigen. Adoptive cell-transfer experiments of site-specific T cells and DNP-primed B cells directly showed that multiple distinct carrier epitopes for T cells could help the antibody responses to the single antigenic epitope for B cells and that a single carrier epitope could help antibody responses to multiple antigenic epitopes. T cells primed with a synthetic peptide SP34-54 (corresponding to sequence 34-54 of HEL) co-operated with DNP-primed B cells on challenge with DNP1-96HEL, but not with DNP1-33HEL.     

Breakpoints in immunoregulation required for Th1 cells to induce diabetes

We describe a novel TCR-transgenic mouse line, TCR7, where MHC class II-restricted, CD4+ T cells are specific for the subdominant H-2b epitope (HEL74-88) of hen egg lysozyme (HEL), and displayed an increased frequency in the thymus and in peripheral lymphoid compartments over that seen in non-transgenic littermate controls. CD4+ T cells responded vigorously to HEL or HEL74-88 epitope presented on APC and could develop into Th1 or Th2 cells under appropriate conditions. Adoptive transfer of TCR7 Ly5.1 T cells into Ly5.2 rat insulin promoter (RIP)-HEL transgenic recipient hosts did not lead to expansion of these cells or result in islet infiltration, although these TCR7 cells could expand upon transfer into mice expressing high levels of HEL in the serum. Islet cell infiltration only occurred when the TCR7 cells had been polarized to either a Th1 or Th2 phenotype prior to transfer, which led to insulitis. Progression from insulitis to autoimmune diabetes only occurred in these recipients when Th1 but not Th2 TCR7 cells were transferred and CTLA-4 signaling was simultaneously blocked. These findings show that regulatory pathways such as CTLA-4 can hold in check already differentiated autoreactive effector Th1 cells, to inhibit the transition from tolerance to autoimmune diabetes.     

Ig-alpha cytoplasmic truncation renders immature B cells more sensitive to antigen contact

To study the function of Ig-alpha in the selection of autoreactive B cells, we have analyzed mb-1 cytoplasmic truncation mutant mice (mb-1delta(c)/delta(c)), which coexpress transgenes encoding hen egg lysozyme (HEL) and HEL-specific immunoglobulin.We demonstrate that in the presence of soluble HEL (sHEL) and dependent on the mb-1delta(c) mutation, most immature B cells bearing the HEL-specific Ig transgene undergo rearrangements of endogenous kappa light chains, resulting in loss of HEL specificity. Moreover, immature B cells from Ig-alpha mutant mice respond to BCR cross-linking with an exaggerated and prolonged calcium response and induction of protein tyrosine phosphorylation. Our data imply a negative signaling role for Ig-alpha in immature B cells.     

Dose-dependent T cell tolerance to an immunodominant self peptide

We have previously described a model of tolerance to self peptides in a mouse transgenic (Tg) line producing secreted hen egg-white lysozyme (HEL). The HEL cDNA was placed under the control of a ubiquitous promoter expressed early in embryogenesis, so that HEL should be present in Tg mice throughout the development of the immune system. Since individual HEL Tg mice express different amounts of serum HEL, we were previously able to show that H-2^d mice with HEL blood level > 10 ng/ml are tolerant to HEL and to the immunodominant (ID) peptide 108–116. However, autoreactive T lymphocytes recognizing the HEL subdominant (SD) peptides 74–96 and 1–18 still persist and the SD-specific responses disappear at higher blood HEL concentrations. In the present work, we have studied HEL Tg H-2^d mice with HEL serum levels < 10 ng/ml (HEL-low Tg animals). We find that 50% of Tg animals with HEL blood concentration < 2 ng/ml are responsive to HEL in T cell proliferation assays, although these responses are lower than those seen in non-Tg control mice. The HEL-specific T lymphocytes react only with 15-mer overlapping peptides encompassing the single H-2^d ID region of HEL (residues 102–122); whereas the 9-mer minimal ID peptide 108–116, which strongly triggers non-Tg T cells, is unable to stimulate auto-reactive T cells in vitro from HEL-low Tg mice. Altogether, our results suggest that T lymphocytes specific for the minimal ID peptide are deleted or inactivated, while T cell clones of lower affinity and reacting with epitopes on longer peptides persist. Thus, the high affinity ID peptide-specific T cell clones can be negatively selected even in the presence of low amounts of HEL.     

The major histocompatibility complex haplotypes dictate and the background genes fine-tune the dominant versus the cryptic response profile of a T-cell determinant within a native antigen: relevance to disease susceptibility and vaccination

The immune system of a healthy individual responds vigorously to foreign microbial antigens. However, all potentially immunogenic regions (determinants) within an antigen are not functionally of equal relevance in mediating host immunity against the pathogen. Moreover, some of these antigenic determinants are well processed and presented (immunodominant), while others are not revealed (cryptic) from the native antigen. Nevertheless, cryptic determinants are good immunogens in the pre-processed peptide form. Defining the factors influencing the dominance versus the crypticity of antigenic determinants is critical to advancing our understanding of the individual variations in host immunity to infection, autoantigens and vaccination. In this study based on a model antigen, hen eggwhite lysozyme (HEL), we describe that the major histocompatibility complex (MHC) haplotypes imprint and the non-MHC genes modify the dominance versus the crypticity of a specific antigenic determinant. Both the H-2(q)- and the H-2(d)-bearing mice raised potent response to native HEL, but responded differently to its determinant region 57-78, which was dominant in the H-2(q) but cryptic in the H-2(d) mice. The H-2(q)- but not the H-2(d)-bearing mice of three different genetic backgrounds yielded patterns of graded reactivity to epitope 57-78 showing the fine-tuning effect of the non-MHC genes. Interestingly, the F1 (H-2(q) x H-2(d)) mice retained the dominant response profile of the H-2(q) parent regardless of the contributing gender, and also responded to a new sub-determinant 61-75. These results highlight the genetic factors influencing the dominance/crypticity of a specific antigenic determinant.     

Modulation of antigen-specific immune responses by the oral administration of a traditional medicine,bo-yang-hwan-o-tang

Bo-yang-hwan-o-tang (BHT) has long been used to treat cancer in traditional Korean medicine and is believed to have immune-modulating activity. This study investigated the effect of BHT on the induction of antigen-specific immune responses using hen egg-white lysozyme (HEL) as a model antigen system. Oral administration of BHT enhanced both HEL-specific humoral and lymphocyte proliferative responses in HEL low-responder mice. Feeding BHT to the mice increased INF-gamma levels, but did not change IL-4 levels. Interestingly, however, the oral BHT feeding significantly increased HEL-specific antibodies of the IgG1, IgG2b, and IgG3 subtypes, which are associated with the direct stimulation of B cells. This indicates that BHT treatment enhances anti-HEL-specific humoral immune responses via the direct stimulation of B lymphocytes rather than by selective priming of specific subtypes of the helper T-cell population. This conclusion was supported by in vitro experiments, in which the presence of BHT significantly augmented B-cell mitogen-mediated proliferation of mouse splenocytes. This augmentation was closely associated with a glycoprotein with a molecular weight of around 100 kDa. The results suggest that BHT modulates antigen-specific immune responses, and might be used as a therapeutic agent for patients who need enhanced immune function.     

In vivo activation of quiescent B cells by anti-immunoglobulin. II. Production of lysozyme-specific monoclonal antibodies of desired isotype

We wished to determine whether injection of mice with anti-isotype antibody would be a means to regulate in vivo isotype expression and to obtain hybridomas secreting monoclonal antibodies (mAbs) of desired antigen specificity and isotype. Treatment with a rat mAb (7D2) reactive with both the IgG2a and IgG2b isotypes of mouse Ig resulted in large increases in the serum concentrations of mouse IgG2(a + b). Moreover, injection of antigen-7D2 conjugates had a profound effect on the isotype distribution of hybridomas subsequently obtained from these animals. Thus, while greater than 95% of anti-hen eggwhite lysozyme (HEL) mAbs prepared from mice immunized with HEL alone were of the IgG1 isotype, 12/15 (80%) of the mAbs from mice injected with HEL-7D2 conjugates were of the IgG2a or IgG2b isotype. When tested for effector functions using HEL-coated erythrocytes, the mAbs showed the expected activities, i.e., the IgG2, but not IgG1 anti-HEL mAbs were able to fix complement, bind protein A, and mediate antibody-dependent cytotoxicity and phagocytosis. These results indicate that in vivo immunization with anti-isotype-antigen conjugates can be used to produce hybridomas of predetermined antigen and isotype specificities.     

Dominant recognition of a cross-reactive B-cell epitope in Mycobacterium leprae 10 K antigen by immunoglobulin G1 antibodies across the disease spectrum in leprosy

Mycobacterium leprae-specific immunoglobulin G1 (IgG1) antibodies in patients with leprosy show a direct correlation with bacterial load (rho=0.748; P<0002) suggesting that IgG1 B-cell responses may be surrogate markers of disease progression. To investigate if this upregulation was a general feature of IgG1 responses to all M. leprae (ML) antigens, we analysed responses to several recombinant purified ML heat-shock proteins (HSP). Three recombinant HSPs (ML10 K, ML 18 K and ML 65 K) were tested for their ability to induce various IgG subclasses in patients with either the lepromatous (LL/BL, n=26) or tuberculoid form (BT/TT, n=39) of the disease as well as in healthy households (HC, n=14) and endemic controls (EC=19). Our major findings were: (1) selective augmentation of IgG1 antibody responses to ML10 K; (2) recognition of a restricted number of epitopes across the disease spectrum and healthy controls by IgG1 antibodies; (3) dominant recognition of cross-reactive epitopes which were common to both ML and MT 10 K. This response was not related to contamination with endotoxin. Epitope mapping using 15-mer overlapping peptides spanning the ML 10 000 MW revealed an immunodominant IgG1 binding peptide (aa41-55) in patients as well as healthy controls. This peptide is a shared epitope with M. tuberculosis 10 K suggesting that postswitched IgG1 B cells recognizing this epitope rather than naive B cells are being expanded.     

Immunoregulation of lysozyme-specific suppression. II. Hen egg-white lysozyme-specific monoclonal suppressor T cell factor suppresses the afferent phase of delayed-type hypersensitivity and induces second-order suppressor T cells

Culture supernatant from a monoclonal T cell lymphoma line (LH8-105) obtained by radiation leukemia virus-induced transformation of hen egg-white lysozyme (HEL)-specific suppressor T lymphocytes is able, when injected into mice, to specifically suppress the delayed-type hypersensitivity (DTH) reaction induced by HEL. The suppressor T cell factor (TsF) exhibits fine antigenic specificity since it suppresses the DTH response induced by HEL without affecting the DTH response induced by ring-necked pheasant egg-white lysozyme (REL), a lysozyme closely related to HEL. Conversely, LH8-105 TsF is able to suppress the DTH response induced by human lysozyme, distantly related to HEL but sharing a common epitope critical for induction of suppressive activity. The fine antigenic specificity of LH8-105 TsF for a restricted epitope on the HEL molecule is confirmed by binding to HEL but not to REL immunosorbents. This TsF also bears I-J determinants, as demonstrated by binding to monoclonal anti-I-J immunosorbents, and it suppresses the afferent but not the efferent phase of the DTH response to HEL. The afferent suppression is controlled by genes apparently mapping in the I-J subregion of the H-2 complex since I-J-incompatible mice are not suppressed by LH8-105 TsF injection. This inducer-type TsF induces second-order effector suppressor T cells only in HEL-primed mice indicating the primary role of antigen, in association with H-2 (I-J) products, in the afferent portion of this suppressive circuit.     

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.     

Immunodominant protein epitopes II. The primary antibody response to hen egg white lysozyme requires and focuses upon a unique N-terminal epitope

The antibody response to a defined protein antigen, hen egg white lysozyme (HEL) has been investigated using an aminopeptidase-treated HEL molecule, des-1,2,3-HEL (AP-HEL). Surprisingly, removal of these three N-terminal residues eliminates an epitope which is a dominant B cell determinant recognized in the primary antibody response to HEL. Thus, the initial antibody response focuses on a very small region of the molecule. Even more striking is the observation that removal of this epitope markedly reduces the immunogenicity of HEL. Therefore, the epitope is not only the focus of the primary antibody response, but is essential for the initiation of the response. This report demonstrates that a selective mechanism must be activated during the response to this protein antigen. Of the multitude of B cell determinants present on HEL, only a limited number are focused upon by the immune system.     

Analysis of lysozyme-specific immune responses by synthetic peptides. I. Characterization of antibody and T cell-mediated responses to the N-terminal peptide of hen egg-white lysozyme

The immunological reactivity against the N-terminal region of hen egg-white lysozyme (HEL) has been investigated by a synthetic peptide (PHEL) comprising residue 1-18 of HEL and by an analogue peptide (PREL) in which phenylalanine at position 3 is substituted by tyrosine. Both peptides are immunogenic in (C57BL/10 X DBA/2)F1 mice genetically responder to HEL. In C57BL/6 mice, genetically nonresponder to HEL, PREL induces anti-peptide antibodies that also bind to PHEL whereas PHEL is not immunogenic. Thus, a single amino acid substitution in a synthetic peptide converts a nonresponder mouse strain into a responder one. Anti-PHEL antibodies demonstrate a higher binding to HEL than anti-PREL antibodies, indicating that phenylalanine at position 3 is important for induction of anti-peptide antibodies able to recognize native HEL. At the T cell level the two peptides show very high bidirectional cross-reactivity between themselves and with HEL for interleukin 2 production, antigen-specific proliferation and delayed-type hypersensitivity response, whereas conservation of phenylalanine at position 3 is required for induction of suppressor cells cross-reactive with HEL. This indicates that the N-terminal region of HEL contains epitope(s) able to induce the same level of helper T cell activity as the native HEL molecule. However, helper T cells do not discriminate between PHEL and PREL whereas phenylalanine at position 3 is critical for HEL-specific suppressor T cell induction.