Hen Egg-white Lysozyme Peptides Research Articles

Autophagy in antigen-presenting cells results in presentation of citrullinated peptides to CD4 T cells

Antibody responses to citrullinated self-proteins are found in autoimmunities, particularly in rheumatoid arthritis, where they serve as a diagnostic indicator. We show here that processing of the protein hen egg-white lysozyme (HEL) resulted in citrullination of peptides presented on class II MHC molecules by antigen-presenting cells. The presentation of the citrullinated peptides but not of the unmodified peptides was associated with autophagy. Dendritic cells (DCs), macrophages, and thymic DCs presented citrullinated peptides constitutively. Their treatment with 3-methyladenine (3MA) blocked presentation of citrullinated HEL peptides, but presentation of unmodified peptides was not affected. Presentation of citrullinated peptides was not detected on B cells or B lymphoma cells under normal culture conditions. In B cells, engagement of the B cell antigen receptor was required for presentation of the citrullinated peptides, also inhibited by 3MA. B lymphoma-expressing HEL cells presented citrullinated peptides only after brief serum starvation. This presentation was reduced by 3MA or by reduction in Atg5 expression. Presentation of the unmodified peptides was not changed. The findings indicate a linkage between autophagy and autoreactivity through the generation of this neo-epitope.

Presentation of citrullinated peptides by class II histocompatibility molecules is associated with autophagy. (100.19)

Abstract Autoimmune responses to citrullinated proteins are associated with rheumatoid arthritis; however, little is known of the mechanism of citrullination by the antigen presenting cells (APC) of the immune system. Using T cell hybridomas as probes, we found constitutive presentation by class II-Major Histocompatibility Complex (MHC) molecules of a dominant citrullinated peptide from hen egg-white lysozyme (HEL) by either macrophages or dendritic cells. Treatment of APC with 3-Methyladenine (3MA) blocked presentation of the citrullinated peptides after processing unmodified HEL, but presentation of unmodified peptides was not at all affected. In contrast presentation of citrullinated peptides by splenic B cells or B lymphoma cells was not detected under normal culture conditions. However, B cell lines presented citrullinated peptides after serum starvation in a process affected by 3MA treatment or by reducing the expression of the autophagy protein Atg5. Higher levels of citrulline in naturally processed peptides from serum starved B cell lines were detected biochemically. Primary B cells presented citrullinated HEL peptides after engagement of their B cell receptor by antigen or by using anti-immunoglobulin antibodies. Our findings demonstrate a role for autophagy in citrullination of antigen during processing by APC and indicate a link in B cells between engagement of their antigen receptor and presentation of citrullinated peptides.

Dendritic Cells Process and Present Antigens Across A Range of Maturation States

We isolated dendritic cells (DC) from lymphoid organs of mice bearing a transgene for a membrane-bound form of the model protein hen egg white lysozyme (HEL). DC from the spleen had a lower representation of costimulatory molecules and class II MHC molecules than those isolated from lymph nodes and thymi. Splenic DC were capable of further maturation by in vivo treatment of mice with LPS. The immature DC from spleen processed HEL and displayed the chemically dominant epitope as evidenced by FACS analysis. These immature DC also presented this epitope to CD4(+) T cells. Splenic DC from another transgenic mouse (ML-5) containing serum HEL also showed the ability to process and present Ag despite low levels of circulating HEL. In vitro-derived DC from the bone marrow (bone marrow-derived DC) of mHEL mice also displayed immature to mature features and in both cases displayed HEL peptides as well as SDS-stable MHC class II molecules. Immature bone marrow-derived DC also processed exogenous HEL. We conclude that the DC sets normally found in tissue show a scale of maturation features but even the most immature process and present peptides by MHC class II molecules.

Chemical Identification of a Low Abundance Lysozyme Peptide Family Bound to I-Ak Histocompatibility Molecules

The processing by antigen-presenting cells (APC) of the protein hen egg-white lysozyme (HEL) results in the selection of a number of peptide families by the class II major histocompatibility complex (MHC) molecule, I-A(k). Some of these families are expressed in very small amounts, in the order of a few picomoles/10(9) APC. We detected these peptides from an extract of class II MHC molecules by using monoclonal anti-peptide antibodies to capture the MHC-bound peptides prior to their examination by HPLC tandem mass spectrometry. Here, we have identified several members of a family of peptides encompassing residues 20-35, which represent less than 1% of the total HEL peptides. Binding analysis indicated that the core segment of the family was represented by residues 24-32 (SLGNWVCAA). Asn-27 (shown in boldface) is the main MHC-binding residue, mapped as interacting with the P4 pocket of the I-A(k) molecule. Analysis of several T cell hybridomas indicated that three residues contacted the T cell receptor: Tyr-23 (P-1), Leu-25 (P3), and Trp-28 (P5). The HEL peptides isolated from the APC extract were sulfated on Tyr-23, but further analysis showed that this modification did not occur physiologically but took place during the peptide isolation.

Distinct recognition by two subsets of T cells of an MHC class II-peptide complex.

We examine here the nature of the differential recognition by CD4+ T cells of a single peptide from hen-egg white lysozyme (HEL) presented by I-A(k) class II MHC molecules. Two subsets of T cells (called A and B) interact with the same peptide, each in unique ways that reflect the nature of the complex of peptide and MHC. We show that the A and B set of T cells can be distinguished by their functional interaction with the three T cell receptor (TCR) contact residues of the bound peptide. The dominant peptide of HEL selected from processing is bound in a single register where a critical TCR contact residue is situated about the middle of the core segment of the peptide: all T cells establish functional contact with it. Three sets of T cells, however, can be distinguished by their differential recognition of two TCR contacts situated at the amino and carboxyl sides of the central TCR contact residue. Type A T cells, the conventional cells that see the peptide after processing of HEL, need to recognize all three TCR contact residues. In contrast, the type B T cells recognize the peptide given exogenously, but not when processed: these T cells recognize either one of the peripheral TCR contact residues, indicating a much more flexible interaction of peptide with I-A(k) molecules. We discuss the mode of generation of the various T cells and their biological relevance.

Quantitative Analysis of the T Cell Repertoire that Escapes Negative Selection

Mice expressing hen egg-white lysozyme (HEL) as a transgene are unresponsive to immunization with the HEL protein. Profound tolerance was found even in situations where the amounts of I-A k-peptide complexes was 100 or less per APC. Among the few T cells that escaped tolerance, we did not observe differential responses to the different HEL epitopes, perhaps because of the very high sensitivity of the negative selection process. The same HEL transgenic mice that did not respond to HEL responded to immunization with the 46–61 peptide of HEL. These peptide-specific T cells that escaped negative selection belonged to a set that reacted with a particular conformer of the HEL peptide-I-A k (type B). The presence of type B reactive T cells should be considered in autoimmunity.

Endogenous and exogenous forms of the same antigen are processed from different pools to bind MHC class II molecules in endocytic compartments.

The current studies were carried out to examine the basis for the differences in the antigenic peptides generated from exogenous and endogenous forms of hen egg white lysozyme (HEL). The role of different intracellular compartments in the generation and binding of HEL peptides derived from two endogenous forms of HEL, either secreted (sHEL) or retained in the endoplasmic reticulum (ER, KDELHEL), presented by MHC class II molecules was examined and compared to exogenous HEL. Initially it was found that antigen-presenting cells bearing both intracellular forms of HEL generated and presented a number of IAk-restricted HEL epitopes to T cell hybridomas, although sHEL was processed more efficiently than KDEL-HEL. There were differences, however, for some determinants between endogenous and exogenous HEL. At equivalent antigen-presenting efficiencies, endogenous HEL-bearing cells displayed a lower surface density of IAk-bound HEL-52-61-related peptides than cells pulsed with exogenous HEL, as detected by a specific monoclonal antibody. Neither endogenous HEL degradation nor peptide binding to MHC class II molecules occurred in the ER. Processing of sHEL and KDELHEL appears to take place either in a post-trans-Golgi network acidic compartment or in the cytosol, whereas peptide binding to MHC class II molecules occurs in endocytic compartments. Furthermore, the peptides generated were derived from an endogenous source rather than from secreted and re-endocytosed HEL. Thus, processing of endogenous HEL is from a different pool than exogenous HEL and occurs in different compartments.

Endogenous and exogenous forms of the same antigen are processed from different pools to bind MHC class II molecules in endocytic compartments

The current studies were carried out to examine the basis for the differences in the antigenic peptides generated from exogenous and endogenous forms of hen egg white lysozyme (HEL). The role of different intracellular compartments in the generation and binding of HEL peptides derived from two endogenous forms of HEL, either secreted (sHEL) or retained in the endoplasmic reticulum (ER, KDELHEL), presented by MHC class II molecules was examined and compared to exogenous HEL. Initially it was found that antigen-presenting cells bearing both intracellular forms of HEL generated and presented a number of IAk-restricted HEL epitopes to T cell hybridomas, although s HEL was processed more efficiently than KDELHEL. There were differences, however, for some determinants between endogeneous and exogenous HEL. At equivalent antigen-presenting efficiencies, endogenous HEL-bearing cells displayed a lower surface density of IAk-bound HEL-52-61-related peptides than cells pulsed with exogenous HEL, as detected by a specific monoclonal antibody. Neither endogenous HEL degradation nor peptide binding to MHC class II molecules occurred in the ER. Processing of sHEL and KDELHEL appears to take place either in a post-trans-Golgi network acidic compartment or in the cytosol, whereas peptide binding to MHC class II molecules occurs in endocytic compartments . Furthermore, the peptides generated were derived from an endogenous source rather than from secreted and re-endocytosed HEL. Thus, processing of endogenous HEL is from a different pool than exogenous HEL and occurs in different compartments.

Isolation and Quantitation of a Minor Determinant of Hen Egg White Lysozyme Bound to I-Ak by Using Peptide-Specific Immunoaffinity

We report here the identification and quantitation of a minor epitope from hen egg white lysozyme (HEL) isolated from the class II MHC molecule I-Ak of APCs. We isolated and concentrated the peptides from the I-Ak extracts by a peptide-specific mAba, followed by their examination by electrospray mass spectrometry. This initial step improved the isolation, recovery, and quantitation and allowed us to identify 13 different minor peptides using the Ab specific for the HEL tryptic fragment 34-45. The HEL peptides varied on both the amino and carboxy termini. The shortest peptide was a 13-mer (residues 33-45), and the longest peptide was a 19-mer (residues 31-49). The two most abundant were 31-47 (1.3 pmol) and 31-46 (1 pmol), while the least abundant were 31-45 (40 fmol) and 32-45 (4 fmol). Only 0.3% of the total class II molecules were occupied by this family of HEL peptides. The amount of the 31-47 peptide, the predominant member of this series, was 22 times lower than that of 48-62, the major epitope of HEL. The 31-47 peptide bound about 20-fold weaker to I-Ak compared with the dominant 48-62 peptide. Thus, the lower abundance of the minor epitope correlated with its weaker binding strength.

Antigen receptor engagement delivers a stop signal to migrating T lymphocytes.

We investigated the role of the T cell antigen receptor (TcR) in control of T cell migration in an in vitro system. We used T cells from transgenic mice bearing a TcR for the lysozyme peptide 48-62 bound to I-A(k) (3A9). T cells from the 3A9 TcR transgenic mice crawled on purified intercellular adhesion molecule-1 substrates, but strikingly, stopped upon interaction with the physiological ligand, i.e., the mouse I-A(k) with covalently attached hen egg white lysozyme peptide residues 48-62 complex. TcR-triggered stopping was reversible by treatment with adhesion-strengthening phorbol esters. The microtubule organizing center of stopped cells was positioned adjacent to the site of stable cell anchorage. Direct conversion of lymphocyte function associated-1 to the high-affinity conformation with antibodies also stopped T cells in a similar manner to antigen. Thus, physiological TcR engagement triggers a stop signal through lymphocyte function associated-1. We propose that the stop signal is an early and essential event in T cell activation that also will play an important role in control of T cell migration.

Induction of antigen-presenting capacity in tumor cells upon infection with non-replicating recombinant vaccinia virus encoding murine MHC class II and costimulatory molecules

The possibility of inducing antigen-presenting capacity in cells normally lacking such capacity, currently represents a major goal in vaccine research. To address this issue we attempted to generate ‘artificial’ APC able to stimulate CD4 + T cell responses when tumor cells were infected with a single, recombinant, vaccinia virus (rVV) containing the two genes encoding murine MHC class II I-A k and a third gene encoding the murine B7-1 (mB7-1) costimulatory molecule. To minimize the cytopathic effect and to improve safety, in view of possible in vivo applications, we made this rVV replication incompetent by Psoralen and long wave UV treatment. Tumor cells infected with rVV encoding I-A k alone, pulsed with hen egg white lysozyme peptide (HEL 46–61), induced IL-2 secretion by an antigen-specific T hybridoma. Tumor cells infected with the rVV encoding mB7-1 provided costimulation for activating resting CD4 + T cells in the presence of ConA. Tumor cells infected with the rVV encoding I-A k and mB7-1, and pulsed with chicken ovotransferrin peptide (conalbumin 133–145), induced a significantly higher response in a specific Th 2 cell clone (D10.G4.1) as compared to cells infected with rVV encoding I-A k molecules only. Thus, this replication incompetent rVV represents a safe, multiple gene, vector system able to confer in one single infection step effective APC capacity to non-professional APCs.

DR alpha: E beta heterodimers in DRA transgenic mice hinder expression of E alpha: E beta molecules and are more efficient in antigen presentation.

HLA-DRA transgenic (tg) mice on H-2d background were constructed to study assembly, expression and function of DR alpha: E beta class II heterodimers when an alternate E alpha chain is available. Cytofluorimetric analysis and immunoprecipitation studies demonstrate that the majority (90%) of E beta d molecules on class II-positive splenocytes from DRA-tg mice are associated with DR alpha rather than E alpha chains. To characterize the functional role of the interspecies as compared with the wild-type I-E molecules, MHC restriction and T cell epitope immunodominance of synthetic peptides spanning the entire sequence of 65 kDa heat shock protein (hsp) from Mycobacterium tuberculosis were determined in hsp-primed DRA-tg and DBA/2 mice. A similar pattern of responsiveness was observed in both strains, but hsp epitopes recalled a higher response in DRA-tg as compared with DBA/2 mice. A panel of T cell hybridomas specific for two hsp peptides or a hen egg white lysozyme peptide presented by both DR alpha: E beta d and E alpha d: E beta d was studied in detail. Surprisingly, DR alpha: E beta d dimers present these peptides more efficiently than E alpha d: E beta d, even when the TCR was selected in mice expressing only E alpha d: E beta d molecules. The higher efficiency of antigen presentation by DR alpha: E beta d dimers does not appear to depend on increased binding affinity for peptides, as demonstrated by competition for antigen presentation, nor on increased efficiency in the interaction with CD4 molecules. Rather, the higher efficiency of antigen presentation could be explained by a more effective ligand-TCR interaction. This is consistent with molecular modeling based on the class II structure, indicating that 16 out of 17 substitutions between the first domain of E alpha d and DR alpha chains ile outside the peptide binding groove and are potentially available for interaction with the TCR.

Cathepsin D, but not cathepsin B, releases T cell stimulatory fragments from lysozyme that are functional in the context of multiple murine class II MHC molecules

In this study, the major endosomal/lysosomal proteases cathepsin D and cathepsin B were tested on their ability to release T cell stimulatory peptides from hen egg white lysozyme (HEL) in vitro. Whereas neither enzyme could cleave unreduced HEL under mild conditions, reduced HEL was readily cleaved by cathepsin D but not by cathepsin B. Instead, cathepsin B was found to be very active in the trimming of HEL peptides after their release by cathepsin D. Following high-performance liquid chromatography (HPLC) fractionation, cathepsin D-released HEL fragments were screened for recognition by HEL-specific T cells from three strains of mice, i.e. B10.A (H-2a), C57BL/6 (H-2b) and BALB/c (H-2d). Peptides in a large number of different HPLC fractions triggered significant T cell responses in all three strains. Interestingly, the response profiles of T cells from the three different strains showed marked similarities. Also, several individual synthetic HEL sequences corresponding to selected cathepsin D-released fragments were recognized by murine T cells in the context of all three major histocompatibility complex (MHC) haplotypes tested. Our data suggest that cathepsin D rather than cathepsin B may play a central role in the initial release of HEL fragments during endosomal/lysosomal processing. The relatively long HEL fragments released by cathepsin D, containing about 20-30 amino acid residues, are significantly more promiscuous in murine class II MHC binding than the shorter synthetic HEL sequences previously employed by others for the delineation of HEL epitopes. Extensive documentation of HEL epitopes in previous investigations indicate that this promiscuity cannot be explained by simply assuming that longer peptides contain additional epitopes. Rather, an increased peptide length by itself appears to promote promiscuous MHC binding.

Public and private V beta T cell receptor repertoires against hen egg white lysozyme (HEL) in nontransgenic versus HEL transgenic mice.

We have previously produced a transgenic mouse line for hen egg lysozyme (HEL), an experimental model for analyzing tolerance to self-antigens at the peptide level. We have now characterized transgenic mice with HEL blood levels below 2 ng/ml, where significant T cell proliferative responses to HEL and its immunodominant peptide were observed. This HEL-low transgenic model was chosen because it mimics physiological conditions in which autoreactive T lymphocytes, recognizing self-components expressed at very low levels, persist without inducing a break in tolerance. Furthermore, in H-2d mice, HEL-specific T lymphocytes are triggered by a single immunodominant region, allowing us to compare the HEL-specific T cell V beta repertoires of transgenic and nontransgenic animals against a single peptide presented as self or foreign, respectively. We found that a V beta 8.2-D beta 1-J beta 1.5 rearrangement is found in response to HEL in all nontransgenic mice, whereas this V beta-restricted response is absent in HEL-low transgenic animals. At the nucleotide level, this rearrangement results from the trimming of the genomic segments during VDJ or DJ joining, without N additions, suggesting that the dominant rearrangement is selected early during fetal or neonatal life, before the expression of terminal deoxynucleotidyl transferase. In HEL-low transgenic mice, no dominant rearrangements are found as alternatives to the one observed in normal mice. Instead, each transgenic animal uses a different set of V beta-J beta combinations in its response to the immunodominant HEL peptide. In nontransgenic mice, besides the dominant V beta 8.2-D beta 1-J beta 1.5 combination, minor V beta repertoires were found which differed in each animal and were distinct from the rearrangements used by individual transgenic mice. These findings suggest that the T cell response to an immunodominant peptide involves a "public" V beta repertoire found in all animals and a "private" one which is specific to each individual.

Sympathetic Regulation of T-Helper Cell Function

The role of sympathetic neurotransmission in regulation of T-cell function was examined in mice. After in vivo priming with a model protein antigen, hen egg white lysozyme (HEL), the specific proliferative recall response of lymph node cells (LNCs) was examined. The release of endogenous catecholamines by amphetamine (5 mg/kg, 45 min prior to sacrifice) markedly inhibited the proliferative response of LNCs. Combined alpha- and beta-adrenergic blockade (phentolamine, 1.0 mg/kg + propranolol, 2.5 mg/kg) prevented this effect of amphetamine on proliferation. In vitro, the alpha-adrenergic agonist phenylephrine, but not the beta-agonist isoproterenol, inhibited proliferation in a dose-dependent manner, up to 80%. The effect of phenylephrine was blocked by the alpha-adrenoceptor antagonist phentolamine. The effects of catecholamines appear to be mediated at the level of antigen processing/presentation: Amphetamine given prior to sacrifice inhibited interleukin 2 production when irradiated spleen cells were used to present HEL and HEL-related peptides to HEL-specific T-cell hybridomas. In summary, the sympathetic nervous system seems to inhibit antigen processing/presentation and, indirectly, T-helper responses.

Identification of two distinct properties of class II major histocompatibility complex-associated peptides.

We have examined the interactions of various peptides with the mouse class II major histocompatibility complex molecule I-Ak. The peptides were derived from the model protein hen egg white lysozyme (HEL). The immunodominant peptide of HEL is a 10-mer, residues 52-61. Our previous work established that this sequence contains the key residues for binding and presentation to T cells. Now we show that the binding of this 10-mer sequence resulted in complexes of I-Ak and peptide that, in SDS/PAGE (without boiling the protein), rapidly dissociated from the component alpha and beta chains. The binding interactions were studied in vitro, by incubating purified I-Ak and radiolabeled peptide, or ex vivo, by using antigen-presenting cells incubated with peptides. Peptides with additional residues at either the amino or carboxyl terminus behaved dramatically differently. Complexes of I-Ak with the longer peptides were stable to SDS/PAGE. Very few amino acid additions result in the change from unstable to stable complexes. The important issue here is that when cultured with HEL, antigen-presenting cells selected the HEL peptides containing the 52-61 sequences that favored stability [Nelson, C. A., Roof, R. W., McCourt, D. W. & Unanue, E. R. (1992) Proc. Natl., Acad. Sci. USA 89, 7380-7383]. Also, from other studies, such sequences correlate with a high immunogenicity of the peptide. We conclude that there are structural features of peptides that change the stability of the class II molecule and that are independent of the "core" peptide seen by the T cells.

Processing and presentation of intact hen egg-white lysozyme by dendritic cells.

Dendritic cells in lymphoid tissues are of key importance as highly specialized antigen-presenting cells for the induction of T lymphocyte responses. Conflicting results have been published regarding antigen processing of intact proteins by dendritic cells. We now report that highly purified dendritic cells isolated from H-2k mouse spleens very efficiently generated immunogenic fragments of intact hen egg-white lysozyme (HEL) protein to present to an I-Ak-restricted T hybridoma cell line, specific for HEL peptide 46-61. Dendritic cells required 100 times less HEL protein than lipopolysaccharide-induced B cell blasts for effective presentation. Uptake of 125I-labeled HEL protein by dendritic cells and inhibition of presentation of HEL protein by chloroquine treatment was observed. This indicates an endocytotic process and the involvement of acidified compartments. Since the supernatant of dendritic cells, that were incubated with intact HEL protein, contained immunogenic fragments, further evidence for processing of HEL protein by dendritic cells was obtained. When HEL protein was covalently coupled to beads, dendritic cells were not able to ingest these beads, but could still process HEL protein for presentation. This suggests cell surface processing of HEL protein, although internalization of HEL protein released from the beads cannot be excluded. Taken together, these data show that H-2k dendritic cells are capable of processing and presenting intact HEL protein.

Identification of the naturally processed form of hen egg white lysozyme bound to the murine major histocompatibility complex class II molecule I-Ak.

A murine B-cell lymphoma bearing the class II major histocompatibility complex molecule I-Ak was cultured with the protein antigen hen egg white lysozyme (HEL). The I-Ak molecules were purified, and their associated peptides were extracted for characterization. Five HEL peptides were identified. Four contained the 10 amino acid residues HEL 52-61 (DYGILQINSR) but were heterogeneous in length and flanking residues. This core sequence is known to confer a high binding affinity for I-Ak. One additional peptide contained the amino acid residues HEL 48-60. These data demonstrate that the HEL epitope containing residues 52-61 is the most abundant HEL epitope presented on the major histocompatibility complex of the antigen-presenting cells and consequently explains its immunodominance.

Selective immunosuppression by administration of major histocompatibility complex (MHC) class II-binding peptides. I. Evidence for in vivo MHC blockade preventing T cell activation.

Draining lymph node cells (LNC) from mice immunized with hen egg white lysozyme (HEL) display at their surface antigen-MHC complexes able to stimulate, in the absence of any further antigen addition, HEL peptide-specific, class II-restricted T cell hybridomas. Chloroquine addition to these LNC cultures fails to inhibit antigen presentation, indicating that antigenic complexes of class II molecules and HEL peptides are formed in vivo. MHC class II restriction of antigen presentation by LNC from HEL-primed mice was verified by the use of anti-class II monoclonal antibodies. Coinjection of HEL and the I-Ak-binding peptide HEL 112-129 in mice of H-2k haplotype inhibits the ability of LNC to stimulate I-Ak-restricted, HEL 46-61-specific T cell hybridomas. Similar results are obtained in mice coinjected with the HEL peptides 46-61 and 112-129. Inhibition of T hybridoma activation can also be observed using as antigen-presenting cells irradiated, T cell-depleted LNC from mice coinjected with HEL 46-61 and HEL 112-129, ruling out the possible role of either specific or nonspecific suppressor T cells. Inhibition of T cell proliferation is associated with MHC-specific inhibition of antigen presentation and with occupancy by the competitor of class II binding sites, as measured by activation of peptide-specific T cell hybridomas. These results demonstrate that administration of MHC class II binding peptide competitors selectively inhibits antigen presentation to class II-restricted T cells, indicating competitive blockade of class II molecules in vivo.

Crystallization of the Fab fragments of anti-peptide monoclonal antibodies and a complex with peptide

The antigen-binding fragments of four monoclonal antibodies that cross-react with both the “loop” peptide of hen egg-white lysozyme (residues 57 to 84) against which they were raised, and with the native protein (HEL) have been crystallized. One of these fragments also crystallizes as a complex with the peptide antigen.