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Identification of the Immunodominant T Cell Epitope of p38, a Major Egg Antigen, and Characterization of the Epitope-Specific Th Responsiveness During Murine Schistosomiasis Mansoni¹

Department of Immunology and Microbiology, Wayne State University School of Medicine, Detroit, MI 48201

	Abstract

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A recently cloned major Schistosoma mansoni egg Ag p38 induced and elicited strong Th1-type responsiveness in mice of H-2^k haplotype. Now, we have identified the immunodominant T cell epitope of p38 and analyzed the dynamics of epitope-specific Th responsiveness during murine schistosomiasis mansoni. Overlapping recombinant and synthetic peptides that encompassed the full-length 354 amino acid of p38 were tested for proliferation and cytokine production in peptide- or p38-sensitized mice. The immunodominant T cell epitope of p38 that elicited pulmonary granuloma formation was localized within peptide P4 (amino acids 235–249). The P4-specific cytokine response of splenocytes that had been sensitized s.c. with p38, P4 or soluble egg Ags in IFA, or i.p. with 3000 eggs was predominantly as the Th1 type, with strong IL-2 and IFN-, but trace amounts of IL-4 and IL-5 secretion. At 6.5 wk of infection, splenocytes and mesenteric lymph node cells responded to p38/P4 peptides with predominantly Th1-type responsiveness. This response did not switch to a Th2-type pattern from 8 wk onwards; rather, it underwent down-modulation. Moreover, the hepatic granuloma lymphocytes at 6.5 wk responded to p38/P4 predominantly with Th1-type cytokine production, indicating that they participate in early granuloma formation. From 8 wk onwards an immune deviation to the p38-specific response was observed that was manifested by rising IgG1, IgE, and IgG2a Ab production as opposed to declining Th1- and Th2-type cytokine secretion.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Infection with the helminth Schistosoma mansoni leads to a chronic inflammatory response that is manifested by peripheral blood eosinophilia, hyperimmunoglobulin E, and eosinophil-rich granuloma formation around parasite eggs (1, 2). This typical inflammatory response is dependent upon the activation of CD4⁺ helper T lymphocytes (3, 4) that are specific for the soluble egg Ags (SEA)³ (5) secreted by the living miracidia within eggs. Although the CD4⁺ T cell response to the total SEA has been well studied during the murine schistosomal infection, only limited information is available about the T cell-related immunogenicity of the individual components of SEA peptides (6, 7, 8, 9, 10, 11, 12). Recently, several laboratories have isolated and cloned a 38- to 40 kDa-sized major egg immunogen (13, 14, 15, 16, 17). Northern blot analysis showed that its encoding mRNA is one of the most abundant messages in the eggs (13). Molecular cloning of the gene elucidated that this peptide has 354 amino acid (aa) residues, and it shares extensive homologies with the family of small heat shock proteins (13). In infected mice, the immune response to the p38 fraction was found to be egg stage-specific, because T cell proliferation and granuloma formation could only be induced after egg deposition (9, 10). Moreover, the T cell response against the p38 fraction that was similar to unseparated SEA underwent down-modulation as the disease progressed into the chronic stage (11). In contrast, the serum Ab response against p38 has been sustained in most strains of mice with chronic schistosomiasis mansoni and was also detected in the majority of human patients (13). Both native p38 and the cloned recombinant peptide proved to be potent immunogens; they sensitized CBA/J^k mice for a Th1-type immune response with or without the aid of IFA and stimulated the splenic lymphocytes of acutely infected mice to a predominantly Th1-type response. In addition, presensitization with p38 induced the early (4 day) pulmonary mononuclear granuloma formation around Ag-coupled beads or parasite eggs (17). More recently, panels of T cell hybridomas specific to SEA and a set of polyclonal lymph node cells from acutely infected mice of H-2^k and H-2^b haplotypes were used to show that the strong CD4⁺ cell response was restricted to the H-2^k haplotype and represented a risk factor for the development of severe disease in murine schistosomiasis mansoni (18).

In light of the significant granuloma-inducing properties of p38, the present study was undertaken to define its immunodominant epitope(s) and to characterize the type and dynamics of Th-derived cytokine secretion during immunization or infection. Using step-wise truncated recombinant and overlapping synthetic peptides, we report that CBA/J^k mice respond mainly to one immunodominant epitope localized between aa 235 and 249. Cytokine secretion analysis of the T cell response shows that this 15-mer immunodominant peptide elicits the same predominantly Th1 cytokine response as the full-length p38. Interestingly, Th lymphocytes display a down-modulation in cytokine production that is accompanied by rising Th1- and Th2-type Ab production over the course of the infection, in response to p38 and its immunodominant peptide.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Mice and infection

Female CBA/J^k mice (The Jackson Laboratory, Bar Harbor, ME) were used in all experiments. Mice were infected s.c. with 25 cercariae of the Puerto Rican strain of S. mansoni for the cytokine study or with 200 cercariae for egg harvest.

Egg isolation and SEA preparation

Schistosome egg isolation and SEA preparation were performed as previously described (5).

Preparation of recombinant and synthetic peptides

The p38 peptide was produced in Escherichia coli that carried the recombinant pGEX vector with the isopropyl-ß-D-thiogalactoside-inducible gene for the expression of the glutathione S-transferase (GST)-p38 fusion protein. The fusion protein was purified by a bulk GST-purification module (Pharmacia, Piscataway, NJ) as described previously (17). The truncated peptides were constructed by preparing the desired DNA fragments, either by restriction enzyme digestion of full-length DNA within the pGEX vector or by primer-directed PCR, followed by the insertion of the DNA fragments into the pGEX-4T-3 vector and transformation of the B21 strain of E. coli. The colonies of the transformants that were grown with ampicillin in agar medium were screened for the expression of the new protein by SDS-PAGE and were selected based on the m.w. of the new fusion protein and the specific recognition by rabbit anti-p38 polyclonal Ab during Western blotting. In some selected transformants, the proper orientation of the desired DNA fragments in the pGEX vector was confirmed by DNA sequencing (Biomedical Research Core Facilities, University of Michigan, Ann Arbor, MI). The large-scale preparation of the truncated peptides was conducted by the recommended purification module (Pharmacia). After isopropyl-ß-D-thiogalactoside induction, several liters of the bacterial cultures were harvested by centrifugation, and the pellet was suspended in PBS. The cell suspension was sonicated to release the intracellular proteins. The bacterial lysate was further dissolved with 1% Triton X-100 and cleared by centrifugation. The GST-peptide fusion protein was isolated by affinity-binding on a glutathione-Sepharose 4B column (Pharmacia). After three washes, the peptide was released from the bead-bound fusion protein by specific cleavage with thrombin. The peptide solution was then mixed with n-octyl-ß-D-glucopyranoside and run through Polymyxin B-bound agarose to remove endotoxin contamination (19). After dialysis against PBS, the peptide was filter sterilized, and the peptide content was determined by the Bradford method (Bio-Rad Laboratories, Richmond, CA). The lack of endotoxin content was verified by an assay with an Endotect kit (ICN Biochemicals, Aurora, OH); the kit showed no detectable level of the toxin.

The short overlapping peptides P1 through P4 were either synthesized commercially (Chiron Mimotopes, Raleigh, NC) or synthesized ourselves using a Dupont Rapid Amide Multiple Peptide Synthesis solid-phase peptide synthesizer (kindly provided by R. H. Swanborg, Wayne State University School of Medicine, Detroit, MI).

Immunization of mice

Groups of mice were sensitized s.c. at the base of the tail with 3 µg of p38, 5 µg of synthetic peptide, or 50 µg of SEA emulsified in IFA, or they were sensitized i.p. with 3000 schistosome eggs.

Cell preparation

Spleens or mesenteric lymph nodes (MLNs) from at least three Ag-immunized or schistosome-infected mice were removed aseptically, and single-cell suspensions were prepared after the removal of RBCs by hypotonic lysis. For granuloma cell (GrC) isolation, livers from eight mice that had been infected for 6.5 wk or three mice that had been infected for 8 wk were pooled and dispersed with collagenase as previously described (20). Cells were resuspended in RPMI 1640 supplemented with 2 mM L-glutamine, 50 U/ml of penicillin and 50 µg/ml streptomycin, 10 mM HEPES, 0.2 mM sodium pyruvate, 50 µM 2-ME, and 10% FCS. In some experiments, CD4⁺ T cells were enriched by positive selection with biotinylated anti-Thy-1.2 Ab. Briefly, 1 x 10⁸ cells were incubated for 30 min at 4°C with 1/20 anti-CD8 hybridoma supernatant. Subsequently, cells were washed twice, resuspended in 15% Low Tox-M rabbit complement (Accurate Chemical, Westbury, NY), and incubated for 30 min at 37°C. After a thorough wash, cells were coated with biotinylated anti-Thy-1.2 Ab at 4°C for 30 min, and Thy-1.2⁺ T cells were isolated by plating on a nitro-avidin-coated flask. The nitro-avidin was prepared in our laboratory according to the published procedure (21). This purification process resulted in 70% pure CD4⁺ T cells as determined by immunofluorescence using an FITC-conjugated anti-CD4 mAb.

Proliferation assays

A concentration of 1 x 10⁶ cells per ml was incubated in triplicate in 96-well flat-bottom tissue culture plates at 37°C in 5% CO₂ in the absence or presence of SEA (50 µg/ml), recombinant peptides (5 µg/ml), or synthetic peptides (5 µg/ml). After 3 days of incubation, 0.5 µCi [³H]TdR was added. After 18 h, the cells were harvested on a Tomtec Harvester 96 (Orange, CT), and incorporated thymidine was measured in a beta counter. The data were expressed as the stimulation index (calculated as mean cpm in cultures with stimulus/mean cpm in control cultures without stimulus).

Elicitation and measurement of pulmonary granulomas

Groups of mice were sensitized s.c. at the base of the tail with 3 µg of p38 in IFA in 0.2 ml. At 7 days after sensitization, they were injected i.v. with 2500 Sepharose 4B beads that were covalently bound with p38 or P1 to P4 peptides. The peptide-bound beads were prepared freshly as previously described (11) with CNBr-activated Sepharose 4B (Pharmacia), which couples ligands containing primary amino groups. After 4 days, lungs were perfused, inflated with 10% buffered formalin, and removed. Fixed and paraffin-embedded lungs were sectioned, stained with hematoxylin and eosin, and examined by light microscopy. Granuloma areas were measured using a Microcomp Integrated Image Analysis System (Southern Micro Instruments, Atlanta, GA). An average of 30 lesions was measured per lung.

Determination of Ag-specific cytokine production

A concentration of 5 x 10⁶ cells per ml was incubated with SEA (50 µg/ml), p38 (5 µg/ml), and the immunodominant P4 peptide (5 µg/ml). Supernatants were collected at 24 h for IL-2 determination and at 48 h for IFN-, IL-4, and IL-5 measurements.

IL-2 and IL-4 levels in the culture supernatants were determined using the IL-2-dependent CTLL-20 cell line (a generous gift from Dr. Frank Fitch, University of Chicago, Chicago, IL) and the IL-4-dependent CT.4S cell line (kindly provided by Dr. William Paul, National Institutes of Health, Bethesda, MD), respectively. The specificity of these assays was confirmed by the complete abrogation of proliferative responses with anti-IL-2 (clone no. S4B6; kindly provided by DNAX Corporation, Palo Alto, CA) or anti-IL-4 (clone no. 11B11; provided by Dr. William Paul). Standard curves for IL-2 or IL-4 were generated using murine rIL-2 (generously donated by Cetus, Emeryville, CA) or rIL-4 (generously donated by Immunex, Seattle, WA). IFN- and IL-5 were measured by ELISA using paired Abs with and without biotinylation (purchased from PharMingen, San Diego, CA) and streptavidin-alkaline phosphatase (AP) conjugate (Sigma, St. Louis, MO). Color was developed using nitrophenyl diamine diethanolamine as a substrate, and the OD of each well was measured at 405 nm. Standard curves for IFN- and IL-5 were completed using a dilution of recombinant cytokine (rIFN- was generously donated by Genentech; rIL-5 was purchased from PharMingen).

Determination of Ag-specific Ab isotype response

The sera of mice at different ages of infection were collected, and the level of SEA-specific total IgG and of p38- and P4-specific total IgG, IgG1, IgE, and IgG2a was determined by ELISA. The coating Ag was SEA at 5 µg/ml, p38 at 5 µg/ml, or P4 at 1 µg/ml. After preliminary results, the serum samples were diluted to 1:800 for total IgG, 1:400 for IgG1 and IgG2a, and 1:100 for IgE testing. The detecting Abs were rat anti-mouse IgG, IgG1, or IgG2a (PharMingen) that were paired with AP-conjugated goat anti-rat Ab (Organon Teknika, Westchester, PA) for total IgG, IgG1, and IgG2a, and rabbit anti-mouse IgE that was paired with biotinylated sheep anti-rabbit Ab and streptavidin-AP conjugate for IgE measurement. Color was developed using nitrophenyl diamine diethanolamine as a substrate, and the OD of each well was measured at 405 nm.

Statistical analysis

An unpaired Student’s t test was used to analyze the granuloma elicitation data. Data were determined to be significant at p < 0.05.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Localization of the immunodominant T cell epitope(s) on p38 peptide

Proliferation. The T cell response to a protein Ag generally focuses on a limited number of potential T cell epitopes (22). Therefore, our strategy for identifying T cell epitopes on p38 was to obtain overlapping peptides that encompass the full length of the molecule and test their immunoreactivities. As shown in Figure 1A, seven truncated recombinant peptides were prepared. Preliminary results indicated strong activity with peptide aa 215–258. Therefore, the peptide was further analyzed by the preparation of three overlapping, synthetic peptides, P1 through P3 (Fig. 1B). Because of the cross-reactivity between peptides P2 and P3 (data not shown), the peptide P4 was synthesized. The proliferative responses of the p38-sensitized splenic lymphocytes to each of the truncated and synthetic peptides are shown in Figure 1C. The p38-sensitized splenic cells responded strongly to SEA, full-length p38, and the mid region of the peptide (aa 172–294 and aa 215–258), but showed either no response or a borderline proliferative response to the N-terminal (aa 1–177) and C-terminal regions (aa 289–354) of p38. The weak activity of p38-sensitized splenic cells to the C-terminal region of p38 was also seen with low IL-2 and IFN- cytokine production (data not shown), indicating the existence of some minor T cell epitope(s). Furthermore, p38-primed splenic cells did not respond to P1, responded partially to P2, and responded fully to P3 and P4. Dose-response experiments using P4 peptide in amounts ranging from 0.01 to 10 µg/ml showed a maximal lymphoproliferative response at 5 µg/ml. The 0.2 µg/ml concentration of P4, which corresponds in molarity to 5 µg/ml of p38 peptide, gave suboptimal results. With splenocytes from egg-sensitized mice, similar patterns of proliferative responses were observed for each peptide (data not shown). Thus, the immunodominant T cell epitope of p38 appeared to reside in the 15-aa, long P4 fragment (aa 235–249).

View larger version (23K): [in this window] [in a new window]   FIGURE 1. The aa sequences of recombinant and synthetic peptides and their capacity to stimulate proliferative responses. A, Full-length (1–354 aa) and overlapping recombinant peptide fragments of p38 peptide. B, P1 to P4 synthetic peptides and aa sequences that were prepared to define the epitope of the aa 215–258 fragment. C, Proliferative responses of p38-sensitized splenocytes stimulated by SEA (50 µg/ml), p38 (5 µg/ml), and its derived peptides (5 µg/ml). The results expressed as the stimulation index are the mean ± SEM of three separate experiments. In each experiment, splenic lymphocytes were pooled from three mice that had been immunized s.c. with 3 µg of p38 in IFA 7 days earlier.

View larger version (48K): [in this window] [in a new window]   FIGURE 2. Pulmonary granuloma formation around peptide-coated beads in p38-presensitized mice. Six mice per group were sensitized s.c. with 3 µg of p38 in IFA followed by i.v. injection of 2500 peptide-coated beads at day 7. At 4 days after bead injection, lungs were removed and prepared for histologic staining. The results are the mean granuloma area ± SEM of six mice in each group. The horizontal line indicates the average bead size alone.

To determine the pattern of the cytokine profile elicited by the immunodominant peptide P4, the splenic cells of mice immunized s.c. with 3 µg of p38 in IFA were first examined in vitro for cytokine responses. As shown in Figure 3A, strong IL-2 and IFN- production was evident in response to SEA, p38, and P4, while IL-4 and IL-5 production was seen at minimal levels. This result suggested that p38-specific lymphocytes responded with a predominantly Th1-type cytokine response to p38 and P4 fragments. Next, the P4 peptide was further examined for its immunogenicity and cytokine-eliciting property. Sensitization with 5 µg of P4 in IFA induced a Th1-type immune response to P4 as well as to p38 and SEA and did not result in Th2-type cytokine production (Fig. 3B). Using P4 at 0.01 µg or 100 µg did not alter the cytokine production profiles (data not shown).

View larger version (29K): [in this window] [in a new window]   FIGURE 3. Cytokine secretion profiles of p38 or P4 peptide-sensitized splenic cells stimulated by SEA, p38, and P4. Three mice were immunized s.c. with 3 µg of p38 in IFA (A) or 5 µg of P4 in IFA (B). After 7 days, the splenocytes were prepared and assayed for in vitro secretion of cytokine after stimulation with SEA (50 µg/ml), p38 (5 µg/ml), or P4 (5 µg/ml). The cytokine levels are expressed as net mean ± SD of triplicate cultures after subtracting the mean of medium only. The results are representative of five separate experiments with similar results.

View larger version (31K): [in this window] [in a new window]   FIGURE 4. Cytokine secretion profiles of SEA- or egg-sensitized splenic cells stimulated by SEA, p38, and P4 peptides. Three mice were immunized s.c. with 50 µg of SEA in IFA (A) or i.p. with 3000 eggs (B). After 7 days, the splenocytes were prepared and assayed for in vitro secretion of cytokine after stimulation with SEA (50 µg/ml), p38 (5 µg/ml), or P4 (5 µg/ml). The cytokine levels are expressed as net mean ± SD of triplicate cultures after subtracting the mean of medium only. The results are representative of three separate experiments with similar results.

Cytokine response. Mice are exposed to egg Ags from 5 wk onwards of the infection; therefore, we analyzed the cytokine production patterns of splenocytes, MLN cells, and GrCs from the induction to the down-modulation phase of the granulomatous response. A dynamic change in SEA-, p38-, and P4-elicited responses was observed in splenocytes. At 5.5 wk of infection, only the SEA-stimulated splenocytes produced low levels of IFN- (data not shown). With the evolution of the early granuloma response (6.5 wk), a strong Th1-type but a good Th2-type anti-SEA response was seen (Fig. 5). At the peak of the granulomatous response (8 wk), a switch to a Th2 pattern and a decrease in Th1 responsiveness to SEA was observed. At 11 and 16 wk of infection, both Th1- and Th2-type responsiveness to SEA underwent down-modulation. The predominant cytokine produced by splenocytes was IL-5. Interestingly, p38 or its P4 epitope-specific response showed a different pattern. At 6.5 wk of infection, splenic responsiveness to p38 or P4 was predominantly of the Th1 type. Compared with the magnitude of that response, Th2-type cytokine production was minimal. By 8 wk, splenocytes retained their Th1-type responses to both p38 and P4 peptides, albeit with diminished levels of IFN- production. At the down-modulated stage of the granuloma response (11 and 16 wk), p38 and P4 still elicited low but dominant Th1 responses by splenocytes with negligible Th2-type IL-4 and IL-5 production.

View larger version (22K): [in this window] [in a new window]   FIGURE 5. Cytokine responses of splenocytes of mice at different ages of infection that were stimulated with SEA, p38, and P4 peptides. In each experiment, splenic lymphocytes were pooled from at least three mice at 6.5, 8, 11, or 16 wk of infection and stimulated with 50 µg/ml of SEA (A), 5 µg/ml of p38 (B), or 5 µg/ml of P4 (C). The cytokine levels are expressed as pooled mean values ± SEM of three to five separate experiments.

Because we detected substantial Th1-type cytokine secretion to p38 and P4 in splenic and MLN cells of early infected mice, it was of interest to know what type of Th response is induced by p38 or P4 peptides in GrCs. Because the GrC population contains a considerable percentage of eosinophils and mast cells (25) that are potential sources of IL-4 and IL-5 cytokines (26), we examined the cytokine production pattern of both unseparated and CD4⁺ T cell-enriched populations. As shown in Figure 6, the SEA-stimulated GrCs already produced very high levels of IL-4 that were dominant over the Th1 response at 6.5 wk. By contrast, p38 and P4 peptides still elicited a higher early Th1-type cytokine response with weaker Th2-type responsiveness. By 8 wk, SEA-stimulated GrCs switched to a preponderant Th2 pattern as shown by IL-4 secretion, while responsiveness to p38 and P4 sharply decreased in both Th1 and Th2 cytokine production. At 11 and 16 wk of infection, IL-2, IL-4, IL-5, and IFN- cytokine responses to p38/P4 peptide were <10 pM, and no rebound was observed. The p38 and P4 peptide-stimulated CD4⁺ T cell-enriched GrCs that were examined at 6.5 wk showed a predominantly Th1 cytokine profile. The lower level of IL-4 production indicated that GrCs other than CD4⁺ T lymphocytes contributed to the overall level of cytokine secretion. By 8 wk of the infection, the CD4⁺ T cell-enriched GrCs showed minimal levels of both Th1- or Th2-type cytokine secretion in mice.

View larger version (28K): [in this window] [in a new window]   FIGURE 6. Cytokine responses of hepatic GrCs or CD4+-enriched granuloma T cells (GrCD4+) from mice that had been infected for 6.5 or 8 wk that were stimulated with 50 µg/ml of SEA (A), 5 µg/ml of p38 (B), or 5 µg/ml of P4 (C). GrCs were prepared from eight livers of mice that had been infected for 6.5 wk or from three livers of mice that had been infected for 8 wk. The same GrC preparation was used for the enrichment of the CD4+ T cells. The cytokine levels are expressed as net mean ± SD of triplicate cultures, and the results are representative of two separate experiments.

View larger version (17K): [in this window] [in a new window]   FIGURE 7. Anti-p38 and anti-P4 Ab isotype responses during the course of murine schistosomiasis mansoni. Serum samples were diluted to 1:400 for IgG1 and IgG2a determination, and 1:100 for IgE testing. The results are expressed as OD at 405 nm and are the mean ± SEM of five different serum samples at each infection stage.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Over the course of schistosomal infection, a shift of egg-specific Th responses from a predominant Th1- to Th2-type reaction has been demonstrated (28, 29, 30). To better relate the P4 epitope activity to the granulomatous process, we analyzed the cytokine profile of lymphoid cells that had been stimulated with the peptide during the course of infection. At the incipient stage of granuloma formation (6.5 wk), the various peptides of SEA had already induced a mixed Th1 and Th2 response, as shown by the cytokine production profile of splenic cells, MLN cells, or GrCs. The high levels of IL-4 produced by purified granuloma T cells are noteworthy. In contrast, the predominant response induced by p38 and its immunodominant P4 epitope was of the Th1 type, with a weak Th2 component. Thus, we propose that the p38/P4 peptide plays an important role in the induction of the early Th1 stage of the egg granuloma. It has been previously reported that this stage occurs at 6 wk of infection, when GrCs produce IFN- in response to SEA (11). By the time the granulomas had fully matured (8 wk), the SEA peptide-directed response indicated decreased Th1 responsiveness and enhanced Th2 responsiveness. The p38/P4-specific response was more complex. Unseparated or T cell-enriched splenic cells showed a decreasing but still dominant Th1 response, MLN cells showed a stable level of IL-5 production in the face of diminished IFN- secretion, and granuloma T cells showed a very low Th1 and Th2 reaction. The overall pattern here is indicative of a decreasing Th1 response without a switch to enhanced Th2-type cytokine production. This observation is in sharp contrast to the profile elicited by the SEA peptides. These lymphoid organ-related differences in the type of the induced Th cytokine profile underline the complexity of the evolving response and may indicate local influences, such as those exerted by the local Ag concentration, by Ag-presenting dendritic cells regulating the Th1 or Th2 profile of the response (31), and by cytokine-mediated cross regulation (32).

As the infection proceeds to chronicity, the intensity of granulomatous inflammation and overall cytokine production are down-modulated (33, 34). This process was reconfirmed by cytokine data that were obtained from SEA-stimulated splenocytes or MLN cells. It is of interest that at 11 and 16 wk of infection, the p38/P4-elicited Th1 response also diminished, indicating that responsiveness to a strong Th1-inducer peptide also underwent down-modulation. This observation agrees with a previous one showing diminished Th1-type cytokine production to the 32- to 38-kDa fraction of SEA at the chronic stage of infection (11). It is noteworthy that the down-modulated p38/P4-specific cytokine response was not followed by a switch to enhanced Th2-type cytokine production. Therefore, the Th1 to Th2 switch that has been thought to occur with egg deposition (28, 29) does not appear to function at the P4 epitope level of the p38 peptide.

A characteristic trait of the down-modulated granuloma response is the concurrent increase in Ab production to egg Ags (33, 34). An interesting finding of the present study is the rising Ab production to the p38 but not to the P4 epitope during the progress of the disease. The rise commenced at 8 wk, when IFN- production was on the decline, and continued into the chronic stage of the infection. This trend conforms to the phenomenon of immune deviation, in which an inflammatory delayed hypersensitivity response is switched to Ab production (35). This deviation is currently interpreted through the dynamics of Th1 and Th2 cell interaction. The isotypes of the circulating Abs indicated that both Th1- and Th2-type lymphocytes and their cytokines mediated the switch to Ab production of different isotypes. At chronicity, the p38 peptide elicited marginal IL-4 and low IFN- production by splenic and MLN cells. However, the higher levels of IL-4 that were stimulated by various peptides of SEA could have sufficed to mediate the switch to IgG1 and IgE production. Alternatively, such a switch could be induced in activated B cells by non-B/non-T cells that produce IL-4 and provide a costimulatory signal (36, 37). Conversely, the residual levels of IFN- that were produced appeared to be sufficient to mediate IgG2a isotype Ab synthesis. Importantly, the P4 epitope, which we identified as the immunodominant portion of p38 that induced Th1 cell responses, induced negligible Ab production of any isotype throughout the chronic stage of infection. Thus the peptide regions that are outside the aa 235–249 stretch of p38 may serve as B cell epitopes in Th2 cell-mediated Ab production.

Identification of the immunodominant epitope of p38 raises the following question: Why is this sequence of 15 aa able to preferentially induce Th1 cell responsiveness? Recent studies have suggested that the structural properties of immunogenic peptides can contribute to polarized Th phenotype differentiation by binding with variable affinity to the MHC class II groove on the APC (38, 39). The strength of such binding by P4, the participation of accessory signals (40), and the generation of the proper cytokine environment (41) during the induction of the response are some of the mechanisms to explore in future investigations.

In conclusion, the present study identified the immunodominant T cell epitope within the p38 molecule and showed that this P4 epitope participates in the induction of the early Th1-type cytokine and granuloma response in infected mice. Moreover, such a response does not appear to be switched to a preponderant Th2 pattern; instead, it undergoes down-modulation. The p38 peptide appears to play a dual role during the infection, because its B cell epitopic portions induce an increasing Th1- and Th2-type Ab response as the infection progresses; this response amounts to 30% of the total IgG-specific, anti-SEA responses. The significance of such a strong humoral response with regard to granuloma regulation remains to be elucidated. The dynamics of Th cell responsiveness to the Th1 or Th2 cell-inducer peptides may be decisive in the evolution of the pathology of the granuloma response.

	Acknowledgments

 
We thank Joel Whitfield for skillful technical support.

	Footnotes

 
¹ This work was supported by Public Health Service Grant AI-12913 from the National Institute of Allergy and Infectious Diseases, Bethesda, MD.

² Address correspondence and reprint requests to Dr. Dov L. Boros, Department of Immunology and Microbiology, Wayne State University School of Medicine, 540 E. Canfield, Detroit, MI 48201.

³ Abbreviations used in this paper: SEA, soluble egg Ag; aa, amino acid; GST, glutathione S-transferase; MLN, mesenteric lymph node; GrC, granuloma cell; AP, alkaline phosphatase.

Received for publication October 22, 1997. Accepted for publication January 28, 1998.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

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