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The Journal of Immunology, 1999, 163: 3877-3882.
Copyright © 1999 by The American Association of Immunologists

Elucidation and Role of Critical Residues of Immunodominant Peptide Associated with T Cell-Mediated Parasitic Disease1

Hector J. Hernandez and Miguel J. Stadecker2

Department of Pathology, Tufts University School of Medicine, Boston, MA 02111


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Granulomatous inflammation in schistosomiasis is strictly dependent on CD4+ Th lymphocytes sensitized to egg Ags, but its intensity is genetically regulated. C3H and CBA (H-2k) are strains of mice that develop large granulomas; they also strongly respond to the major egg Ag Sm-p40. We now show that the immunodominant epitope recognized by CD4+ Th cells from infected H-2k mice is confined to 13-mer peptide 234–246 (PKSDNQIKAVPAS), which elicits an I-Ak-restricted Th1-type response. Using a panel of alanine-monosubstituted peptides, we identified Asp237 as the main contact residue with I-Ak. On the other hand, three TCR contact residues were essential to stimulate epitope-specific T cell hybridomas: for two hybridomas these were Asn238, Gln239, and Lys241; and for one, Asn238, Lys241, and Pro244. In one instance, alanine substitution for Gln239 generated an antagonist that blocked subsequent stimulation with wild-type peptide. Most importantly, replacement of Asn238, Gln239, or Lys241 caused a profound loss of polyclonal CD4+ T cell reactivity from schistosome-infected mice. This study identifies the critical residues of immunodominant peptide 234–246 involved in the T cell response against the Sm-p40 egg Ag and suggests that suitable altered peptides may be capable of precipitating its down-regulation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Infection with the helminth Schistosoma mansoni leads to granuloma formation around parasite eggs in the liver and intestines. Studies using a murine model have shown that the development of egg granulomas is an immunopathologic reaction critically dependent on CD4+ Th lymphocytes sensitized to schistosomal egg Ags (SEA)3 (1, 2). Stimulation of CD4+ Th cells from schistosome-infected mice with a soluble preparation of SEA results in blastogenesis and production/expression of the cytokines IL-2 and IFN-{gamma} representative of the Th1 subtype, as well as of IL-4, IL-5, and IL-10, representative of the Th2 subtype (3, 4, 5, 6).

The Sm-p40 Ag is a major glycoprotein of SEA of 354 amino acids, first reported and cloned by Nene et al. (7), and then independently described by several laboratories (8, 9, 10). The importance of the Sm-p40 Ag was underscored by our observation that all members of a panel of derived SEA-specific T hybridomas from C3H mice were specific for this Ag, whereas in BL/6 mice, none of the derived SEA-specific T cell hybridomas was specific for Sm-p40 (11). This suggested that a substantial proportion of the anti-SEA T cell repertoire in C3H mice is directed against Sm-p40. Subsequent studies using additional inbred mouse strains demonstrated that the strong CD4+ Th cell proliferative response to Sm-p40 is characteristic of H-2k strains C3H and CBA (12), which are prone to developing large granulomatous lesions (13, 14). These findings raised the intriguing possibility that the intensity of the anti-Sm-p40 Th cell response correlates with the overall magnitude of the resulting immunopathology. Interestingly, the CD4+ Th cell response against the Sm-p40 Ag in infected mice is predominantly of the Th1 type (10, 12), a subset with demonstrated capacity to mediate granuloma formation (15).

Our initial studies using overlapping synthetic peptides of the Sm-p40 Ag clearly demonstrated that CD4+ Th cells from infected H-2k mice recognized a single immunodominant epitope located in the 30-mer peptide 229–258; two additional subdominant epitopes were recognized by Th cells from mice directly immunized with a fragment of recombinant Sm-p40 (12). In the present work, we determine the minimal peptide containing the dominant epitope, and, using peptides with alanine substitutions, we define and examine the minimal peptide’s contact points with the MHC class II molecule I-Ak, as well as with clonotypic TCRs from specific mono- and polyclonal CD4+ T cell populations. The precise dissection and understanding of each relevant peptide/class II complex is important because it offers the possibility of manipulating the outcome of the T cell response.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Ags and peptides

SEA, prepared as previously described (16), was obtained from the Biomedical Research Institute (Rockville, MD). Recombinant Sm-p40 Ag (rSm-p40), amino acids 94–341, was isolated as a GST fusion protein, released by proteolysis, and purified as described (11).

Three overlapping peptides of peptide 229–258 (12) were synthesized at the Department of Medical and Molecular Parasitology (New York University, New York, NY). These are 17-mer peptides with 10–11 residue overlaps, respectively comprising amino acids 229-QVAVRPKSDNQIKAVPA-245, 235-KSDNQIKAVPASQALVA-251, and 242-AVPASQALVAKGVHGLS-258. A minimal wild-type (WT) 13-mer peptide, 234PKSDNQIKAVPAS-246 (peptide 234–246), as well as eleven peptides with single alanine substitutions (see Fig. 3Go), were synthesized at New York University or at the Tufts University Core Facility (Boston, MA), using, respectively, t-boc and f-moc technology. All peptides were purified by HPLC and their m.w. verified by mass spectrometry.



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  		FIGURE 3. I-Ak restriction of the CD4+ Th cell response to peptide 234–246 and rSm-p40. CD4+ Th cells obtained from 7.5- to 8-wk-infected CBA mice were cultured for 96 h with normal APC and 0.05 µM of peptide 234–246 (left side), or 10 µg/ml of rSm-p40 (right side), in the presence of blocking mAb against I-Ak or I-Ek, and proliferative responses were assessed after 96 h of culture. Data from mean cpm of triplicate determinations ± 1 SEM are expressed as percent of control cultures without mAb.

 
Infection of mice

Female C3HeB/FeJ (C3H, H-2k), CBA/J (CBA, H-2k), BALB/cJ (BALB/c, H-2d), DBA/1J (DBA/1, H-2q), and C57BL/6J (BL/6, H-2b) mice, 6–8 wk old, were purchased from The Jackson Laboratory (Bar Harbor, ME). Some mice were infected i.p. with 70 cercariae of S. mansoni (Puerto Rico strain). Cercariae were shed from infected Biomphalaria glabrata snails provided to us by the Biomedical Research Institute, under National Institutes of Health-National Institute of Allergy and Infectious Diseases Contract N01-AI-55260.

CD4+ Th cell populations

Mesenteric lymph nodes from 7.5- to 8-wk-infected mice were removed aseptically. Single cell suspensions were prepared, and RBC were eliminated by hypotonic lysis. CD4+ Th cells were purified by negative selection, by passing the lymph node cells through a nylon wool column, followed by a first incubation for 30 min at 4°C in the presence of mAbs from hybridoma M5/114.15.2 (ATCC TIB 120; American Type Culture Collection, Manassas, VA) against I-Ek/I-Ab/I-Ed/I-Aq, J11d.2 (ATCC TIB 183) against heat-stable Ag, and 3.155 (ATCC TIB 211) against CD8, and a second incubation for 30 min at 37°C in the presence of 15% rabbit C (Pel-Freez Biologicals, Roger, AK). The mAbs were present in hybridoma culture supernatants, which were used at concentrations of 1:6.7, 1:10, and 1:30, respectively. mAb and C treatments were repeated once more, and dead cells were eliminated by density gradient separation. FACS analysis determined that the resulting cell populations were >95% CD4+.

T cell hybridomas

T cell hybridomas were derived from SEA-sensitized C3H mice, as described previously (11). Briefly, draining lymph node cells, cultured with 20 µg/ml SEA for 3 days and with 40 U/ml recombinant human IL-2 for 2 additional days, were fused with the AKR thymoma BW5147 (17). Following incubation in hypoxanthine-aminopterin-thymidine (HAT) and HT media, and expansion, the hybridomas were selected for antigenic specificity against SEA and rSm-p40. All hybridomas were demonstrated to be monoclonal, and clonally distinct, by Southern blot analysis. The T cell hybridomas Ci39, Ci59, and Ci99, used in this study, have been previously shown to display I-Ak-restricted responses to an epitope mapped within Sm-p40 peptide 229–258 (12).

Poly- and monoclonal T cell responses

Proliferative responses and cytokine production by polyclonal CD4+ Th cells were determined as previously described (12). To assess proliferation, 1.5 x 105 purified CD4+ Th cells plus 3 x 105 normal irradiated (3000 R) splenic APC were incubated in 200 µl of medium for 96 h in the presence of Ags or peptides, as indicated. The cells were pulsed during the last 24 h of culture with 0.5 µCi [3H]TdR, and incorporation into DNA was measured by scintillation counting. In some experiments, proliferative responses were assessed in the presence of blocking mAbs against I-Ak and I-Ek molecules. These mAbs were secreted into culture supernatants, respectively, by hybridomas 10-2.16 (ATCC TIB 93) and 14-4-4S (ATCC HB 32), and tested at indicated concentrations.

The cytokines IL-2 and IL-4 were measured by ELISA in supernatants from 1 x 106 purified CD4+ Th cells plus 4 x 106 (3000 R) irradiated normal splenic APC, cultured for 48 h in 1 ml of medium in the presence of the indicated Ags or peptides. Cytokine-specific capture and detection mAbs, as well as standard cytokines and protocols were obtained from PharMingen (San Diego, CA).

T cell hybridoma responses were determined following stimulation with Ags or peptides in the presence of syngeneic irradiated splenic APC, as described (11). Positive responses were judged from the ability of 24-h culture supernatants to support the proliferation of cytokine-dependent HT-2 indicator cells.

Determination of peptide binding to I-Ak

Each of the various peptides was tested for its ability to competitively inhibit the binding of a 125I-labeled standard peptide to the purified I-Ak molecule (18). A relative inhibitory capacity (RIC) was determined for each peptide as the amount needed to inhibit 50% of the labeled peptide’s binding. Each obtained value was normalized against the activity of a standard unlabeled peptide. The binding strength is expressed as 1/RIC (RIC-1), such that a small value is indicative of strong binding, and a large value represents weak binding.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Definition and properties of minimal epitope peptide 234–246

Previous work demonstrated that the dominant epitope of the Sm-p40 Ag recognized by CD4+ Th cells from infected H-2k mice resided within 30-mer peptide 229–258. To further define the minimal stimulatory peptide, three synthetic overlapping 17 mers, 229–245, 235–251, and 242–258, were used to stimulate the I-Ak-restricted T cell hybridoma Ci39, with known specificity for peptide 229–258 (12). As shown in Fig. 1Go, 17-mers 229–245 and 234–251 strongly stimulated hybridoma Ci39, while 17-mer 242–258 failed to do so; identical findings were obtained with polyclonal CD4+ Th cells from infected CBA mice (data not shown). These findings clearly placed the epitope within segment KSDNQIKAVPA, which is the sequence common to 17-mers 229–245 and 234–251.



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  		FIGURE 1. T cell hybridoma responses to overlapping 17-mer peptides 229–245, 235–251, and 242–258. The epitope-specific T cell hybridoma Ci39 (9 x 105 cells/well) was cultured together with 1.8 x 106 normal irradiated (3000 R) splenic APC in the presence of 0.5 µg/ml of 17-mer peptides 229–245, 235–251, and 242–258, as well as with 10 µg/ml of rSm-p40. Cytokine responses in 24-h culture supernatants were assessed by measuring the cytokine-dependent proliferation of HT-2 indicator cells by [3H]TdR incorporation, as described in Materials and Methods. Bars reflect mean cpm of triplicate cultures ± 1 SEM.

 
Based on the preceding results, the 13-mer peptide 234–246 with the sequence PKSDNQIKAVPAS was synthesized, and its stimulatory capabilities were tested on CD4+ Th cells from various strains of schistosome-infected mice. The results shown in Fig. 2Go demonstrate that peptide 234–246 stimulated CD4+ Th cells from H-2k strains C3H and CBA, but not cells from the non-H-2k strains BL/6, BALB/c, and DBA/1; the level of stimulation was comparable to that exerted by rSm-p40. By comparison, SEA stimulated CD4+ Th cells in all groups, with both H-2k strains giving the highest responses. Similarly, cytokine analysis of supernatants from polyclonal CD4+ Th cells revealed that peptide 234–246, like rSm-p40, elicited IL-2, but not IL-4, production in C3H and CBA, but not in BL/6, BALB/c, and DBA/1 mice, while SEA stimulated IL-2 and IL-4 production in all strains. These findings indicate that peptide 234–246, like the Sm-p40 Ag itself, elicits an H-2k-restricted Th cell response of the Th-1 type. The proliferation of CD4+ Th cells from infected CBA mice, in the presence of irradiated normal syngeneic APC and of either peptide 234–246 or rSm-p40, was inhibited by mAbs against I-Ak, but not against I-Ek (Fig. 3Go), thus determining I-Ak as the restricting MHC molecule.



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  		FIGURE 2. CD4+ Th cell proliferative and cytokine responses elicited by minimal 13-mer peptide 234–246. CD4+ Th cells were obtained from mesenteric lymph nodes 7.5–8 wk after infection of the indicated mouse strains and cultured in the presence of normal syngeneic APC and 0.5 µg/ml of 13-mer peptide 234–246 (13 mer in figure), or 10 µg/ml rSm-p40, or 40 µg/ml of SEA, as described in Material and Methods. Proliferative responses (left side) were assessed after 96 h of culture, including a final 24-h pulse with [3H]TdR, and are expressed as cpm of triplicate cultures ± 1 SEM. The cytokines IL-2 and IL-4 (right side) were measured by ELISA in supernatants from 48-h cultures. Bars represent means of triplicate determinations ± 1 SEM.

 
Binding of peptide 234–246 and of alanine-substituted peptides to I-Ak

To formally document the binding of peptide 234–246 to the restricting I-Ak molecule and specifically determine the relative contribution of each amino acid toward the binding strength, 11 peptides were synthesized in which each residue was substituted for alanine. The experiment shown in Fig. 4Go assessed the binding of peptide 234–246 and the alanine-substituted peptides to purified I-Ak, by measuring their ability to compete for the binding of a labeled standard peptide of high affinity. Peptide 234–246 bound strongly to I-Ak with a RIC-1 value of 5.5. Likewise, 10 of the 11 alanine-substituted peptides bound I-Ak with comparable strength (RIC-1 values ranging from 5.6 to 17.7). However, the peptide with alanine substituting for Asp237 (D237A) failed to bind (RIC-1 > 400), thereby defining Asp237 as the primary anchor residue for I-Ak.



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  		FIGURE 4. Binding of peptide 234–246 and alanine-substituted peptides to I-Ak. The binding of the peptides to solubilized I-Ak was performed as described in Materials and Methods. The RIC, expressed as RIC-1, represents the amount of peptide needed to inhibit by 50% the binding of a labeled standard peptide.

 
Identification and role of TCR contact residues of peptide 234–246

Having established the peptide’s principal contact residue with I-Ak, the following experiments were conducted to determine the amino acid(s) critical for T cell stimulation. For this purpose, we first used T cell hybridomas Ci39, Ci59, and Ci99, with demonstrated capacity to recognize Sm-p40’s dominant T cell epitope (12). These T cell hybridomas were stimulated in the presence of APC with peptide 234–246 as well as each of the 11 alanine-substituted peptides, and a positive cytokine response was measured with the aid of HT-2 indicator cells. Results in Fig. 5Go demonstrate that alanine-substitution of Asn238 (N238A) and Lys241 (K241A) resulted in the complete loss of stimulation of all three T cell hybridomas. In contrast, substitution of Gln239 (Q239A) caused the loss of stimulation of hybridomas Ci39 and Ci59, and substitution of Pro244 (P244A) caused the loss of stimulation of hybridoma Ci99. As expected, alanine-replacement of MHC anchor residue Asp237 (D237A) also caused a significant, but not complete, drop in stimulation of all hybridomas.



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  		FIGURE 5. T cell hybridoma responses to peptide 234–246 and alanine-substituted peptides. The epitope-specific T cell hybridomas Ci39, Ci59, and Ci99 were cultured together with normal APC in the presence of peptide 234–246 and 11 alanine-substituted peptides, and 24-h cytokine responses were assessed by measuring the cytokine-dependent proliferation of HT-2 indicator cells, as described in Fig. 1Go. Bars reflect stimulation of hybridomas with optimal discriminatory concentrations of the peptides, which were 1 µM, 5 nM, and 5 µM for hybridomas Ci39, Ci59, and Ci99, respectively. Background values with no peptide were subtracted. Experiments with each hybridoma were repeated up to four times with similar results.

 
To explore the effects of residue substitutions on T cell function, cells were stimulated following exposure to altered peptides to which they do not respond. Specifically, in the experiment shown in Fig. 6Go, T cell hybridoma Ci39 was preincubated with peptides N238A, Q239A, and K241A, and subsequently challenged with peptide 234–246. Results clearly show that among the three nonstimulatory peptides, Q239A rendered the hybridoma refractory to respond to the WT peptide, suggesting that only Q239A has the properties of a true antagonist.



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  		FIGURE 6. Identification of Q239A as an antagonist peptide. The T cell hybridoma Ci39 was incubated with irradiated splenic APC and the nonstimulatory peptides N238A, Q239A, and K241A at indicated concentrations, as described in Fig. 1Go. After 6 h, peptide 234–246 was added to each well to a final concentration of 5 µM, and cultures proceeded for an additional 24 h. At this point, culture supernatants were obtained to measure the cytokine-dependent proliferation of HT-2 indicator cells. Results from mean cpm of triplicate determinations ± 1 SEM are expressed as percent of control cultures, which were stimulated with peptide 234–246 but received no analogue peptide at time 0. Background value from cultures without added peptide 234–246 was subtracted. This experiment was repeated three times with similar results.

 
Critical residues of peptide 234–246 involved in the stimulation of polyclonal CD4+ Th cells

To validate the results obtained with individual monoclonal T cells, we examined the CD4+ Th cell proliferative response from 7.5- to 8-wk-infected CBA mice to peptide 234–246 and the 11 alanine-substituted peptides. Strikingly, peptides with alanine substitutions at 238, 239, and 241 failed to stimulate the polyclonal T cells; by comparison, the peptide with alanine substitution at 244 elicited a reduced response. Similarly, the T cell response to D237A was markedly inhibited (Fig. 7Go). Moreover, cytokine analysis of the polyclonal CD4+ Th cell response to peptide 234–246 and the alanine-substituted peptides revealed that IL-2 was produced in a manner that entirely paralleled the proliferative response (Fig. 8Go); in no case was there secretion of IL-4 in response to any of the peptides.



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  		FIGURE 7. CD4+ Th cell proliferative response to peptide 234–246 and alanine-substituted peptides. CD4+ Th cells obtained from 7.5- to 8-wk-infected CBA mice were cultured for 96 h together with normal APC in the presence of peptide 234–246 and 11 alanine-substituted peptides, as described in Fig. 2Go. Bars represent stimulation of the T cells with the optimal discriminatory concentration of 0.05 µM and are expressed as mean cpm of triplicate cultures ± 1 SEM. Background value with no peptide was subtracted. This experiment was repeated three times with similar results.

 


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  		FIGURE 8. CD4+ Th cell cytokine response to peptide 234–246 and alanine-substituted peptides. CD4+ Th cells obtained from 7.5- to 8-wk-infected CBA mice were cultured for 96 h together with normal APC in the presence of 0.05 µM peptide 234–246 and 11 alanine-substituted peptides, 10 µg/ml rSm-p40, and 40 µg/ml SEA, as described in Fig. 2Go. The cytokines IL-2 and IL-4 (right side) were measured by ELISA in supernatants from 48-h cultures. Bars represent means of triplicate determinations ± 1 SEM. Background value with no peptide was subtracted. This experiment was repeated three times with similar results.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Previous work determined that schistosomal egg granuloma formation is strictly dependent on egg Ag-specific CD4+ Th cells. Importantly, in the large granuloma-forming H-2k mice C3H and CBA, an inordinate proportion of the anti-SEA CD4+ Th cell repertoire appears to be directed against the Sm-p40 Ag (11). Furthermore, of three T cell epitopes detected with specific T cell hybridomas and polyclonal T cells, an epitope within peptide 229–258 is the target of the bulk of the anti-Sm-p40 CD4+ Th cell response in schistosome-infected mice (12, 19). The current work was undertaken to further analyze the CD4+ Th cell response against this immunodominant epitope.

Our study demonstrated that the immunodominant epitope of the Sm-p40 Ag is confined to minimal peptide 234–246 and that, like the parent Ag, peptide 234–246 elicits a strong I-Ak-restricted Th1-type response in CD4+ Th cells from schistosome-infected C3H and CBA mice. This Th1 polarization (10, 12) is by itself of great interest, as it persists in a milieu progressively dominated by Th2-type cytokines. The basis of this peculiar response is not known, but may pertain to Ag abundance, relative avidity for MHC molecules, and relative dependence on costimulatory signals.

Binding studies measuring the interaction of peptide 234–246 and the alanine-monosubstitutes with I-Ak clearly identified Asp237 as the main anchor residue for the P1 site of I-Ak, with Ile240, Ala242, Val243, and Ala245 possibly serving as secondary binding residues. On the other hand, three TCR contact residues were found to be critical for the stimulation of individual T cell hybridomas. Asn238 and Lys241 were necessary for all three hybridomas, Gln239 was necessary for two, and Pro244 for one. Interestingly, alanine substitution of Gln239, but not of Asn238 or Lys241, rendered T cell hybridoma Ci39 refractory to subsequent stimulation with WT peptide 234–246. This observation entails practical importance and warrants the search for antagonist peptides (see later) capable of attenuating the T cell response to the Sm-p40 egg Ag.

Surprisingly, alanine substitution of Asn238, Gln239, and Lys241 also caused a striking loss of stimulation of polyclonal Sm-p40-specific CD4+ Th cells obtained from infected mice, while the substitution of Pro244 had a lesser effect; the same substitute peptides also caused a parallel drop in IL-2 secretion. These findings suggest that the hybridomas are representative of the total specific T cell population and clearly indicate that, collectively, Asn238, Gln239, and Lys241 represent the key TCR contact residues required for the unabridged polyclonal T cell response against immunodominant peptide 234–246. As expected, replacement of MHC anchor residue Asp237 also caused a reduction, but not an abrogation, of T cell stimulation, further suggesting that peptide D237A retained a weak capacity to bind I-Ak via secondary anchor residues. Because of the possible role of flanking residues in the outcome of the T cell response (20), we investigated the effect of adding the WT residues Val232 and Arg233 at the N terminus, or Gln247 and Ala248 at the C terminus of peptide 234–246, and found that these 15 mers did not significantly alter the response of either the hybridomas or the polyclonal CD4+ Th cells (data not shown).

Although critical contact residues vary greatly in relation to the corresponding MHC and TCR molecules (21), our results with peptide 234–246 are in agreement with other foreign or autologous peptides isolated from I-Ak, in which an aspartic acid (or asparagine) close to the amino terminus similarly serves as the main (P1) anchor residue (18, 22). Furthermore, the TCR contact residues of peptide 234–246 are displayed in a manner comparable to that established for hen egg lysozyme’s peptide 50–62 (23). Based on the I-Ak binding studies and the T cell responses, Fig. 9Go represents a hypothetical model depicting a section with Sm-p40 peptide 234–246 in the crystallographically determined structure of I-Ak. The figure illustrates the major I-Ak anchor residue Asp237 (D), as well as the TCR contact residues Asn238 (N), Gln239 (Q), Lys241 (K), and Pro244 (P).



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  		FIGURE 9. Three-dimensional model of Sm-p40 peptide 234–246 in I-Ak. The image represents a section showing peptide 234–246 in I-Ak. It is based on the crystallographically determined structure of I-Ak containing peptide 50–62 from hen egg lysozyme (HEL) (23 ). The sequence of HEL peptide 50–62 STDYGILQINSRW was replaced with that of Sm-p40 peptide 234–246 PKSDNQIKAVPAS. Side chains of the altered amino acids were arranged arbitrarily in energetically favorable positions. No further energy minimization was performed, so the backbone remains positioned as in HEL. Side chain replacement and rotamer optimization were performed with Swiss PDB Viewer (Nicolas Guex, Glaxo Wellcome Experimental Research, Geneva, Switzerland), and images were generated with RasMol (Roger Sayle, Metaphorics, Santa Fe, NM) and labeled with LViewPro.

 
Single amino acid substitutions have proved to be useful, as in this case for the analysis of peptide interactions with MHC molecules and TCRs. However, some of these altered peptides have by themselves been found to exert profound biological effects on the corresponding T cell populations, variously resulting in T cell unresponsiveness (24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35) or in reduced or altered cytokine secretion (36, 37, 38, 39, 40). Most importantly, such in vitro effects have been extended to in vivo systems, in which altered peptides significantly ameliorated the outcome of T cell-mediated autoimmune disease (41, 42, 43, 44, 45, 46, 47, 48). Whether a similar approach can be useful in circumstances where the T cell response is directed against a limited amount of immunodominant epitopes from a foreign Ag, such as Sm-p40, is presently unknown and must await further experimentation.

The significance of our study lies in the fact that peptide 234–246 is derived from a highly immunogenic egg Ag, and that it represents the main target of the anti-Sm-p40 response in schistosome-infected H-2k mice. Additional interest in the anti-Sm-p40 T cell response resides in its apparent association with enhanced pathology, although the ultimate magnitude of the schistosome infection is clearly influenced by unrelated genetic factors (14, 49).


   Acknowledgments
 
We thank Dr. Emil Unanue for performing the binding studies, for helpful advice, and for critically reading the manuscript; and Dr. Eric Martz for generating the color three-dimensional image.


   Footnotes
 
1 This work was supported in part by U.S. Public Service Grant 18919, and by United Nations Development Program/World Bank World Health Organization Special Program for Research and Training in Tropical Diseases. Schistosome-infected snails were supplied through National Institutes of Health-National Institute of Allergy and Infectious Diseases Contract N01-AI-55270. Recombinant Sm-p40 Ag was produced with the generous help of the GRASP Digestive Disease Center (National Institute of Diabetes and Digestive and Kidney Diseases, P30 DK34928). Back

2 Address correspondence and reprint requests to Dr. Miguel J. Stadecker, Department of Pathology, Tufts University School of Medicine, 136 Harrison Avenue, Boston, MA 02111. E-mail address: Back

3 Abbreviations used in this paper: SEA, schistosomal egg Ags; WT, wild type; RIC, relative inhibitory capacity. Back

Received for publication April 8, 1999. Accepted for publication July 16, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Mathew, R. C., D. L. Boros. 1986. Anti-L3T4 antibody treatment suppresses hepatic granuloma formation and abrogates Ag-induced interleukin-2 production in Schistosoma mansoni infection. Infect. Immun. 54:820.[Abstract/Free Full Text]
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