INFECTION AND IMMUNITY, Mar. 1991, p. 941-948
Vol. 59, No. 3
0019-9567/91/030941-08$02.00/0 Copyright X) 1991, American Society for Microbiology
Splenic and Granuloma T-Lymphocyte Responses to Fractionated Soluble Egg Antigens of Schistosoma mansoni-Infected Mice NICHOLAS W. LUKACS AND DOV L. BOROS* Department of Immunology and Microbiology, Wayne State University School of Medicine, Detroit, Michigan 48201 Received 24 September 1990/Accepted 14 December 1990
Soluble egg antigens (SEA) secreted by the eggs of Schistosoma mansoni worms induce a T-cell-mediated granulomatous response that is principally responsible for the pathology of the disease. In the present study sodium dodecyl sulfate-polyacrylamide gel electrophoresis-separated SEA proteins were divided into nine fractions (200 kDa), electroeluted, and utilized in in vitro lymphoproliferation assays. T-cell-enriched spleen cells from acutely infected mice responded to all nine fractions, while those from chronically infected mice responded to only the 50- to 56- and the 60- to 66-kDa fractions. Depletion of the CD4+ T-cell subset among acute and chronic-infection spleen cells abrogated the response. Depletion of the CD8+ T-cell population resulted in increased proliferation in response to fractions by acute-infection T cells and facilitated responsiveness to hitherto-inactive SEA fractions in chronic-infection T cells. Acute-infection CD4+ granuloma T cells responded to the 40- to 46-, 50- to 56-, 70to 90-, 93- to 125-, and >200-kDa fractions, while the chronic-infection granuloma T cells responded only to the >200-kDa fraction of SEA. Selective depletion of the CD4+ T-cell subset when acute-infection granuloma lymphocytes were tested abrogated proliferation, whereas subset depletions when chronic-infection granuloma cells were tested indicated that both CD4+ and CD8+ T cells respond to the >200-kDa fraction. The present study reveals differences between acute- and chronic-infection splenic and granuloma T cells in the pattern of T-cell blastogenic responses to fractionated SEA.
T-cell-mediated responses to SEA-derived antigens at the acute and chronic stages of disease has not yet been done. In the present study, we utilized electrophoretic separation and electroelution for the fractionation of crude SEA preparations and compared the in vitro proliferative responses of splenic and granuloma T lymphocytes at the acute and chronic stages of infection. We present data that demonstrate differences in the pattern of lymphoproliferation between spleen and granuloma T lymphocytes and their subsets at both stages of infection. These data may reflect differences in local versus peripheral responses to SEA.
The pathology of Schistosoma mansoni infection is attributed mostly to the T-cell-mediated host immune response to disseminating eggs that are lodged in the liver and intestines of the infected hosts (4, 37, 40, 41). The miracidia within the eggs secrete soluble egg antigens (SEA) that induce a T-cellmediated granulomatous response. In the murine system, the peak intensity of the granulomatous inflammatory response occurs between 8 and 10 weeks postinfection. After 12 to 16 weeks, the intensity of the response gradually diminishes, and by 20 weeks of infection, the granulomas are immunologically down-modulated by a circuitry of antigenspecific suppressor T cells and their soluble factors (1, 5, 13, 14, 16, 17, 21, 30, 35, 36). Crude SEA isolated from homogenized eggs have been shown to induce and elicit granuloma formation and dermal responses in experimental animals (7). This preparation is very heterogeneous and contains numerous proteins, glycoproteins, polysaccharides, and glycolipids (3, 9, 44). In the past, several attempts have been made to characterize the function of the different antigenic moieties of SEA. Primary separation of SEA by concanavalin A affinity chromatography produced three different serologically active (glyco)protein antigens (34). The glycoprotein fractions of SEA also elicited in vitro splenic T-cell proliferative responses (10). By using preparative electrophoresis and affinity chromatography, a number of negatively or positively charged SEA fractions were shown to induce granuloma formation (6). Although a number of studies have characterized isolated glycoproteins from crude SEA by serological (20, 22, 33, 34, 42) and dermal (6, 8, 29, 33) reactivity, as well as granuloma induction-elicitation capacity (6, 29, 42), a comparison of splenic and granuloma *
MATERIALS AND METHODS Mice. Female CBA/J (H-2k) mice purchased from Jackson Laboratories, Bar Harbor, Maine, were used throughout the study. The mice were maintained under standard laboratory care.
Infection. Mice 6 to 8 weeks old were infected subcutaneously with 25 cercariae of the Puerto Rican strain of S. mansoni and were examined 8 and 20 weeks after the infection. Preparation of SEA. Eggs were isolated from mice infected with 200 cercariae (15). SEA were prepared as previously described (7). Antibodies and reagents. For phenotypic characterizations of Thyl.2+ cells, a fluorescein isothiocyanate-labeled antiThyl.2 mouse monoclonal antibody (MAb; Becton Dickinson, Mountain View, Calif.) was used. For Lyt-2.1+ and L3T4+ cell depletion, the immunoglobulin M and G2a MAbs (3.155.2 [ATCC TIB211] and GK1.5 [ATCC TIB207], respectively; American Type Culture Collection, Rockville,
Corresponding author. 941
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LUKACS AND BOROS
Md.) were used. B cells were depleted by a rabbit antimouse immunoglobulin serum specific for heavy and light chains (Miles Scientific, Naperville, Ill.). T-cell subsets were depleted with the respective MAb and rabbit complement (Low-Tox-M diluted 1:20 in cytotoxicity medium; Cedarlane, Ontario, Canada). Electrophoresis and electroelution of SEA fractions. SEA prepared from eggs of S. mansoni-infected hamsters was supplied through the courtesy of Clint Carter of Vanderbilt University, Nashville, Tenn. SEA was separated on a 12% sodium dodecyl sulfate (SDS)-polyacrylamide gel (100 ,ug per lane) under nonreduced conditions (27). The use of a 12% gel allowed the separation of antigens ranging from 20 to >200 kDa. To ensure that no proteins were excluded in the 12% polyacrylamide gels, 7.5% gels were also used for separation. Such gels revealed no additional proteins of higher molecular weights. For the preparation of electroeluted protein fractions, the polyacrylamide gel was stained (0.1% Coomassie blue in 40% methanol-10% acetic acid) and bands were identified. In addition, a lane containing standard molecular weight markers was also stained. The Coomassie blue-stained bands of SEA were used as a reference to localize the SEA proteins on an unstained acrylamide gel. The unstained lanes were cut into nine regions comprising the 200-kDa fractions. The proteins were electroeluted from gel slices with 8 to 10 mA per gel fraction at 4°C for 3 to 4 h (Bio-Rad, Richmond, Calif.). The effluents of a particular fraction were pooled from three consecutive elutions and dialyzed overnight in phosphatebuffered saline (pH 7.2) by using dialysis tubing with a 12- to 14-kDa exclusion. The dialyzed fractions were sterilized with a 0.22-,um-pore-size syringe filter, and the protein concentration was determined with a Bio-Rad protein assay kit. The protein concentration of each fraction was adjusted to 6 to 8 p,g/ml. A blank elution consisting of polyacrylamide gel devoid of proteins was also processed and used as a control fraction. The individual fractions were separated by SDS-polyacrylamide gel electrophoresis (PAGE) on a 12% gel and silver stained (Quick-Silver; Amersham) to ascertain that only the Coomassie blue-stained proteins were present in each fraction. Spleen cell preparation. Single-cell suspensions were prepared from spleens of normal and acutely and chronically infected mice. Erythrocytes were lysed with Tris-ammonium chloride, and the normal and acute-infection cells were passed over nylon wool columns (26). The relative percentage of acute-infection T cells in the nylon wool nonadherent fraction was 80 to 90% as determined by Thyl.2 MAb and immunofluorescence staining. Because the response of the spleen T cells from the chronically infected mice could not be enriched by nylon wool columns, B cells were depleted from spleen cells of chronically infected mice by treatment with rabbit anti-mouse immunoglobulin serum and complement. After depletion of the B cells, the percentage of T cells rose from a range of 30 to 35% to a range of 50 to
55%. Granuloma T-cell isolation. Granuloma T cells were isolated from collagenase enzyme-dispersed liver granulomas as previously described (38). The washed granuloma cells were plated at a concentration of 5 x 106 cells per ml onto plastic tissue culture-grade petri dishes for 60 to 90 min for removal of macrophages. Wright's stain revealed that the nonadherent fraction of granuloma cells contained 5% macrophages, 12 to 15% lymphocytes, and 65 to 75% eosino-
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25-30 [ 2) in the presence of all nine SEA fractions. The 32to 38- and 93- to 125-kDa fractions elicited the strongest response (PI of 5.8 and 4.9, respectively). The indices for the other seven fractions (200 kDa) ranged from 2.5 to 4.0. In contrast, the T-cell-enriched population from chronically infected mice significantly (E/C > 2) proliferated in response to only the 40- to 46- and 50- to 56-kDa fractions. The other seven fractions (200 kDa) did not elicit significant proliferative responses (PI of 1.3 to 1.7). To demonstrate the specificity of the response to the SEA fractions, all nine fractions were tested with splenic T cells from normal mice. As the results in Fig. 2 demonstrate, normal splenic T cells did not significantly proliferate (E/C < 2) in response to any of the SEA fractions. Proliferation of acute- and chronic-infection splenic T-cell subsets to SEA fractions. To determine the reactivity of different T-cell subsets to the fractions of SEA that elicited proliferation, T-cell-enriched populations were treated with either anti-L3T4 or anti-Lyt 2 antibody and complement. The results in Fig. 3A show that the PI of L3T4+-depleted acute-infection T cells to the 200-kDa fractions significantly decreased (P < 0.05) compared with the unseparated T-cell response. Conversely, elimination of the Lyt 2+ subset significantly increased the PI in cultures stimulated with the 32- to 38-, 40- to 46-, 60- to 66-, 70- to 90-, and 93- to 125-kDa fractions (P < 0.05). Figure 3B shows that the PI of L3T4+-depleted chronicinfection splenic T cells stimulated with the 40- to 46- and 50to 56-kDa fractions significantly decreased. Depletion of the Lyt 2+ population caused a significant increase in PI only in response to the 50- to 56-kDa fraction (P < 0.05 when compared with the unseparated T-cell response). Therefore, we tested whether depletion of the Lyt 2+ subset would significantly increase the proliferation of chronic-infection splenic T cells in response to those fractions which hitherto had showed no reactivity (E/C < 2). The results in Fig. 4 show that the 200-kDa fractions, which did not elicit proliferation from the whole T-cell population, elicited significant proliferation after Lyt 2+ cell depletion (PI of 2.2 to 3.2). Comparison of acute- and chronic-infection granuloma T-cell and T-cell subset responsiveness to SEA fractions. Isolated granuloma T cells were examined for reactivity to the nine SEA fractions. As Fig. 5 illustrates, acute-infection granuloma T cells proliferated to the 93- to 125- and the >200-kDa fractions (PI of 2.2 and 2.5), whereas chronicinfection granuloma T cells responded to only the >200-kDa SEA fraction (PI of 2.0). Selective depletion of the L3T4+ population from acute-infection granuloma T cells significantly abrogated proliferation to the 93- to 125- and >200kDa fractions (PI of 1.3 to 1.5; P < 0.05 when compared with unseparated T-cell populations) (Fig. 6). After removal of the Lyt 2+ subset, the response to the same two fractions remained unchanged (P > 0.05). T lymphocytes from the modulated granulomas showed no decrease in proliferation in response to the >200-kDa fraction after depletion of either the L3T4+ or the Lyt 2+ subset of T cells (PI of 1.5 and 1.6; P > 0.05). However, double depletion of both L3T4+ and Lyt 2+ T-cell subsets significantly decreased proliferation in response to the >200-kDa fraction from a PI of 3.0 to a PI of 1.6. Because unresponsiveness in chronic-infection splenic T cells was alleviated after elimination of Lyt 2+ cells, the granuloma T cells were similarly treated and then were exposed to the nonstimulatory fractions of SEA. Figure 7A shows that after Lyt 2+ subset depletion, the acute-infection granuloma T-cell responses to the 40- to 46-, 50- to 56-, and 70- to 90-kDa fractions increased (PI of >2.0). A similar increase in proliferation was not seen in the Lyt 2-depleted chronic-infection granuloma T-cell population (Fig. 7B).
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FIG. 6. Effect of T-cell subset depletion on granuloma T-cell responsiveness to SEA fractions. Bars represent means of three experiments for acute-infection cell preparations and means of two experiments for chronic-infection cell preparations. Background proliferation ranged from 8,000 to 16,000 cpm for acute-infection and 4,300 to 6,300 for chronic-infection granuloma T-cell subset depletions. In each experiment, liver granulomas pooled from at least three mice were used. Symbols: E, granuloma T cell; X, L3T4 T-cell depletion; Lyt 2 T-cell depletion. SEM, Standard error of the mean. U,
DISCUSSION In this study we compared splenic and granuloma T-cell proliferative responses of acutely and chronically infected mice to SEA fractions. This was accomplished by the use of nonreducing conditions during SDS-PAGE separation that ensured identification of proteins in their native configurations. The total number of separated protein and glycoprotein bands obtained was comparable to that reported earlier (9). Separated, electroeluted proteins proved to be useful reagents because their soluble forms allowed quantitation and interfraction comparisons of lymphocyte proliferation. Comparison of proliferative profiles of splenic T cells from acutely and chronically infected mice revealed both quantitative and qualitative differences. All pooled antigen fractions ranging from 200 kDa in size elicited proliferation by acute-infection splenic T lymphocytes, whereas only the 40- to 46- and 50- to 56-kDa fractions elicited proliferation by chronic-infection splenic T cells (Fig. 2). Moreover, the response was much greater in cultures of cells from acutely infected mice than in those of cells from chronically infected mice. This confirms earlier observations obtained with crude, unseparated SEA (16). Depletion of the CD4+ T-cell population significantly decreased proliferation in response to SEA fractions in both acute- and chronic-infection splenic T-cell populations, indi-
cating that the CD4+ subset is the major responder to the various fractionated antigens contained within SEA (Fig. 3). The demonstration that CD8+ subset-depleted chronic-infection splenic T cells could respond to all of the SEA fractions (Fig. 4) indicates that the acute- and chronicinfection CD4+ T cells demonstrate qualitatively similar patterns of antigen responsiveness. Depletion of CD8+ T cells enriched the CD4+ T-cell population and may have decreased competition for interleukin-2 (45) and other growth factors, thereby increasing responsiveness of the CD4+ T-cell population. Alternatively, increased CD4+ T-cell responses after removal of CD8+ T cells may suggest a regulatory role for CD8+ T cells. This concept is supported by several studies implying a role for CD8+ T cells as suppressor cells in egg granulomatous responses (11, 14, 18, 19, 38) and production of inflammatory migration inhibition factor (12, 13). In contrast to the reactivity of acute- and chronic-infection splenic T cells, granuloma T lymphocytes showed a more limited range of antigen reactivity. Acute-infection CD4+ granuloma T cells demonstrated significant responses to only the mid- and higher-molecular-weight fractions (40 to 46, 50 to 56, 70 to 90, 93 to 125, and >200 kDa). Selective subset depletion again showed that CD4+ T cells constitute the major antigen-reactive subset in the vigorous granuloma. T lymphocytes from modulated chronic-infection granulomas
T-CELL REACTIVITY TO SEA FRACTIONS
VOL. 59, 1991
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FIG. 7. Effect of Lyt 2+ subset depletion on acute-infection (A) and chronic-infection (B) granuloma T-cell responsiveness to SEA fractions. Bars represent means of three experiments for acuteinfection cell preparations and means of two experiments for chronic-infection cell preparations. Background proliferation after depletions ranged from 6,800 to 17,000 cpm for acute-infection cells and from 3,800 to 7,000 cpm for chronic-infection granuloma cells. In each experiment, liver granulomas pooled from at least three mice Lyt 2 T-cell deplewere used. Symbols: 1, granuloma T cell; tion. SEM, Standard error of the mean. U,
showed very limited antigen responsiveness, having proliferated in response to only the >200-kDa protein fraction (Fig. 5). Because only depletion of both CD4+ and CD8+ T-cell subsets abrogated responses to the >200-kDa fraction, we conclude that both subsets proliferate in response to this moiety within the chronic-infection granuloma T lymphocytes. The limited range of antigen proliferation demonstrated by modulated granuloma lymphocytes is striking. Whether this range is caused by acquired hyporesponsiveness (23, 31, 39) or active down-regulation by CD8+ lymphocytes in the modulated granuloma (38) needs further investigation. However, if active regulation of CD4+ T-cell proliferation is operative within modulated granuloma cells, it is not readily demonstrated by selective depletion of the CD8+ T-cell subset.
947
The fact that splenic lymphocytes have a broader range of antigen recognition than their granuloma counterparts is noteworthy. This difference may be due in part to differences in previous antigen exposure. Granuloma T cells are thought to have been exposed only to locally secreted egg antigens, whereas putative cross-reactive splenic T cells that had been exposed to various antigens from the developmental stages of the parasite (43) may also respond to SEA. This assumption is based on data which demonstrated antibody crossreactivity between schistosomal and egg antigen epitopes (2). More relevant, both a protective MAb (25) and chronicinfection sera (32) cross-reacted with the >200-kDa fraction of egg and schistosomal antigen. In the past, several studies investigated granulomatous (29, 34, 42), dermal (8, 29, 33), or lymphoproliferative (34) responses to SEA fractions. Without exception, several fractions of SEA were found to possess biological activity. In a recent study, acute- and chronic-infection lymphocyte responsiveness to SEA moieties separated by isoelectric focusing was compared (24). Data showed that acute-infection lymph node lymphocytes expressed greater reactivity to acidic SEA moieties, whereas chronic-infection cells responded better to the near-neutral antigenic fractions. These biologically active moieties ranged from 27 to >200 kDa in size. The present study confirms and extends the foregoing observations, demonstrating that with the progress of the infection, both qualitative and quantitative changes occur in the pattern of antigen responsiveness in both splenic and granuloma T cells. The initial separation of SEA into nine broad fractions has allowed a comparison of splenic and granuloma helpereffector T-cell proliferation at the acute and chronic stages of infection. Definition by blastogenesis of T-cell reactivity to SEA fractions is the first step in identifying the antigen(s) that participates in granuloma formation and regulation. Further resolution of the antigenic fractions into homogeneous moieties will facilitate the characterization of the role of purified antigens in the granulomatous process. ACKNOWLEDGMENT This work was supported by Public Health Service grant Al12913. REFERENCES 1. Abe, T., and D. G. Colley. 1984. Modulation of Schistosoma mansoni egg-induced granuloma formation. III. Evidence for an anti-idiotypic, I-J-positive, I-J-restricted, soluble T suppressor factor. J. Immunol. 132:2084-2088. 2. Bickle, Q. D., M. J. Ford, and B. J. Andrews. 1983. Studies on the development of anti-schistosomular surface antibodies by mice exposed to irradiated cercariae, adults and/or eggs of S. mansoni. Parasite Immunol. 5:499-505. 3. Boctor, F. N., T. E. Nash, and A. W. Cheever. 1979. Isolation of a polysaccharide antigen from Schistosoma mansoni eggs. J. Immunol. 122:39-43. 4. Boros, D. L. 1989. Immunopathology of Schistosoma mansoni infection. Clin. Microbiol. Rev. 2:250-269. 5. Boros, D. L., R. P. Pelley, and K. S. Warren. 1975. Spontaneous modulation of granulomatous hypersensitivity in schistosomiasis mansoni. J. Immunol. 114:1437-1441. 6. Boros, D. L., R. Tomford, and K. S. Warren. 1977. Induction of granulomatous and elicitation of cutaneous sensitivity by partially purified SEA of Schistosoma mansoni. J. Immunol. 118: 373-376. 7. Boros, D. L., and K. S. Warren. 1970. Delayed hypersensitivity granuloma formation and dermal reaction induced and elicited by a soluble factor isolated from Schistosoma mansoni eggs. J. Exp. Med. 132:488-507.
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8. Brown, A. P., H. G. Remold, K. S. Warren, and J. R. David. 1977. Partial purification of antigens from eggs of Schistosoma mansoni that elicit delayed hypersensitivity. J. Immunol. 119: 1275-1278. 9. Carter, C. E., and D. G. Colley. 1978. An electrophoretic analysis of Schistosoma mansoni soluble egg antigen preparation. J. Parasitol. 64:385-389. 10. Carter, C. E., and D. G. Colley. 1979. Partial purification of Schistosoma mansoni soluble egg antigen with Con A-Sepharose chromatography. J. Immunol. 122:2204-2209. 11. Chensue, S. W., and D. L. Boros. 1979. Modulation of granulomatous hypersensitivity. I. Characterization of T lymphocytes involved in the adoptive suppression of granuloma formation in Schistosoma mansoni-infected mice. J. Immunol. 123:1409-1414. 12. Chensue, S. W., D. L. Boros, and C. S. David. 1980. Regulation of granulomatous inflammation in murine schistosomiasis: in vitro characterization of T lymphocyte subsets involved in the production and suppression of migration inhibition factor (MIF). J. Exp. Med. 157:1398-1412. 13. Chensue, S. W., D. L. Boros, and C. S. David. 1983. Regulation of granulomatous inflammation in murine schistosomiasis. II. T suppressor cell-derived, I-C subregion-encoded soluble suppressor factor mediates regulation of lymphokine production. J. Exp. Med. 157:219-230. 14. Chensue, S. W., S. R. Wellhausen, and D. L. Boros. 1981. Modulation of granulomatous hypersensitivity. II. Participation of Lyl+ and Ly2+ T lymphocytes in the suppression of granuloma formation and lymphokine production in Schistosoma mansoni infected mice. J. Immunol. 127:363-367. 15. Coker, C. M., and F. von Lichtenberg. 1956. A revised method for isolation of Schistosoma mansoni eggs for biological experimentation. Proc. Soc. Exp. Biol. Med. 92:780-782. 16. Colley, D. G. 1975. Immune responses to a soluble schistosomal egg antigen preparation during chronic primary infection with Schistosoma mansoni. J. Immunol. 115:150-156. 17. Colley, D. G., F. A. Lewis, and C. W. Todd. 1979. Adoptive suppression of granuloma formation by T lymphocytes and by lymphoid cells sensitive to cyclophosphamide. Cell. Immunol. 46:192-207. 18. Doughty, B. L., and S. M. Phillips. 1982. Delayed hypersensitivity granuloma formation around Schistosoma mansoni eggs in vitro. I. Definition of the model. J. Immunol. 128:30-36. 19. Doughty, B. L., and S. M. Phillips. 1982. Delayed hypersensitivity granuloma formation around Schistosoma mansoni eggs in vitro. II. Regulatory T cell subsets. J. Immunol. 128:37-42. 20. Dunne, D. W., S. Lucas, Q. Bickle, S. Pearson, L. Madgwick, J. Bain, and M. J. Doenhoff. 1981. Identification and partial purification of an antigen w, from Schistosoma mansoni eggs which is putatively hepatotoxic in T-cell deprived mice. Trans. R. Soc. Trop. Med. Hyg. 75:54-71. 21. Green, W. F., and D. G. Colley. 1981. Modulation of Schistosoma mansoni egg-induced granuloma formation: I-J restriction of T cell-mediated suppression in a chronic parasitic infection. Proc. Nati. Acad. Sci. USA 78:1152-1156. 22. Hamburger, J., S. Lustigman, T. K. A. Siongok, J. H. Ouma, and A. A. F. Mahmoud. 1982. Characterization of a purified glycoprotein from Schistosoma mansoni eggs: specificity, stability, and the involvement of carbohydrate and peptide moieties in its serological activity. J. Immunol. 128:1864-1869. 23. Hang, L. M., D. L. Boros, and K. S. Warren. 1974. Induction of
immunological hyporesponsiveness to granulomatous hypersensitivity in Schistosoma mansoni infection. J. Infect. Dis. 130: 515-522. 24. Harn, D. A., K. Danko, J. J. Quinn, and M. J. Stadecker. 1989. Schistosoma mansoni: the host immune response to egg antigens. I. Partial characterization of cellular and humoral responses to pl fractions of soluble egg antigen. J. Immunol. 142:2061-2066. 25. Harn, D. A., M. Mitsuyama, and J. R. David. 1984. Schistosoma mansoni: anti-egg monoclonal antibodies protect against cercarial challenge in vivo. J. Exp. Med. 159:1371-1387. 26. Julius, M. H., E. Simpson, and L. A. Herzenberg. 1973. A rapid
INFECT. IMMUN.
method for the isolation of functional thymus-derived murine lymphocytes. Eur. J. Immunol. 3:645-649. 27. Laemmli, U. K. 1971. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227:680-685. 28. Lee, S. P., N. G. Stoker, K. A. Grant, Z. T. Handzel, R. Hussain, K. P. W. J. McAdam, and H. M. Dockrell. 1989. Cellular immune responses of leprosy contacts to fractionated Mycobacterium leprae antigens. Infect. Immun. 57:2475-2480. 29. Lustigman, S., A. A. F. Mahmoud, and J. Hamburger. 1985. Glycoproteins in soluble egg antigen of Schistosoma mansoni: isolation, characterization, and elucidation of their immunochemical and immunopathological relation to the major egg glycoprotein (MEG). J. Immunol. 134:1961-1967. 30. Mathew, R. C., and D. L. Boros. 1986. Regulation of granulomatous inflammation in murine schistosomiasis. III. Recruitment of antigen-specific I-J' T suppressor cells of the granulomatous response by I-J' soluble suppressor factor. J. Immunol. 136:1093-1099. 31. Nossal, G. J. V. 1989. Immunological tolerance: collaboration between antigen and lymphokines. Science 245:147-153. 32. Omer-Ali, P. O., S. R. Smithers, Q. Bickle, S. M. Phillips, D. Harn, and A. J. G. Simpson. 1988. Analysis of the antiSchistosoma mansoni surface antibody response during murine infection and its potential contribution to protective immunity. J. Immunol. 140:3273-3279. 33. Owhashi, M., Y. Horii, J. Imai, A. Ishii, and Y. Nawa. 1986. Purification and physicochemical characterization of Schistosoma mansoni egg allergen recognized by mouse sera obtained at an acute stage of infection. Int. Arch. Allergy Appl. Immunol. 81:129-135. 34. Pelley, R. P., R. J. Pelley, J. Hamburger, P. A. Peters, and K. S. Warren. 1976. Schistosoma mansoni soluble egg antigens. I. Identification and purification of three major antigens, and the employment of radioimmunoassay for their further characterization. J. Immunol. 117:1553-1560. 35. Perrin, P. J., and S. M. Phillips. 1988. The molecular basis of granuloma formation in schistosomiasis. I. A T cell-derived suppressor effector factor. J. Immunol. 141:1714-1719. 36. Perrin, P. J., M. B. Prystowsky, and S. M. Phillips. 1989. The molecular basis of granuloma formation in schistosomiasis. II. Analogies of a T cell-derived suppressor effector factor to the T cell receptor. J. Immunol. 142:985-991. 37. Phillips, S. M., and P. Lammie. 1986. Immunopathology of granuloma formation and fibrosis in schistosomiasis. Parasitol. Today 2:296-302. 38. Ragheb, S., and D. L. Boros. 1989. Characterization of granuloma T lymphocyte function from Schistosoma mansoni-infected mice. J. Immunol. 142:3239-3246. 39. Sercarz, E., A. Oki, and G. Gammon. 1989. Central versus peripheral tolerance: clonal inactivation versus suppressor T cells, the second half of the 'Thirty Years War'. Immunol. Suppl. 2:9-14. 40. Warren, K. S. 1982. The secret of immunopathogenesis of schistosomiasis: in vivo models. Immunol. Rev. 61:189-213. 41. Warren, K. S., E. 0. Domingo, and R. B. T. Cowan. 1967. Granuloma formation around schistosome eggs as a manifestation of delayed hypersensitivity. Am. J. Pathol. 51:735-756. 42. Weiss, J. B., W. S. Aronstein, and M. Strand. 1987. Schistosoma mansoni: stimulation of artificial granuloma formation in vivo by carbohydrate determinants. Exp. Parasitol. 64:228-236. 43. Weiss, J. B., and M. Strand. 1985. Characterization of developmentally regulated epitopes of Schistosoma mansoni egg glycoprotein antigens. J. Immunol. 135:1421-1429. 44. Weiss, J. B., J. L. Magnani, and M. Strand. 1986. Identification of Schistosoma mansoni glycolipids that share immunogenic carbohydrate epitopes with glycoproteins. J. Immunol. 136: 4275-4282. 45. Yamashita, T., and D. L. Boros. 1990. Changing patterns of lymphocyte proliferation, IL-2 production, utilization and IL-2 receptor expression in mice infected with Schistosoma mansoni. J. Immunol. 145:724-731.
Vol. 59, No. 3
0019-9567/91/030941-08$02.00/0 Copyright X) 1991, American Society for Microbiology
Splenic and Granuloma T-Lymphocyte Responses to Fractionated Soluble Egg Antigens of Schistosoma mansoni-Infected Mice NICHOLAS W. LUKACS AND DOV L. BOROS* Department of Immunology and Microbiology, Wayne State University School of Medicine, Detroit, Michigan 48201 Received 24 September 1990/Accepted 14 December 1990
Soluble egg antigens (SEA) secreted by the eggs of Schistosoma mansoni worms induce a T-cell-mediated granulomatous response that is principally responsible for the pathology of the disease. In the present study sodium dodecyl sulfate-polyacrylamide gel electrophoresis-separated SEA proteins were divided into nine fractions (200 kDa), electroeluted, and utilized in in vitro lymphoproliferation assays. T-cell-enriched spleen cells from acutely infected mice responded to all nine fractions, while those from chronically infected mice responded to only the 50- to 56- and the 60- to 66-kDa fractions. Depletion of the CD4+ T-cell subset among acute and chronic-infection spleen cells abrogated the response. Depletion of the CD8+ T-cell population resulted in increased proliferation in response to fractions by acute-infection T cells and facilitated responsiveness to hitherto-inactive SEA fractions in chronic-infection T cells. Acute-infection CD4+ granuloma T cells responded to the 40- to 46-, 50- to 56-, 70to 90-, 93- to 125-, and >200-kDa fractions, while the chronic-infection granuloma T cells responded only to the >200-kDa fraction of SEA. Selective depletion of the CD4+ T-cell subset when acute-infection granuloma lymphocytes were tested abrogated proliferation, whereas subset depletions when chronic-infection granuloma cells were tested indicated that both CD4+ and CD8+ T cells respond to the >200-kDa fraction. The present study reveals differences between acute- and chronic-infection splenic and granuloma T cells in the pattern of T-cell blastogenic responses to fractionated SEA.
T-cell-mediated responses to SEA-derived antigens at the acute and chronic stages of disease has not yet been done. In the present study, we utilized electrophoretic separation and electroelution for the fractionation of crude SEA preparations and compared the in vitro proliferative responses of splenic and granuloma T lymphocytes at the acute and chronic stages of infection. We present data that demonstrate differences in the pattern of lymphoproliferation between spleen and granuloma T lymphocytes and their subsets at both stages of infection. These data may reflect differences in local versus peripheral responses to SEA.
The pathology of Schistosoma mansoni infection is attributed mostly to the T-cell-mediated host immune response to disseminating eggs that are lodged in the liver and intestines of the infected hosts (4, 37, 40, 41). The miracidia within the eggs secrete soluble egg antigens (SEA) that induce a T-cellmediated granulomatous response. In the murine system, the peak intensity of the granulomatous inflammatory response occurs between 8 and 10 weeks postinfection. After 12 to 16 weeks, the intensity of the response gradually diminishes, and by 20 weeks of infection, the granulomas are immunologically down-modulated by a circuitry of antigenspecific suppressor T cells and their soluble factors (1, 5, 13, 14, 16, 17, 21, 30, 35, 36). Crude SEA isolated from homogenized eggs have been shown to induce and elicit granuloma formation and dermal responses in experimental animals (7). This preparation is very heterogeneous and contains numerous proteins, glycoproteins, polysaccharides, and glycolipids (3, 9, 44). In the past, several attempts have been made to characterize the function of the different antigenic moieties of SEA. Primary separation of SEA by concanavalin A affinity chromatography produced three different serologically active (glyco)protein antigens (34). The glycoprotein fractions of SEA also elicited in vitro splenic T-cell proliferative responses (10). By using preparative electrophoresis and affinity chromatography, a number of negatively or positively charged SEA fractions were shown to induce granuloma formation (6). Although a number of studies have characterized isolated glycoproteins from crude SEA by serological (20, 22, 33, 34, 42) and dermal (6, 8, 29, 33) reactivity, as well as granuloma induction-elicitation capacity (6, 29, 42), a comparison of splenic and granuloma *
MATERIALS AND METHODS Mice. Female CBA/J (H-2k) mice purchased from Jackson Laboratories, Bar Harbor, Maine, were used throughout the study. The mice were maintained under standard laboratory care.
Infection. Mice 6 to 8 weeks old were infected subcutaneously with 25 cercariae of the Puerto Rican strain of S. mansoni and were examined 8 and 20 weeks after the infection. Preparation of SEA. Eggs were isolated from mice infected with 200 cercariae (15). SEA were prepared as previously described (7). Antibodies and reagents. For phenotypic characterizations of Thyl.2+ cells, a fluorescein isothiocyanate-labeled antiThyl.2 mouse monoclonal antibody (MAb; Becton Dickinson, Mountain View, Calif.) was used. For Lyt-2.1+ and L3T4+ cell depletion, the immunoglobulin M and G2a MAbs (3.155.2 [ATCC TIB211] and GK1.5 [ATCC TIB207], respectively; American Type Culture Collection, Rockville,
Corresponding author. 941
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LUKACS AND BOROS
Md.) were used. B cells were depleted by a rabbit antimouse immunoglobulin serum specific for heavy and light chains (Miles Scientific, Naperville, Ill.). T-cell subsets were depleted with the respective MAb and rabbit complement (Low-Tox-M diluted 1:20 in cytotoxicity medium; Cedarlane, Ontario, Canada). Electrophoresis and electroelution of SEA fractions. SEA prepared from eggs of S. mansoni-infected hamsters was supplied through the courtesy of Clint Carter of Vanderbilt University, Nashville, Tenn. SEA was separated on a 12% sodium dodecyl sulfate (SDS)-polyacrylamide gel (100 ,ug per lane) under nonreduced conditions (27). The use of a 12% gel allowed the separation of antigens ranging from 20 to >200 kDa. To ensure that no proteins were excluded in the 12% polyacrylamide gels, 7.5% gels were also used for separation. Such gels revealed no additional proteins of higher molecular weights. For the preparation of electroeluted protein fractions, the polyacrylamide gel was stained (0.1% Coomassie blue in 40% methanol-10% acetic acid) and bands were identified. In addition, a lane containing standard molecular weight markers was also stained. The Coomassie blue-stained bands of SEA were used as a reference to localize the SEA proteins on an unstained acrylamide gel. The unstained lanes were cut into nine regions comprising the 200-kDa fractions. The proteins were electroeluted from gel slices with 8 to 10 mA per gel fraction at 4°C for 3 to 4 h (Bio-Rad, Richmond, Calif.). The effluents of a particular fraction were pooled from three consecutive elutions and dialyzed overnight in phosphatebuffered saline (pH 7.2) by using dialysis tubing with a 12- to 14-kDa exclusion. The dialyzed fractions were sterilized with a 0.22-,um-pore-size syringe filter, and the protein concentration was determined with a Bio-Rad protein assay kit. The protein concentration of each fraction was adjusted to 6 to 8 p,g/ml. A blank elution consisting of polyacrylamide gel devoid of proteins was also processed and used as a control fraction. The individual fractions were separated by SDS-polyacrylamide gel electrophoresis (PAGE) on a 12% gel and silver stained (Quick-Silver; Amersham) to ascertain that only the Coomassie blue-stained proteins were present in each fraction. Spleen cell preparation. Single-cell suspensions were prepared from spleens of normal and acutely and chronically infected mice. Erythrocytes were lysed with Tris-ammonium chloride, and the normal and acute-infection cells were passed over nylon wool columns (26). The relative percentage of acute-infection T cells in the nylon wool nonadherent fraction was 80 to 90% as determined by Thyl.2 MAb and immunofluorescence staining. Because the response of the spleen T cells from the chronically infected mice could not be enriched by nylon wool columns, B cells were depleted from spleen cells of chronically infected mice by treatment with rabbit anti-mouse immunoglobulin serum and complement. After depletion of the B cells, the percentage of T cells rose from a range of 30 to 35% to a range of 50 to
55%. Granuloma T-cell isolation. Granuloma T cells were isolated from collagenase enzyme-dispersed liver granulomas as previously described (38). The washed granuloma cells were plated at a concentration of 5 x 106 cells per ml onto plastic tissue culture-grade petri dishes for 60 to 90 min for removal of macrophages. Wright's stain revealed that the nonadherent fraction of granuloma cells contained 5% macrophages, 12 to 15% lymphocytes, and 65 to 75% eosino-
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25-30 [ 2) in the presence of all nine SEA fractions. The 32to 38- and 93- to 125-kDa fractions elicited the strongest response (PI of 5.8 and 4.9, respectively). The indices for the other seven fractions (200 kDa) ranged from 2.5 to 4.0. In contrast, the T-cell-enriched population from chronically infected mice significantly (E/C > 2) proliferated in response to only the 40- to 46- and 50- to 56-kDa fractions. The other seven fractions (200 kDa) did not elicit significant proliferative responses (PI of 1.3 to 1.7). To demonstrate the specificity of the response to the SEA fractions, all nine fractions were tested with splenic T cells from normal mice. As the results in Fig. 2 demonstrate, normal splenic T cells did not significantly proliferate (E/C < 2) in response to any of the SEA fractions. Proliferation of acute- and chronic-infection splenic T-cell subsets to SEA fractions. To determine the reactivity of different T-cell subsets to the fractions of SEA that elicited proliferation, T-cell-enriched populations were treated with either anti-L3T4 or anti-Lyt 2 antibody and complement. The results in Fig. 3A show that the PI of L3T4+-depleted acute-infection T cells to the 200-kDa fractions significantly decreased (P < 0.05) compared with the unseparated T-cell response. Conversely, elimination of the Lyt 2+ subset significantly increased the PI in cultures stimulated with the 32- to 38-, 40- to 46-, 60- to 66-, 70- to 90-, and 93- to 125-kDa fractions (P < 0.05). Figure 3B shows that the PI of L3T4+-depleted chronicinfection splenic T cells stimulated with the 40- to 46- and 50to 56-kDa fractions significantly decreased. Depletion of the Lyt 2+ population caused a significant increase in PI only in response to the 50- to 56-kDa fraction (P < 0.05 when compared with the unseparated T-cell response). Therefore, we tested whether depletion of the Lyt 2+ subset would significantly increase the proliferation of chronic-infection splenic T cells in response to those fractions which hitherto had showed no reactivity (E/C < 2). The results in Fig. 4 show that the 200-kDa fractions, which did not elicit proliferation from the whole T-cell population, elicited significant proliferation after Lyt 2+ cell depletion (PI of 2.2 to 3.2). Comparison of acute- and chronic-infection granuloma T-cell and T-cell subset responsiveness to SEA fractions. Isolated granuloma T cells were examined for reactivity to the nine SEA fractions. As Fig. 5 illustrates, acute-infection granuloma T cells proliferated to the 93- to 125- and the >200-kDa fractions (PI of 2.2 and 2.5), whereas chronicinfection granuloma T cells responded to only the >200-kDa SEA fraction (PI of 2.0). Selective depletion of the L3T4+ population from acute-infection granuloma T cells significantly abrogated proliferation to the 93- to 125- and >200kDa fractions (PI of 1.3 to 1.5; P < 0.05 when compared with unseparated T-cell populations) (Fig. 6). After removal of the Lyt 2+ subset, the response to the same two fractions remained unchanged (P > 0.05). T lymphocytes from the modulated granulomas showed no decrease in proliferation in response to the >200-kDa fraction after depletion of either the L3T4+ or the Lyt 2+ subset of T cells (PI of 1.5 and 1.6; P > 0.05). However, double depletion of both L3T4+ and Lyt 2+ T-cell subsets significantly decreased proliferation in response to the >200-kDa fraction from a PI of 3.0 to a PI of 1.6. Because unresponsiveness in chronic-infection splenic T cells was alleviated after elimination of Lyt 2+ cells, the granuloma T cells were similarly treated and then were exposed to the nonstimulatory fractions of SEA. Figure 7A shows that after Lyt 2+ subset depletion, the acute-infection granuloma T-cell responses to the 40- to 46-, 50- to 56-, and 70- to 90-kDa fractions increased (PI of >2.0). A similar increase in proliferation was not seen in the Lyt 2-depleted chronic-infection granuloma T-cell population (Fig. 7B).
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FIG. 6. Effect of T-cell subset depletion on granuloma T-cell responsiveness to SEA fractions. Bars represent means of three experiments for acute-infection cell preparations and means of two experiments for chronic-infection cell preparations. Background proliferation ranged from 8,000 to 16,000 cpm for acute-infection and 4,300 to 6,300 for chronic-infection granuloma T-cell subset depletions. In each experiment, liver granulomas pooled from at least three mice were used. Symbols: E, granuloma T cell; X, L3T4 T-cell depletion; Lyt 2 T-cell depletion. SEM, Standard error of the mean. U,
DISCUSSION In this study we compared splenic and granuloma T-cell proliferative responses of acutely and chronically infected mice to SEA fractions. This was accomplished by the use of nonreducing conditions during SDS-PAGE separation that ensured identification of proteins in their native configurations. The total number of separated protein and glycoprotein bands obtained was comparable to that reported earlier (9). Separated, electroeluted proteins proved to be useful reagents because their soluble forms allowed quantitation and interfraction comparisons of lymphocyte proliferation. Comparison of proliferative profiles of splenic T cells from acutely and chronically infected mice revealed both quantitative and qualitative differences. All pooled antigen fractions ranging from 200 kDa in size elicited proliferation by acute-infection splenic T lymphocytes, whereas only the 40- to 46- and 50- to 56-kDa fractions elicited proliferation by chronic-infection splenic T cells (Fig. 2). Moreover, the response was much greater in cultures of cells from acutely infected mice than in those of cells from chronically infected mice. This confirms earlier observations obtained with crude, unseparated SEA (16). Depletion of the CD4+ T-cell population significantly decreased proliferation in response to SEA fractions in both acute- and chronic-infection splenic T-cell populations, indi-
cating that the CD4+ subset is the major responder to the various fractionated antigens contained within SEA (Fig. 3). The demonstration that CD8+ subset-depleted chronic-infection splenic T cells could respond to all of the SEA fractions (Fig. 4) indicates that the acute- and chronicinfection CD4+ T cells demonstrate qualitatively similar patterns of antigen responsiveness. Depletion of CD8+ T cells enriched the CD4+ T-cell population and may have decreased competition for interleukin-2 (45) and other growth factors, thereby increasing responsiveness of the CD4+ T-cell population. Alternatively, increased CD4+ T-cell responses after removal of CD8+ T cells may suggest a regulatory role for CD8+ T cells. This concept is supported by several studies implying a role for CD8+ T cells as suppressor cells in egg granulomatous responses (11, 14, 18, 19, 38) and production of inflammatory migration inhibition factor (12, 13). In contrast to the reactivity of acute- and chronic-infection splenic T cells, granuloma T lymphocytes showed a more limited range of antigen reactivity. Acute-infection CD4+ granuloma T cells demonstrated significant responses to only the mid- and higher-molecular-weight fractions (40 to 46, 50 to 56, 70 to 90, 93 to 125, and >200 kDa). Selective subset depletion again showed that CD4+ T cells constitute the major antigen-reactive subset in the vigorous granuloma. T lymphocytes from modulated chronic-infection granulomas
T-CELL REACTIVITY TO SEA FRACTIONS
VOL. 59, 1991
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FIG. 7. Effect of Lyt 2+ subset depletion on acute-infection (A) and chronic-infection (B) granuloma T-cell responsiveness to SEA fractions. Bars represent means of three experiments for acuteinfection cell preparations and means of two experiments for chronic-infection cell preparations. Background proliferation after depletions ranged from 6,800 to 17,000 cpm for acute-infection cells and from 3,800 to 7,000 cpm for chronic-infection granuloma cells. In each experiment, liver granulomas pooled from at least three mice Lyt 2 T-cell deplewere used. Symbols: 1, granuloma T cell; tion. SEM, Standard error of the mean. U,
showed very limited antigen responsiveness, having proliferated in response to only the >200-kDa protein fraction (Fig. 5). Because only depletion of both CD4+ and CD8+ T-cell subsets abrogated responses to the >200-kDa fraction, we conclude that both subsets proliferate in response to this moiety within the chronic-infection granuloma T lymphocytes. The limited range of antigen proliferation demonstrated by modulated granuloma lymphocytes is striking. Whether this range is caused by acquired hyporesponsiveness (23, 31, 39) or active down-regulation by CD8+ lymphocytes in the modulated granuloma (38) needs further investigation. However, if active regulation of CD4+ T-cell proliferation is operative within modulated granuloma cells, it is not readily demonstrated by selective depletion of the CD8+ T-cell subset.
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The fact that splenic lymphocytes have a broader range of antigen recognition than their granuloma counterparts is noteworthy. This difference may be due in part to differences in previous antigen exposure. Granuloma T cells are thought to have been exposed only to locally secreted egg antigens, whereas putative cross-reactive splenic T cells that had been exposed to various antigens from the developmental stages of the parasite (43) may also respond to SEA. This assumption is based on data which demonstrated antibody crossreactivity between schistosomal and egg antigen epitopes (2). More relevant, both a protective MAb (25) and chronicinfection sera (32) cross-reacted with the >200-kDa fraction of egg and schistosomal antigen. In the past, several studies investigated granulomatous (29, 34, 42), dermal (8, 29, 33), or lymphoproliferative (34) responses to SEA fractions. Without exception, several fractions of SEA were found to possess biological activity. In a recent study, acute- and chronic-infection lymphocyte responsiveness to SEA moieties separated by isoelectric focusing was compared (24). Data showed that acute-infection lymph node lymphocytes expressed greater reactivity to acidic SEA moieties, whereas chronic-infection cells responded better to the near-neutral antigenic fractions. These biologically active moieties ranged from 27 to >200 kDa in size. The present study confirms and extends the foregoing observations, demonstrating that with the progress of the infection, both qualitative and quantitative changes occur in the pattern of antigen responsiveness in both splenic and granuloma T cells. The initial separation of SEA into nine broad fractions has allowed a comparison of splenic and granuloma helpereffector T-cell proliferation at the acute and chronic stages of infection. Definition by blastogenesis of T-cell reactivity to SEA fractions is the first step in identifying the antigen(s) that participates in granuloma formation and regulation. Further resolution of the antigenic fractions into homogeneous moieties will facilitate the characterization of the role of purified antigens in the granulomatous process. ACKNOWLEDGMENT This work was supported by Public Health Service grant Al12913. REFERENCES 1. Abe, T., and D. G. Colley. 1984. Modulation of Schistosoma mansoni egg-induced granuloma formation. III. Evidence for an anti-idiotypic, I-J-positive, I-J-restricted, soluble T suppressor factor. J. Immunol. 132:2084-2088. 2. Bickle, Q. D., M. J. Ford, and B. J. Andrews. 1983. Studies on the development of anti-schistosomular surface antibodies by mice exposed to irradiated cercariae, adults and/or eggs of S. mansoni. Parasite Immunol. 5:499-505. 3. Boctor, F. N., T. E. Nash, and A. W. Cheever. 1979. Isolation of a polysaccharide antigen from Schistosoma mansoni eggs. J. Immunol. 122:39-43. 4. Boros, D. L. 1989. Immunopathology of Schistosoma mansoni infection. Clin. Microbiol. Rev. 2:250-269. 5. Boros, D. L., R. P. Pelley, and K. S. Warren. 1975. Spontaneous modulation of granulomatous hypersensitivity in schistosomiasis mansoni. J. Immunol. 114:1437-1441. 6. Boros, D. L., R. Tomford, and K. S. Warren. 1977. Induction of granulomatous and elicitation of cutaneous sensitivity by partially purified SEA of Schistosoma mansoni. J. Immunol. 118: 373-376. 7. Boros, D. L., and K. S. Warren. 1970. Delayed hypersensitivity granuloma formation and dermal reaction induced and elicited by a soluble factor isolated from Schistosoma mansoni eggs. J. Exp. Med. 132:488-507.
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INFECT. IMMUN.
method for the isolation of functional thymus-derived murine lymphocytes. Eur. J. Immunol. 3:645-649. 27. Laemmli, U. K. 1971. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227:680-685. 28. Lee, S. P., N. G. Stoker, K. A. Grant, Z. T. Handzel, R. Hussain, K. P. W. J. McAdam, and H. M. Dockrell. 1989. Cellular immune responses of leprosy contacts to fractionated Mycobacterium leprae antigens. Infect. Immun. 57:2475-2480. 29. Lustigman, S., A. A. F. Mahmoud, and J. Hamburger. 1985. Glycoproteins in soluble egg antigen of Schistosoma mansoni: isolation, characterization, and elucidation of their immunochemical and immunopathological relation to the major egg glycoprotein (MEG). J. Immunol. 134:1961-1967. 30. Mathew, R. C., and D. L. Boros. 1986. Regulation of granulomatous inflammation in murine schistosomiasis. III. Recruitment of antigen-specific I-J' T suppressor cells of the granulomatous response by I-J' soluble suppressor factor. J. Immunol. 136:1093-1099. 31. Nossal, G. J. V. 1989. Immunological tolerance: collaboration between antigen and lymphokines. Science 245:147-153. 32. Omer-Ali, P. O., S. R. Smithers, Q. Bickle, S. M. Phillips, D. Harn, and A. J. G. Simpson. 1988. Analysis of the antiSchistosoma mansoni surface antibody response during murine infection and its potential contribution to protective immunity. J. Immunol. 140:3273-3279. 33. Owhashi, M., Y. Horii, J. Imai, A. Ishii, and Y. Nawa. 1986. Purification and physicochemical characterization of Schistosoma mansoni egg allergen recognized by mouse sera obtained at an acute stage of infection. Int. Arch. Allergy Appl. Immunol. 81:129-135. 34. Pelley, R. P., R. J. Pelley, J. Hamburger, P. A. Peters, and K. S. Warren. 1976. Schistosoma mansoni soluble egg antigens. I. Identification and purification of three major antigens, and the employment of radioimmunoassay for their further characterization. J. Immunol. 117:1553-1560. 35. Perrin, P. J., and S. M. Phillips. 1988. The molecular basis of granuloma formation in schistosomiasis. I. A T cell-derived suppressor effector factor. J. Immunol. 141:1714-1719. 36. Perrin, P. J., M. B. Prystowsky, and S. M. Phillips. 1989. The molecular basis of granuloma formation in schistosomiasis. II. Analogies of a T cell-derived suppressor effector factor to the T cell receptor. J. Immunol. 142:985-991. 37. Phillips, S. M., and P. Lammie. 1986. Immunopathology of granuloma formation and fibrosis in schistosomiasis. Parasitol. Today 2:296-302. 38. Ragheb, S., and D. L. Boros. 1989. Characterization of granuloma T lymphocyte function from Schistosoma mansoni-infected mice. J. Immunol. 142:3239-3246. 39. Sercarz, E., A. Oki, and G. Gammon. 1989. Central versus peripheral tolerance: clonal inactivation versus suppressor T cells, the second half of the 'Thirty Years War'. Immunol. Suppl. 2:9-14. 40. Warren, K. S. 1982. The secret of immunopathogenesis of schistosomiasis: in vivo models. Immunol. Rev. 61:189-213. 41. Warren, K. S., E. 0. Domingo, and R. B. T. Cowan. 1967. Granuloma formation around schistosome eggs as a manifestation of delayed hypersensitivity. Am. J. Pathol. 51:735-756. 42. Weiss, J. B., W. S. Aronstein, and M. Strand. 1987. Schistosoma mansoni: stimulation of artificial granuloma formation in vivo by carbohydrate determinants. Exp. Parasitol. 64:228-236. 43. Weiss, J. B., and M. Strand. 1985. Characterization of developmentally regulated epitopes of Schistosoma mansoni egg glycoprotein antigens. J. Immunol. 135:1421-1429. 44. Weiss, J. B., J. L. Magnani, and M. Strand. 1986. Identification of Schistosoma mansoni glycolipids that share immunogenic carbohydrate epitopes with glycoproteins. J. Immunol. 136: 4275-4282. 45. Yamashita, T., and D. L. Boros. 1990. Changing patterns of lymphocyte proliferation, IL-2 production, utilization and IL-2 receptor expression in mice infected with Schistosoma mansoni. J. Immunol. 145:724-731.
