Splenic and Granuloma T-Lymphocyte Responses to Fractionated Soluble Egg Antigens of Schistosoma mansoni-infected Mice

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1 INFECTION AND IMMUNITY, Mar. 1991, p /91/3941-8$2./ Copyright X) 1991, American Society for Microbiology Vol. 59, No. 3 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 4821 Received 24 September 199/Accepted 14 December 199 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 (<21, 25 to 3, 32 to 38, 4 to 46, 5 to 56, 6 to 66, 7 to 9, 93 to 125, and >2 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 5- to 56- and the 6- 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 4- to 46-, 5- to 56-, 7- to 9-, 93- to 125-, and >2-kDa fractions, while the chronic-infection granuloma T cells responded only to the >2-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 >2-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. 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, 4, 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 1 weeks postinfection. After 12 to 16 weeks, the intensity of the response gradually diminishes, and by 2 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, 3, 35, 36). Crude SEA isolated from homogenized eggs have been shown to induce and elicit granuloma formation and dermal responses in eperimental 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 (1). 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 (2, 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 * Corresponding author. 941 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. 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 eamined 8 and 2 weeks after the infection. Preparation of SEA. Eggs were isolated from mice infected with 2 cercariae (15). SEA were prepared as previously described (7). Antibodies and reagents. For phenotypic characterizations of Thyl.2+ cells, a fluorescein isothiocyanate-labeled anti- Thyl.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 ( [ATCC TIB211] and GK1.5 [ATCC TIB27], respectively; American Type Culture Collection, Rockville, Downloaded from on January 25, 219 by guest

2 942 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-To-M diluted 1:2 in cytotoicity 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 (1,ug per lane) under nonreduced conditions (27). The use of a 12% gel allowed the separation of antigens ranging from 2 to >2 kda. To ensure that no proteins were ecluded 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 (.1% Coomassie blue in 4% methanol-1% 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 <21, 25- to 3-, 3- to 38-, 4- to 46-, 5- to 56-, 6- to 66-, 7- to 9-, 93- to 125-, and >2-kDa fractions. The proteins were electroeluted from gel slices with 8 to 1 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 eclusion. The dialyzed fractions were sterilized with a.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 8 to 9% 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 3 to 35% to a range of 5 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 16 cells per ml onto plastic tissue culture-grade petri dishes for 6 to 9 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- > 2 _u [ 6-66k _Q" r, =,I _ l-: [ <21 [ ~92 fi4 42 _t 3 _t 21 INFECT. IMMUN. FIG. 1. Coomassie blue staining of SDS-PAGE-separated crude SEA (left) and molecular weight standards (right). SEA was loaded on a 4% stacking gel and a 12% separating gel at 1 Lg per lane. Numbers at the left of the gel indicate the division into fractions of separated SEA for the electroelution process. phils. Granuloma T cells could not be further enriched, as previously demonstrated (38). Phenotype determination. T-cell-enriched spleen cells and granuloma T cells from acutely and chronically infected mice were treated with anti-l3t4 or anti-lyt 2 MAb and complement to deplete CD4+ and CD8+ T cells, respectively. Cells at a concentration of 2 17/ml were incubated with the individual antibodies for 3 to 6 min at 4 C and pelleted in a refrigerated centrifuge. The pelleted cells were then suspended and incubated for 45 to 6 min in complement (1/2 dilution). At the end of the incubation, cells were washed twice in Hanks balanced salt solution and resuspended at 4 16 cells per ml for spleen cells and 8 16 cells per ml for granuloma cells. Proliferation assays. Separated T-cell populations were suspended in RPMI 164 supplemented with 5% fetal calf serum (GIBCO Laboratories, Grand Island, N.Y.), i-5 M 2-mercaptoethanol (Eastman Organic Chemical, Rochester, N.Y.), 2 mm sodium pyruvate, 2 mm HEPES (N-2-hydroyethylpiperazine-N'-2-ethanesulfonic acid) buffer, 2 mm glutamine, 1 U of penicillin per ml, and 1,ug of streptomycin per ml (all from M. A. Bioproducts, Walkersville, Md.). Cells were plated in triplicate into 96-well flat-bottom plates (Corning Glass Works, Corning, N.Y.) at a concentration of 4 1' cells per ml for spleen cells and 8 16 cells per ml for granuloma cells in a volume of 5,u (respectively, 2 15 or 4 15 per well). The eluted SEA fractions to be tested were added at 5,lI per well. The volume of each well was raised to 2,ul with medium. Downloaded from on January 25, 219 by guest

3 VOL. 59, 1991 T-CELL REACTIVITY TO SEA FRACTIONS 943 ce L U = +1 3tS 3o E ~~~~ -~ ~ CY Nl C')V ) C- Y FIG. 2. Comparison of acute-infection (), chronic-infection (U), and normal (U) splenic T-lymphocyte responsiveness to SEA fractions. Background proliferation responses of unstimulated acute- and chronic-infection cells ranged from 2,5 to 1, and 3,2 to 11, cpm, respectively. Normal proliferation was 4,1 cpm. Acute-infection cell responses to unseparated SEA ranged from 5, to 1,, chronic-infection cell responses ranged from 33, to 5, cpm, and normal spleen cell response was 5,8 cpm. Each bar represents the mean of three eperiments for the acute- and chronic-infection cells or a representive eperiment for normal spleen response. In each eperiment, spleens pooled from at least three mice were used. SEM, Standard error of the mean. The proliferative responses of acute-infection splenic T cells to hamster and murine SEA were comparable. Thus, to save hamster-derived SEA for fractionation, murine SEA was used as a positive control. Negative controls consisted of cells alone and cells with the blank gel control. After 4 days of culture at 37 C in 5% C2, the cells were pulsed with 1,Ci of [3H]thymidine (ICN Radiochemicals, Irvine, Calif.) for 12 to 16 h and harvested onto glass wool filters (type A/E; Gelman Sciences Inc., Ann Arbor, Mich.). The dried filters were immersed in 1 ml of scintillation cocktail (Research Products International, Mount Prospect, Ill.) and measured for radioactivity in a liquid scintillation counter (Beckman Instruments, Irvine, Calif.). To compare group profiles of reactivity to different fractions, the results are epressed as stimulation indices. Significant proliferation to a particular fraction was defined as at least a twofold increase in counts of [3H]thymidine per minute over the response of the blank control (eperimental results/control results [E/C]. 2) (28). Results from three eperiments were pooled and epressed as the mean proliferation inde (PI). Statistical analysis. An unpaired Student's t test was used to eamine the T-cell subset depletion data. P <.5 was considered a significant difference in T-cell depletion eperiments. RESULTS Fractionation of SEA by SDS-PAGE separation and electroelution. Crude SEA preparation (1,ug of protein per well) was separated by SDS-PAGE and fractionated by electroelution based on molecular weight distribution. Figure 1 shows that hamster-derived SEA could be separated into 15 to 22 different protein bands, as demonstrated by Coomassie blue staining of the 12% gel. Similar banding patterns were seen with murine-derived SEA. Furthermore, proliferation of acute-infection splenic T cells in response to hamster (94,93 cpm) and murine (85,82 cpm) SEA was comparable. The protein bands were divided into nine different fractions (<21, 25 to 3, 32 to 38, 4 to 46, 5 to 56, 6 to 66, 7 to 9, 93 to 125, and >2 kda) by using a stained reference lane to localize the protein bands. Each fraction contained two to four visible Coomassie blue-stained bands (Fig. 1). The SEA fractions retained biological activity when kept at 4 C for 2 weeks but lost significant activity after a single freeze-thaw cycle. Comparison of acute- and chronic-infection splenic T-cell responsiveness to SEA fractions. T-cell-enriched splenic cells from acutely and chronically infected mice were tested for proliferative responses to SEA fractions. Figure 2 demonstrates that acute-infection T cells proliferated significantly Downloaded from on January 25, 219 by guest

4 944 LUKACS AND BOROS C ) - M +I c ll) (n A B FIG. 3. Effect of T-cell subset depletion on acute-infection (A) and chronic-infection (B) splenic T-cell responses to SEA fractions. Bars represent the means of three eperiments. Background proliferation ranged from 3,4 to 11, and 2,5 to 13, cpm for acute- and chronic-infection T-cell-subset-depleted populations, respectively. In each eperiment, spleens pooled from at least three mice were used. Symbols:, splenic T cells; U, L3T4 T-cell depletion; Lyt 2 T-cell depletion. SEM, Standard error of the mean. 1 (E/C > 2) in the presence of all nine SEA fractions. The 32- to 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 (<21, 25 to 3, 32 to 38, 6 to 66, 7 to 9, 93 to 125, and >2 kda) ranged from 2.5 to 4.. In contrast, the T-cell-enriched population from chronically infected mice significantly (E/C > 2) proliferated in response to only the 4- to 46- and 5- to 56-kDa fractions. The other seven fractions (<21, 25 to 3, 32 to 38, 6 to 66, 7 to 9, 93 T INFECT. IMMUN. to 125, and >2 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 <21, 25- to 3-, 32- to 38-, 4- to 46-, 5- to 56-, 6- to 66-, 7- to 9-, 93- to 125-, and >2-kDa fractions significantly decreased (P <.5) 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-, 4- to 46-, 6- to 66-, 7- to 9-, and 93- to 125-kDa fractions (P <.5). Figure 3B shows that the PI of L3T4+-depleted chronicinfection splenic T cells stimulated with the 4- to 46- and 5- to 56-kDa fractions significantly decreased. Depletion of the Lyt 2+ population caused a significant increase in PI only in response to the 5- to 56-kDa fraction (P <.5 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 <21-, 25- to 3-, 32- to 38-, 6- to 66-, 7- to 9-, 93- to 125-, and >2-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 eamined for reactivity to the nine SEA fractions. As Fig. 5 illustrates, acute-infection granuloma T cells proliferated to the 93- to 125- and the >2-kDa fractions (PI of 2.2 and 2.5), whereas chronicinfection granuloma T cells responded to only the >2-kDa SEA fraction (PI of 2.). Selective depletion of the L3T4+ population from acute-infection granuloma T cells significantly abrogated proliferation to the 93- to 125- and >2- kda fractions (PI of 1.3 to 1.5; P <.5 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 >.5). T lymphocytes from the modulated granulomas showed no decrease in proliferation in response to the >2-kDa fraction after depletion of either the L3T4+ or the Lyt 2+ subset of T cells (PI of 1.5 and 1.6; P >.5). However, double depletion of both L3T4+ and Lyt 2+ T-cell subsets significantly decreased proliferation in response to the >2-kDa fraction from a PI of 3. to a PI of 1.6 Ḃecause 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 eposed to the nonstimulatory fractions of SEA. Figure 7A shows that after Lyt 2+ subset depletion, the acute-infection granuloma T-cell responses to the 4- to 46-, 5- to 56-, and 7- to 9-kDa fractions increased (PI of >2.). A similar increase in proliferation was not seen in the Lyt 2-depleted chronic-infection granuloma T-cell population (Fig. 7B). Downloaded from on January 25, 219 by guest

5 ollil =- +1 co oci)~ ~~~ 2 D O U CY CO) C) CD Y ) N N; CY) C.) w7d-.a r-~~~~~~~~~~~~ FIG. 4. Effect of Lyt 2 subset depletion on chronic-infection splenic T-cell responsiveness to SEA fractions. Bars represent the means of three eperiments. Background proliferation ranged from 3, to 12, cpm. In each eperiment, spleens pooled from at least three mice were used. Symbols: E, splenic T cells; E, Lyt 2 T-cell depletion. SEM, Standard error of the mean. 3 Downloaded from a).+ _la Cl - C:3.- 2 on January 25, 219 by guest - N c) Go.) CD s CD U) CD so ) Net V N cn C.) U) CD r- U) N\ _ C) as A FIG. 5. Comparison of acute-infection () and chronic-infection (E) granuloma T-cell responsiveness to SEA fractions. Background proliferation for acute- and chronic-infection cells ranged from 7,3 to 16, and 4, to 7, cpm, respectively. Responses to whole SEA ranged from 45, to 5, and 1, to 2,, respectively. Bars represent the means of three eperiments. In each eperiment, liver granulomas pooled from at least three mice were used. SEM, Standard error of the mean. 945

6 946 LUKACS AND BOROS INFECT. IMMUN. 1) C 4-3- T T CU. nw C,) +1 CU E 2 o Acute A >2 >2 T Chronic FIG. 6. Effect of T-cell subset depletion on granuloma T-cell responsiveness to SEA fractions. Bars represent means of three eperiments for acute-infection cell preparations and means of two eperiments for chronic-infection cell preparations. Background proliferation ranged from 8, to 16, cpm for acute-infection and 4,3 to 6,3 for chronic-infection granuloma T-cell subset depletions. In each eperiment, liver granulomas pooled from at least three mice were used. Symbols: E, granuloma T cell; X, L3T4 T-cell depletion; U, Lyt 2 T-cell depletion. SEM, Standard error of the mean. 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 <2 to >2 kda in size elicited proliferation by acute-infection splenic T lymphocytes, whereas only the 4- to 46- and 5- 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, indicating 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 (4 to 46, 5 to 56, 7 to 9, 93 to 125, and >2 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 Downloaded from on January 25, 219 by guest

7 VOL. 59, 1991 ) V _ a W Ul - +l L. r = 4) 76 e a.- C: _ +1 C = 2 A - N c, ( (D CD (D a - col\ O O OD O 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 eperiments for acuteinfection cell preparations and means of two eperiments for chronic-infection cell preparations. Background proliferation after depletions ranged from 6,8 to 17, cpm for acute-infection cells and from 3,8 to 7, cpm for chronic-infection granuloma cells. In each eperiment, liver granulomas pooled from at least three mice were used. Symbols: 1, granuloma T cell; U, Lyt 2 T-cell depletion. SEM, Standard error of the mean. showed very limited antigen responsiveness, having proliferated in response to only the >2-kDa protein fraction (Fig. 5). Because only depletion of both CD4+ and CD8+ T-cell subsets abrogated responses to the >2-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. B T-CELL REACTIVITY TO SEA FRACTIONS 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 eposure. Granuloma T cells are thought to have been eposed only to locally secreted egg antigens, whereas putative cross-reactive splenic T cells that had been eposed 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 >2-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 eception, 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 epressed 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 >2 kda in size. The present study confirms and etends 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 Al REFERENCES 1. Abe, T., and D. G. Colley 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: Bickle, Q. D., M. J. Ford, and B. J. Andrews Studies on the development of anti-schistosomular surface antibodies by mice eposed to irradiated cercariae, adults and/or eggs of S. mansoni. Parasite Immunol. 5: Boctor, F. N., T. E. Nash, and A. W. Cheever Isolation of a polysaccharide antigen from Schistosoma mansoni eggs. J. Immunol. 122: Boros, D. L Immunopathology of Schistosoma mansoni infection. Clin. Microbiol. Rev. 2: Boros, D. L., R. P. Pelley, and K. S. 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