CLINICAL
IMMUNOLOGY
AND
IMMUNOPATHOLOGY
Vol. 65, No. 3, December, pp. 325-329, 1992
BRIEF COMMUNICATION Human EBV-Transformed Lymphocytes of Patients with Schistosoma japonicum Infection Secrete Idiotypically Related Immunoregulatory Antibodies THOMAS
Department *Department
F. KRESINA,
of Medicine, of Immunology,
LAURA W. CHEEVER,~MONIQUE CHIREAU,~JOAN JOHNSON, BERNADETTERAMIREZ,* PIERRE PETERS, AND G. RICHARDOLDS Miriam Hospital, Brown University International Health Institute, Research Institute for Tropical Medicine, Alabang Muntinlupa,
Lymphocytes derived from the peripheral blood of individuals infected with Schistosomajaponica were transformed in uittw with Ebstein-Barr virus (EBV). Serological characterization of antibody molecules revealed both antigen reactive (idiotypic) and anti-idiotypic transformants. One idiotypic EBV transformant, LOZC2, describesa major crossreactive idiotype associated with anti-antigen binding molecules. Other antibody populations expressing idiotypic cross-reactivity were derived from separate individuals showing shared idiotypy in 5’.japonicum field study populations in the Republic of Philippines. Both idiotypic and antiidiotypic molecules suppressed parasite antigen-driven blastogenesisof heterologous human peripheral blood lymphocytes. The data show a serologically related immunoregulatory immune network in patients in the Republic of the Philippines which is serologically distinct from idiotypy expressedin other selectedS. japonicum endemic areas in the Far East. o MU Academic PIW, IIIC. INTRODUCTION
Schistosomiasis is a helminthic infection characterized by granulomatous inflammation around deposited parasite eggs (1). This inflammatory response has been characterized as a delayed-type hypersensitivity response to soluble egg antigen(s) mediated by T cells. Modulation of granulomatous inflammation occurs, at least in murine models of disease, as the disease progresses from acute to chronic stages. In murine Schistosoma juponicum infection, modulation is mediated in part by suppressive serum components (2-4). Adoptive transfer of serum from chronically infected mice reduces granulomatous inflammation and portal pres’ Current address: Department of Medicine, University of California, San Francisco, CA 94143. ’ Current address: Department of Obstetrics and Gynecology, Yale University School of Medicine, New Haven, CT 06520.
Providence, Metro Manila,
Rhode Island 02906; and Republic of the Philippines
sure of acutely infected recipient animals. Zn vitro suppression of soluble egg antigen (SEA)-induced blastogenesis has been demonstrated by anti-SEA (idiotypic) and anti-idiotypic antibody molecules (4). A major cross-reactive idiotype @J-CR&,& was noted to be expressed on murine anti-SEA antibody molecules in acute infection. Anti-idiotypic (anti-SJ-GRIM) murine antibody molecules were expressed in chronic infection at the time of reduced granulomatous inflammation (5). Furthermore, in uiuo administration of antiidiotypic molecules revealed granulomas with reduced areas of inflammation. Such data from animal studies have inferred a role for immune network components in human Schistosomiusis juponica infection. Recent studies (6, 7) from the laboratory have focused on the human immune repertoire of individuals infected with S. japonicum from both the Philippines and the People’s Republic of China. These studies have shown differing antigenic profiles of antibodies derived from individuals in two endemic regions of these countries. Additional studies (8) have shown that the prevalence of hepatomegaly on Jishan Island (People’s Republic of China) is significantly higher than that found in the Philippines. These observations taken together cause one to question the role of antibody molecules present in the infected populations in immunoregulation of chronic disease. In addition, it is unknown if these molecules are serologically related. To this point, our recent studies (6, 7) have shown that both antibodies derived from EBV-transformed splenic cells of a Chinese patient with S. juponicum and a derived human monoclonal antibody modulate in vitro S. japonicum splenic cell responses. However, no studies have addressed the serological relatedness of these immunoregulatory Chinese antibodies and comparable Philippine-derived molecules. Thus, the present studies describes idiotypic and anti-idiotypic antibodies derived from EBV-transformed peripheral
325 0090-1229/92
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Copyright 0 1992 by Academic Press, Inc.
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326
BRIEF
COMMUNICATION
blood lymphocytes from Philippine patients with S. juponicum. The immunoregulatory function of these antibodies is described using an in vitro human blastogenesis assay. In addition, their serological relatedness to Chinese S. juponicum idiotypy is presented. MATERIALS
AND
METHODS
Patients. Individuals were identified as S. juponicum infected as part of the Republic of Philippines
Schistosomiasis Control Program which has been part of a g-year study of infected villages on the island of Leyte. Antigens. CFI mice (Charles River Breeding Laboratories, Cambridge, MA) were infected with 50 cercariae of the Philippine strain of S. juponicum at Lowell University (Lowell, MA) provided by National Institute of Allergy and Immune Disease Supply Contract AI-02636. At 8-12 weeks the livers and intestines were removed and the parasite worms and eggs were isolated and processed for antigen as described previously (9). S. juponicum adult worms were obtained by liver perfusion of these animals and a freeze-thawed homogenate extract was utilized to obtain antigen as described (9). EBV transformation. Human peripheral blood lymphocytes were obtained in Leyte, Republic of the Philippines, as part of the National Schistosomiasis Control Program and village surveillance. Lymphocytes were separated on Sepracel, frozen in liquid nitrogen, shipped to Brown University on dry ice, and EBVtransformed as routinely performed in the laboratory (6, 7). In brief, lymphocytes were incubated in EBV containing culture medium derived from the B95-8 marmoset cell line. Cells were plated in 24-well plates at a density of 2 x lo5 cells/well and incubated for 7 days prior to feeding. Cultures containing B cell blasts were screened for anti-antigen (idiotypic) binding molecules by an ELISA (4,7) and for anti-idiotypic binding by a idiotype-inhibition assay (10). ELISA assay. Ninety-six-well microtiter plates were coated with either 2 pg/well of SWAP (soluble worm antigenic protein) or 1 p,g/well of SEA diluted in 100 ~1 of PBS and incubated overnight at 4°C. Plates were subsequently washed with PBS 0.05% Tween 20 and blocked with 200 kl/well of 5% BSA in PBS overnight at 4°C. One hundred microliters of EBV culture supernatant was added per well, and plates were incubated for 2 hr at room temperature. Plates were washed with PBS in 0.05% Tween 20 and 200 ~1 of a 1:2000 dilution of horseradish peroxidase-conjugated goat anti-human IgG was added per well. Following a 40-min incubation at room temperature, plates were washed with PBS-0.05% Tween 20, substrate was added, and the optical density was read at 492 nm. Idiotype-inhibition
assay.
Anti-idiotypic
activity
was measured as the inhibition of binding of LO2C2 (idiotype) to SWAP (antigen) by competitive ELISA (10). Limiting dilutions of LO2C2 were preincubated with 50 ~1 culture supernatant from different EBV cell lines for 2 hr at room temperature. The samples were subsequently incubated in antigen-coated microtiter plates as previously described. Inhibition binding curves were generated from the serial dilution of culture supernate. The percentage of idiotype recognized by anti-idiotypic antibody was calculated from a standard curve using serial dilutions of LO2C2 antibody. Duplicate samples were utilized in each experiment, each performed twice. Lymphocyte-proliferation assay. Individuais noted to have previous schistosome infection based on the g-year follow-up study were used as cell donors (11). PBL were isolated using Seprocil and incubated on the island of Leyte in a 37°C incubator. Medium contained RPM1 1640 (MA Bioproducts, Walkerville KS) with 10% heat-inactivated fetal calf serum supplemented with antibiotics, L-glutamine, and Na biocarbonate buffer. Aliquots of 2 x lo5 cells in 200 ~1 of medium were added to 96-well microtiter plates. To each well with 5 pg/ml SEA (final concentration) or 10 kg/ml SWAP (final concentration) or PHA (10 kg/ml), streptolysin-0 (SLO, 1:lOOO dilution) and antibody derived from EBV-transformed cells were added. Cultures were incubated for 4 days, pulsed for 18 hr with 1 p.Ci c3Hlthymidine, and harvested in a mash semiautomated cell harvester. Proliferation was determined as mean percentage inhibition of isotope uptake of triplicate samples. Replicate experiments were performed as noted based on the availability of donor cells.
Antibodies secreted by EBVWestern blot analysis. transformed cells (LO2C2) were analyzed for antigen binding by immunoblot as described previously (7). In brief, soluble worm antigens from the S. juponicum subspecies from the Philippines were separated using a 10% polyacrylamide-SDS gel. Antibody binding was detailed by using a 1:lOO dilution of horseradish peroxidase-conjugated goat anti-human immunoglobulin overnight at room temperature followed by incubation with 4-chloro-1-naphthol. RESULTS
AND
DISCUSSION
As part of a continuing Schistosomiasis National Control Program in the Republic of the Philippines (111, inhabitants of the island of Leyte were routinely screened for schistosome infection on a yearly basis. Individuals with differing disease parameters were identified and peripheral blood lymphocytes were obtained and EBV-transformed. Two individuals coded LO2 and L012, respectively, were noted to have active infections based on positive Kato smears showing 10 and 40 eggs per gram of stool. Patient LO2 was a 30-
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year-old male with no hepatosplenomegaly but a history of previous infections. Patient LO12 was a 25 year-old female with hepatosplenomegaly. Patients LO14 and LO16 exhibited negative Kato smears indicating a lack of mature infection. LO14 was a 54-yearold male with a history of previous infections, and LO16 was a 30-year-old female with a similar history of multiple infections. Anti-antigen or idiotypic antibodies were derived from EBV-transformed PBL from patients L02, L012, and L014. Anti-idiotypic antibodies were derived from EBV transformation of PBL from patients LO12 and L016. The serology of these idiotypic and antiidiotypic antibodies is presented in Table 1. Four idiotypic (anti-antigen) antibodies are represented: LO2C2 (derived from patient LO2), L012AC4 and L012BC3 (derived from patient LO12), and L014BC2 (derived from patient LO14). LO2C2 exhibited the highest ELISA readings for both SWAP and SEA. In addition, on Western blot analysis this polyclonal EBV transformant exhibited an antigen binding profile virtually identical to that of pooled serum samples from acutely infected individuals (Figure 1). Although LO2C2 was derived from an acutely infected individual, it was surprising to find an EBV-transformed population derived from a single patient which reflected the antigen binding characteristics of serum pools. However, EBV activation or transformation occurs only in immature B cells expressing the EBV receptor. Thus, samples transformed from individuals could express and expand the antibody repertoire derived from immature B cells not fully differentiated into plasma cells by acute infection. LO2C2 was chosen as a standard idiotypic reagent (anti-antigen antibody population) to test for antiidiotype binding activity. The reasons are: (1) LO2C2 exhibited an antigen binding pattern comparable to that of acutely infected human serum (Fig. 1); (2)
12 3 FIG. 1. Immunoblot analysis using S. juponicum soluble worm antigens. Lane 1 was developed using antibodies derived from nonschistosome-reactive EBV-transformed cell line; lane 2 was developed using antibodies derived from the LO2C2 EBV-transformed cell line; lane 3 was developed using pooled serum from acutely infected individuals. Antibodies derived from the EBV-transformed cell line and those of acutely infected individuals recognized similar antigens by this analysis.
LO2C2 was derived from an acutely infected patient; and (3) in the mouse system the major cross-reactive idiotype is expressed on antibodies found in acute infection (4, 5). Thus, if a cross-reactive idiotype was expressed on LO2C2 antibodies, this idiotype expression would parallel that previously characterized in murine S. juponicum infection. Three anti-idiotypic antibodies, LOlBABl and L012BD4 (derived from patient L012) and L016AC2 (derived from patient LO16), were identified. As shown in Table 1, LOlBABl and L012BD4 maximally inhibited the binding of LO2C2 to antigen at levels of 45 2 3 and 33 + 7, respectively.
TABLE 1
Serology of Idiotypic and Anti-idiotypic
Human Antibodies” Anti-idiotype (% inhibition of idiotype binding)d
EBV-transformed cell line LO2C2 L012AC4 L012BC3 L014BC2 LOlPABl L012BD4 L016AC2
Idiotype’ Serology*
SWAP
SEA
Idiotypic Idiotypic Idiotypic Idiotypic Anti-idiotypic Anti-idiotypic Anti-idiotypic
0.750 0.080 0.080 0.0 N/A N/A N/A
0.500 0.028 0.226 0.035 N/A N/A N/A
Philippine idiotype N/A N/A N/A N/A 45 33 72
Chinese idiotype N/A N/A N/A N/A 8 0 12
a Antibodies derived from EBV transformation of PBL from individuals infected with S. japonicum. b Idiotypic anti-SEA or anti-SWAP binding antibodies as determined by ELISA; anti-idiotypic antibodies exhibited no reading in idiotype ELISA assay, but inhibited binding of idiotype to antigen. e Data presented as optical density reading in ELISA. N/A-not applicable; no readings in assay. d Data presented as inhibition of binding of LO2C2 to antigen in anti-idiotype assay. N/A-not applicable; no inhibition in assay.
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BRIEF COMMUNICATION
These values are maximal inhibitory levels derived from binding curves utilizing excess anti-idiotypes. Thus, quantitatively these anti-idiotypes describe a minor cross-reactive idiotype expressed on LO2C2 antibodies. L016AC2 maximally inhibited ligand binding at a level of 72 2 lo%, thus quantitatively describing a major cross-reactive idiotype expressed on LO2C2 antibodies. It is important to note that in these latter studies both idiotypic and anti-idiotypic antibodies were derived from separate individuals with differing disease parameters. Thus, these data show the expression of an immune network composed of idiotypic and anti-idiotypic antibodies in different infected individuals in the Philippines. Further studies were performed to detail the serological relatedness between anti-idiotypes LOlBABl, L012BD4, and L016AC2 and the idiotypy expressed on anti-antigen immunoregulatory antibodies of individuals with S. juponicum infection in the People’s Republic of China (6). As shown in Table 1, the antiidiotypes which recognized a minor or major level of idiotypy on Philippine antibodies barely recognized Chinese idiotypy. Levels of recognition of the Chinese idiotype of these anti-idiotypes ranged from 0 to 12%. Thus, at least with these initial studies there appears to be little serologic cross-reactivity between antiantigen (S. juponicum) antibodies derived from patients in China and those derived from patients in the Philippines. These data agree with a previous observation of nonidentity in these antibody populations based on antigen binding of S. juponicum SWAP and SEA (7). Antibodies derived from the EBV-transformed cell lines were subsequently tested for secretion of molecules which inhibited antigen (SWAP or SEA) and mitogen-induced blastogenesis. Human lymphoid cells derived from patients with chronic schistosomiasis were utilized as effector cells to determine inhibition of antigen-induced proliferation. As shown in Table 2, idiotypic EBV-derived antibodies and specifically purified serum antibodies inhibited antigen- and mitogeninduced lymphocyte responses. Serum antibodies and idiotypic antibodies from LO2C2, L012AC4, and L012BC3 inhibited in the range of G-85% U’ < 0.05) of SWAP-induced blastogenesis. For these samples SEA-induced blastogenesis was inhibited in the range of 30-80% (P < 0.05). Mitogen-induced blastogenesis was also inhibited by specifically purified acute or chronically derived antibodies and LO2C2 and L012BC3. These data suggest a nonspecific suppression of blastogenesis mediated by idiotypic antibodies. Not all antibodies derived from EBV-transformed cell were immunoregulatory. L014BC2 showed nonsignificant levels of antigen-induced inhibition (9 and 23%, P = NS) and virtually no inhibition of mitogen-induced 13H]thymidine uptake. Idiotype EBV transformants also suppressed mitogen-induced [3H]thymidine uptake of human lymphocytes from noninfected individ-
TABLE 2
Suppression of S. japonicum Antigen-Induced Blastogenesis” ‘ic Suppression Antigen Sampleb Idiotypic Acute sera (children) Acute sera (adults) LO2C2 L012AC4 L012BC3 L014BC2 Anti-idiotypic Chronic sera (children) Chronic sera (adults) LOlBABl L012BD4 L016AC2
Mitogen
SWAP
SEA
PHA
SLO
73 + 5
29 2 16’
55 t 14
NT”
55 i 9 8Oi 11 85 f 6 77 i- 15 91-3
39 56 79 74 23
65 77 21 62 0
71 F 4
55 + 6
44 Ifr 10
NT
80 84 84 87
65 45 74 80
0 20 -+ 4” 34 2 22’ 4 -t 3’
NT NT NT NT
-t 2 i+
10 12 2 3
t t 2 t ‘-
+ t + t
12 17 3 2 6’
4 11 9 5
z ? i+
8 10 20 8
NT 65 :t 12 0 73 i 9 4 2 2’
n Human lymphocytes derived from two chronically infected individuals were incubated with individual samples and antigen or mitogen. Cells were cultured as described in the text. Data are for cells from chronically infected patients and are presented as ?SD of in. hibition of [3H]thymidine uptake of triplicate samples replicated at least twice based on availability of effector cells. b Sample inhibitor used in blastogenesis assay. Pooled serum samples were derived from acutely infected children (~7 years age, with record of first infection, no hepatic pathology) and adults (>30 years age, 2 years prior, no infection with positive infection in 1989, no hepatic pathology). Pooled serum samples were derived from chronically infected children (~7 years old, recorded infection with hepatic pathology) and adults (>30 years old, recorded infection with hepatic pathology. These samples were specifically purified using SWAP/ SEA-coupled agarose affinity chromatography. Antigen: SEA Csoluble egg antigens), 5 pgiml; SWAP (soluble worm antigens), 5 kg/ml. Mitogens: PHA (phytohemagglutinin). 50 kg/ml; SLO (streptolysin0). 1:lOOO dilution. ’ Not statistically significant suppression of blastogenesis by Student’s t test, P > 0.05. L3H]Thymidine incorporation by cells on antigen stimulation was ( ISD) SEA, 1054 -t 253 cpm; SWAP, 2394 z 740 cpm; PHA, 22,883 t 2415; SLO, 689 t 115. Nonspecific inhibition was determined by utilizing normal human serum and antibodies for EBV-transformed lymphocytes of S. mansoni patients and ranged from 3 to 7% in all assays. d NT, not tested.
uals (30-75%, P < 0.05). Schistosome specificity controls for idiotype-mediated suppression were performed with S. mansoni human monoclonal antibody F96 and EBV transformant llE2 (data not shown). These molecules did not inhibit S. juponicum SEA- or SWAP-induced blastogenesis. Anti-idiotype-mediated suppression of human antigen-induced blastogenesis is also shown in Table 2. SWAP-induced lymphocyte responses for serum antibody and EBV transformants were inhibited in the range of 70-90% (P < 0.05) and SEA-induced responses
329
BRIEF COMMUNICATION
in the range of 45-80% (P < 0.05). PHA responses were partially inhibited by serum antibody and antibody from EBV transformants LOlBABl and L012BD4 (P = NS). No inhibition of mitogen responses was noted in serum from chronically infected adults and L016AC2. Anti-idiotypic EBV transformants also did not suppress (O-9%, P = NS) mitogen-induced blastogenesis of human lymphocytes from noninfected individuals. From these limited data it can be noted that the most immunosuppressive anti-idiotypes also inhibited the idiotype-antigen binding interaction to the greatest degree. Thus, specific anti-idiotype-mediated suppression may correlate with the degree of serological expression of the human major cross-reactive idiotype associated with LO2C2. To date no studies have addressed immune network interactions in human S. japonicum infection. However, the existence of idiotype and anti-idiotype has been shown in experimentally induced and human S. munsoni infection. In murine S. mansoni SEA-specific anti-idiotypic population of molecules has been described (12). In murine and human S. munsoni, idiotype-specific antibodies stimulated anti-idiotypicinduced cellular blastogenesis (13). However, the regulatory role of these serologically based interactions remains to be elucidated. A potential role for auto-antiidiotypic antibodies in the regulation of resistance to S. mansoni infection has been reported (14). In addition, in vitro idiotypic manipulation of granulomatous inflammation expressed in schistosomiasis mansoni has been reported (15). These studies support the present data and implicate immune network immunoregulation in human schistosomiasis. The data show that antibodies derived from EBV transformants from different Philippine individuals with S. japonicum infection are serologically related and immunoregulatory. These immunoregulatory antibodies, however, are unrelated to immunoregulatory antibodies expressing Chinese S. japonicum-specific idiotypes. These data further support the dichotomy of the Philippine and Chinese immune repertoires in S. japonicum infection. ACKNOWLEDGMENTS Informed consent was obtained from patients where applicable. The present study was approved by the Miriam Hospital Human Use Institution Review Board and followed the human experimentation guidelines of the U.S. Department of Health, National Institutes of Health. The data were presented in abstract form at the 1991 National Meeting of the American Association of Physicians/American Society of Clinical Investigation/American Federation for Clinical Research, Clin. Res. 39,328A, 1991. This work was supported in part by NIH Grants ROl-AI 25167 and P-50-AI-30601. Received May 1, 1992; accepted with revision August 24, 1992
REFERENCES 1. Warren, K. S., Domingo, E. O., and Cowan, R. B. T., Granuloma formation around schistosome eggs as a manifestation of delayed hypersensitivity. Am. J. Pathol. 51, 735-741, 1967. 2. Olds, G. R., Olveda, R., Tracy, J. W., and Mahmoud, A. A. F., Adoptive transfer of modulation of granuloma formation and hepatosplenic disease in murine schistosomiasis japonica by serum from chronically infected mice. J. Zmmunol. 128, 13911393, 1982. 3. Garb, K. S., Stavitsky, A. B., Olds, G. R., Tracy, J. W., and Mahmoud, A. A. F., Immune regulation in murine Schistosomiasis japonica: Inhibition of in vitro antigen- and mitogen-induced cellular responses by splenocyte culture supernates and by purified fractions from serum of chronically infected mice. J. Zmmunol.
129, 2752-2758,1982.
4. Olds, G. R., and Kresina, T. F., Network interaction in Schistosomn japonicum infection: Identification and characterization of a serologically distinct immunoregulatory auto-anti-idiotypic antibody population. J. Clin. Invest. 76, 2338-2347, 1985. 5. Kresina, T. F., and Olds, G. R., Concomitant cellular and humoral expression of a regulatory cross-reactive idiotype in acute schistosoma japonicum infection. Infect. Zmmun. 53, 90-94, 1986.
6. Kresina, T. F., Guan, H. X., Posner, M., Wisnewski, A., and Olds, G. R., An immunoregulatory human monoclonal antibody in Schistosomiasis japonica. Hum. Antib. Hybridomus 2, 4245, 1991. 7. Kresina, T. F., Guan, H. X., Posner, M., Ramirez, B., Olds, G. R., Comparison of immune repertoires of Chinese and Philippine patients with Schistosoma japonicum. Infect. Zmmun. 59, 46984700,199l. 8. Wiest, P. M., Wu, G., Zhang, S., Yuan, J., Peters, P. A. S., McGarvey, S., Tso, M., Olveda, T. R., and Olds, G. R., Morbidity due to Schistosomiasis japonica in the People’s Republic of China. Trans. R. Sot. Trap. Med. Hyg. 86, 47-50, 1992. 9. Boros, D. L., and Warren, K. S., Delayed hypersensitivity-type granuloma formation and dermal reaction induced and elicited by a soluble factor isolated from Schistosoma mansoni eggs. J. Exp. Med. 132, 488-507, 1970. 10. Herylyn, D., Wettendortf, M., Schmoll, E., Iliopoulos, D., Schedel, I., Dreikhausen, U., Raab, R., Ross, A. H., Jakscheh, Scriba, M., and Koprowski, H., Anti-idiotype immunization of cancer patients: Modulation of the immune response. Proc. Natl. Acad. Sci. USA 84, 8055-8059, 1987. 11. Olveda, R., Wiest, P. M., and Olds, G. R., A six year prospective study of the effect of praziquantel on Schistosomiasis japonica. Clin. Res. 36, 622A, 1988. 12. Powell, M. R., and Colley, D. G., Demonstration of splenic autoanti-idiotypic plague-forming cells in mice infected with schistosoma mansoni. J. Zmmunol. 134, 4146-4145, 1985. 13. Parra, J. C., Lina, M. S., Gazzinelli, G., and Colley, D. G., Immune responses during human Schistosomiasis mansoni XV anti-idiotypic T cells can recognize and respond to anti-SEA idiotype directly. J. Zmmunol. 140, 2401-2404, 1988. 14. Phillips, S. M., Perrin, P. J., Walker, D. J., Fathelbab, N. G., Linette, G. P., and Idris, M. A., The regulation of resistance to Schistosomn mansoni by auto-anti-idiotypic immunity. J. Zmmunol. 141, 1728-1733, 1988. 15. Parra, J. C., Gazzinelli, G., Goes, A. M., Moyes, R. B., Rocha, R., Colley, D. G., and Doughty, B. L., Granulomatous hypersensitivity to Schistosoma mansoni egg antigens in human schistosomiasis. II. In vitro granuloma modulation induced by polyclonal idiotypic antibodies. J. Zmmunol. 147, 3949-3954, 1991.
IMMUNOLOGY
AND
IMMUNOPATHOLOGY
Vol. 65, No. 3, December, pp. 325-329, 1992
BRIEF COMMUNICATION Human EBV-Transformed Lymphocytes of Patients with Schistosoma japonicum Infection Secrete Idiotypically Related Immunoregulatory Antibodies THOMAS
Department *Department
F. KRESINA,
of Medicine, of Immunology,
LAURA W. CHEEVER,~MONIQUE CHIREAU,~JOAN JOHNSON, BERNADETTERAMIREZ,* PIERRE PETERS, AND G. RICHARDOLDS Miriam Hospital, Brown University International Health Institute, Research Institute for Tropical Medicine, Alabang Muntinlupa,
Lymphocytes derived from the peripheral blood of individuals infected with Schistosomajaponica were transformed in uittw with Ebstein-Barr virus (EBV). Serological characterization of antibody molecules revealed both antigen reactive (idiotypic) and anti-idiotypic transformants. One idiotypic EBV transformant, LOZC2, describesa major crossreactive idiotype associated with anti-antigen binding molecules. Other antibody populations expressing idiotypic cross-reactivity were derived from separate individuals showing shared idiotypy in 5’.japonicum field study populations in the Republic of Philippines. Both idiotypic and antiidiotypic molecules suppressed parasite antigen-driven blastogenesisof heterologous human peripheral blood lymphocytes. The data show a serologically related immunoregulatory immune network in patients in the Republic of the Philippines which is serologically distinct from idiotypy expressedin other selectedS. japonicum endemic areas in the Far East. o MU Academic PIW, IIIC. INTRODUCTION
Schistosomiasis is a helminthic infection characterized by granulomatous inflammation around deposited parasite eggs (1). This inflammatory response has been characterized as a delayed-type hypersensitivity response to soluble egg antigen(s) mediated by T cells. Modulation of granulomatous inflammation occurs, at least in murine models of disease, as the disease progresses from acute to chronic stages. In murine Schistosoma juponicum infection, modulation is mediated in part by suppressive serum components (2-4). Adoptive transfer of serum from chronically infected mice reduces granulomatous inflammation and portal pres’ Current address: Department of Medicine, University of California, San Francisco, CA 94143. ’ Current address: Department of Obstetrics and Gynecology, Yale University School of Medicine, New Haven, CT 06520.
Providence, Metro Manila,
Rhode Island 02906; and Republic of the Philippines
sure of acutely infected recipient animals. Zn vitro suppression of soluble egg antigen (SEA)-induced blastogenesis has been demonstrated by anti-SEA (idiotypic) and anti-idiotypic antibody molecules (4). A major cross-reactive idiotype @J-CR&,& was noted to be expressed on murine anti-SEA antibody molecules in acute infection. Anti-idiotypic (anti-SJ-GRIM) murine antibody molecules were expressed in chronic infection at the time of reduced granulomatous inflammation (5). Furthermore, in uiuo administration of antiidiotypic molecules revealed granulomas with reduced areas of inflammation. Such data from animal studies have inferred a role for immune network components in human Schistosomiusis juponica infection. Recent studies (6, 7) from the laboratory have focused on the human immune repertoire of individuals infected with S. japonicum from both the Philippines and the People’s Republic of China. These studies have shown differing antigenic profiles of antibodies derived from individuals in two endemic regions of these countries. Additional studies (8) have shown that the prevalence of hepatomegaly on Jishan Island (People’s Republic of China) is significantly higher than that found in the Philippines. These observations taken together cause one to question the role of antibody molecules present in the infected populations in immunoregulation of chronic disease. In addition, it is unknown if these molecules are serologically related. To this point, our recent studies (6, 7) have shown that both antibodies derived from EBV-transformed splenic cells of a Chinese patient with S. juponicum and a derived human monoclonal antibody modulate in vitro S. japonicum splenic cell responses. However, no studies have addressed the serological relatedness of these immunoregulatory Chinese antibodies and comparable Philippine-derived molecules. Thus, the present studies describes idiotypic and anti-idiotypic antibodies derived from EBV-transformed peripheral
325 0090-1229/92
$4.00
Copyright 0 1992 by Academic Press, Inc.
All rights of reproduction
in any form reserved.
326
BRIEF
COMMUNICATION
blood lymphocytes from Philippine patients with S. juponicum. The immunoregulatory function of these antibodies is described using an in vitro human blastogenesis assay. In addition, their serological relatedness to Chinese S. juponicum idiotypy is presented. MATERIALS
AND
METHODS
Patients. Individuals were identified as S. juponicum infected as part of the Republic of Philippines
Schistosomiasis Control Program which has been part of a g-year study of infected villages on the island of Leyte. Antigens. CFI mice (Charles River Breeding Laboratories, Cambridge, MA) were infected with 50 cercariae of the Philippine strain of S. juponicum at Lowell University (Lowell, MA) provided by National Institute of Allergy and Immune Disease Supply Contract AI-02636. At 8-12 weeks the livers and intestines were removed and the parasite worms and eggs were isolated and processed for antigen as described previously (9). S. juponicum adult worms were obtained by liver perfusion of these animals and a freeze-thawed homogenate extract was utilized to obtain antigen as described (9). EBV transformation. Human peripheral blood lymphocytes were obtained in Leyte, Republic of the Philippines, as part of the National Schistosomiasis Control Program and village surveillance. Lymphocytes were separated on Sepracel, frozen in liquid nitrogen, shipped to Brown University on dry ice, and EBVtransformed as routinely performed in the laboratory (6, 7). In brief, lymphocytes were incubated in EBV containing culture medium derived from the B95-8 marmoset cell line. Cells were plated in 24-well plates at a density of 2 x lo5 cells/well and incubated for 7 days prior to feeding. Cultures containing B cell blasts were screened for anti-antigen (idiotypic) binding molecules by an ELISA (4,7) and for anti-idiotypic binding by a idiotype-inhibition assay (10). ELISA assay. Ninety-six-well microtiter plates were coated with either 2 pg/well of SWAP (soluble worm antigenic protein) or 1 p,g/well of SEA diluted in 100 ~1 of PBS and incubated overnight at 4°C. Plates were subsequently washed with PBS 0.05% Tween 20 and blocked with 200 kl/well of 5% BSA in PBS overnight at 4°C. One hundred microliters of EBV culture supernatant was added per well, and plates were incubated for 2 hr at room temperature. Plates were washed with PBS in 0.05% Tween 20 and 200 ~1 of a 1:2000 dilution of horseradish peroxidase-conjugated goat anti-human IgG was added per well. Following a 40-min incubation at room temperature, plates were washed with PBS-0.05% Tween 20, substrate was added, and the optical density was read at 492 nm. Idiotype-inhibition
assay.
Anti-idiotypic
activity
was measured as the inhibition of binding of LO2C2 (idiotype) to SWAP (antigen) by competitive ELISA (10). Limiting dilutions of LO2C2 were preincubated with 50 ~1 culture supernatant from different EBV cell lines for 2 hr at room temperature. The samples were subsequently incubated in antigen-coated microtiter plates as previously described. Inhibition binding curves were generated from the serial dilution of culture supernate. The percentage of idiotype recognized by anti-idiotypic antibody was calculated from a standard curve using serial dilutions of LO2C2 antibody. Duplicate samples were utilized in each experiment, each performed twice. Lymphocyte-proliferation assay. Individuais noted to have previous schistosome infection based on the g-year follow-up study were used as cell donors (11). PBL were isolated using Seprocil and incubated on the island of Leyte in a 37°C incubator. Medium contained RPM1 1640 (MA Bioproducts, Walkerville KS) with 10% heat-inactivated fetal calf serum supplemented with antibiotics, L-glutamine, and Na biocarbonate buffer. Aliquots of 2 x lo5 cells in 200 ~1 of medium were added to 96-well microtiter plates. To each well with 5 pg/ml SEA (final concentration) or 10 kg/ml SWAP (final concentration) or PHA (10 kg/ml), streptolysin-0 (SLO, 1:lOOO dilution) and antibody derived from EBV-transformed cells were added. Cultures were incubated for 4 days, pulsed for 18 hr with 1 p.Ci c3Hlthymidine, and harvested in a mash semiautomated cell harvester. Proliferation was determined as mean percentage inhibition of isotope uptake of triplicate samples. Replicate experiments were performed as noted based on the availability of donor cells.
Antibodies secreted by EBVWestern blot analysis. transformed cells (LO2C2) were analyzed for antigen binding by immunoblot as described previously (7). In brief, soluble worm antigens from the S. juponicum subspecies from the Philippines were separated using a 10% polyacrylamide-SDS gel. Antibody binding was detailed by using a 1:lOO dilution of horseradish peroxidase-conjugated goat anti-human immunoglobulin overnight at room temperature followed by incubation with 4-chloro-1-naphthol. RESULTS
AND
DISCUSSION
As part of a continuing Schistosomiasis National Control Program in the Republic of the Philippines (111, inhabitants of the island of Leyte were routinely screened for schistosome infection on a yearly basis. Individuals with differing disease parameters were identified and peripheral blood lymphocytes were obtained and EBV-transformed. Two individuals coded LO2 and L012, respectively, were noted to have active infections based on positive Kato smears showing 10 and 40 eggs per gram of stool. Patient LO2 was a 30-
327
BRIEF COMMUNICATION
year-old male with no hepatosplenomegaly but a history of previous infections. Patient LO12 was a 25 year-old female with hepatosplenomegaly. Patients LO14 and LO16 exhibited negative Kato smears indicating a lack of mature infection. LO14 was a 54-yearold male with a history of previous infections, and LO16 was a 30-year-old female with a similar history of multiple infections. Anti-antigen or idiotypic antibodies were derived from EBV-transformed PBL from patients L02, L012, and L014. Anti-idiotypic antibodies were derived from EBV transformation of PBL from patients LO12 and L016. The serology of these idiotypic and antiidiotypic antibodies is presented in Table 1. Four idiotypic (anti-antigen) antibodies are represented: LO2C2 (derived from patient LO2), L012AC4 and L012BC3 (derived from patient LO12), and L014BC2 (derived from patient LO14). LO2C2 exhibited the highest ELISA readings for both SWAP and SEA. In addition, on Western blot analysis this polyclonal EBV transformant exhibited an antigen binding profile virtually identical to that of pooled serum samples from acutely infected individuals (Figure 1). Although LO2C2 was derived from an acutely infected individual, it was surprising to find an EBV-transformed population derived from a single patient which reflected the antigen binding characteristics of serum pools. However, EBV activation or transformation occurs only in immature B cells expressing the EBV receptor. Thus, samples transformed from individuals could express and expand the antibody repertoire derived from immature B cells not fully differentiated into plasma cells by acute infection. LO2C2 was chosen as a standard idiotypic reagent (anti-antigen antibody population) to test for antiidiotype binding activity. The reasons are: (1) LO2C2 exhibited an antigen binding pattern comparable to that of acutely infected human serum (Fig. 1); (2)
12 3 FIG. 1. Immunoblot analysis using S. juponicum soluble worm antigens. Lane 1 was developed using antibodies derived from nonschistosome-reactive EBV-transformed cell line; lane 2 was developed using antibodies derived from the LO2C2 EBV-transformed cell line; lane 3 was developed using pooled serum from acutely infected individuals. Antibodies derived from the EBV-transformed cell line and those of acutely infected individuals recognized similar antigens by this analysis.
LO2C2 was derived from an acutely infected patient; and (3) in the mouse system the major cross-reactive idiotype is expressed on antibodies found in acute infection (4, 5). Thus, if a cross-reactive idiotype was expressed on LO2C2 antibodies, this idiotype expression would parallel that previously characterized in murine S. juponicum infection. Three anti-idiotypic antibodies, LOlBABl and L012BD4 (derived from patient L012) and L016AC2 (derived from patient LO16), were identified. As shown in Table 1, LOlBABl and L012BD4 maximally inhibited the binding of LO2C2 to antigen at levels of 45 2 3 and 33 + 7, respectively.
TABLE 1
Serology of Idiotypic and Anti-idiotypic
Human Antibodies” Anti-idiotype (% inhibition of idiotype binding)d
EBV-transformed cell line LO2C2 L012AC4 L012BC3 L014BC2 LOlPABl L012BD4 L016AC2
Idiotype’ Serology*
SWAP
SEA
Idiotypic Idiotypic Idiotypic Idiotypic Anti-idiotypic Anti-idiotypic Anti-idiotypic
0.750 0.080 0.080 0.0 N/A N/A N/A
0.500 0.028 0.226 0.035 N/A N/A N/A
Philippine idiotype N/A N/A N/A N/A 45 33 72
Chinese idiotype N/A N/A N/A N/A 8 0 12
a Antibodies derived from EBV transformation of PBL from individuals infected with S. japonicum. b Idiotypic anti-SEA or anti-SWAP binding antibodies as determined by ELISA; anti-idiotypic antibodies exhibited no reading in idiotype ELISA assay, but inhibited binding of idiotype to antigen. e Data presented as optical density reading in ELISA. N/A-not applicable; no readings in assay. d Data presented as inhibition of binding of LO2C2 to antigen in anti-idiotype assay. N/A-not applicable; no inhibition in assay.
328
BRIEF COMMUNICATION
These values are maximal inhibitory levels derived from binding curves utilizing excess anti-idiotypes. Thus, quantitatively these anti-idiotypes describe a minor cross-reactive idiotype expressed on LO2C2 antibodies. L016AC2 maximally inhibited ligand binding at a level of 72 2 lo%, thus quantitatively describing a major cross-reactive idiotype expressed on LO2C2 antibodies. It is important to note that in these latter studies both idiotypic and anti-idiotypic antibodies were derived from separate individuals with differing disease parameters. Thus, these data show the expression of an immune network composed of idiotypic and anti-idiotypic antibodies in different infected individuals in the Philippines. Further studies were performed to detail the serological relatedness between anti-idiotypes LOlBABl, L012BD4, and L016AC2 and the idiotypy expressed on anti-antigen immunoregulatory antibodies of individuals with S. juponicum infection in the People’s Republic of China (6). As shown in Table 1, the antiidiotypes which recognized a minor or major level of idiotypy on Philippine antibodies barely recognized Chinese idiotypy. Levels of recognition of the Chinese idiotype of these anti-idiotypes ranged from 0 to 12%. Thus, at least with these initial studies there appears to be little serologic cross-reactivity between antiantigen (S. juponicum) antibodies derived from patients in China and those derived from patients in the Philippines. These data agree with a previous observation of nonidentity in these antibody populations based on antigen binding of S. juponicum SWAP and SEA (7). Antibodies derived from the EBV-transformed cell lines were subsequently tested for secretion of molecules which inhibited antigen (SWAP or SEA) and mitogen-induced blastogenesis. Human lymphoid cells derived from patients with chronic schistosomiasis were utilized as effector cells to determine inhibition of antigen-induced proliferation. As shown in Table 2, idiotypic EBV-derived antibodies and specifically purified serum antibodies inhibited antigen- and mitogeninduced lymphocyte responses. Serum antibodies and idiotypic antibodies from LO2C2, L012AC4, and L012BC3 inhibited in the range of G-85% U’ < 0.05) of SWAP-induced blastogenesis. For these samples SEA-induced blastogenesis was inhibited in the range of 30-80% (P < 0.05). Mitogen-induced blastogenesis was also inhibited by specifically purified acute or chronically derived antibodies and LO2C2 and L012BC3. These data suggest a nonspecific suppression of blastogenesis mediated by idiotypic antibodies. Not all antibodies derived from EBV-transformed cell were immunoregulatory. L014BC2 showed nonsignificant levels of antigen-induced inhibition (9 and 23%, P = NS) and virtually no inhibition of mitogen-induced 13H]thymidine uptake. Idiotype EBV transformants also suppressed mitogen-induced [3H]thymidine uptake of human lymphocytes from noninfected individ-
TABLE 2
Suppression of S. japonicum Antigen-Induced Blastogenesis” ‘ic Suppression Antigen Sampleb Idiotypic Acute sera (children) Acute sera (adults) LO2C2 L012AC4 L012BC3 L014BC2 Anti-idiotypic Chronic sera (children) Chronic sera (adults) LOlBABl L012BD4 L016AC2
Mitogen
SWAP
SEA
PHA
SLO
73 + 5
29 2 16’
55 t 14
NT”
55 i 9 8Oi 11 85 f 6 77 i- 15 91-3
39 56 79 74 23
65 77 21 62 0
71 F 4
55 + 6
44 Ifr 10
NT
80 84 84 87
65 45 74 80
0 20 -+ 4” 34 2 22’ 4 -t 3’
NT NT NT NT
-t 2 i+
10 12 2 3
t t 2 t ‘-
+ t + t
12 17 3 2 6’
4 11 9 5
z ? i+
8 10 20 8
NT 65 :t 12 0 73 i 9 4 2 2’
n Human lymphocytes derived from two chronically infected individuals were incubated with individual samples and antigen or mitogen. Cells were cultured as described in the text. Data are for cells from chronically infected patients and are presented as ?SD of in. hibition of [3H]thymidine uptake of triplicate samples replicated at least twice based on availability of effector cells. b Sample inhibitor used in blastogenesis assay. Pooled serum samples were derived from acutely infected children (~7 years age, with record of first infection, no hepatic pathology) and adults (>30 years age, 2 years prior, no infection with positive infection in 1989, no hepatic pathology). Pooled serum samples were derived from chronically infected children (~7 years old, recorded infection with hepatic pathology) and adults (>30 years old, recorded infection with hepatic pathology. These samples were specifically purified using SWAP/ SEA-coupled agarose affinity chromatography. Antigen: SEA Csoluble egg antigens), 5 pgiml; SWAP (soluble worm antigens), 5 kg/ml. Mitogens: PHA (phytohemagglutinin). 50 kg/ml; SLO (streptolysin0). 1:lOOO dilution. ’ Not statistically significant suppression of blastogenesis by Student’s t test, P > 0.05. L3H]Thymidine incorporation by cells on antigen stimulation was ( ISD) SEA, 1054 -t 253 cpm; SWAP, 2394 z 740 cpm; PHA, 22,883 t 2415; SLO, 689 t 115. Nonspecific inhibition was determined by utilizing normal human serum and antibodies for EBV-transformed lymphocytes of S. mansoni patients and ranged from 3 to 7% in all assays. d NT, not tested.
uals (30-75%, P < 0.05). Schistosome specificity controls for idiotype-mediated suppression were performed with S. mansoni human monoclonal antibody F96 and EBV transformant llE2 (data not shown). These molecules did not inhibit S. juponicum SEA- or SWAP-induced blastogenesis. Anti-idiotype-mediated suppression of human antigen-induced blastogenesis is also shown in Table 2. SWAP-induced lymphocyte responses for serum antibody and EBV transformants were inhibited in the range of 70-90% (P < 0.05) and SEA-induced responses
329
BRIEF COMMUNICATION
in the range of 45-80% (P < 0.05). PHA responses were partially inhibited by serum antibody and antibody from EBV transformants LOlBABl and L012BD4 (P = NS). No inhibition of mitogen responses was noted in serum from chronically infected adults and L016AC2. Anti-idiotypic EBV transformants also did not suppress (O-9%, P = NS) mitogen-induced blastogenesis of human lymphocytes from noninfected individuals. From these limited data it can be noted that the most immunosuppressive anti-idiotypes also inhibited the idiotype-antigen binding interaction to the greatest degree. Thus, specific anti-idiotype-mediated suppression may correlate with the degree of serological expression of the human major cross-reactive idiotype associated with LO2C2. To date no studies have addressed immune network interactions in human S. japonicum infection. However, the existence of idiotype and anti-idiotype has been shown in experimentally induced and human S. munsoni infection. In murine S. mansoni SEA-specific anti-idiotypic population of molecules has been described (12). In murine and human S. munsoni, idiotype-specific antibodies stimulated anti-idiotypicinduced cellular blastogenesis (13). However, the regulatory role of these serologically based interactions remains to be elucidated. A potential role for auto-antiidiotypic antibodies in the regulation of resistance to S. mansoni infection has been reported (14). In addition, in vitro idiotypic manipulation of granulomatous inflammation expressed in schistosomiasis mansoni has been reported (15). These studies support the present data and implicate immune network immunoregulation in human schistosomiasis. The data show that antibodies derived from EBV transformants from different Philippine individuals with S. japonicum infection are serologically related and immunoregulatory. These immunoregulatory antibodies, however, are unrelated to immunoregulatory antibodies expressing Chinese S. japonicum-specific idiotypes. These data further support the dichotomy of the Philippine and Chinese immune repertoires in S. japonicum infection. ACKNOWLEDGMENTS Informed consent was obtained from patients where applicable. The present study was approved by the Miriam Hospital Human Use Institution Review Board and followed the human experimentation guidelines of the U.S. Department of Health, National Institutes of Health. The data were presented in abstract form at the 1991 National Meeting of the American Association of Physicians/American Society of Clinical Investigation/American Federation for Clinical Research, Clin. Res. 39,328A, 1991. This work was supported in part by NIH Grants ROl-AI 25167 and P-50-AI-30601. Received May 1, 1992; accepted with revision August 24, 1992
REFERENCES 1. Warren, K. S., Domingo, E. O., and Cowan, R. B. T., Granuloma formation around schistosome eggs as a manifestation of delayed hypersensitivity. Am. J. Pathol. 51, 735-741, 1967. 2. Olds, G. R., Olveda, R., Tracy, J. W., and Mahmoud, A. A. F., Adoptive transfer of modulation of granuloma formation and hepatosplenic disease in murine schistosomiasis japonica by serum from chronically infected mice. J. Zmmunol. 128, 13911393, 1982. 3. Garb, K. S., Stavitsky, A. B., Olds, G. R., Tracy, J. W., and Mahmoud, A. A. F., Immune regulation in murine Schistosomiasis japonica: Inhibition of in vitro antigen- and mitogen-induced cellular responses by splenocyte culture supernates and by purified fractions from serum of chronically infected mice. J. Zmmunol.
129, 2752-2758,1982.
4. Olds, G. R., and Kresina, T. F., Network interaction in Schistosomn japonicum infection: Identification and characterization of a serologically distinct immunoregulatory auto-anti-idiotypic antibody population. J. Clin. Invest. 76, 2338-2347, 1985. 5. Kresina, T. F., and Olds, G. R., Concomitant cellular and humoral expression of a regulatory cross-reactive idiotype in acute schistosoma japonicum infection. Infect. Zmmun. 53, 90-94, 1986.
6. Kresina, T. F., Guan, H. X., Posner, M., Wisnewski, A., and Olds, G. R., An immunoregulatory human monoclonal antibody in Schistosomiasis japonica. Hum. Antib. Hybridomus 2, 4245, 1991. 7. Kresina, T. F., Guan, H. X., Posner, M., Ramirez, B., Olds, G. R., Comparison of immune repertoires of Chinese and Philippine patients with Schistosoma japonicum. Infect. Zmmun. 59, 46984700,199l. 8. Wiest, P. M., Wu, G., Zhang, S., Yuan, J., Peters, P. A. S., McGarvey, S., Tso, M., Olveda, T. R., and Olds, G. R., Morbidity due to Schistosomiasis japonica in the People’s Republic of China. Trans. R. Sot. Trap. Med. Hyg. 86, 47-50, 1992. 9. Boros, D. L., and Warren, K. S., Delayed hypersensitivity-type granuloma formation and dermal reaction induced and elicited by a soluble factor isolated from Schistosoma mansoni eggs. J. Exp. Med. 132, 488-507, 1970. 10. Herylyn, D., Wettendortf, M., Schmoll, E., Iliopoulos, D., Schedel, I., Dreikhausen, U., Raab, R., Ross, A. H., Jakscheh, Scriba, M., and Koprowski, H., Anti-idiotype immunization of cancer patients: Modulation of the immune response. Proc. Natl. Acad. Sci. USA 84, 8055-8059, 1987. 11. Olveda, R., Wiest, P. M., and Olds, G. R., A six year prospective study of the effect of praziquantel on Schistosomiasis japonica. Clin. Res. 36, 622A, 1988. 12. Powell, M. R., and Colley, D. G., Demonstration of splenic autoanti-idiotypic plague-forming cells in mice infected with schistosoma mansoni. J. Zmmunol. 134, 4146-4145, 1985. 13. Parra, J. C., Lina, M. S., Gazzinelli, G., and Colley, D. G., Immune responses during human Schistosomiasis mansoni XV anti-idiotypic T cells can recognize and respond to anti-SEA idiotype directly. J. Zmmunol. 140, 2401-2404, 1988. 14. Phillips, S. M., Perrin, P. J., Walker, D. J., Fathelbab, N. G., Linette, G. P., and Idris, M. A., The regulation of resistance to Schistosomn mansoni by auto-anti-idiotypic immunity. J. Zmmunol. 141, 1728-1733, 1988. 15. Parra, J. C., Gazzinelli, G., Goes, A. M., Moyes, R. B., Rocha, R., Colley, D. G., and Doughty, B. L., Granulomatous hypersensitivity to Schistosoma mansoni egg antigens in human schistosomiasis. II. In vitro granuloma modulation induced by polyclonal idiotypic antibodies. J. Zmmunol. 147, 3949-3954, 1991.
