Immunity to schistosomes Andr R. Capron Unit~ M i x t e INSERM CNRS, Institut Pasteur, Lille, France
Significant advances have been made during the past years in our knowledge of schistosomiasis, the second major parasitic disease in the world. The highlights provide a good illustration of the recent progress made in our understanding of immunity in human populations and the regulation of the immune response, and in the approach toward a vaccine.
Current Opinion in Immunology 1992, 4:419-424
Introduction Schistosomiasis is a chronic and debilitating parasitic disease affecting 200 million people throughout the world and is responsible for at least 500 000 deaths per year. Infection is characterized by the presence of adult worms in the portal and mesenteric veins of humans and various mammalian species, as part of a complex migratory cycle initiated by cutaneous penetration of infective larvae (cercariae) shed by infected fresh water snails. The infective larvae transform into schistosomula in the skin of appropriate hosts and, over several weeks, develop into sexually mature, egg-laying worms. It is agreed that pathology, in this disease, is related to the deposition of numerous parasite eggs in host tissues [ 1]. Unlike protozoan parasites such as P l a s m o d i u m sp. schistosomes do not replicate within their vertebrate hosts. Therefore, a partial, non-sterilizing naturally acquired, or vaccine induced immunity can strongly decrease human pathology and transmission levels in endemic areas. Among the parasites used as models by immunologists, schistosomes have probably provided the broadest experimental approaches. They have illustrated the existence of novel effector and regulatory mechanisms, now confirmed in man, that may be of interest not only in the field of parasitic diseases but also as more general immunological processes. As a result of this increase of our basic knowledge, progress made during the last few years toward successful immunization allows us to consider that, in terms of human vaccinations, schistosomiasis may now be the most promising candidate among the major parasitic diseases.
Effector mechanisms and immunity in human populations Although contrasting results can be obtained according to the experimental model used (rats or mice for instance) it is generally agreed that antibodydependent mechanisms play a major role in the expression of acquired resistance to schistosome infection. The major role of antibodies in protective immunity is to induce cytotoxic destruction of schistosomulum targets and although the significance of complement-fixing antibodies is not yet resolved, antibody-dependent cell-mediated cytotoxicity (ADCC) appears to be the main mechanism of killing parasites both in rat and human schistosomiasis (A Capron, M Capron, JP Dessaint, Proceedings of the 4th International Congress of Immunology, Paris, July 1980, pp 782-793). Extensive studies based on in vitro c~otoxicity assays and in vivo passive transfer experiments have revealed the existence of novel ADCC systems involving inflammatory cells (macrophages, eosinophils, platelets) as cellular partners, and IgE or anaphylactic subclasses of IgG as humoral components [2]. These findings in experimental and human infections pointing to the key role of anaphylactic antibodies, particularly IgE, in defence against schistosomes, have led to a re-evaluation of their general biologic significance. So far this has only been considered in the context of allergy, but it is now clear that IgE and anaphylactic antibodies might represent essential participants in the complex response to parasitic infections [3]. At the same time, evidence was accumulated both in rat and man that expression of immunity might depend not only on 'effector' isotypes but also on blocking isotypes directed against the same epitopes [4,5].
Abbreviations ADCC--antibody-dependent cell-mediated cytotoxicity; DAF--decay accelerating factor; DTH--delayed type hypersensitivity; IFN--interferon; IL--interleukin; mAb~monoclonal antibody; MHC--major histocompatibility complex; POMC--proopiomelanocortin; R--receptor; SEA--soluble egg antigen; Sm28 GST~. mansoni glutathione S-transferase; Th--T helper; TNF~tumor necrosis factor; TPI--triose phosphate isomerase.
(~) Current Biology Ltd ISSN 0952-7915
419
420
Immunityto infection Following the pioneering immunoepidemiological studies of Butterworth and colleagues in Kenya [6], two independent studies have now, for the first time in human populations, given support to these concepts, essentially based on experimental observations [7"*,8"]. After .treatment in an endemic area of the Gambia for Schistosoma h a e m a t o b i u m , Hagan et al. [7"'] have demonstrated a significant correlation between the production of antischistosome IgE antibodies and the acquisition of immunity during reinfection. They have also shown that anti-schistosome IgG4 antibodies, potentially blocking IgE effector pathways, are correlated with susceptibility to reinfection, supporting the idea that they might be responsible for delaying the development of protective immunity. Other studies have been performed by Dessein and his colleagues [8"] in Brazil in adolescents with high or low resistance to infection by S. m a n s o n L The comparison of IgE antibodies between the two groups showed that IgE levels were on average six- to eightfold higher in the sera of the most resistant adolescents, but that there was no difference in patterns of antigen recognition between study groups. In contrast to IgE, anti-larval IgG or IgM levels were either similar in both groups or higher in the least resistant subjects. More recently (A Dessein, personal communication), it has been shown that the association of low levels of IgE antibodies to S. m a n s o n i with high IgG4 levels resulted in an increase of over 100-fold in susceptibility to reinfection. Similar observations have now been made in Kenya, in a community infected by S. m a n s o n i [9o], and there is now converging evidence supporting a role for IgE in human resistance to infection by schistosomes. Confirming experimental evidence, a new dimension of IgE function and its significance in the human immune response has now appeared. It can be considered that a number of clinically significant allergic responses to environmental antigens may reflect the inappropriate activation of immunological circuits or effector pathways; these pathways are ordinarily initiated by helminths and confer resistance whereas other effector mechanisms appear to be of low efficiency. The mathematical analysis of age prevalence data for human infection with S. h a e m a t o b i u m has also demonstrated patterns of variation that are consistent with the epidemiological effects of acquired immunity predicted by mathematical models [10]. Of particular interest in the context of studies of human populations, is the recently acquired evidence that there is a codominant major gene controlling human susceptibility/resistance to infection by S. m a n s o n L Parameter estimates indicate a frequency of 0.20-0.25 for the deleterious allele; thus, about 5% of the population under study appeared to be predisposed to high infections, 60% are resistant, and 35% have an intermediate level of resistance [11oo]. These findings provide a genetic basis for earlier observations on the lower resistance and the predisposition to reinfection of certain individuals. In addition to the detection of a major gene effect, the data suggest that immunity to S. m a n s o n i develops progressively during childhood reaching a maximum around the age of
puberty, confirming the observations of Hagan and Butterworth discussed above [6].
Immunoregulation, T-cell subsets and cytokines Recent advances in our understanding of CD4 + T helper (Th) lymphocyte function have allowed a re-evaluation of some of the immunologic characteristics of helminth infection, in particular schistosomiasis [12]. The pivotal discovery, from work on mouse T-cell clones, that cytokine production tends to be coordinated along two main lines by two types of Th cell termed Thl and Th2, has generated a series of elegant studies in the mouse model of experimental schistosomiasis that have shed light on some of the regulatory mechanisms involved in immunity and/or pathology. Following the initial discovery by Sher and his colleagues [13"] of a functional dichotomy in the CD4 + T-cell response to S. m a n s o n i , further studies by the same group have led to the theory that schistosome worms stimulate Thl-cell responses that are down-regulated by egg-induced Th2 cell responses. In the context of experimental infection, it has been shown that, like eosinophil and IgE levels, interleukin (IL)-4 and IL-5 production by Th2 cells increases at about the time mature infection and egg production begins, suggesting a causal link between eggs and Th2 cell responses [13.,14.o]. Consistent with this is the finding that little IL4 or IL5 is produced by antigen-stimulated T cells from mice carrying prepatent or single sex infections in which eggs are not produced [15"]. However, injection of eggs into immunized mice resulted in a reduction of antigen- and mitogen-stimulated Thl-cell function accompanied by a coincident expression of Th2-cell responses. Vaccination of mice with optimally irradiated cercariae remains a favourite model in many laboratories for inducing high levels of protective immunity, which is accompanied by the generation of interferon (IFN)-7 producing T lymphocytes. Current work has indicated that the success of irradiated vaccines depends not only on the generation of a population of antigen-specific CD4 + T cells in the skin-draining lymph nodes but also on recruitement of these cells to the lungs before challenge [16]. Lymphocytes recruited to the lungs produce abundant quantities of IFN-7 and IL-3 on stimulation with larval antigen and appear to act as memory/effector cells. It is suggested, in this context, that when antigen released from challenge parasites is presented to these cells, they respond by secreting cytokines that mediate the formation of cellular aggregates around the parasites, blocking their onward migration [17-]. In the context of the vaccination of mice with attenuated larval stages, it was further shown that vaccinated animals primarily responded with Thl lymphokines and that, in contrast to the situation in infected mice, larval schistosome antigens tend to stimulate stronger Thl-cell responses than egg antigens. These findings are supported by parallel studies indicating that protective immunity to S. m a n s o n i i n mice, induced by irradiated cercariae, is dependent on L3T4 +, IL-2 receptor
Immunity to schistosomes Capron (IL-2R) + lymphocytes, is negatively regulated by Lyt-2+ cells [18]. This study suggests that IL-4 does not affect the development of resistance in this particular model [18]. Further, using selective depletion by appropriate monoclonal antibodies (mAbs) of either the T-cell subpopulation bearing the high affinity IL-2R or IL-4R, the same authors have also shown, in the rat model, that protective immunity is dependent upon IL-2R-bearing T lymphocytes and is regulated by OX8 + cells, but is not absolutely contingent upon IL-4 [19]. The demonstration of coordinated responses regarding Thl and Th2 activities in the mouse model of schistosome infection has also stimulated a number of studies concerning the orchestration and relative contribution of the various cytokines involved. Granuloma development seems to involve a sequential production of cytokines resuiting from the participation of several Th-cell populations [20]. There is some indication that antigen-specific MHC-restricted, local delayed type hypersensitivity (DTH) reactions as well as granuloma formation can be mediated by soluble egg antigen (SEA)-specific Thl-cell clones [21]. However, administration of anti-IL-4 antibodies to mice synchronously developing granulomas dramatically reduced granuloma size, whereas antibodies to IFN-y had no effect [22]. Egg-induced granuloma formation has been classically characterized as a DTH response mediated by CD4 + T lymphocytes, and only Thl lymphocytes, are currently believed to mediate DTH responses. However, a recent and elegant study based on an in vivo molecular analysis of Thl and Th2 activity that quantified endogenous steady state IL-2 mRNA and IL-4 mRNA levels in granulomatous livers and isolated granulomatous lesions, directly challenges this hypothesis. The study [23"] shows that IL4-producing Th2 lymphocytes are active in, and possibly integral to, the granulomatous, DTH response, characteristic of schistosome infection in the liver. Although the factors underlying the down-regulation of Thl responses have not been definitively determined, an important candidate is IL-10; this cytokine is secreted by Th2 cells (as well as mast cells and Lyl + B cells) and has, among other activities, an inhibitory role on cytokine (IFN-y and IL-2) synthesis by Thl cells, as well as by CD8 + lymphocytes. It is of interest to mention that the first evidence for a possible function of IL-10 in immunoregulation in vivo has been provided by the study of the mouse model of schistosome infection [24"]. This study provided convincing evidence indicating that CD4 + cells from schistosome infected mice produce large quantities of IL-10, and that this response is both temporally and mechanistically linked to the down-regulation in Thl cytokine synthesis seen in the same animals. Taken together, these results indicate that, at least in the mouse model of schistosome infection, expression of both protective immunity and of pathological reactions is regulated by a complex network of coordinated responses leading to the concept of a Th cross-regulatory circuit [24.]. However, when confronted with other experimental evidence and recent immunoepidemiological observations in human populations (summarized in the first part of this review), the interpretation of these results might
appear rather confusing and deserves some general comments. Firstly, although they represent dominant responses in infected mice, there is no evidence that either IgE or eosinophils are involved in the acquired resistance to superinfection demonstrated by these animals [12]. Several studies [25-27] have shown that 'immunity' in this model is largely non-specific in nature and due in part to pathological changes in the hepatic vasculature precipitated by the granulomatous response [12]. In addition, extensive studies in rodent models [28], have given support to the concept of a 'belt and braces' strategy of the effector mechanisms of immune defence. In the rat for instance, IgG2a participates significantly together with IgE in the expression of resistance, and IgG2a ADCC and other non Th2-cell-dependent cellular responses would not be expected to be affected by anti-IL-4 antibodies. Secondly, Thl and Th2 cell associated immune reactivities appear to be largely dependent on single versus multiple antigen stimulations, among other factors. In this respect, the recent results reported by the group of James [29"] are of particular interest. It has been shown that whereas cell-mediated mechanisms associated with induction of CD4 + Thl-cell reactivity predominate in singly vaccinated mice, further vaccination stimulates Th2-cell responses such as enhanced IgG1 production that also contribute to protective immunity [29~ These results are compatible with the earlier demonstration of the protective role of IgGt antibodies in mice [30], as well as with the age-dependent acquisition of immunity, following multiple exposures to parasite infection, in human populations. Thirdly, there is increasing evi dence that multiple cytokines can be secreted by cells other than Thl or Th2 cells. The recent demonstration that eosinophils, a pivotal cell population in the immune response to schistosomes, express the IL-5 gene [31,o], and secrete IL-5 in response to various stimuli (M Capron, personal communication) provides an example of the existence of regulatory networks, independent from the Thl-Th2 cell cross-reactive circuit. Along the same lines, it was recently demonstrated that tumor necrosis factor (TNF)
Significant advances have been made during the past years in our knowledge of schistosomiasis, the second major parasitic disease in the world. The highlights provide a good illustration of the recent progress made in our understanding of immunity in human populations and the regulation of the immune response, and in the approach toward a vaccine.
Current Opinion in Immunology 1992, 4:419-424
Introduction Schistosomiasis is a chronic and debilitating parasitic disease affecting 200 million people throughout the world and is responsible for at least 500 000 deaths per year. Infection is characterized by the presence of adult worms in the portal and mesenteric veins of humans and various mammalian species, as part of a complex migratory cycle initiated by cutaneous penetration of infective larvae (cercariae) shed by infected fresh water snails. The infective larvae transform into schistosomula in the skin of appropriate hosts and, over several weeks, develop into sexually mature, egg-laying worms. It is agreed that pathology, in this disease, is related to the deposition of numerous parasite eggs in host tissues [ 1]. Unlike protozoan parasites such as P l a s m o d i u m sp. schistosomes do not replicate within their vertebrate hosts. Therefore, a partial, non-sterilizing naturally acquired, or vaccine induced immunity can strongly decrease human pathology and transmission levels in endemic areas. Among the parasites used as models by immunologists, schistosomes have probably provided the broadest experimental approaches. They have illustrated the existence of novel effector and regulatory mechanisms, now confirmed in man, that may be of interest not only in the field of parasitic diseases but also as more general immunological processes. As a result of this increase of our basic knowledge, progress made during the last few years toward successful immunization allows us to consider that, in terms of human vaccinations, schistosomiasis may now be the most promising candidate among the major parasitic diseases.
Effector mechanisms and immunity in human populations Although contrasting results can be obtained according to the experimental model used (rats or mice for instance) it is generally agreed that antibodydependent mechanisms play a major role in the expression of acquired resistance to schistosome infection. The major role of antibodies in protective immunity is to induce cytotoxic destruction of schistosomulum targets and although the significance of complement-fixing antibodies is not yet resolved, antibody-dependent cell-mediated cytotoxicity (ADCC) appears to be the main mechanism of killing parasites both in rat and human schistosomiasis (A Capron, M Capron, JP Dessaint, Proceedings of the 4th International Congress of Immunology, Paris, July 1980, pp 782-793). Extensive studies based on in vitro c~otoxicity assays and in vivo passive transfer experiments have revealed the existence of novel ADCC systems involving inflammatory cells (macrophages, eosinophils, platelets) as cellular partners, and IgE or anaphylactic subclasses of IgG as humoral components [2]. These findings in experimental and human infections pointing to the key role of anaphylactic antibodies, particularly IgE, in defence against schistosomes, have led to a re-evaluation of their general biologic significance. So far this has only been considered in the context of allergy, but it is now clear that IgE and anaphylactic antibodies might represent essential participants in the complex response to parasitic infections [3]. At the same time, evidence was accumulated both in rat and man that expression of immunity might depend not only on 'effector' isotypes but also on blocking isotypes directed against the same epitopes [4,5].
Abbreviations ADCC--antibody-dependent cell-mediated cytotoxicity; DAF--decay accelerating factor; DTH--delayed type hypersensitivity; IFN--interferon; IL--interleukin; mAb~monoclonal antibody; MHC--major histocompatibility complex; POMC--proopiomelanocortin; R--receptor; SEA--soluble egg antigen; Sm28 GST~. mansoni glutathione S-transferase; Th--T helper; TNF~tumor necrosis factor; TPI--triose phosphate isomerase.
(~) Current Biology Ltd ISSN 0952-7915
419
420
Immunityto infection Following the pioneering immunoepidemiological studies of Butterworth and colleagues in Kenya [6], two independent studies have now, for the first time in human populations, given support to these concepts, essentially based on experimental observations [7"*,8"]. After .treatment in an endemic area of the Gambia for Schistosoma h a e m a t o b i u m , Hagan et al. [7"'] have demonstrated a significant correlation between the production of antischistosome IgE antibodies and the acquisition of immunity during reinfection. They have also shown that anti-schistosome IgG4 antibodies, potentially blocking IgE effector pathways, are correlated with susceptibility to reinfection, supporting the idea that they might be responsible for delaying the development of protective immunity. Other studies have been performed by Dessein and his colleagues [8"] in Brazil in adolescents with high or low resistance to infection by S. m a n s o n L The comparison of IgE antibodies between the two groups showed that IgE levels were on average six- to eightfold higher in the sera of the most resistant adolescents, but that there was no difference in patterns of antigen recognition between study groups. In contrast to IgE, anti-larval IgG or IgM levels were either similar in both groups or higher in the least resistant subjects. More recently (A Dessein, personal communication), it has been shown that the association of low levels of IgE antibodies to S. m a n s o n i with high IgG4 levels resulted in an increase of over 100-fold in susceptibility to reinfection. Similar observations have now been made in Kenya, in a community infected by S. m a n s o n i [9o], and there is now converging evidence supporting a role for IgE in human resistance to infection by schistosomes. Confirming experimental evidence, a new dimension of IgE function and its significance in the human immune response has now appeared. It can be considered that a number of clinically significant allergic responses to environmental antigens may reflect the inappropriate activation of immunological circuits or effector pathways; these pathways are ordinarily initiated by helminths and confer resistance whereas other effector mechanisms appear to be of low efficiency. The mathematical analysis of age prevalence data for human infection with S. h a e m a t o b i u m has also demonstrated patterns of variation that are consistent with the epidemiological effects of acquired immunity predicted by mathematical models [10]. Of particular interest in the context of studies of human populations, is the recently acquired evidence that there is a codominant major gene controlling human susceptibility/resistance to infection by S. m a n s o n L Parameter estimates indicate a frequency of 0.20-0.25 for the deleterious allele; thus, about 5% of the population under study appeared to be predisposed to high infections, 60% are resistant, and 35% have an intermediate level of resistance [11oo]. These findings provide a genetic basis for earlier observations on the lower resistance and the predisposition to reinfection of certain individuals. In addition to the detection of a major gene effect, the data suggest that immunity to S. m a n s o n i develops progressively during childhood reaching a maximum around the age of
puberty, confirming the observations of Hagan and Butterworth discussed above [6].
Immunoregulation, T-cell subsets and cytokines Recent advances in our understanding of CD4 + T helper (Th) lymphocyte function have allowed a re-evaluation of some of the immunologic characteristics of helminth infection, in particular schistosomiasis [12]. The pivotal discovery, from work on mouse T-cell clones, that cytokine production tends to be coordinated along two main lines by two types of Th cell termed Thl and Th2, has generated a series of elegant studies in the mouse model of experimental schistosomiasis that have shed light on some of the regulatory mechanisms involved in immunity and/or pathology. Following the initial discovery by Sher and his colleagues [13"] of a functional dichotomy in the CD4 + T-cell response to S. m a n s o n i , further studies by the same group have led to the theory that schistosome worms stimulate Thl-cell responses that are down-regulated by egg-induced Th2 cell responses. In the context of experimental infection, it has been shown that, like eosinophil and IgE levels, interleukin (IL)-4 and IL-5 production by Th2 cells increases at about the time mature infection and egg production begins, suggesting a causal link between eggs and Th2 cell responses [13.,14.o]. Consistent with this is the finding that little IL4 or IL5 is produced by antigen-stimulated T cells from mice carrying prepatent or single sex infections in which eggs are not produced [15"]. However, injection of eggs into immunized mice resulted in a reduction of antigen- and mitogen-stimulated Thl-cell function accompanied by a coincident expression of Th2-cell responses. Vaccination of mice with optimally irradiated cercariae remains a favourite model in many laboratories for inducing high levels of protective immunity, which is accompanied by the generation of interferon (IFN)-7 producing T lymphocytes. Current work has indicated that the success of irradiated vaccines depends not only on the generation of a population of antigen-specific CD4 + T cells in the skin-draining lymph nodes but also on recruitement of these cells to the lungs before challenge [16]. Lymphocytes recruited to the lungs produce abundant quantities of IFN-7 and IL-3 on stimulation with larval antigen and appear to act as memory/effector cells. It is suggested, in this context, that when antigen released from challenge parasites is presented to these cells, they respond by secreting cytokines that mediate the formation of cellular aggregates around the parasites, blocking their onward migration [17-]. In the context of the vaccination of mice with attenuated larval stages, it was further shown that vaccinated animals primarily responded with Thl lymphokines and that, in contrast to the situation in infected mice, larval schistosome antigens tend to stimulate stronger Thl-cell responses than egg antigens. These findings are supported by parallel studies indicating that protective immunity to S. m a n s o n i i n mice, induced by irradiated cercariae, is dependent on L3T4 +, IL-2 receptor
Immunity to schistosomes Capron (IL-2R) + lymphocytes, is negatively regulated by Lyt-2+ cells [18]. This study suggests that IL-4 does not affect the development of resistance in this particular model [18]. Further, using selective depletion by appropriate monoclonal antibodies (mAbs) of either the T-cell subpopulation bearing the high affinity IL-2R or IL-4R, the same authors have also shown, in the rat model, that protective immunity is dependent upon IL-2R-bearing T lymphocytes and is regulated by OX8 + cells, but is not absolutely contingent upon IL-4 [19]. The demonstration of coordinated responses regarding Thl and Th2 activities in the mouse model of schistosome infection has also stimulated a number of studies concerning the orchestration and relative contribution of the various cytokines involved. Granuloma development seems to involve a sequential production of cytokines resuiting from the participation of several Th-cell populations [20]. There is some indication that antigen-specific MHC-restricted, local delayed type hypersensitivity (DTH) reactions as well as granuloma formation can be mediated by soluble egg antigen (SEA)-specific Thl-cell clones [21]. However, administration of anti-IL-4 antibodies to mice synchronously developing granulomas dramatically reduced granuloma size, whereas antibodies to IFN-y had no effect [22]. Egg-induced granuloma formation has been classically characterized as a DTH response mediated by CD4 + T lymphocytes, and only Thl lymphocytes, are currently believed to mediate DTH responses. However, a recent and elegant study based on an in vivo molecular analysis of Thl and Th2 activity that quantified endogenous steady state IL-2 mRNA and IL-4 mRNA levels in granulomatous livers and isolated granulomatous lesions, directly challenges this hypothesis. The study [23"] shows that IL4-producing Th2 lymphocytes are active in, and possibly integral to, the granulomatous, DTH response, characteristic of schistosome infection in the liver. Although the factors underlying the down-regulation of Thl responses have not been definitively determined, an important candidate is IL-10; this cytokine is secreted by Th2 cells (as well as mast cells and Lyl + B cells) and has, among other activities, an inhibitory role on cytokine (IFN-y and IL-2) synthesis by Thl cells, as well as by CD8 + lymphocytes. It is of interest to mention that the first evidence for a possible function of IL-10 in immunoregulation in vivo has been provided by the study of the mouse model of schistosome infection [24"]. This study provided convincing evidence indicating that CD4 + cells from schistosome infected mice produce large quantities of IL-10, and that this response is both temporally and mechanistically linked to the down-regulation in Thl cytokine synthesis seen in the same animals. Taken together, these results indicate that, at least in the mouse model of schistosome infection, expression of both protective immunity and of pathological reactions is regulated by a complex network of coordinated responses leading to the concept of a Th cross-regulatory circuit [24.]. However, when confronted with other experimental evidence and recent immunoepidemiological observations in human populations (summarized in the first part of this review), the interpretation of these results might
appear rather confusing and deserves some general comments. Firstly, although they represent dominant responses in infected mice, there is no evidence that either IgE or eosinophils are involved in the acquired resistance to superinfection demonstrated by these animals [12]. Several studies [25-27] have shown that 'immunity' in this model is largely non-specific in nature and due in part to pathological changes in the hepatic vasculature precipitated by the granulomatous response [12]. In addition, extensive studies in rodent models [28], have given support to the concept of a 'belt and braces' strategy of the effector mechanisms of immune defence. In the rat for instance, IgG2a participates significantly together with IgE in the expression of resistance, and IgG2a ADCC and other non Th2-cell-dependent cellular responses would not be expected to be affected by anti-IL-4 antibodies. Secondly, Thl and Th2 cell associated immune reactivities appear to be largely dependent on single versus multiple antigen stimulations, among other factors. In this respect, the recent results reported by the group of James [29"] are of particular interest. It has been shown that whereas cell-mediated mechanisms associated with induction of CD4 + Thl-cell reactivity predominate in singly vaccinated mice, further vaccination stimulates Th2-cell responses such as enhanced IgG1 production that also contribute to protective immunity [29~ These results are compatible with the earlier demonstration of the protective role of IgGt antibodies in mice [30], as well as with the age-dependent acquisition of immunity, following multiple exposures to parasite infection, in human populations. Thirdly, there is increasing evi dence that multiple cytokines can be secreted by cells other than Thl or Th2 cells. The recent demonstration that eosinophils, a pivotal cell population in the immune response to schistosomes, express the IL-5 gene [31,o], and secrete IL-5 in response to various stimuli (M Capron, personal communication) provides an example of the existence of regulatory networks, independent from the Thl-Th2 cell cross-reactive circuit. Along the same lines, it was recently demonstrated that tumor necrosis factor (TNF)
