IL-4 and IL-10 are essential for immunosuppression induced by high molecular weight proteins from Ascaris suum

The extract from Ascaris suum worms (Asc) impairs Th1 and Th2 responses to a non-related antigen, i.e. ovalbumin (OVA). Its suppressive capacity is due to high molecular weight components present in a gel filtration fraction (PI). This fraction is able to elicit IL-4 and IL-10 secretion. Interestingly enough, it induces anti-PI non-anaphylactic IgG1 synthesis through the action of IL-12/IFN-gamma. Here, we investigated the down-regulation of the immune response to OVA by PI in IL-12, IFN-gamma, IL-4 or IL-10 C57BL/6 knockout mice immunized with OVA+PI in adjuvant. OVA-induced delayed-type hypersensitivity (DTH) reactions, secretion of IL-2 and IFN-gamma, and IgG1, IgG2c and IgE antibody production were suppressed by PI in wild-type mice, as well as in IL-12- or IFN-gamma-deficient mice. In contrast, PI had no effect on anti-OVA IgE production and DTH, and induced only a partial suppression of IgG1 and IFN-gamma in IL-10(-/-) mice. The experiments also showed that IL-4 was involved in the PI-induced suppression of IgG2c antibodies and IL-2 secretion. Finally, down-regulation of IFN-gamma was not seen in mice lacking both IL-4 and IL-10, i.e. IL-4(-/-) mice treated with anti-IL-10 antibodies before immunization. These results exclude the participation of IL-12 and IFN-gamma in PI-induced immunosuppression, and highlight the essential role of IL-10 in the suppression of OVA-specific Th2-related parameters, as well as the cooperation between IL-10 and IL-4 in the suppression of Th1-related parameters.     

Chimeras of labile toxin one and cholera toxin retain mucosal adjuvanticity and direct Th cell subsets via their B subunit

Native cholera toxin (nCT) and the heat-labile toxin 1 (nLT) of enterotoxigenic Escherichia coli are AB5-type enterotoxins. Both nCT and nLT are effective adjuvants that promote mucosal and systemic immunity to protein Ags given by either oral or nasal routes. Previous studies have shown that nCT as mucosal adjuvant requires IL-4 and induces CD4-positive (CD4+) Th2-type responses, while nLT up-regulates Th1 cell production of IFN-gamma and IL-4-independent Th2-type responses. To address the relative importance of the A or B subunits in CD4+ Th cell subset responses, chimeras of CT-A/LT-B and LT-A/CT-B were constructed. Mice nasally immunized with CT-A/LT-B or LT-A/CT-B and the weak immunogen OVA developed OVA-specific, plasma IgG Abs titers similar to those induced by either nCT or nLT. Both CT-A/LT-B and LT-A/CT-B promoted secretory IgA anti-OVA Ab, which established their retention of mucosal adjuvant activity. The CT-A/LT-B chimera, like nLT, induced OVA-specific mucosal and peripheral CD4+ T cells secreting IFN-gamma and IL-4-independent Th2-type responses, with plasma IgG2a anti-OVA Abs. Further, LT-A/CT-B, like nCT, promoted plasma IgG1 more than IgG2a and IgE Abs with OVA-specific CD4+ Th2 cells secreting high levels of IL-4, but not IFN-gamma. The LT-A/CT-B chimera and nCT, but not the CT-A/LT-B chimera or nLT, suppressed IL-12R expression and IFN-gamma production by activated T cells. Our results show that the B subunits of enterotoxin adjuvants regulate IL-12R expression and subsequent Th cell subset responses.     

Role of IFN-gamma in Th1 differentiation: IFN-gamma regulates IL-18R alpha expression by preventing the negative effects of IL-4 and by inducing/maintaining IL-12 receptor beta 2 expression

Two key events occur during the differentiation of IFN-gamma-secreting Th1 cells: up-regulation of IL-12Rbeta2 and IL-12-driven up-regulation of IL-18Ralpha. We previously demonstrated that IL-12-driven up-regulation of IL-18Ralpha expression is severely impaired in IFN-gamma(-/-) mice. However, it was unclear from these studies how IFN-gamma influenced IL-18Ralpha since IFN-gamma alone had no direct effect on IL-18Ralpha expression. In the absence of IL-4, IL-12-dependent up-regulation of IL-18Ralpha/IL-12Rbeta2 was independent of IFN-gamma. However, in the presence of IL-4, IFN-gamma functions to limit the negative effects of IL-4 on both IL-18Ralpha and IL-12Rbeta2. Neutralization of IL-4 restored IL-12-driven up-regulation of IL-18Ralpha/IL-12Rbeta2 in an IFN-gamma-independent fashion. In the absence of both IL-12 and IL-4, IFN-gamma up-regulates IL-12beta2 expression and primes IFN-gamma-producing Th1 cells. When T cells were primed in the presence of IL-4, no correlation was found between the levels of expression of the IL-18Ralpha or the IL-12Rbeta2 and the capacity of these cells to produce IFN-gamma, suggesting that IL-4 may also negatively affect IL-12-mediated signal transduction and thus Th1 differentiation. These data clarify the role of IFN-gamma in regulation of IL-18Ralpha/IL-12Rbeta2 during both IL-12-dependent and IL-12-independent Th1 differentiation.     

Topical superantigen exposure induces epidermal accumulation of CD8+ T cells, a mixed Th1/Th2-type dermatitis and vigorous production of IgE antibodies in the murine model of atopic dermatitis

Patients with atopic dermatitis (AD) have repeated cutaneous exposure to both environmental allergens and superantigen-producing strains of Staphylococcus aureus. We used a murine model of AD to investigate the role of staphylococcal enterotoxin B (SEB) in the modulation of allergen-induced skin inflammation. Mice were topically exposed to SEB, OVA, a combination of OVA and SEB (OVA/SEB), or PBS. Topical SEB and OVA/SEB exposure induced epidermal accumulation of CD8+ T cells and TCRVbeta8+ cells in contrast to OVA application, which induced a mainly dermal infiltration of CD4+ cells. SEB and OVA/SEB exposure elicited a mixed Th1/Th2-associated cytokine and chemokine expression profile within the skin. Restimulation of lymph node cells from OVA- and OVA/SEB-exposed mice with OVA elicited strong production of IL-13 protein, whereas substantial amounts of IFN-gamma protein were detected after SEB stimulation of cells derived from SEB- or OVA/SEB-exposed mice. Topical SEB treatment elicited vigorous production of SEB-specific IgE and IgG2a Abs and significantly increased the production of OVA-specific IgE and IgG2a Abs. The present study shows that topical exposure to SEB provokes epidermal accumulation of CD8+ T cells, a mixed Th2/Th1 type dermatitis and vigorous production of specific IgE and IgG2a Abs, which can be related to the chronic phase of atopic skin inflammation.     

IFN-gamma is necessary but not sufficient for anti-CD40 antibody-mediated inhibition of the Th2 response to Schistosoma mansoni eggs

The injection of Schistosoma mansoni eggs into the footpads of mice results in a localized Th2 cytokine response and tissue eosinophilia. We examined whether treatment with CD40-activating Abs would block the development of Th2 cytokine responses and eosinophilic tissue pathology in this model. Seven days after C57BL/6 mice were injected with eggs and the FGK45 anti-CD40 Ab, Ag-specific synthesis of IL-4, IL-5, and IL-13 in lymph node culture was reduced (>10-fold) relative to control mice treated with eggs and rat IgG. In contrast, IFN-gamma and IL-12 were increased in both culture supernatants and in the serum. Similar changes in lymph node cytokine mRNA were observed in vivo, and tissue eosinophilia was reduced nearly 20-fold. Th2 cytokine responses in anti-CD40-treated IFN-gamma-/- and IL-12 p40-/- C57BL/6 mice were unaffected, although anti-CD40 induced high levels of systemic and local IFN-gamma production in both wild-type and IL-12 p40-/- mice. We conclude that CD40-activating treatments strongly reverse the immune phenotype generated in response to a classic, Th2-biasing stimulus and stimulate IFN-gamma through a novel IL-12-independent pathway. This model for Th1-deviating immune therapy may have relevance to the treatment of Th2-dependent diseases in general.     

IL-2 receptor blockade inhibits late,but not early,IFN-gamma and CD40 ligand expression in human T cells: disruption of both IL-12-dependent and -independent pathways of IFN-gamma production

mAbs directed against the alpha-chain (Tac/CD25) of the IL-2R are an emerging therapy in both transplantation and autoimmune disease. However, the mechanisms underlying their therapeutic efficacy have not been fully elucidated. Therefore, we examined the effect of IL-2R blockade on Th1 and Th2 cytokine production from human PBMC. Addition of a humanized anti-Tac Ab (HAT) to activated PBMC cultures inhibited IFN-gamma production from CD4 and CD8 T cells by 80-90%. HAT partially inhibited production of TNF-alpha and completely inhibited production of IL-4, IL-5, and IL-10. Furthermore, IL-12, a central regulatory cytokine that induces IFN-gamma, was undetectable in treated cultures. As T cell-dependent induction of IL-12 is regulated via CD40/CD40 ligand (CD40L) interactions, we examined the effect of HAT on CD40L expression. We found CD40L expression to be biphasic with an early (6 h) peak that is CD28/IL-2-independent, but a later peak (48 h) being CD28/IL-2-dependent and inhibited by HAT. Similarly, IFN-gamma production at 6 h was CD28/IL-2-independent but CD28/IL-2-dependent and inhibited by HAT at 48 h. Nonetheless, addition of rCD40L or exogenous IL-12 to HAT-treated cultures could not restore IFN-gamma production. The IFN-gamma deficit in such cultures appears to be due to a direct inhibition by HAT of IL-12-independent IFN-gamma production from T cells rather than altered expression of either the IL-12Rbeta1 or IL-12Rbeta2 chains. These data demonstrate that IL-2 plays a critical role in the regulation of Th1 and Th2 responses and impacts both IL-12-dependent and -independent IFN-gamma production.     

Normal induction of oral tolerance in the absence of a functional IL-12-dependent IFN-gamma signaling pathway.

There is considerable evidence that regulatory cytokines play an important role in mediating the systemic tolerance found after oral administration of protein Ags. Although most existing work has focused on cytokines such as IL-4, IL-10, and TGF-beta, recent evidence from TCR transgenic systems suggests that the induction of oral tolerance is accompanied by priming of Ag-specific IFN-gamma production. IFN-gamma has also been implicated as a mediator of T cell tolerance in other models in vivo and in vitro, including that induced by aerosol administration of protein. We show here that feeding tolerogenic doses of OVA primes for IFN-gamma production in the spleen of mice with a normal T cell repertoire. However, depleting IFN-gamma at the time of feeding OVA had no effect on the induction of tolerance. In addition, tolerance was induced normally in both IFN-gamma receptor knockout (IFN-gammaR-/-) and IL-12 p40 knockout (IL-12-/-) mice. This was the case for all components of the systemic immune response and also with a variety of feeding protocols, including those believed to induce distinct regulatory mechanisms. We conclude that IL-12-dependent IFN-gamma-mediated regulation does not play an essential role in oral tolerance.     

IL-12-independent Th1-type immune responses to respiratory viral infection: requirement of IL-18 for IFN-gamma release in the lung but not for the differentiation of viral-reactive Th1-type lymphocytes

We demonstrated that IL-12 was induced during primary or secondary pulmonary adenoviral infection in wild-type (wt) mice. However, cellular responses were not compromised in the lungs of IL-12-/- mice. The level of IFN-gamma in the lung was similar in wt and IL-12-/- mice during pulmonary viral infection. Upon Ag stimulation in vitro, lymphocytes from draining lymph nodes or spleen of infected IL-12-/- mice released large amounts of IFN-gamma, but not IL-4, which were comparable to those released by wt lymphocytes. Furthermore, a predominantly IgG2a response to adenoviral infection was unimpaired in IL-12-/- mice. These significant anti-adenoviral Th1-type responses in IL-12-/- mice led to an efficient clearance of virus-infected cells in the lung. Whether IL-18 was involved in IL-12-independent anti-adenoviral immune responses was investigated. Abrogation of endogenous IL-18 by an Ab resulted in diminished IFN-gamma release and lymphocytic infiltrate in the lung during adenoviral infection. Nevertheless, the development of lymphocytes of the Th1 phenotype was unimpaired in the absence of both IL-12 and IL-18. In contrast to their intact ability to mount Th1-type responses to viral infection, IL-12-/- mice suffered impaired Th1-type immune responses to pulmonary mycobacterial infection. Our findings suggest that IL-12, although induced, is not required for Th1-type responses to respiratory viral infection, in contrast to mycobacterial infection. IL-18 is required for the optimal release of IFN-gamma in the lung during viral infection, but is not required for the generation of virus-reactive Th1-type lymphocytes. Th1 differentiation during respiratory adenoviral infection may involve molecules different from IL-12 or IL-18.     

IL-23 compensates for the absence of IL-12p70 and is essential for the IL-17 response during tuberculosis but is dispensable for protection and antigen-specific IFN-gamma responses if IL-12p70 is available

IL-12p70 induced IFN-gamma is required to control Mycobacterium tuberculosis growth; however, in the absence of IL-12p70, an IL-12p40-dependent pathway mediates induction of IFN-gamma and initial bacteriostatic activity. IL-23 is an IL-12p40-dependent cytokine containing an IL-12p40 subunit covalently bound to a p19 subunit that is implicated in the induction of CD4 T cells associated with autoimmunity and inflammation. We show that in IL-23 p19-deficient mice, mycobacterial growth is controlled, and there is no diminution in either the number of IFN-gamma-producing Ag-specific CD4 T cells or local IFN-gamma mRNA expression. Conversely, there is an almost total loss of both IL-17-producing Ag-specific CD4 T cells and local production of IL-17 mRNA in these mice. The absence of IL-17 does not alter expression of the antimycobacterial genes, NO synthase 2 and LRG-47, and the absence of IL-23 or IL-17, both of which are implicated in mediating inflammation, fails to substantially affect the granulomatous response to M. tuberculosis infection of the lung. Despite this redundancy, IL-23 is required to provide a moderate level of protection in the absence of IL-12p70, and this protection correlates with a requirement for IL-23 in the IL-12p70-independent induction of Ag-specific, IFN-gamma-producing CD4 T cells. We also show that IL-23 is required for the induction of an IL-17-producing Ag-specific phenotype in naive CD4 T cells in vitro and that absence of IL-12p70 promotes an increase in the number of IL-17-producing Ag-specific CD4 T cells both in vitro and in vivo.     

Dendritic cell-derived IL-12 promotes B cell induction of Th2 differentiation: a feedback regulation of Th1 development.

B cells convert what are normally conditions for Th1 differentiation into an environment suitable for Th2 development. This capacity is dependent on CD40 as B cells from CD40-/- mice do not elicit Th2 differentiation. To elucidate the basis of this effect, we surveyed cytokine RNA made by naive B cells after activation with anti-Ig and anti-CD40. Resting B cells make TGF-beta message only, however, 4 days after activation, RNA encoding IL-6, IL-10, and TNF-alpha was found. The expression of these messages was accelerated by 2 days in the presence of IL-12. The relevance of these observations to T cell differentiation was investigated: addition of OVA peptide to splenic cells from DO.11.10 transgenic mice causes most T cells to make IFN-gamma. Coactivation of B cells in these cultures reduces the number of IFN-gamma-producing T cells and increases the number synthesizing IL-4. Abs to IL-6 and IL-10 block the IL-4 enhancement. Dissection of the component APC demonstrated that interaction of B cells with IL-12-producing dendritic cells is crucial for B cell-mediated IL-4 enhancement: Thus, B cells preactivated in the presence of dendritic cells from IL-12-/- mice show little IL-4-inducing activity when used to activate T cells. This immune regulation is initiated by IL-12 and therefore represents a feedback loop to temper its own dominant effect (IFN-gamma induction).     

IL-12p40-dependent agonistic effects on the development of protective innate and adaptive immunity against Salmonella enteritidis.

To study a potential IL-12p40-dependent but IL-12p75-independent agonistic activity regulating the immune response against Salmonella Enteritidis, the course of infection in IL-12p35-deficient mice (IL-12p35(-/-), capable of producing IL-12p40) was compared with that of IL-12p40(-/-) mice. Mice lacking IL-12p40 revealed a higher mortality rate and higher bacterial organ burden than mice capable of producing IL-12p40. This phenotype was found in both genetically susceptible (BALB/c, Ity(s)) and resistant mice (129Sv/Ev, Ity(r)) indicating Ity-independent mechanisms. The more effective control of bacteria in the IL-12p35(-/-) mice was associated with elevated serum IFN-gamma and TNF-alpha levels. In contrast, IL-12p40(-/-) mice showed reduced IFN-gamma production, which was associated with significantly elevated serum IgE levels. Early during infection (days 3 and 4 postinfection), as well as late (day 20 postinfection), the number of infected phagocytes was strongly increased in the absence of IL-12p40 indicating impaired bactericidal activity when IL-12p40 was missing. Liver histopathology revealed a decreased number of mononuclear granulomas in IL-12p40(-/-) mice. Depletion of CD4(+) or CD8(+) T lymphocytes in vivo suggested that both T cell subpopulations contribute to the IL-12p40-dependent protective functions. Analysis of IL-12p40 vs IL-23p19 mRNA expression revealed an up-regulation of only IL-12p40 mRNA during Salmonella infection. Together these data indicate that IL-12p40 can induce protective mechanisms during both the innate and the adaptive type 1 immune response in Salmonella infection. This novel activity of IL-12p40 complements the well described dominant and essential role of IL-12p75 in protective immunity to Salmonella infection.     

Prevention of autoantibody-mediated Graves'-like hyperthyroidism in mice with IL-4, a Th2 cytokine

Graves' hyperthyroidism has long been considered to be a Th2-type autoimmune disease because it is directly mediated by autoantibodies against the thyrotropin receptor (TSHR). However, several lines of evidence have recently challenged this concept. The present study evaluated the Th1/Th2 paradigm in Graves' disease using a recently established murine model involving injection of adenovirus expressing the TSHR (AdCMVTSHR). Coinjection with adenovirus expressing IL-4 (AdRGDCMVIL-4) decreased the ratio of Th1/Th2-type anti-TSHR Ab subclasses (IgG2a/IgG1) and suppressed the production of IFN-gamma by splenocytes in response to TSHR Ag. Importantly, immune deviation toward Th2 was accompanied by significant inhibition of thyroid-stimulating Ab production and reduction in hyperthyroidism. However, in a therapeutic setting, injection of AdRGDCMVIL-4 alone or in combination with AdCMVTSHR into hyperthyroid mice had no beneficial effect. In contrast, coinjection of adenoviruses expressing IL-12 and the TSHR promoted the differentiation of Th1-type anti-TSHR immune responses as demonstrated by augmented Ag-specific IFN-gamma secretion from splenocytes without changing disease incidence. Coinjection of adenoviral vectors expressing IL-4 or IL-12 had no effect on the titers of anti-TSHR Abs determined by ELISA or thyroid-stimulating hormone-binding inhibiting Ig assays, suggesting that Ab quality, not quantity, is responsible for disease induction. Our observations demonstrate the critical role of Th1 immune responses in a murine model of Graves' hyperthyroidism. These data may raise a cautionary note for therapeutic strategies aimed at reversing Th2-mediated autoimmune responses in Graves' disease in humans.     

PAS-1, a protein affinity purified from Ascaris suum worms, maintains the ability to modulate the immune response to a bystander antigen

Helminth infections and parasite components have potent immunomodulatory effects on a host's immune system. In the present study, we investigated the effect of PAS-1, a protein component of Ascaris suum adult worms recognized by a monoclonal antibody (MAIP-1), on humoral and cell-mediated responses to a bystander antigen (ovalbumin [OVA]). MAIP-1 recognized only one of the three polypeptide chains of PAS-1, but neutralized the suppressive effect of the whole worm extract on OVA-specific antibody production. PAS-1 inhibited antibody production against a T-cell-dependent, but not a T-cell-independent, antigen in a dose-dependent way. IgM, IgG1, IgG2b, and also IgE and anaphylactic IgG1 levels were downregulated. In addition, PAS-1 inhibited OVA-specific delayed type hypersensitivity reactions in the footpad of mice, showing a potent immunosuppressive activity on both Th1 and Th2 responses that seems to be mediated by the induction of large amounts of IL-10 and IL-4. Indeed, PAS-1-specific spleen cells secreted sevenfold more IL-10 and threefold more IL-4 than OVA-specific cells in response to in vitro restimulation with the respective antigens. In conclusion, we showed that PAS-1, a single protein component from A. suum, maintains all its immunosuppressive properties.     

Prevention of allergen-specific, Th2-biased immune responses in vivo: role of increased IL-12 and IL-18 responsiveness

The factors that control development of adaptive responses to exogenous Ag remain incompletely understood. An ability to selectively direct immunity toward a specific phenotype would be of clinical benefit in numerous immunological disorders. Administration of chemically modified allergen glutaraldehyde-polymerized OVA (OA-POL) leads to >90% reductions in murine IgE and >500-fold increases in IgG2c responses that develop upon subsequent immunization with native Ag. In the present study, we examine the mechanisms underlying this reorientation of the type 2 dominant response that would normally develop. Lack of endogenous IL-12 or IFN-gamma results in markedly reduced induction of IgG2c responses following OA-POL treatment, but only IFN-gamma(-/-) mice demonstrate reduced capacity to prevent IgE induction. This indicates that while both IL-12 and IFN-gamma are critical promoters of type 1 immunity, only IFN-gamma is required to maximally inhibit development of type 2 immune responses. Compared with OVA-immunized mice, CD69(+) T cells from OA-POL-immunized mice demonstrate elevated IL-12Rbeta(2), IL-18Ralpha, and IL-18Rbeta mRNA levels, as well as increased IFN-gamma production in response to rIL-12 or rIL-18 stimulation. Collectively, these data indicate that preventing induction of type 2 immune responses is critically dependent on altered T cell responsiveness to these cytokines. The finding that targeted, Ag-specific manipulation of IL-12 and IL-18 responsiveness can be used to shape the phenotype of the dominant immune response that develops suggests that specifically targeting IL-12 and IL-18 receptor expression may offer clinical options for clinical prophylaxis or intervention.     

IL-12-independent IFN-gamma production by T cells in experimental Chagas' disease is mediated by IL-18.

IL-12p35-deficient (IL-12p35(-/-)) mice were highly susceptible to Trypanosoma cruzi infection and succumbed during acute infection, demonstrating the crucial importance of endogenous IL-12 in resistance to experimental Chagas' disease. Delayed immune responses were observed in mutant mice, although comparable IFN-gamma and TNF-alpha blood levels as in wild-type mice were detected 2 wk postinfection. In vivo and in vitro analysis demonstrated that T cells, but not NK cells, were recruited to infected organs. Analysis of mice double deficient in the recombinase-activating gene 2 (RAG2) and IL-12p35, as well as studies involving T cell depletion, identified CD4(+) T cells as the cellular source for IL-12-independent IFN-gamma production. IL-18 was induced in IL-12p35(-/-) mice and was responsible for IFN-gamma production, as demonstrated by in vivo IL-18 neutralization studies. In conclusion, evidence is presented for an IL-12-independent IFN-gamma production in experimental Chagas' disease that is T cell and IL-18 dependent.     

Immunoprevention of mammary carcinoma in HER-2/neu transgenic mice is IFN-gamma and B cell dependent

A vaccine combining IL-12 and allogeneic mammary carcinoma cells expressing p185(neu) completely prevents tumor onset in HER-2/neu transgenic BALB/c mice (NeuT mice). The immune protection elicited was independent from CTL activity. We now formally prove that tumor prevention is mainly based on the production of anti-p185(neu) Abs. In the present studies, NeuT mice were crossed with knockout mice lacking IFN-gamma production (IFN-gamma(-/-)) or with B cell-deficient mice (microMT). Vaccination did not protect NeuT-IFN-gamma(-/-) mice, thus confirming a central role of IFN-gamma. The block of Ab production in NeuT-microMT mice was incomplete. About one third of NeuT-microMT mice failed to produce Abs and displayed a rapid tumor onset. By contrast, those NeuT-microMT mice that responded to the vaccine with a robust production of anti-p185(neu) Ab displayed a markedly delayed tumor onset. In these NeuT-microMT mice, the vaccine induced a lower level of IgG2a and IgG3 and a higher level of IgG2b than in NeuT mice. Moreover, NeuT-microMT mice failed to produce anti-MHC class I Abs in response to allogeneic H-2(q) molecules present in the cell vaccine. These findings show that inhibition of HER-2/neu carcinogenesis depends on cytokines and specific Abs, and that a highly effective vaccine can rescue Ab production even in B cell-deficient mice.     

Absence of IFN-gamma or IL-12 has different effects on experimental myasthenia gravis in C57BL/6 mice

Immunization with acetylcholine receptor (AChR) causes experimental myasthenia gravis (EMG). Th1 cells facilitate EMG development. IFN-gamma and IL-12 induce Th1 responses: we investigated whether these cytokines are necessary for EMG development. We immunized wild-type (WT) C57BL/6 mice and IFN-gamma and IL-12 knockout mutants (IFN-gamma-/-, IL-12-/-) with Torpedo AChR (TAChR). WT and IFN-gamma-/- mice developed EMG with similar frequency, IL-12-/-mice were resistant to EMG. All strains synthesized anti-AChR Ab that were not IgM or IgE. WT mice had anti-AChR IgG1, IgG2b, and IgG2c, IFN-gamma-/- mice had significantly less IgG2c, and IL-12-/- mice less IgG2b and IgG2c. All mice had IgG bound to muscle synapses, but only WT and IFN-gamma-/- mice had complement; WT mice had both IgG2b and IgG2c, IFN-gamma-/- only IgG2b, and IL-12-/- neither IgG2b nor IgG2c. CD4+ cells from all AChR-immunized mice proliferated in response to AChR and recognized similar epitopes. After stimulation with TAChR, CD4+ cells from IFN-gamma-/- mice secreted less IL-2 and similar amounts of IL-4 and IL-10 as WT mice. CD4+ cells from IL-12-/- mice secreted less IFN-gamma, but more IL-4 and IL-10 than WT mice, suggesting that they developed a stronger Th2 response to TAChR. The EMG resistance of IL-12-/- mice is likely due to both reduction of anti-TAChR Ab that bind complement and sensitization of modulatory Th2 cells. The reduced Th1 function of IFN-gamma-/- mice does not suffice to reduce all complement-fixing IgG subclasses, perhaps because as in WT mice a protective Th2 response is missing.