Interleukin-4 transgenic mice of resistant background are susceptible to Leishmania major infection

The outcome of cutaneous leishmaniasis is dependent on the balance of Th1 and Th2 cells. In the murine model, Th1 cells are host-protective whereas the Th2 cells are disease-promoting. However, the in vivo role of interleukin-4 (IL-4), a signature product of Th2 cells, is uncertain. We compared the course of Leishmania major infection in the genetically resistant 129/Sv mice and the mutant 129/Sv mice transgenic for the murine IL-4 gene under the control of the immunoglobulin heavy chain enhancer and promoter. We report here that in contrast to their wild-type parents, the IL-4 transgenic mice are susceptible to L. major infection. This is associated with the development of inexorably progressive lesions and parasite loads. Spleen cells from infected transgenic mice produced significantly higher levels of IL-4 but lower amounts of interferon-γ when stimulated in vitro with leishmanial antigens compared to those from infected normal 129/Sv mice. Furthermore, sera from the infected transgenic mice contained higher levels of IL-4 and IgE than the sera of infected normal 129/Sv mice. These results, therefore, establish in a new animal model that IL-4 promotes disease development in murine cutaneous leishmaniasis.     

CXCR3-/- mice mount an efficient Th1 response but fail to control Leishmania major infection

Chemokines play a critical role in recruitment of leukocytes to the site of infection, which is essential for host defense. We analyzed the role of CXC chemokine receptor 3 (CXCR3) in the control of cutaneous leishmaniasis using CXCR3-/- C57BL/6 mice. We found that Leishmania major-infected CXCR3-/- mice mount an efficient Th1 response as evident by markedly increased serum levels of Th1-associated IgG2a and significant production of IFN-gamma and IL-12 by the draining lymph node cells, restrict systemic spread of infection, but fail to control parasite replication at the site of infection and develop chronic non-healing lesions. Furthermore, the inability of CXCR3-/- mice to control cutaneous L. major growth was associated with fewer CD4+ and CD8+ T cells and significantly lower levels of IFN-gamma in their lesions as compared to CXCR3+/+ mice. These results demonstrate that CXCR3 plays a critical role in the host defense against cutaneous leishmaniasis caused by L. major. Furthermore, they also suggest that the susceptibility of CXCR3-/- mice to L. major is due to impaired CD4+ and CD8+ T cell trafficking and decreased production of IFN-gamma at the site of infection rather than to their inability to mount a parasite-specific Th1 response.     

Impaired Th1 responses in mice deficient in Epstein-Barr virus-induced gene 3 and challenged with physiological doses of Leishmania major

Protection against Leishmania major is dependent on IL-12 release from L. major-infected dendritic cells (DC) that induce IFN-gamma-producing Th1/Tc1 cells. IL-27, a novel member of the IL-12 family, is a heterodimer composed of p28 and IL-12p40-related Epstein-Barr virus-induced gene 3 (EBI3), and was shown to be produced by DC. In this study, we utilized EBI3-deficient mice to investigate the role of IL-27 in leishmaniasis using physiological low-dose infections that mimic natural transmissions. Lesions in EBI3(-/-) mice were significantly larger between weeks 3 and 10 post infection, reaching up to approximately threefold increased lesion volumes compared to wild types. In parallel, dermal lesions of EBI3(-/-) mice contained greater parasite numbers, reaching a peak load that was 2-log higher than in C57BL/6 mice. However, lesions in EBI3(-/-) and wild-type mice resolved after 12 weeks. At early time points, the antigen-specific cytokine response in EBI3(-/-) lymph nodes showed increased levels of IL-4, IL-10 and IL-13 and decreased IFN-gamma production. IL-27 production was restricted to the DC population, since the majority of EBI3 expression in lymph nodes of infected mice was found in CD11c(+) cells. In conclusion, our data show that DC-derived IL-27 is critical for the timely initiation of efficient anti-parasite Th1 immunity early in infections.     

Virulent or avirulent (dhfr-ts-) Leishmania major elicit predominantly a type-1 cytokine response by human cells in vitro

In this study we have compared the immune response of normal human cells cultured in vitro to two virulent strains of Leishmania major (CC1 and LV39), and to an avirulent vaccine strain (dhfr-ts-) made by targeted deletion of the essential gene DHFR-TS. We utilized an in vitro system in which naive T cells from normal human donors were primed with autologous Leishmania-infected macrophages. All three parasites infected macrophages and transformed into amastigotes within the cells. However, whereas LV39 and CC1 replicated in macrophages, dhfr-ts- did not. When peripheral blood lymphocytes (PBL) were stimulated with autologous macrophages infected with any of the three parasites, the lymphocytes produced a type-1-biased cytokine response. Finally, addition of IL-12 during the first stimulation period increased the production of interferon-gamma but decreased IL-5 secretion. On the other hand, anti-IL-12 resulted in the opposite effect.     

Accessory signaling by CD40 for T cell activation: induction of Th1 and Th2 cytokines and synergy with interleukin-12 for interferon-γ production

The interaction of CD40 ligand (CD40L) on activated T cells with CD40 on B cells, monocytes and dendritic cells is essential for humoral immunity and for up-regulation of antigen-presenting cell (APC) functions, as a result of signaling through CD40. There are also some indications that after interaction with CD40, CD40L can directly signal T cells. In this study we demonstrate that upon stimulation of human peripheral blood T cells through the T cell receptor (TCR)/CD3 complex, CD40/CD40L interaction strongly enhances the production of Th1 cytokines such as interleukin (IL)-2 and interferon (IFN)-γ and Th2 cytokines such as IL-4, IL-5 and IL-10 by a direct effect on T cells. Furthermore, CD40/CD40L interaction synergizes with IL-12 in selectively enhancing IFN-γ production by purified anti-CD3-stimulated T cells. These effects were observed at both the protein and the mRNA level. Both CD4⁺ and CD8⁺ T cells were able to produce IFN-γ in the presence of helper signals from IL-12 and CD40, although CD8⁺ T cells were less active. Since CD40/CD40L interaction also up-regulates IL-12 production and B7 expression by APC, our results suggest that CD40/CD40L interaction is bidirectional, and promotes activation of both APC and T cells.     

Interleukin-12 increases interleukin-4 production by established human ThO and Th2-like T cell clones

Interleukin (IL)-12 is a potent inducer of cell-mediated immunity: it favors the generation of interferon (IFN)-γ-producing T cells, increases IFN-γ production by T cells and natural killer cells and prevents the generation of a Th2 response in murine in vivo models. Nevertheless, the effects of IL-12 on an established Th2 response remain poorly documented. In the present paper, we analyzed the effect of IL-12 on the profile of lymphokines produced by established IL-4-producing Th0 and Th2-like human T cell clones (TCC) and by polyclonal T cells. We found that IL-12 (i) enhances, as previously reported, IFN-γ production by Th0 TCC and, to a smaller extent, by Th2-like TCC, (ii) increases the proliferation of Th0 and Th2-like TCC and (iii) unexpectedly, synergizes with T cell receptor-associated or nonspecific stimuli in increasing IL-4 production by these TCC. Thus, IL-12 potentiates the production of IFN-γ and also of IL-4 by established IL-4-producing TCC. Although IL-12 has been widely reported to induce a Th1 response and to prevent the development of a Th2 response in vivo, IL-12 may on the contrary potentiate an established Th2 response in humans.     

The induction of a protective response in Leishmania major-infected BALB/c mice with anti-CD40 mAb

A protective immune response to the intracellular parasite Leishmania major requires the development of a Th1 CD4⁺ T cell phenotype. We demonstrate herein that BALB/c mice, which normally develop a susceptible Th2 response to L. major infection, are protected when co-injected with an agonistic anti-murine CD40 mAb. Anti-CD40 mAb-mediated protection in this system was found to be T cell dependent, since it was not observed in C57BL/ 6 × 129 mice that were rendered T cell deficient (TCR β^–/– × TCR δ^–/–) and L. major susceptible. Anti-CD40 mAb stimulation of L. major-infected BALB/c mice was accompanied by increased IL-12 and IFN-γ production in draining lymphnodes, analyzed either by direct expression, or in an antigen-specific in vitro recall assay. The protective role of these cytokines was indicated by the finding that anti-CD40 mAb-mediated protection of L. major-infected BALB/c mice could be reversed by co-treating the animals with neutralizing anti-IL-12 and/or anti-IFN-γ mAb. Collectively, these data suggest that BALB/c mice develop a protective Th1 CD4⁺ T cell response to L. major infection when co-injected with anti-CD40 mAb. While the CD40-CD40L interaction has been previously shown to be vital in the control of murine Leishmaniasis, the current study establishes in vivo that anti-CD40 mAb treatment alone is sufficient to protect BALB/c mice from L. majorinfection and raises the possibility of utilizing this approach for vaccination strategies.     

Thymic selection and peptide-induced activation of T cell receptor-transgenic CD8 T cells in interleukin-2-deficient mice

The requirement for interleukin-2 (IL-2) in repertoire selection and peripheral activation of CD8 T cells was tested in mice rendered IL-2 deficient by gene targeting and expressing a transgenic T cell receptor (TcR) (F5) specific for influenza nucleoprotein (NP) 366-374 + H-2D^b. Positive selection of the transgenic F5 TcR into the CD8 compartment proceeded normally. Both in vivo and in vitro, the antigenic peptide induced depletion of immature thymocytes and proliferation of mature CD8 T cells regardless of the presence of an intact IL-2 gene. In contrast, cytotoxic T lymphocyte (CTL) activity was only generated by T cells from IL-2⁺ F5 transgenic mice. Exogenous IL-2 was able to fully restore the CTL response of IL-2^?/? responder cells in vitro. Thus, both in vivo and in vitro, clonal expansion of CD8 T cells can proceed in the absence of IL-2, whereas in peptide-immunized F5 transgenic mice, induction of cytotoxic effector function is IL-2 dependent.     

Normal clonal expansion but impaired Fasmediated cell death and anergy induction in interleukin-2-deficient mice

Despite a normal development of all major lymphoid subsets, with time, interleukin-2 (IL-2)-deficient mice develop a fatal immunopathology. The disease phenotype is characterized by lymphoadenopathy, splenomegaly, T cell infiltration of various organs, overproduction of a number of cytokines and autoantibody formation. Phenotypically, CD4⁺ and CD8⁺ T cells exhibit features characteristic of antigenically experienced cells. The accumulation of cells with a memory phenotype together with the previous suggestion of an involvement of IL-2 in the termination phase of immune responses prompted us to study the fate of superantigen-reactive T cells in IL-2-deficient mice in comparison to their IL-2-producing littermates. We show that expansion in vivo of CD4⁺ and, to a lesser extent, CD8⁺ T cells reactive to the superantigens staphylococcal enterotoxin A and B (SEA and SEB) proceeds normally in the absence of IL-2, but that fewer CD4⁺ cells are subsequently deleted. The residual superantigenreactive cells fail to become anergic as measured by proliferation in vitro in response to the same superantigen. T cell blasts generated in vitro from lymph node cells of IL-2-deficient mice by superantigen stimulation in the absence of exogenous IL-2 also fail to become anergic. In contrast to cells from IL-2-producing littermates, they do not exhibit Fas-induced apoptosis when cultured on anti-Fas antibody-coated plates, although Fas expression by IL-2-deficient cells is normal or even elevated compared to the IL-2-producing control cells. The data suggest that activation of T cells in the absence of IL-2 fails to generate a signal which is necessary to activate the apoptotic pathway and thus leads to an accumulation of antigen-experienced cells and the chronic inflammatory responses observed in IL-2-deficient mice.     

c-Rel promotes type 1 and type 17 immune responses during Leishmania major infection

Recent studies demonstrated the crucial role of c‐Rel in directing Treg lineage commitment and its involvement in T helper 1 (Th1) cell‐mediated autoimmune inflammation. We thus wondered whether these opposite functions of c‐Rel influence the course of antiparasitic immune responses against Leishmania major, an accepted model for the impact of T‐cell subsets on disease outcome. Here we show that c‐Rel‐deficient (rel^?/?) mice infected with L. major displayed dramatically exacerbated leishmaniasis and enhanced parasite burdens. In contrast to WT mice, IFN‐γ and IL‐17 production in response to L. major antigens was severely impaired in rel^?/? mice. Reconstitution of Rag1^?/? T‐cell deficient mice with rel^?/? CD4⁺ T cells followed by L. major infection demonstrated that c‐Rel‐deficient T cells mount normal Th1 responses and are able to contain the infection. Similarly, Th1 differentiation of naïve CD4⁺ cells in vitro was normal. Notably, a selective defect in IL‐12 and IL‐23 production was observed in rel^?/? DCs compared with their WT counterparts. In conclusion, our data suggest that the expression of c‐Rel in myeloid cells is essential for clearance of L. major and that this c‐Rel‐mediated effect is dominant over the lack of Tregs.     

Resistant mice lacking interleukin-12 become susceptible to Trypanosoma cruzi infection but fail to mount a T helper type 2 response

Interleukin-12 (IL-12) is essential to resistance to Trypanosoma cruzi infection because it stimulates the synthesis of interferon-gamma (IFN-gamma) that activates macrophages to a parasiticidal effect. Investigation of mice deprived of IL-12 genes (IL-12 knockout mice) has confirmed the important role of IL-12 and IFN-gamma in controlling parasitism in T. cruzi infection. However, it has not yet been addressed whether a shift towards a T helper type 2 (Th2) pattern of cytokine response occurred in these mice that might have contributed to the aggravation of the infection caused by IL-12 deprivation. We examined the course of T. cruzi (Y strain) infection and the regulation of cytokine responses and nitric oxide production in C57BL/6 IL-12 p40-knockout mice. The mutant mice were extremely susceptible to the infection as evidenced by increased parasitaemia, tissue parasitism and mortality in comparison with the control C57BL/6 mouse strain (wild-type) that is resistant to T. cruzi. A severe depletion of parasite-antigen-specific IFN-gamma response, without an increase in IL-4 or IL-10 production, accompanied by reduced levels of nitric oxide production was observed in IL-12 knockout mice. We found no evidence of a shift towards a Th2-type cytokine response. In IL-12 knockout mice, the residual IFN-gamma production is down-regulated by IL-10 but not by IL-4 and nitric oxide production is stimulated by tumour necrosis factor-alpha. Parasite-specific immunoglobulin G1 antibody levels were similar in IL-12 knockout and wild-type mice, whereas IL-12 knockout mice had much higher levels of immunoglobulin G2b.     

Interleukin-12/T cell stimulating factor,a cytokine with multiple effects on T helper type 1 (Th1) but not on Th2 cells

At least two subsets of CD4⁺ T helper cell lymphocytes termed T_h1 and T _h, 2 exist in the mouse and probably in humans. They are characterized by the secretion of different lymphokines and by their functional behavior. Dysregulated expansion of one or the other subset may be one reason for the development of certain diseases. Thus, it is of importance to define the signals involved in the differentiation and activation of the two T_h cell subsets. It is known and has been confirmed in this report that the cytokine interleukin (IL)-1 acts onT_h2 cells but not on T_h1 cells. We now report that a previously identified cytokine which was provisionally termed T cell stimulating factor is identical with IL-12 and exhibits a reciprocal behaviour to IL-1. IL-12 has several effects on T_h1 cells. It can induce the proliferation of certain T_h1 cells in combination with IL-2. Synthesis of interferon (IFN)-γ by T_h1 cells can be triggered by IL-2 plus IL-12. In contrast to the IFN-γ production observed after T cell receptor (TcR) CD3 stimulation of T_h1 cells with lectin Concanavalin A the IFN-γ production induced by IL-12+IL-2 is insensitive to the immunosuppressive drug cyclosporin A. Furthermore, IL-12 enhances the TcR/CD3-induced synthesis of IFN-γ of several T_h1 clones. Finally, IL-12 (+ IL-2) induces homotypic cell aggregation of T_h1 clones. This type of cell aggregation depends on the participation of LFA-1 and ICAM-1 molecules. In all activation systems with T_h1 cells no effect of IL-1 was demonstrable. In contrast, only IL-1 but not IL-12 served as a co-stimulatory signal for several T_h2 cell lines activated via the TcR/CD3 complex.     

Deviation of pancreas-infiltrating cells to Th2 by interleukin-12 antagonist administration inhibits autoimmune diabetes

Nonobese diabetic (NOD) mice develop spontaneous insulin-dependent diabetes mellitus (IDDM), and the pancreas-infiltrating T cells invariably show a Th1 phenotype. We demonstrated here that the interleukin (IL)-12 antagonist (p40)₂ can deviate the default Th1 development of naive T cell receptor (TCR)-transgenic CD4⁺ cells to the Th2 pathway in vitro. Although (p40)₂ does not modify the cytokine profile of polarized Th1 cells, it prevents further recruitment of CD4⁺ cells into the Th1 subset. To study the involvement of Th1 and Th2 cells in the initiation and progression of IDDM, we targeted endogenous IL-12 by administration of (p40)₂ in NOD mice. (p40)₂ administration to NOD mice inhibits interferon-γ but not IL-10 production in response to lipopolysaccharide (LPS) or to the putative autoantigen IA-2. Serum immunoglobulin isotypes determined after (p40)₂ treatment indicate an increase in Th2 and a decrease in Th1 helper activity. Administration of (p40)₂ from 3 weeks of age onwards, before the onset of insulitis, results in the deviation of pancreas-infiltrating CD4⁺ but not CD8⁺ cells to the Th2 phenotype as well as in the reduction of spontaneous and cyclophosphamide-accelerated IDDM. After treating NOD mice with (p40)₂ from 9 weeks of age, when insulitis is well established, few Th2 and a reduced percentage of Th1 cells are found in the pancreas. This is associated with a slightly decreased incidence of spontaneous IDDM, but no protection from cyclophosphamide-accelerated IDDM. In conclusion, deviation of pancreas-infiltrating CD4⁺ cells to Th2 is associated with protection from IDDM. However, targeting IL-12 after the onset of insulitis, when the pancreas contains polarized Th1 cells, is not sufficient to induce an effective immune deviation able to significantly modify the course of disease.     

Establishment of resistance to Leishmania major infection in susceptible BALB/c mice requires parasite-specific CD8+ T cells

Although CD4⁺ T cells are generally accepted to be responsiblefor the determination of resistance to infection in experimentalmurine cutaneous leishmanlasis, a contribution of CD8⁺ lymphocytesto immunity can be demonstrated under certain well-defined conditions.Normally highly susceptible BALB/c mice can be rendered resistantto infection with Leishmania major promastigotes by a singleinjection of monoclonal anti-CD4 antibodies at the beginningof infection. Mice treated in such a way can heal their primarycutaneous lesions and acquire immunity to subsequent challengeinfection. Both the resolution of the primary infection andthe induced state of immunityto reinfection in these mice isshown to be dependent upon the anti-leishmanial effector functionsof CD8⁺ T cells. Furthermore, in contrast to control infectedBALB/c mice, which are unable to mount a delayed-type hypersensitivity(DTH) response to viable parasites, mice cured as a result oftreatment with anti-CD4 antibodies in vivo exhibit a strongDTH response, which can be significantly reduced by injectionof either anti-CD4 or anti-CD8 monoclonal antibodies prior toantigenic challenge with viable promastigotes. Moreover, increasednumbers of specific CD8⁺ T cells, able to transferLeishmania-specificDTH responses, were found in lymphold organs of BALB/c micerendered resistant to infection by immunointervention with anti-CD4monoclonal antibodies at the beginning of infection. Neutralizationin vivo of interleukin 4 during the course of infection in BALB/cmice also enables these otherwise susceptible mice to resolvetheir cutaneous lesions and to decrease the parasite burdenin infected tissues. CD8⁺ T cells are required for both of thesebeneficial effects. Taken together, these results indicate thatin the immune BALB/c mouse, as in the normally resistant CBAmouse, CD8⁺ lymphocytes are involved in the elimination of L.major and in the establishment and maintenance of immunity againstinfection with this parasite.     

Parasite dose determines the Th1/Th2 nature of the response to Leishmania major independently of infection route and strain of host or parasite

Leishmania major causes cutaneous leishmaniasis in mice and man. Infection of mice with relatively low or high numbers of parasites leads respectively to parasite containment, associated with a Th1, cell-mediated response, or progressive disease, associated with a Th2, antibody response in all circumstances studied. These include different parasite strains, different routes of infection, and different hosts previously classified as susceptible, resistant or of intermediate susceptibility. This dose dependency appears to reflect a general rule. We argue that this rule may allow the design of a vaccination strategy that is effective among a genetically diverse population, and that it imposes severe constraints upon proposals for the nature of the “decision criterion” determining whether antigen induces a Th1 or Th2 response.     

The role of IL-12 in the maintenance of an established Th1 immune response in experimental leishmaniasis

IL-12 initiates the development of cell-mediated immunity by promoting the differentiation of naive T cells into the Th1 phenotype, and is essential in the development of a Th1 immune response to the intracellular protozoan parasite, Leishmania major. The present study investigated whether IL-12 is also required for the maintenance and effector function of an established Th1 immune response in L. major -infected mice. While neutralization of IL-12 com promised the ability of a leishmanial antigen-reactive Th1 cell clone to produce IFN-γ in vitro, lymphnode cells taken from 2-week L. major -infected mice were able to secrete IFN-γ in an IL-12-independent manner. However, when a short-term T cell line was established in vitro from lymph node cells, the production of IFN-γ again became IL-12 dependent. These results suggest that other factors may compensate for IL-12 in vivo in promoting IFN-γ production during L. major infection. To directly assess if IL-12 was required in vivo for resistance to L. major, we studied the effect of IL-12 neutralization on both a primary and secondary L. major infection in C3H mice. L. major infection in C3H mice is characterized by the development of a small lesion that heals by 8 weeks, and these animals are resistant to reinfection. As previously reported, administration of anti-IL-12 monoclonal antibody (mAb) during a primary infection led to severe disease. However, mice that had healed from a primary infection with L. major and were treated with anti-IL-12 mAb were as resistant as control animals. These findings suggest that once Th1 cells have developed, their effector function in vivo is independent of IL-12, and that this independence is not due to an intrinsic property of the T cell, but to the microenvironment created by the infection.     

Deficiency of prolactin‐inducible protein leads to impaired Th1 immune response and susceptibility to Leishmania major in mice

Although the strategic production of prolactin‐inducible protein (PIP) at several ports of pathogen entry into the body suggests it might play a role in host defense, no study has directly implicated it in immunity against any infectious agent. Here, we show for the first time that PIP deficiency is associated with reduced numbers of CD4⁺ T cells in peripheral lymphoid tissues and impaired CD4⁺ Th1‐cell differentiation in vitro. In vivo, CD4⁺ T cells from OVA‐immunized, PIP‐deficient mice showed significantly impaired proliferation and IFN‐γ production following in vitro restimulation. Furthermore, PIP‐deficient mice were highly susceptible to Leishmani major infection and failed to control lesion progression and parasite proliferation. This susceptibility was associated with impaired NO production and leishmanicidal activity of PIP KO macrophages following IFN‐γ and LPS stimulation. Collectively, our findings implicate PIP as an important regulator of CD4⁺ Th1‐cell‐mediated immunity.     

Interleukin-12 promotes a chronic intestinal nematode infection

Resistance and susceptibility to the intestinal parasite Trichuris muris has been shown to be due to a dominant T helper 2 (Th2) and a dominant Th1 response, respectively. The factors determining the initial polarization of the immune response remain largely unresolved, although the cytokine environment at the time of antigen presentation clearly plays an essential role. Interleukin (IL)-12, a cytokine produced mainly by macrophages, dendritic cells, and other monocytes has been shown to be important in driving a strong Th1 response by stimulating the production of interferon (IFN)-γ from natural killer and Th0 cells and therefore forms a link between the innate and adaptive immune system. IL-12 has been shown to play an important role in resistance to a number of intracellular pathogens, including Listeria and Leishmania. It has also been proposed as an anti-tumor agent and for use in the treatment of HIV. Conversely, IL-12 has been shown to prolong the survival of Nippostrongylus brasiliensis and to accelerate autoimmunity. Our studies demonstrate that by driving a strong Th1 response, IL-12 promotes chronic T. muris infection when given to normally resistant BALB/K mice. Parasite-specific IgG2a, a Th1 parameter of infection, was greatly up-regulated, whereas some Th2 parameters of infection were down-regulated. IL-12 treatment could be delayed until 1 week after infection had started and still promote a strong Th1 response. The actions of IL-12 in promoting a chronic infection were IFN-γ dependent as an anti-IFN-γ mAb abrogated the effects of IL-12.     

Th1/Th2 cytokine gene expression after mercuric chloride in susceptible and resistant rat strains

Mercuric chloride (HgCl₂) has contrasting effects on different rat strains: susceptible strains, e.g. Brown Norway (BN) develop polyclonal B cell activation, multiple autoantibodies and widespread tissue injury. Lewis (LEW) rats are resistant: no autoimmune response occurs after HgCl₂; instead, there is immunosuppression. We have previously shown, by fully quantitative polymerase chain reaction (PCR), up-regulation of interleukin-4 (IL-4) gene expression in HgCl₂-treated BN rats, implicating Th2 cells in the autoimmune syndrome. Involvement of the reciprocal Th1 subset, producing interferon-γ (IFN-γ), in resistance of LEW rats to HgCl₂ has been suggested. We now report extensive analysis of Th1 and Th2 cytokine gene expression in spleen and lymph nodes of susceptible (BN) and resistant (LEW) rats after HgCl₂. IL-4 and IFN-γ were analyzed by quantitative PCR, other cytokines were assessed using semiquantitative PCR: the relative merits of these two techniques are discussed. We show pronounced up-regulation of IL-4 and more modest up-regulation of IFN-γ in BN rats, but no up-regulation of either in LEW rats. Baseline levels of IFN-γ were higher in LEW rats. Semi-quantitative PCR showed increased expression of IL-2, IL-6 and IL-10 in BN; in LEW rats only IL-10 was increased. There was no marked change in IL-5, IL-13 or transforming growth factor-β (TGF-β) in either strain. These data further support the key role of IL-4 in HgCl₂-induced autoimmunity, and suggest that failure of up-regulation of IL-4, together with higher baseline IFN-γ expression, accounts for resistance of LEW rats to HgCl₂. However, neither IFN-γ nor TGF-β can be implicated in HgCl₂-induced immunosuppression in the LEW rat in vivo: our data suggest a role for IL-10 in this phenomenon.     

Development in vitro of human CD4+ thymocytes into functionally mature Th2 cells. Exogenous interleukin-12 is required for priming thymocytes to produce both Th1 cytokines and interleukin-10

Fresh postnatal thymocyte cell suspensions were directly cloned under limiting dilution conditions with either phytohemagglutinin or toxic shock syndrome toxin-1 (TSST-1), a bacterial superantigen. Cultures contained allogenic irradiated feeder cells and interleukin (IL)-2, in the absence or presence of exogenous IL-4, interferon (IFN)-γ or IL-12. The resulting CD4⁺ T cell clones generated under these different experimental conditions were then analyzed for their ability to produce IL-2, IL-4, IL-5, IL-10, IFN-γ and tumor necrosis factor (TNF)-β in response to stimulation with phorbol 12-myristate 13-acetate (PMA)+anti-CD3 monoclonal antibody or PMA + ionomycin. Different from T cell clones generated from peripheral blood, virtually all CD4⁺ T cell clones generated from human thymocytes produced high concentrations of IL-2, IL-4 and IL-5, but no IFN-γ, TNF-β or IL-10. Moreover, after activation, these clones expressed on their surface membrane both CD30 and CD40 ligand, but not the product of lymphocyte activation gene (LAG)-3, and provided strong helper activity for IgE synthesis by allogeneic B cells. The Th2 cytokine pattern could not be modified by the addition of IFN-γ. However, upon addition of exogenous IL-12, the resulting CD4⁺ thymocyte clones produced TNF-β, IFN-γ, and IL-10 in addition to IL-4 and IL-5. These results suggest that CD4⁺ human thymocytes have the potential to develop into cells producing the Th2 cytokines IL-4 and IL-5, whereas the ability to produce both Th1 cytokines and IL-10 is acquired only after priming with IL-12.