Genetic mapping of a murine locus controlling development of T helper 1/T helper 2 type responses.

Genetic background of the T cell can influence T helper (Th) phenotype development, with some murine strains (e.g., B10.D2) favoring Th1 development and others (e.g., BALB/c) favoring Th2 development. Recently we found that B10.D2 exhibit an intrinsically greater capacity to maintain interleukin 12 (IL-12) responsiveness under neutral conditions in vitro compared with BALB/c T cells, allowing for prolonged capacity to undergo IL-12-induced Th1 development. To begin identification of the loci controlling this genetic effect, we used a T-cell antigen receptor-transgenic system for in vitro analysis of intercrosses between BALB/c and B10.D2 mice and have identified a locus on murine chromosome 11 that controls the maintenance of IL-12 responsiveness, and therefore the subsequent Th1/Th2 response. This chromosomal region is syntenic with a locus on human chromosome 5q31.1 shown to be associated with elevated serum IgE levels, suggesting that genetic control of Th1/Th2 differentiation in mouse, and of atopy development in humans, may be expressed through similar mechanisms.

Local production of the p40 subunit of interleukin 12 suppresses T-helper 1-mediated immune responses and prevents allogeneic myoblast rejection.

The p40 subunit of interleukin 12 (IL-12p40) has been known to act as an IL-12 antagonist in vitro. We here describe the immunosuppressive effect of IL-12p40 in vivo. A murine myoblast cell line, C2C12, was transduced with retro-virus vectors carrying the lacZ gene as a marker and the IL-12p40 gene. IL-12p40 secreted from the transfectant inhibited the IL-12-induced interferon gamma (IFN-gamma) production by splenocytes in vitro. Survival of C2C12 transplanted into allogeneic recipients was substantially prolonged when transduced with IL-12p40. Cytokine (IL-2 and IFN-gamma) production and cytotoxic T lymphocyte induction against allogeneic C2C12 were impaired in the recipients transplanted with the IL-12p40 transfectant. Delayed-type hypersensitivity response against C2C12 was also diminished in the IL-12p40 recipients. Furthermore, serum antibodies against beta-galactosidase of the T-helper 1-dependent isotypes (IgG2 and IgG3) were decreased in the IL-12p40 recipients. These results indicate that locally produced IL-12p40 exerts a potent immunosuppressive effect on T-helper 1-mediated immune responses that lead to allograft rejection. Therefore, IL-12p40 gene transduction would be useful for preventing the rejection of allografts and genetically modified own cells that are transduced with potentially antigenic molecules in gene therapy.

Effects of TH1 and TH2 cytokines on CD8+ cell response against human immunodeficiency virus: implications for long-term survival.

CD8+ cells from long-term survivors [LTS; infected with human immunodeficiency virus (HIV) for 10 or more years and having CD4+ cell counts of > or = 500 cells per microliters] have a 3-fold greater ability to suppress HIV replication than do CD8+ cells from patients who have progressed to disease (progressors) during the same time period. A change in the pattern of cytokines produced in the host from those that typically favor cell-mediated immunity (T helper 1, TH1 or type 1) to those that down-regulate it (T helper 2, TH2 or type 2) was investigated as a cause of this reduced CD8+ cell anti-HIV function. Treatment of CD8+ cells from LTS with the TH1 cytokine interleukin (IL)-2 enhanced their anti-HIV activity, whereas exposure of these cells to TH2 cytokines IL-4 or IL-10 reduced their ability to suppress HIV replication and to produce IL-2. IL-2 could prevent and reverse the inhibitory effects of IL-4 and IL-10. Moreover, prolonged exposure of CD8+ cells from some progressors to IL-2 improved the ability of these cells to suppress HIV replication. These observations support previous findings suggesting that strong CD8+ cell responses play an important role in maintaining an asymptomatic state in HIV infection. The data suggest that the loss of CD8+ cell suppression of HIV replication associated with disease progression results from a shift in cytokine production within the infected host from a TH1 to a TH2 pattern. Modulation of these cytokines could provide benefit to HIV-infected individuals by improving their CD8+ cell anti-HIV activity.

Gene therapy in allergic encephalomyelitis using myelin basic protein-specific T cells engineered to express latent transforming growth factor-β1

A myelin basic protein (MBP)-specific BALB/c T helper 1 (Th1) clone was transduced with cDNA for murine latent transforming growth factor-β1 (TGF-β1) by coculture with fibroblasts producing a genetically engineered retrovirus. When SJL x BALB/c F1 mice, immunized 12–15 days earlier with proteolipid protein in complete Freund’s adjuvant, were injected with 3 × 106 cells from MBP-activated untransduced cloned Th1 cells, the severity of experimental allergic encephalomyelitis (EAE) was slightly increased. In contrast, MBP-activated (but not resting) latent TGF-β1-transduced T cells significantly delayed and ameliorated EAE development. This protective effect was negated by simultaneously injected anti-TGF-β1. The transduced cells secreted 2–4 ng/ml of latent TGF-β1 into their culture medium, whereas control cells secreted barely detectable amounts. mRNA profiles for tumor necrosis factor, lymphotoxin, and interferon-γ were similar before and after transduction; interleukin-4 and -10 were absent. TGF-β1-transduced and antigen-activated BALB/c Th1 clones, specific for hemocyanin or ovalbumin, did not ameliorate EAE. Spinal cords from mice, taken 12 days after receiving TGF-β1-transduced, antigen-activated cells, contained detectable amounts of TGF-β1 cDNA. We conclude that latent TGF-β1-transduced, self-reactive T cell clones may be useful in the therapy of autoimmune diseases.

IL-18, although antiallergic when administered with IL-12, stimulates IL-4 and histamine release by basophils

IL-18 is a proinflammatory cytokine that plays an important role in natural killer cell activation and T helper 1 (Th1) cell responses. Mast cells and basophils are major inducers and effectors of allergic inflammation. Here we show that basophils and mast cells derived by culture of bone marrow cells with IL-3 for 10 days express IL-18Rα chain and that basophils produce large amounts of IL-4 and IL-13 in response to stimulation with IL-3 and IL-18. Injection of IL-12 and IL-18 inhibits IgE production in helminth-infected wild-type mice and abolishes the capacity of their basophils to produce IL-4 and IL-13 in response to stimulation either with IL-3 and IL-18 or with FcɛR cross-linkage. By contrast, this combination of cytokines actually increases IgE levels in helminth-infected IFN-γ−/− mice and enhances IL-4 and IL-13 production by their basophils. Furthermore, injection of IL-18 alone enhances basophil production of IL-4 and histamine both in wild-type and IFN-γ−/− mice. Thus, IL-18 has the potential to stimulate basophils but, when given with IL-12, exhibits an antiallergic action in vivo.

Enhanced resistance in STAT6-deficient mice to infection with ectromelia virus

We inoculated BALB/c mice deficient in STAT6 (STAT6−/−) and their wild-type (wt) littermates (STAT6+/+) with the natural mouse pathogen, ectromelia virus (EV). STAT6−/− mice exhibited increased resistance to generalized infection with EV when compared with STAT6+/+ mice. In the spleens and lymph nodes of STAT6−/− mice, T helper 1 (Th1) cytokines were induced at earlier time points and at higher levels postinfection when compared with those in STAT6+/+ mice. Elevated levels of NO were evident in plasma and splenocyte cultures of EV-infected STAT6−/− mice in comparison with STAT6+/+ mice. The induction of high levels of Th1 cytokines in the mutant mice correlated with a strong natural killer cell response. We demonstrate in genetically susceptible BALB/c mice that the STAT6 locus is critical for progression of EV infection. Furthermore, in the absence of this transcription factor, the immune system defaults toward a protective Th1-like response, conferring pronounced resistance to EV infection and disease progression.

The Ets transcription factor ERM is Th1-specific and induced by IL-12 through a Stat4-dependent pathway

Interleukin 12 (IL-12)-induced T helper 1 (Th1) development requires Stat4 activation. However, antigen-activated Th1 cells can produce interferon γ (IFN-γ) independently of IL-12 and Stat4 activation. Thus, in differentiated Th1 cells, factors regulated by IL-12 and Stat4 may be involved in IFN-γ production. Using subtractive cloning, we identified ERM, an Ets transcription factor, to be a Th1-specific, IL-12-induced gene. IL-12-induction of ERM occurred in wild-type and Stat1-deficient, but not Stat4-deficient, T cells, suggesting ERM is Stat4-inducible. Retroviral expression of ERM did not restore IFN-γ production in Stat4-deficient T cells, but augmented IFN-γ expression in Stat4-heterozygous T cells. Ets factors frequently regulate transcription via cooperative interactions with other transcription factors, and ERM has been reported to cooperate with c-Jun. However, in the absence of other transcription factors, ERM augmented expression of an IFN-γ reporter by only 2-fold. Thus, determining the requirement for ERM in Th1 development likely will require gene targeting.

Identification of HLA class II-restricted determinants of Mycobacterium tuberculosis-derived proteins by using HLA-transgenic, class II-deficient mice

T helper 1 cells play a major role in protective immunity against mycobacterial pathogens. Since the antigen (Ag) specificity of CD4+ human T cells is strongly controlled by HLA class II polymorphism, the immunogenic potential of candidate Ags needs to be defined in the context of HLA polymorphism. We have taken advantage of class II-deficient (Ab0) mice, transgenic for either HLA-DRA/B1*0301 (DR3) or HLA-DQB1*0302/DQA*0301 (DQ8) alleles. In these animals, all CD4+ T cells are restricted by the HLA molecule. We reported previously that human DR3-restricted T cells frequently recognize heat shock protein (hsp)65 of Mycobacterium tuberculosis, and only a single hsp65 epitope, p1–20. DR3.Ab0 mice, immunized with bacillus Calmette–Guérin or hsp65, developed T cell responses to M. tuberculosis, and recognized the same hsp65 epitope, p1–20. Hsp65-immunized DQ8.Ab0 mice mounted a strong response to bacillus Calmette–Guérin but not to p1–20. Instead, we identified three new DQ8-restricted T cell epitopes in the regions 171–200, 311–340, and 411–440. DR3.Ab0 mice immunized with a second major M. tuberculosis protein, Ag85 (composed of 85A, 85B, and 85C), also developed T cell responses against only one determinant, 85B p51–70, that was identified in this study. Importantly, subsequent analysis of human T cell responses revealed that HLA-DR3+, Ag85-reactive individuals recognize exactly the same peptide epitope as DR3.Ab0 mice. Strikingly, both DR3-restricted T cell epitopes represent the best DR3-binding sequences in hsp65 and 85B, revealing a strong association between peptide-immunodominance and HLA binding affinity. Immunization of DR3.Ab0 with the immunodominant peptides p1–20 and p51–70 induced T cell reactivity to M. tuberculosis. Thus, for two different Ags, T cells from DR3.Ab0 mice and HLA-DR3+ humans recognize the same immunodominant determinants. Our data support the use of HLA-transgenic mice in identifying human T cell determinants for the design of new vaccines.

An NO derivative of ursodeoxycholic acid protects against Fas-mediated liver injury by inhibiting caspase activity

Caspases are key mediators in liver inflammation and apoptosis. In the present study we provide evidence that a nitric oxide (NO) derivative of ursodeoxycholic acid (UDCA), NCX-1000 ([2-(acetyloxy)benzoic acid 3-(nitrooxymethyl)phenyl ester]), protects against liver damage in murine models of autoimmune hepatitis induced by i.v. injection of Con A or a Fas agonistic antibody, Jo2. Con A administration causes CD4+ T lymphocytes to accumulate in the liver and up-regulates FasL expression, resulting in FasL-mediated cytotoxicity. Cotreating mice with NCX-1000, but not with UDCA, protected against liver damage induced by Con A and Jo2, inhibited IL-1β, IL-18, and IFN-γ release and caspase 3, 8, and 9 activation. Studies on HepG2 cells demonstrated that NCX-1000, but not UDCA, directly prevented multiple caspase activation induced by Jo2. Incubating HepG2 cells with NCX-1000 resulted in intracellular NO formation and a DTT-reversible inhibition of proapoptotic caspases, suggesting that cysteine S-nitrosylation was the main mechanism responsible for caspase inhibition. Collectively, these data suggest that NCX-1000 protects against T helper 1-mediated liver injury by inhibiting both the proapoptotic and the proinflammatory branches of the caspase superfamily.

Copolymer 1 induces T cells of the T helper type 2 that crossreact with myelin basic protein and suppress experimental autoimmune encephalomyelitis

The synthetic amino acid copolymer copolymer 1 (Cop 1) suppresses experimental autoimmune encephalomyelitis (EAE) and is beneficial in multiple sclerosis. To further understand Cop 1 suppressive activity, we studied the cytokine secretion profile of various Cop 1-induced T cell lines and clones. Unlike T cell lines induced by myelin basic protein (MBP), which secreted either T cell helper type 1 (Th1) or both Th1 and Th2 cytokines, the T cell lines/clones induced by Cop 1 showed a progressively polarized development toward the Th2 pathway, until they completely lost the ability to secrete Th1 cytokines. Our findings indicate that the polarization of the Cop 1-induced lines did not result from the immunization vehicle or the in vitro growing conditions, but rather from the tendency of Cop 1 to preferentially induce a Th2 response. The response of all of the Cop 1 specific lines/clones, which were originated in the (SJL/J×BALB/c)F1 hybrids, was restricted to the BALB/c parental haplotype. Even though the Cop 1-induced T cells had not been exposed to the autoantigen MBP, they crossreacted with MBP by secretion of interleukin (IL)-4, IL-6, and IL-10. Administration of these T cells in vivo resulted in suppression of EAE induced by whole mouse spinal cord homogenate, in which several autoantigens may be involved. Secretion of anti-inflammatory cytokines by Cop 1-induced suppressor cells, in response to either Cop 1 or MBP, may explain the therapeutic effect of Cop 1 in EAE and in multiple sclerosis.

DNA immunization circumvents deficient induction of T helper type 1 and cytotoxic T lymphocyte responses in neonates and during early life

The relative deficiency of T helper type 1 (Th1) and cytotoxic T lymphocyte (CTL) responses in early life is associated with an increased susceptibility to infections by intracellular microorganisms. This is likely to reflect a preferential polarization of immature CD4 T cells toward a Th2 rather than a Th1 pattern upon immunization with conventional vaccines. In this report, it is shown that a single immunization within the first week of life with DNA plasmids encoding viral (measles virus hemagglutinin, Sendai virus nucleoprotein) or bacterial (C fragment of tetanus toxin) vaccine antigens can induce adult-like Th1 or mixed Th1/Th2 responses indicated by production of IgG2a vaccine-specific antibodies and preferential secretion of interferon-γ (IFN-γ) compared with interleukin (IL)-5 by antigen-specific T cells, as well as significant CTL responses. However, in spite of this potent Th1-driving capacity, subsequent DNA immunization was not capable of reverting the Th2-biased responses induced after early priming with a recombinant measles canarypox vector. Thus, DNA vaccination represents a novel strategy capable of inducing Th1 or mixed Th1/Th2 and CTL responses in neonates and early life, providing it is performed prior to exposure to Th2-driving conventional vaccine antigens.

Immunization with live attenuated simian immunodeficiency virus induces strong type 1 T helper responses and β-chemokine production

Immunization with live attenuated simian immunodeficiency virus (SIV) strains has proved to be one of the most effective strategies to induce protective immunity in the SIV/macaque model. To better understand the role that CD4+ T helper responses may play in mediating protection in this model, we characterized SIV-specific proliferative and cytokine responses in macaques immunized with live attenuated SIV strains. Macaques chronically infected with live attenuated SIV had strong proliferative responses to SIV proteins, with stimulation indices of up to 74. The magnitude of the proliferative response to SIV Gag varied inversely with the degree of attenuation; Gag-specific but not envelope-specific responses were lower in animals infected with more highly attenuated SIV strains. SIV-specific stimulation of lymphocytes from vaccinated macaques resulted in secretion of interferon-γ, IL-2, regulated-upon-activation, normal T cells expressed and secreted (RANTES), macrophage inflammatory protein (MIP)-1α, and MIP-1β but not IL-4 or IL-10. Intracellular flow cytometric analysis documented that, in macaques vaccinated with SIVmac239Δnef, up to 2% of all CD4+T cells were specific for SIV p55. The ability of live attenuated SIV to induce a strong, sustained type 1 T helper response may play a role in the success of this vaccination approach to generate protection against challenge with wild-type SIV.

Sequential cytokine dynamics in chronic rejection of rat renal allografts: roles for cytokines RANTES and MCP-1.

Chronic rejection, the most important cause of long-term graft failure, is thought to result from both alloantigen-dependent and -independent factors. To examine these influences, cytokine dynamics were assessed by semiquantitative competitive reverse transcriptase-PCR and by immunohistology in an established rat model of chronic rejection lf renal allografts. Isograft controls develop morphologic and immunohistologic changes that are similar to renal allograft changes, although quantitatively less intense and at a delayed speed; these are thought to occur secondary to antigen-independent events. Sequential cytokine expression was determined throughout the process. During an early reversible allograft rejection episode, both T-cell associated [interleukin (IL) 2, IL-2 receptor, IL-4, and interferon gamma] and macrophage (IL-1 alpha, tumor necrosis factor alpha, and IL-6) products were up-regulated despite transient immunosuppression. RANTES (regulated upon activation, normal T-cell expressed and secreted) peaked at 2 weeks; intercellular adhesion molecule (ICAM-1) was maximally expressed at 6 weeks. Macrophage products such as monocyte chemoattractant protein (MCP-1) increased dramatically (to 10 times), presaging intense peak macrophage infiltration at 16 weeks. In contrast, in isografts, ICAM-1 peaked at 24 weeks. MCP-1 was maximally expressed at 52 weeks, commensurate with a progressive increase in infiltrating macrophages. Cytokine expression in the spleen of allograft and isograft recipients was insignificant. We conclude that chronic rejection of kidney allografts in rats is predominantly a local macrophage-dependent event with intense up-regulation of macrophage products such as MCP-1, IL-6, and inducible nitric oxide synthase. The cytokine expression in isografts emphasizes the contribution of antigen-independent events. The dynamics of RANTES expression between early and late phases of chronic rejection suggest a key role in mediating the events of the chronic process.

Switch from a type 2 to a type 1 T helper cell response and cure of established Leishmania major infection in mice is induced by combined therapy with interleukin 12 and Pentostam.

Successful treatment in allergic, autoimmune, and infectious diseases often requires altering the nature of a detrimental immune response mediated by a particular CD4+ T helper (Th) cell subset. While several factors contribute to the development of CD4+ Th1 and Th2 cells, the requirements for switching an established response are not understood. Here we use infection with Leishmania major as a model to investigate those requirements. We report that treatment with interleukin 12 (IL-12), in combination with the antimony-based leishmanicidal drug Pentostam, induces healing in L. major-infected mice and that healing is associated with a switch from a Th2 to a Th1 response. The data suggest that decreasing antigen levels may be required for IL-12 to inhibit a Th2 response and enhance a Th1 response. These observations are important for treatment of nonhealing forms of human leishmaniasis and also demonstrate that in a chronic infectious disease an inappropriate Th2 response can be switched to an effective Th1 response.

The chemokine monocyte chemoattractant protein-1 induces functional responses through dimerization of its receptor CCR2

Cytokines interact with hematopoietin superfamily receptors and stimulate receptor dimerization. We demonstrate that chemoattractant cytokines (chemokines) also trigger biological responses through receptor dimerization. Functional responses are induced after pairwise crosslinking of chemokine receptors by bivalent agonistic antichemokine receptor mAb, but not by their Fab fragments. Monocyte chemoattractant protein (MCP)-1-triggered receptor dimerization was studied in human embryonic kidney (HEK)-293 cells cotransfected with genes coding for the CCR2b receptor tagged with YSK or Myc sequences. After MCP-1 stimulation, immunoprecipitation with Myc-specific antibodies revealed YSK-tagged receptors in immunoblotting. Receptor dimerization also was validated by chemical crosslinking in both HEK-293 cells and the human monocytic cell line Mono Mac 1. Finally, we constructed a loss-of-function CCR2bY139F mutant that acted as a dominant negative, blocking signaling through the CCR2 wild-type receptor. This study provides functional support for a model in which the MCP-1 receptor is activated by ligand-induced homodimerization, allowing discussion of the similarities between bacterial and leukocyte chemotaxis.