T1/ST2 is preferentially expressed on murine Th2 cells, independent of interleukin 4, interleukin 5, and interleukin 10, and important for Th2 effector function

T helper (Th) cells can be categorized according to their cytokine expression. The differential induction of Th cells expressing Th1 and/or Th2 cytokines is key to the regulation of both protective and pathological immune responses. Cytokines are expressed transiently and there is a lack of stably expressed surface molecules, significant for functionally different types of Th cells. Such molecules are of utmost importance for the analysis and selective functional modulation of Th subsets and will provide new therapeutic strategies for the treatment of allergic or autoimmune diseases. To this end, we have identified potential target genes preferentially expressed in Th2 cells, expressing interleukin (IL)-4, IL-5, and/or IL-10, but not interferon-γ. One such gene, T1/ST2, is expressed stably on both Th2 clones and Th2-polarized cells activated in vivo or in vitro. T1/ST2 expression is independent of induction by IL-4, IL-5, or IL-10. T1/ST2 plays a critical role in Th2 effector function. Administration of either a mAb against T1/ST2 or recombinant T1/ST2 fusion protein attenuates eosinophilic inflammation of the airways and suppresses IL-4 and IL-5 production in vivo following adoptive transfer of Th2 cells.

CD8+ T cells control the TH phenotype of MBP-reactive CD4+ T cells in EAE mice

Trimolecular interactions between the T cell antigen receptor and MHC/peptide complexes, together with costimulatory molecules and cytokines, control the initial activation of naïve T cells and determine whether the helper precursor cell differentiates into either T helper (TH)1 or TH2 effector cells. We now present evidence that regulatory CD8+ T cells provide another level of control of TH phenotype during further evolution of immune responses. These regulatory CD8+ T cells are induced by antigen-triggered CD4+ TH1 cells during T cell vaccination and, in vitro, distinguish mature TH1 from TH2 cells in a T cell antigen receptor Vβ-specific and Qa-1-restricted manner. In vivo, protection from experimental autoimmune encephalomyelitis (EAE) induced by T cell vaccination depends on CD8+ T cells, and myelin basic protein-reactive TH1 Vβ8+ clones, but not TH2 Vβ8+ clones, used as vaccine T cells, protect animals from subsequent induction of EAE. Moreover, in vivo depletion of CD8+ T cells during the first episode of EAE results in skewing of the TH phenotype toward TH1 upon secondary myelin basic protein stimulation. These data provide evidence that CD8+ T cells control autoimmune responses, in part, by regulating the TH phenotype of self-reactive CD4+ T cells.

T helper type 2 inflammatory disease in the absence of interleukin 4 and transcription factor STAT6

An important signaling pathway for the differentiation of T helper type 2 (TH2) cells from uncommitted CD4 T cell precursors is activation of the STAT6 transcription factor by interleukin 4 (IL-4). The protooncogene BCL-6 is also involved in TH2 differentiation, as BCL-6 −/− mice develop an inflammation of the heart and lungs associated with an overproduction of TH2 cells. Surprisingly, IL-4 −/− BCL-6 −/− and STAT6 −/− BCL-6 −/− double-mutant mice developed the same TH2-type inflammation of the heart and lungs as is characteristic of BCL-6 −/− mice. Furthermore, a TH2 cytokine response developed in STAT6 −/− BCL-6 −/− and IL-4 −/− BCL-6 −/− mice after immunization with a conventional antigen in adjuvant. In contrast to these in vivo findings, STAT6 was required for the in vitro differentiation of BCL-6 −/− T cells into TH2 cells. BCL-6, a transcriptional repressor that can bind to the same DNA binding motifs as STAT transcription factors, seems to regulate TH2 responses in vivo by a pathway independent of IL-4 and STAT6.

IL-4 inhibits osteoclast formation through a direct action on osteoclast precursors via peroxisome proliferator-activated receptor γ1

IL-4 is a pleiotropic immune cytokine secreted by activated TH2 cells that inhibits bone resorption both in vitro and in vivo. The cellular targets of IL-4 action as well as its intracellular mechanism of action remain to be determined. We show here that IL-4 inhibits receptor activator of NF-κB ligand-induced osteoclast differentiation through an action on osteoclast precursors that is independent of stromal cells. Interestingly, this inhibitory effect can be mimicked by both natural as well as synthetic peroxisome proliferator-activated receptor γ1 (PPARγ1) ligands and can be blocked by the irreversible PPARγ antagonist GW 9662. These findings suggest that the actions of IL-4 on osteoclast differentiation are mediated by PPARγ1, an interpretation strengthened by the observation that IL-4 can activate a PPARγ1-sensitive luciferase reporter gene in RAW264.7 cells. We also show that inhibitors of enzymes such as 12/15-lipoxygenase and the cyclooxygenases that produce known PPARγ1 ligands do not abrogate the IL-4 effect. These findings, together with the observation that bone marrow cells from 12/15-lipoxygenase-deficient mice retain sensitivity to IL-4, suggest that the cytokine may induce novel PPARγ1 ligands. Our results reveal that PPARγ1 plays an important role in the suppression of osteoclast formation by IL-4 and may explain the beneficial effects of the thiazolidinedione class of PPARγ1 ligands on bone loss in diabetic patients.

Inhibition of NF-AT-dependent transcription by NF-kappa B: implications for differential gene expression in T helper cell subsets.

Activation of individual CD4+ T cells results in differential lymphokine expression: interleukin 2 (IL-2) is preferentially produced by T helper type 1 (TH1) cells, which are involved in cell-mediated immune responses, whereas IL-4 is synthesized by TH2 cells, which are essential for humoral immunity. The Ca(2+)-dependent factor NF-ATp plays a key role in the inducible transcription of both these lymphokine genes. However, while IL2 expression requires the contribution of Ca(2+)- and protein kinase C-dependent signals, we report that activation of human IL4 transcription through the Ca(2+)-dependent pathway is diminished by protein kinase C stimulation in Jurkat T cells. This phenomenon is due to mutually exclusive binding of NF-ATp and NF-kappa B to the P sequence, an element located 69 bp upstream of the IL4 transcription initiation site. Human IL4 promoter-mediated transcription is downregulated in Jurkat cells stimulated with the NF-kappa B-activating cytokine tumor necrosis factor alpha and suppressed in RelA-overexpressing cells. In contrast, protein kinase C stimulation or RelA overexpression does not affect the activity of a human IL4 promoter containing a mouse P sequence, which is a higher-affinity site for NF-ATp and a lower-affinity site for RelA. Thus, competition between two general transcriptional activators, RelA and NF-ATp, mediates the inhibitory effect of protein kinase C stimulation on IL4 expression and may contribute to differential gene expression in TH cells.

Interleukin 12 suppresses autoantibody production by reversing helper T-cell phenotype in hepatitis B e antigen transgenic mice.

Helper T (Th) cells are classified as Th1 or Th2 cells by virtue of cytokine secretion and function as mediators of cellular or humoral immunity, respectively. Cytokines also regulate the differentiation of Th cells. For example, interleukin (IL)-12 promotes Th1 and suppresses Th2 cell development, suggesting that IL-12 may be useful therapeutically in Th2-mediated autoimmune and allergic disorders. Therefore, the effect of systemic IL-12 treatment on in vivo autoantibody synthesis in hepatitis B e antigen (HBeAg)-expressing transgenic mice, which is dependent on self-reactive Th2 cells, was examined. Low-dose IL-12 significantly inhibited autoantibody production by shifting the Th2-mediated response toward Th1 predominance. Additionally, previous studies suggest that a predominance of HBeAg-specific Th2-type cells may contribute to chronicity in hepatitis B virus infection. Therefore, IL-12 may also prove beneficial in modulating the HBeAg-specific Th response to favor viral clearance in chronic hepatitis B virus infection.

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.

Neuropeptides, by direct interaction with T cells, induce cytokine secretion and break the commitment to a distinct T helper phenotype

Searching for nervous system candidates that could directly induce T cell cytokine secretion, I tested four neuropeptides (NPs): somatostatin, calcitonin gene-related peptide, neuropeptide Y, and substance P. Comparing neuropeptide-driven versus classical antigen-driven cytokine secretion from T helper cells Th0, Th1, and Th2 autoimmune-related T cell populations, I show that the tested NPs, in the absence of any additional factors, directly induce a marked secretion of cytokines [interleukin 2 (IL-2), interferon-γ, IL-4, and IL-10) from T cells. Furthermore, NPs drive distinct Th1 and Th2 populations to a “forbidden” cytokine secretion: secretion of Th2 cytokines from a Th1 T cell line and vice versa. Such a phenomenon cannot be induced by classical antigenic stimulation. My study suggests that the nervous system, through NPs interacting with their specific T cell-expressed receptors, can lead to the secretion of both typical and atypical cytokines, to the breakdown of the commitment to a distinct Th phenotype, and a potentially altered function and destiny of T cells in vivo.

A T cell receptor antagonist peptide induces T cells that mediate bystander suppression and prevent autoimmune encephalomyelitis induced with multiple myelin antigens

Experimental autoimmune encephalomyelitis (EAE) induced with myelin proteolipid protein (PLP) residues 139–151 (HSLGKWLGHPDKF) can be prevented by treatment with a T cell receptor (TCR) antagonist peptide (L144/R147) generated by substituting at the two principal TCR contact residues in the encephalitogenic peptide. The TCR antagonist peptide blocks activation of encephalitogenic Th1 helper cells in vitro, but the mechanisms by which the antagonist peptide blocks EAE in vivo are not clear. Immunization with L144/R147 did not inhibit generation of PLP-(139–151)-specific T cells in vivo. Furthermore, preimmunization with L144/R147 protected mice from EAE induced with the encephalitogenic peptides PLP-(178–191) and myelin oligodendrocyte protein (MOG) residues 92–106 and with mouse myelin basic protein (MBP). These data suggest that the L144/R147 peptide does not act as an antagonist in vivo but mediates bystander suppression, probably by the generation of regulatory T cells. To confirm this we generated T cell lines and clones from animals immunized with PLP-(139–151) plus L144/R147. T cells specific for L144/R147 peptide were crossreactive with the native PLP-(139–151) peptide, produced Th2/Th0 cytokines, and suppressed EAE upon adoptive transfer. These studies demonstrate that TCR antagonist peptides may have multiple biological effects in vivo. One of the principal mechanisms by which these peptides inhibit autoimmunity is by the induction of regulatory T cells, leading to bystander suppression of EAE. These results have important implications for the treatment of autoimmune diseases where there are autopathogenic responses to multiple antigens in the target organ.

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.

A nontoxic mutant of cholera toxin elicits Th2-type responses for enhanced mucosal immunity

We have characterized a nontoxic mutant of cholera toxin (CT) as a mucosal adjuvant in mice. The mutant CT was made by substitution of serine with phenylalanine at position 61 of the A subunit (S61F), which resulted in loss of ADP ribosyltransferase activity and toxicity. Mice were intranasally immunized with ovalbumin, tetanus toxoid, or influenza virus either alone or together with mutant CT S61F, native CT, or recombinant CT-B. Mice immunized with these proteins plus S61F showed high serum titers of protein-specific IgG and IgA antibodies that were comparable to those induced by native CT. Further, high protein-specific IgA antibody responses were observed in nasal and vaginal washes, saliva, and fecal extracts as well as increased numbers of IgG and IgA antibody forming cells in cervical lymph nodes and lung tissues of mice intranasally immunized with these proteins and S61F or native CT, but not with recombinant CT-B or protein alone. Both S61F and native CT enhanced the induction of ovalbumin-specific CD4+ T cells in lung and splenic tissues, and these T cells produced a Th2-type cytokine pattern of interleukin 4 (IL-4), IL-5, IL-6, and IL-10 as determined by analysis of secreted proteins and by quantitation of cytokine-specific mRNA. These results have shown that mutant CT S61F is an effective mucosal adjuvant when administrated intranasally and induces mucosal and systemic antibody responses which are mediated by CD4+ Th2-type cells.

High-affinity binding of bioactive glycosylation-inhibiting factor to antigen-primed T cells and natural killer cells

High-affinity binding was demonstrated between suppressor-T-cell-derived bioactive glycosylation-inhibiting factor (GIF) and helper T hybridomas and natural killer cell line cells. Inactive GIF present in cytosol of suppressor T cells and Escherichia coli-derived recombinant human GIF (rhGIF) failed to bind to these cells. However, affinity of rhGIF for the target cells was generated by replacement of Cys-57 in the sequence with Ala or of Asn-106 with Ser or binding of 5-thio-2-nitrobenzoic acid to Cys-60 in the molecule. Such mutations and the chemical modification of rhGIF synergistically increased the affinity of GIF molecules for the target cells. The results indicated that receptors on the target cells recognize conformational structures of bioactive GIF. Equilibrium dissociation constant (Kd) of the specific binding between bioactive rGIF derivatives and high-affinity receptors was 10–100 pM. Receptors for bioactive GIF derivatives were detected on Th1 and Th2 T helper clones and natural killer NK1.1+ cells in normal spleen but not on naive T or B cells. Neither the inactive rGIF nor bioactive rGIF derivatives bound to macrophage and monocyte lines or induced macrophages for tumor necrosis factor α production.

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.

Differential modulation of Th1- and Th2-related cytokine mRNA expression by a synthetic peptide homologous to a conserved domain within retroviral envelope protein.

The influence of a synthetic retroviral peptide, CKS-17, on T helper type 1 (Th1)- or Th2-related cytokines was investigated in human blood mononuclear cells. Cells were stimulated with staphylococcal enterotoxin A, anti-CD3 plus anti-CD28 monoclonal antibodies, or lipopolysaccharide to induce cytokine mRNA. mRNA was detected by a reverse transcription-polymerase chain reaction or Northern blot analysis. CKS-17 down-regulated stimulant-induced mRNA accumulation for interferon gamma (IFN-gamma), interleukin (IL)-2, and p40 heavy and p35 light chains of IL-12, a cytokine that mediates development of Th1 response. CKS-17 up-regulated stimulant-induced mRNA accumulation of IL-10 and did not suppress Th2-related cytokine (IL-4, IL-5, IL-6, or IL-13) mRNA expression. A reverse sequence of CKS-17 peptide, used as a control, showed no such action. Anti-human IL-10 monoclonal antibody blocked ability of CKS-17 to inhibit mRNA accumulation for IFN-gamma but not the CKS-17 suppressive activity of IL-12 p40 heavy chain mRNA. Thus, CKS-17-mediated suppression of IFN-gamma mRNA expression is dependent upon augmentation of IL-10 production by CKS-17. This conserved component of several retroviral envelope proteins, CKS-17, may act as an immunomodulatory epitope responsible for cytokine dysregulation that leads to suppression of cellular immunity.