Independent roles for IL-2 and GATA-3 in stimulating naive CD4+ T cells to generate a Th2-inducing cytokine environment

T cell receptor (TCR) signaling plays an important role in early interleukin (IL)-4 production by naive CD4+ T cells. This "antigen-stimulated" early IL-4 is sufficient for in vitro Th2 differentiation. Here, we provide evidence that early IL-4 production by naive CD4+ T cells stimulated with cognate peptide requires TCR-induced early GATA-3 expression and IL-2 receptor signaling, both of which are controlled by the degree of activation of extracellular signal-regulated kinase (ERK). Stimulation of naive CD4+ T cells from TCR transgenic mice with low concentrations of peptide-induced IL-2-dependent STAT5 phosphorylation, IL-4-independent early GATA-3 expression, and IL-4 production. Neutralization of IL-2 abolished early IL-4 production without affecting early GATA-3 expression. In addition, naive CD4+ T cells from GATA-3 conditional KO mice failed to produce early IL-4 in response to TCR/CD28 stimulation. Stimulation with high concentrations of peptide abrogated early GATA-3 expression and IL-2-dependent STAT5 phosphorylation, and resulted in the failure to produce early IL-4. This high concentration-mediated suppression of early IL-4 production was reversed by blockade of the ERK pathway. A MEK inhibition rescued early GATA-3 expression and responsiveness to IL-2; these cells were now capable of producing early IL-4 and undergoing subsequent Th2 differentiation.     

流式微珠阵列法检测IFN-γ诱导的Jurkat细胞Th1/Th2细胞因子表达

本研究旨在探讨流式微珠阵列法检测Th1／Th2细胞因子表达及IFN-1应用于T淋巴细胞白血病治疗的临床价值。以不同浓度IFN-γ诱导培养Jurkat细胞48小时，用流式微珠阵列法检测培养上清液中IL-2、IL-6、TNF-α和IFN-γ表达，并采用流式细胞术检测膜上IL-2受体（CD25）的表达。结果表明：IFN-γ诱导Jurkat细胞IL-2和TNF-α的表达呈浓度依赖性升高，未检测到IL-6表达，CD25表达明显升高。结论：Jurkat细胞能被IFN-γ诱导高表达Th1细胞因子和CD25，流式微珠阵列法能准确客观地反映Th1／Th2细胞因子表达的动态变化。     

环孢菌素A对慢性再生障碍性贫血患者T-bet、GATA-3、相关信号分子、细胞因子及Th1/Th2的影响

本研究探讨转录因子T-bet、GATA-3及相关信号通路在慢性再生障碍性贫血(CAA)免疫发病中的作用,从Th细胞失衡、转录因子及相关信号通路水平研究环孢菌素(CsA)治疗慢性AA的免疫调节机理。采用实时荧光定量PCR(real-time FQ-PCR)检测慢性AA患者治疗前和CsA治疗6月后外周血单个核细胞(PBMNC)T-bet、GATA-3及STAT4、STAT6mRNA表达;采用流式细胞术、酶联免疫吸附法(ELISA)检测慢性AA患者治疗前和CsA治疗6月后外周血Th1、Th2比例及PBMNC培养上清IFN-γ、IL-12、IL-4水平,并与正常人进行比较。结果表明:慢性AA患者PBMNC T-bet、STAT4mRNA表达、T-bet/GATA-3比值、Th1比例、Th1/Th2比值、PBMNC培养上清IFN-γ、IL-12表达均明显高于正常组(p0.01),经CsA治疗6月后,患者T-bet、STAT4表达、T-bet/GATA-3比值、Th1比例、IFN-γ、IL-12表达均有所下降,但T-bet、STAT4、T-bet/GATA-3比值、Th1比例、IFN-γ表达仍未达到正常水平,GATA-3、STAT6mRNA表达、Th2比例、IL-4表达在治疗前后与正常人比较均无明显差异(p0.05)。结论:IFN-γ/T-bet、IL-12/STAT4通路的异常活化及Th平衡向Th1型偏移在AA免疫异常的发病过程中起到关键的作用;CsA能通过下调IFN-γ/T-bet、IL-12/STAT4通路的异常活化及纠正Th1过度极化而减轻AA异常亢进的细胞免疫,解除造血抑制。     

Selective Expression and Functions of Interleukin 18 Receptor on T Helper (Th) Type 1 but not Th2 Cells

Interleukin (IL)-18 induces interferon (IFN)-γ synthesis and synergizes with IL-12 in T helper type 1 (Th1) but not Th2 cell development. We report here that IL-18 receptor (IL-18R) is selectively expressed on murine Th1 but not Th2 cells. IL-18R mRNA was expressed constitutively and consistently in long-term cultured clones, as well as on newly polarized Th1 but not Th2 cells. IL-18 sustained the expression of IL-12Rβ2 mRNA, indicating that IL-18R transmits signals that maintain Th1 development through the IL-12R complex. In turn, IL-12 upregulated IL-18R mRNA. Antibody against an IL-18R–derived peptide bound Th1 but not Th2 clones. It also labeled polarized Th1 but not Th2 cells derived from naive ovalbumin–T cell antigen receptor-αβ transgenic mice (D011.10). Anti–IL-18R antibody inhibited IL-18– induced IFN-γ production by Th1 clones in vitro. In vivo, anti–IL-18R antibody reduced local inflammation and lipopolysaccharide-induced mortality in mice. This was accompanied by shifting the balance from Th1 to Th2 responses, manifest as decreased IFN-γ and proinflammatory cytokine production and increased IL-4 and IL-5 synthesis. Therefore, these data provide a direct mechanism for the selective effect of IL-18 on Th1 but not Th2 cells. They also show that the synergistic effect of IL-12 and IL-18 on Th1 development may be due to the reciprocal upregulation of their receptors. Furthermore, IL-18R is a cell surface marker distinguishing Th1 from Th2 cells and may be a therapeutic target.     

Two types of mouse T helper cell. IV. Th2 clones secrete a factor that inhibits cytokine production by Th1 clones

A cytokine synthesis inhibitory factor (CSIF) is secreted by Th2 clones in response to Con A or antigen stimulation, but is absent in supernatants from Con A-induced Th1 clones. CSIF can inhibit the production of IL-2, IL-3, lymphotoxin (LT)/TNF, IFN-gamma, and granulocyte-macrophage CSF (GM-CSF) by Th1 cells responding to antigen and APC, but Th2 cytokine synthesis is not significantly affected. Transforming growth factor beta (TGF-beta) also inhibits IFN-gamma production, although less effectively than CSIF, whereas IL-2 and IL-4 partially antagonize the activity of CSIF. CSIF inhibition of cytokine synthesis is not complete, since early cytokine synthesis (before 8 h) is not significantly affected, whereas later synthesis is strongly inhibited. In the presence of CSIF, IFN-gamma mRNA levels are reduced slightly at 8, and strongly at 12 h after stimulation. Inhibition of cytokine expression by CSIF is not due to a general reduction in Th1 cell viability, since actin mRNA levels were not reduced, and proliferation of antigen-stimulated cells in response to IL-2, was unaffected. Biochemical characterization, mAbs, and recombinant or purified cytokines showed that CSIF is distinct from IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IFN-gamma, GM-CSF, TGF-beta, TNF, LT, and P40. The potential role of CSIF in crossregulation of Th1 and Th2 responses is discussed.     

Th2-driven, allergen-induced airway inflammation is reduced after treatment with anti-Tim-3 antibody in vivo

T cell immunoglobulin and mucin domain-containing molecule-3 (Tim-3) is a surface molecule that is preferentially expressed on activated Th1 cells in comparison to Th2 cells. Blockade of Tim-3 has been shown to enhance Th1-driven pathology in vivo, suggesting that blockade of Tim-3 may improve the development of Th2-associated responses such as allergy. To examine the effects of Tim-3 blockade on the Th2 response in vivo, we administered anti-Tim-3 antibody during pulmonary inflammation induced by transfer of ovalbumin (OVA)-reactive Th2 cells, and subsequent aerosol challenge with OVA. In this model, anti-Tim-3 antibody treatment before each airway challenge significantly reduced airway hyperreactivity, with a concomitant decrease in eosinophils and Th2 cells in the lung. We examined Th1 and Th2 cytokine levels in the lung after allergen challenge and found that pulmonary expression of the Th2 cytokine IL-5 was significantly reduced, whereas IFN-gamma levels were significantly increased by anti-Tim-3 antibody treatment. Thus, blocking Tim-3 function has a beneficial effect during pulmonary inflammation by skewing the Th2 response toward that of a Th1 type, suggesting an important role for Tim-3 in the regulation of allergic disease.     

Two types of mouse helper T cell clone. III. Further differences in lymphokine synthesis between Th1 and Th2 clones revealed by RNA hybridization, functionally monospecific bioassays, and monoclonal antibodies

Lymphokine synthesis patterns of a panel of 19 T cell clones have been evaluated, using mRNA hybridization methods to examine 11 different mRNAs induced by Con A. The two types of CD4+ Th cell clone described previously were clearly distinguished by this procedure, and the differences between the two types have now been extended to six induced products. With minor exceptions, only Th1 clones synthesized mRNA for IL-2, IFN-gamma, and lymphotoxin, and only Th2 clones synthesized mRNA for IL-4, IL-5, and another induced gene, P600. Four more induced products were expressed preferentially but not uniquely by one or another type of clone: mRNAs for GM-CSF, TNF, and another induced, secreted product (TY5) were produced in larger amounts by Th1 clones, whereas preproenkephalin was preferentially expressed by Th2 clones. IL-3 was produced in similar amounts by both types of clone. mAbs were used to establish three bioassays that were functionally monospecific for IL-2, IL-3, and IL-4, and a new anti-IFN gamma mAb, XMG1.2, was used to establish an ELISA for IFN-gamma. These four assays were used to show that secreted protein and mRNA levels correlated well for all cell lines. The implications of these findings for normal T cells are discussed.     

C型CpG单链寡核苷酸对哮喘小鼠Th1／Th2相关细胞因子的影响

目的C型CpG—ODN对哮喘小鼠Th1／Th2相关细胞因子的影响，为CpG-ODN进一步应用提供依据。方法利用人工合成C型的CpG-ODN（ODN-M362），将其注射于哮喘小鼠腹腔，采用酶联免疫吸附法（ELISA）检测注射后小鼠的Th1和Th2细胞因子的含量，分析其有无差异性。结果C型CpG-ODN组BALF中EOS数量，血清IL-4水平明显低于对照组和哮喘组（P〈0．01），血清IFN-γ明显升高于对照组和哮喘组（P〈0．01）。结论C型的CpG-ODN，对哮喘小鼠Th1／Th2失衡有明显的逆转作用。     

Interleukin 12 inhibits antigen-induced airway hyperresponsiveness, inflammation, and Th2 cytokine expression in mice

Allergic asthma is characterized by airway hyperresponsiveness and pulmonary eosinophilia, and may be mediated by T helper (Th) lymphocytes expressing a Th2 cytokine pattern. Interleukin (IL) 12 suppresses the expression of Th2 cytokines and their associated responses, including eosinophilia, serum immunoglobulin E, and mucosal mastocytosis. We have previously shown in a murine model that antigen- induced increases in airway hyperresponsiveness and pulmonary eosinophilia are CD4+ T cell dependent. We used this model to determine the ability of IL-12 to prevent antigen-induced increases in airway hyperresponsiveness, bronchoalveolar lavage (BAL) eosinophils, and lung Th2 cytokine expression. Sensitized A/J mice developed airway hyperresponsiveness and increased numbers of BAL eosinophils and other inflammatory cells after single or repeated intratracheal challenges with sheep red blood cell antigen. Pulmonary mRNA and protein levels of the Th2 cytokines IL-4 and IL-5 were increased after antigen challenge. Administration of IL-12 (1 microgram/d x 5 d) at the time of a single antigen challenge abolished the airway hyperresponsiveness and pulmonary eosinophilia and promoted an increase in interferon (IFN) gamma and decreases in IL-4 and IL-5 expression. The effects of IL-12 were partially dependent on IFN-gamma, because concurrent treatment with IL-12 and anti-IFN-gamma monoclonal antibody partially reversed the inhibition of airway hyperresponsiveness and eosinophilia by IL-12. Treatment of mice with IL-12 at the time of a second antigen challenge also prevented airway hyperresponsiveness and significantly reduced numbers of BAL inflammatory cells, reflecting the ability of IL-12 to inhibit responses associated with ongoing antigen-induced pulmonary inflammation. These data show that antigen-induced airway hyperresponsiveness and inflammation can be blocked by IL-12, which suppresses Th2 cytokine expression. Local administration of IL-12 may provide a novel immunotherapy for the treatment of pulmonary allergic disorders such as atopic asthma.     

Regulatory B cells are identified by expression of TIM-1 and can be induced through TIM-1 ligation to promote tolerance in mice

T cell Ig domain and mucin domain protein 1 (TIM-1) is a costimulatory molecule that regulates immune responses by modulating CD4+ T cell effector differentiation. However, the function of TIM-1 on other immune cell populations is unknown. Here, we show that in vivo in mice, TIM-1 is predominantly expressed on B rather than T cells. Importantly, TIM-1 was expressed by a large majority of IL-10-expressing regulatory B cells in all major B cell subpopulations, including transitional, marginal zone, and follicular B cells, as well as the B cell population characterized as CD1d(hi)CD5+. A low-affinity TIM-1-specific antibody that normally promotes tolerance in mice, actually accelerated (T cell-mediated) immune responsiveness in the absence of B cells. TIM-1+ B cells were highly enriched for IL-4 and IL-10 expression, promoted Th2 responses, and could directly transfer allograft tolerance. Both cytokine expression and number of TIM-1+ regulatory B cells (Bregs) were induced by TIM-1-specific antibody, and this was dependent on IL-4 signaling. Thus, TIM-1 is an inclusive marker for IL-10+ Bregs that can be induced by TIM-1 ligation. These findings suggest that TIM-1 may be a novel therapeutic target for modulating the immune response and provide insight into the signals involved in the generation and induction of Bregs.     

Specific inhibition of lymphokine biosynthesis and autocrine growth using antisense oligonucleotides in Th1 and Th2 helper T cell clones

T helper cells have recently been divided into two subsets. The Th1 subset secretes and responds to IL-2 in an autocrine manner. The Th2 subset upon mitogen or antigen stimulation releases IL-4. Here we describe a novel technology that allowed us to confirm this distinction. We have used synthetic oligonucleotides complementary to the 5' end of mouse IL-2 and IL-4 to specifically block the biosynthesis of IL-2 or IL-4 in two murine helper T cell clones from the Th1 or Th2 subset. We show that the antisense IL-2 oligonucleotide inhibited the proliferation of the Th1 clone and had no effect on the Th2 clone. In parallel experiments, the antisense IL-4 oligonucleotide blocked the proliferation of the Th2 clone and not the proliferation of the Th1 clone. The inhibition was significantly reversed in both cases by the addition of the relevant lymphokine (IL-2 in the case of the Th1 clone, IL-4 in the case of the Th2 clone). Northern analysis, using cDNA probes specific for the two lymphokines, showed a decrease in the steady-state level of the relevant lymphokine mRNA, suggesting the specific degradation of the mRNA by an RNase H-like enzymatic activity. This strategy, which allows the specific blockade of the biosynthesis of a lymphokine, could be useful for future studies on the role of each T helper subset in physiological immune responses.     

Evidence for mouse Th1- and Th2-like helper T cells in vivo. Selective reduction of Th1-like cells after total lymphoid irradiation

Purified CD4+ BALB/c spleen T cells obtained 4-6 wk after total lymphoid irradiation (TLI) helped normal syngeneic B cells to produce a vigorous antibody response to TNP keyhole limpet hemocyanin in adoptive cell transfer experiments. However, the same cells failed to transfer delayed-type hypersensitivity to the adoptive hosts as measured by a foot pad swelling assay. In addition, purified CD4+ cells from TLI-treated mice were unable to induce graft vs. host disease in lethally irradiated allogeneic C57BL/Ka recipient mice. In response to mitogen stimulation, unfractionated spleen cells obtained from TLI mice secreted normal levels of IL-4 and IL-5, but markedly reduced levels of IL-2 and INF-gamma. A total of 229 CD4+ clones from spleen cells of both normal and TLI-treated mice were established, and the cytokine secretion pattern from each clone was analyzed. The results demonstrate that the ratio of Th1- and Th2-like clones in the spleens of normal BALB/c mice is 1:0.6, whereas the ratio in TLI mice is approximately 1:7. These results suggest that Th2-like cells recover rapidly (at approximately 4-6 wk) after TLI treatment and account for the early return of antibody helper activity and secretion of IL-4 and IL-5, but Th1-like cells recover more slowly (in approximately 3 mo) after irradiation, and this accounts for the deficit in cell-mediated immunity and the reduced amount of IL-2 and IFN-gamma secretion.