Exclusive Th2 primary effector function in spleens but mixed Th1/Th2 function in lymph nodes of murine neonates

Recent studies have shown that neonatal mice are competent to develop mature, Ag-specific Th1 function in situ. However, under many conditions, Th2 responses dominate in the neonate, while Th1 responses are more prevalent in adults. To compare further the immune responses of neonates and adults, we used the enzyme-linked immunospot method to measure the frequencies of primary Th1/Th2 effectors generated in situ in the spleens and lymph nodes. As assessed by the detection of IFN-gamma- or IL-4-producing cells, adults developed mixed Th1/Th2 responses in both organs. Neonatal lymph nodes contained mature frequencies of IFN-gamma- and IL-4-producing cells. In striking contrast, while mature frequencies of Th2 cells developed in neonatal spleens, virtually no IFN-gamma-secreting cells were detected. Exclusive Th2 function was observed in both BALB/c and C57BL/6 neonates, strains in which the Th2 and Th1 lineages, respectively, are favored in adults. Although Th1 effectors were virtually undetectable, the addition of rIL-12 boosted the frequency of IFN-gamma-secreting cells to adult levels. Therefore, Th1 effectors apparently developed in situ, but Th1 effector function either was not promoted or was inhibited upon subsequent exposure to the Ag in culture. Together, these results indicate that the quality of a primary Th response in neonates is strongly dependent on the site of initial Ag exposure; responses initiated in the lymph nodes are mixed Th1/Th2, whereas responses occurring in the spleen are heavily Th2 biased.     

Schnurri-2 controls memory Th1 and Th2 cell numbers in vivo

Schnurri-2 (Shn-2) is a large zinc-finger containing protein, and it plays a critical role in cell growth, signal transduction and lymphocyte development. In Shn-2-deficient CD4 T cells, the activation of NF-kappaB was up-regulated and their ability to differentiate into Th2 cells was enhanced. We herein demonstrate that Th1 and Th2 memory cells are not properly generated from Shn-2-deficient effector Th1/Th2 cells. Even a week after the transfer of effector Th1/Th2 cells into syngeneic mice, a dramatic decrease in the number of Shn-2-deficient donor T cells was detected particularly in the lymphoid organs. The transferred Shn-2-deficient Th1/Th2 cells express higher levels of the activation marker CD69. No significant defect in the BrdU incorporation in the Shn-2-deficient transferred CD4 T cells was observed. The numbers of apoptotic cells were selectively higher in Shn-2-deficient donor Th1/Th2 cell population. Moreover, Shn-2-deficient effector Th1 and Th2 cells showed an increased susceptibility to cell death in in vitro cultures with increased expression of FasL. Transfer of Th2 effector cells over-expressing the p65 subunit of NF-kappaB resulted in a decreased number of p65-expressing cells in the lymphoid organs. As expected, T cell-dependent Ab responses after in vivo immunization of Shn-2-deficient mice were significantly reduced. Thus, Shn-2 appears to control the generation of memory Th1/Th2 cells through a change in their susceptibility to cell death.     

Human TCR transgenic Bet v 1-specific Th1 cells suppress the effector function of Bet v 1-specific Th2 cells

Pollinosis to birch pollen is a common type I allergy in the Northern Hemisphere. Moreover, birch pollen-allergic individuals sensitized to the major birch pollen allergen Bet v 1 frequently develop allergic reactions to stone fruits, hazelnuts, and certain vegetables due to immunological cross-reactivity. The major T cell epitope Bet v 1(142-153) plays an important role in cross-reactivity between the respiratory allergen Bet v 1 and its homologous food allergens. In this study, we cloned and functionally analyzed a human αβ TCR specific for the immunodominant epitope Bet v 1(142-153). cDNAs encoding TCR α- and β-chains were amplified from a Bet v 1(142-153)-specific T cell clone, introduced into Jurkat T cells and peripheral blood T lymphocytes of allergic and nonallergic individuals, and evaluated functionally. The resulting TCR transgenic (TCRtg) T cells responded in an allergen-specific and costimulation-dependent manner to APCs either pulsed with Bet v 1(142-153) peptide or coexpressing invariant chain::Bet v 1(142-153) fusion proteins. TCRtg T cells responded to Bet v 1-related food and tree pollen allergens that were processed and presented by monocyte-derived dendritic cells. Bet v 1(142-153)-presenting but not Bet v 1(4-15)-presenting artificial APCs coexpressing membrane-bound IL-12 polarized allergen-specific TCRtg T cells toward a Th1 phenotype, producing high levels of IFN-γ. Coculture of such Th1-polarized T cells with allergen-specific Th2-differentiated T cells significantly suppressed Th2 effector cytokine production. These data suggest that human allergen-specific TCR can transfer the fine specificity of the original T cell clone to heterologous T cells, which in turn can be instructed to modulate the effector function of the disease initiating/perpetuating allergen-specific Th2-differentiated T cells.     

Tissue-level regulation of Th1 and Th2 primary and memory CD4 T cells in response to Listeria infection

Ag-specific Th1 and Th2 cytokine-producing CD4 T cells were quantitated in secondary lymphoid and tertiary tissues following oral Listeria monocytogenes infection. Although the response to Listeria was previously believed to be predominantly Th1 like, CD4 T cells producing IL-4 or IL-5 comprised a substantial proportion of the overall primary and memory response. The frequency of IFN-gamma-, IL-4-, or IL-5-producing primary effector or memory CD4 T cells was significantly higher in lung, liver, and intestinal lamina propria (LP) as compared with spleen and lymph node. However, maximum numbers of IL-4- and IL-5-producing cells were detected in the LP several days after the peak of the Th1 response, and IL-5 production was skewed toward the mucosal tissues. Remarkably, the recall response resulted in sustained Th1 and Th2 responses in tertiary, but not lymphoid tissues and long-term retention of Th1 and Th2 memory cells in equal proportions in the LP. Finally, CD40 ligand was essential for induction of IFN-gamma in the spleen and LP, but not in the liver and lung, while the IL-4 response required CD40 ligand only in the spleen. Therefore, the rules governing the effector phenotype, and the overall magnitude of the CD4 response, are regulated at the level of individual tissues.     

Cutting edge: CD94/NKG2 is expressed on Th1 but not Th2 cells and costimulates Th1 effector functions

Th1 and Th2 cells can be phenotypically distinguished by very few cell surface markers. To identify cell surface molecules that are specifically expressed on Th1 cells, we have generated a panel of mAbs that specifically bind the surfaces of murine Th1 but not Th2 cells. One of these Abs identified the NK cell receptor CD94 as a molecule also specifically expressed on the surface of Th1 cells. As in NK cells, CD94 is expressed on Th1 cells together with members of the NKG2 family of molecules, including NKG2A, C, and E. Cross-linking these receptors on differentiated Th1 cells in vitro costimulates proliferation and cytokine production with a potency similar to that obtained by cross-linking CD28. We propose that CD94/NKG2 heterodimers may costimulate effector functions of differentiated Th1 cells.     

IFN-gamma modulates the early development of Th1 and Th2 responses in a murine model of cutaneous leishmaniasis.

Resistance to Leishmania major in mice is associated with the generation of distinct CD4+ Th subsets, termed TH1 and TH2. To define the factors contributing to the genesis of these Th cells, we first investigated when these subsets developed following L. major infection. Lymph node (LN) cells collected 3 days after infection of BALB/c mice secreted IL-4 and IL-5 in vitro, but little IFN-gamma, whereas LN cells from a resistant strain, C3H/HeN, secreted IFN-gamma and no IL-4 or IL-5. Cytokine production was eliminated in both cases by in vivo or in vitro depletion of CD4+ cells, but not after depletion of CD8+ cells. Similar responses were observed after inoculation of killed promastigotes or a soluble leishmanial Ag preparation. These data indicate that the development of Th1- and Th2-like responses can precede lesion formation and does not require a live infection. We next investigated whether IFN-gamma was important in the differentiation of Th1 and Th2 cells. C3H/HeN mice have previously been shown to be susceptible to leishmanial infection after treatment with anti-IFN-gamma. We confirmed this observation and found that the abrogation of resistance was associated with enhanced production of IL-4 and IL-5, and decreased production of IFN-gamma by cells taken from these mice. Conversely, LN cells from BALB/c mice inoculated with parasites plus IFN-gamma produced significantly higher levels of IFN-gamma, and decreased levels of IL-4 and IL-5, than mice infected with parasites alone. Finally, we determined if IFN-gamma might augment vaccine induced immunity. We found that s.c. immunization with soluble leishmanial Ag, the bacterial adjuvant, Corynebacterium parvum and IFN-gamma could protect mice against L. major infection, and that this protection was associated with induction of Th1 responses. From these data we conclude that levels of IFN-gamma at the time of infection or immunization dramatically alters the type of response elicited: high levels of IFN-gamma favor Th1 type responses, whereas low levels promote a Th2 response.     

Cutting edge: IFN-gamma enables APC to promote memory Th17 and abate Th1 cell development

Th1-derived IFN-gamma targets naive T cells and inhibits Th17 development. However, Th1, Th17, and memory but not naive T cells are colocalized in an inflammatory environment. To demonstrate the kinetic relationship between these T cell subsets, we investigated the role of IFN-gamma in regulating the development and balance between Th17 and Th1 in humans. We show that IFN-gamma stimulates B7-H1 expression on APC subsets and abates their Th1 polarization capacity in a B7-H1-dependent manner. Interestingly, IFN-gamma triggers APCs to produce IL-1 and IL-23 and enables them to induce memory Th17 expansion via IL-1 and IL-23 in a B7-H1-independent manner. We propose a novel dynamic between Th1 and Th17 in the course of inflammation as follows: Th1-mediated inflammation is attenuated by IFN-gamma-induced B7-H1 on APCs and is evolved toward Th17-mediated chronic inflammation by IFN-gamma-induced, APC-derived IL-1 and IL-23. Our study challenges the dogma that IFN-gamma suppresses Th17 and enhances Th1 development.     

ICOS expression by activated human Th cells is enhanced by IL-12 and IL-23: increased ICOS expression enhances the effector function of both Th1 and Th2 cells

Previous mouse studies have shown that IL-4 increases the expression of ICOS on activated Th cells, resulting in enhanced ICOS expression on Th2 cells. In this study, we show that ICOS expression on human Th cells is not increased by IL-4, but by IL-12 and by IL-23 instead. Consequently, ICOS expression during IL-12-driven Th1 cell polarization was transiently increased compared with the levels on Th0 cells and IL-4-driven Th2 cells. Addition of IL-12 and/or IL-23 during restimulation increased ICOS expression to the same extent on pre-established Th1, Th2, and Th0 cells, indicating that ICOS levels are not stably imposed by prior polarization. In contrast to the findings in the mouse, IL-4 significantly suppressed the ICOS-enhancing effects of IL-12 and IL-23. The functional consequence of variable ICOS levels was shown in coculture experiments with cells expressing the ICOS-ligand B7-related protein 1 (either transfected Chinese hamster ovary cells or autologous dendritic cells). Ligation of ICOS on 2-day-preactivated effector cells increased their cytokine production to an extent proportional to their ICOS expression levels. As the ICOS-enhancing potentials of IL-12 and IL-23 were maintained for several days after stimulation, both on Th1 and Th2 cells, we propose the concept that local regulation of ICOS expression on activated Th cells by IL-12 and/or IL-23 may provide a powerful means to amplify effector T cell responses in peripheral tissues, independently of the polarized state of the Th cells.     

Lack of interferon-gamma confers impaired neutrophil granulocyte function and imparts prolonged survival of adult filarial worms in murine filariasis

We investigated the role of IFN-gamma in host defense during murine filariasis. Using the fully permissive infection of BALB/c mice with the rodent filaria Litomosoides sigmodontis, we show that interferon (IFN)-gamma is essential for encapsulation of adult filarial worms in inflammatory nodules and for normal worm clearance. IFN-gamma knockout (KO) mice had only one third of the nodules of wild-type mice but displayed a more than twofold increase in worm burden and increased microfilaremia. Neutrophil granulocytes, but not macrophages or eosinophils, appear to directly control worm load and nodule formation. Neutrophils, which we showed earlier to be essential for the encapsulation process in the thoracic cavity, where the worms reside, were diminished at this location in IFN-gamma KO compared to wild-type mice; they also displayed strongly reduced chemotactic and phagocytic activity compared to neutrophils of controls. This argues for a distinct defect in neutrophil activation accounting for the low formation of inflammatory nodules. Tumor necrosis factor-alpha, a major neutrophil-activating cytokine expressed by macrophages in the thoracic cavity around the worms, was highly induced in wild-type but absent in KO mice. Diminished activation of neutrophils seems to be a general hallmark of IFN-gamma KO mice, since neutrophils from uninfected KO mice also showed a reduction in chemotactic and phagocytic activity when induced by casein. In conclusion, these data are the first to define an IFN-gamma-dependent immune effector mechanism in murine filarial infection, i.e. neutrophil-mediated control of the adult worm load.     

菌群失调小鼠感染鼠伤寒沙门菌后Th1和Th2细胞因子的动态研究

目的研究鼠伤寒沙门菌感染小鼠在菌群失调下Th细胞因子的动态变化,以探讨菌群失调对沙门菌感染的免疫机制。方法分别建立菌群失调、感染和空白对照的小鼠模型。各组动物在感染不同时点处死,观察小肠、肝脏和脾脏病理改变。采用流式细胞仪检测脾脏细胞中IFN-γ和IL-4表达,以此代表Th1和Th2细胞。结果菌群失调组病理损害最重,Th1明显增加,表达水平高,Th2变化不大,Th1/Th2比值升高。结论菌群失调后,能够加重小鼠感染鼠伤寒沙门菌引起的的Th1型反应,产生炎症性损伤。     

Th1/Th2-regulated expression of arginase isoforms in murine macrophages and dendritic cells.

Activated murine macrophages metabolize arginine by two alternative pathways involving the enzymes inducible NO synthase (iNOS) or arginase. The balance between the two enzymes is competitively regulated by Th1 and Th2 T helper cells via their secreted cytokines: Th1 cells induce iNOS, whereas Th2 cells induce arginase. Whereas the role of macrophages expressing iNOS as inflammatory cells is well established, the functional competence of macrophages expressing arginase remains a matter of speculation. Two isoforms of mammalian arginases exist, hepatic arginase I and extrahepatic arginase II. We investigated the regulation of arginase isoforms in murine bone marrow-derived macrophages (BMMPhi) in the context of Th1 and Th2 stimulation. Surprisingly, in the presence of either Th2 cytokines or Th2 cells, we observe a specific induction of the hepatic isoform arginase I in BMMPhi. Induction of arginase I was shown on the mRNA and protein levels and obeyed the recently demonstrated synergism among the Th2 cytokines IL-4 and IL-10. Arginase II was detectable in unstimulated BMMPhi and was not significantly modulated by Th1 or Th2 stimulation. Similar to murine BMMPhi, murine bone marrow-derived dendritic cells, as well as a dendritic cell line, up-regulated arginase I expression and arginase activity upon Th2 stimulation, whereas arginase II was never detected. In addition to revealing the unexpected expression of arginase I in the macrophage/monocyte lineage, these results uncover a further intriguing parallelism between iNOS and arginase: both have a constitutive and an inducible isoform, the latter regulated by the Th1/Th2 balance.     

T cell synergy in the primary MLR: proliferative kinetics, effector cell generation, and IL 2 production

A primary rat MLR was initiated, and on each of 8 consecutive days during the evolving culture, an aliquot of cells was separated into its constitutive helper/inducer (W3/25+) and suppressor/cytotoxic (OX8+) T cell subsets by a monoclonal antibody, Degalan-bead immunoadsorbent column technique. This allowed a detailed kinetic analysis of T cell proliferation, the generation of effector cells, and the production of IL 2 by each subset relative to net whole culture supernatant IL 2 activity. The primary MLR demonstrates an early period of helper/inducer cell proliferation, IL 2 production and accumulation, followed by a period of suppressor/cytotoxic cell (OX8+) proliferation and IL 2 consumption during which there are distinct waves of allospecific suppressor, followed by cytotoxic activity. If fresh T cells of the helper/inducer or suppressor/cytotoxic phenotype were preseparated and then cultured alone with irradiated allogeneic stimulator cells, proliferation was noted in both subsets despite no demonstrable IL 2 activity in cultures of the suppressor/cytotoxic cells. Finally, a suppressed primary MLR exhibited proliferative inhibition of both T cell subsets.     

Th1 cytokines and NK cells participate in the development of murine syngeneic graft-versus-host disease.

Syngeneic graft-vs-host disease (SGVHD) is induced by reconstituting lethally irradiated mice with syngeneic bone marrow cells followed by a short course of therapy with the immunosuppressive agent cyclosporine A. Following cessation of cyclosporine A therapy, animals develop clinical symptoms of SGVHD: weight loss, runting, and diarrhea. While it has been suggested that T cells are responsible for the induction and effector phases of SGVHD, the role of nonspecific effector cells and cytokine mediators has yet to be examined in the disease process. Mice with SGVHD had increased levels of message for IL-12p40, IFN-gamma, and TNF-alpha in the target organs of SGVHD as compared with transplant controls and asymptomatic cyclosporine A-treated mice. Concomitant with the increase in Th1 cytokines was an enhanced cellular infiltrate in the target organs of SGVHD mice as determined by histological analysis. To directly examine the role of IL-12 in the development of SGVHD, in vivo neutralization of IL-12 was performed. Treatment of mice with Abs to IL-12 inhibited SGVHD-mediated tissue pathology and mortality. Because IL-12 has been shown to activate both T cells and NK cells to secrete IFN-gamma and to become more cytolytic, studies were initiated to ascertain which lymphocyte populations play a role in the development of murine SGVHD. Depletion of NK cells inhibited clinical symptoms of SGVHD. In contrast, T cell depletion did not alter the disease process. Therefore, these findings collectively demonstrate a role for IL-12 and NK cells in the effector phase of murine SGVHD.     

Differential effects of Th1 versus Th2 cytokines in combination with hypoxia on HIFs and angiogenesis in RA

Introduction

Dysregulated angiogenesis is implicated in the pathogenesis of rheumatoid arthritis (RA). To provide a more profound understanding of arthritis-associated angiogenesis, we evaluated the expression of angiogenesis-modulating genes at onset, peak and declining phases of collagen-induced arthritis (CIA), a well-established mouse model for RA.

Methods

CIA was induced in DBA/1 mice with type II collagen. Functional capillary density in synovial tissue of knee joints was determined by intravital fluorescence microscopy. To assess the ability of arthritic joint homogenates to induce angiogenesis, an endothelial chemotaxis assay and an in vivo matrigel plug assay were employed. The temporal expression profile of angiogenesis-related genes in arthritic paws was analysed by quantitative real-time RT-PCR using an angiogenesis focused array as well as gene specific PCR. Finally, we investigated the therapeutic effect of a monoclonal antibody specifically blocking the binding of VEGF to neuropilin (NRP)-1.

Results

Although arthritic paw homogenates displayed angiogenic activity in vitro and in vivo, and synovia of arthritic paws appeared highly vascularised on histological examination, the functional capillary density in arthritic knee synovia was significantly decreased, whereas capillary diameter was increased. Of the 84 genes analysed, 41 displayed a differential expression in arthritic paws as compared to control paws. Most significant alterations were seen at the peak of clinical arthritis. Increased mRNA expression could be observed for VEGF receptors (Flt-1, Flk-1, Nrp-1, Nrp-2), as well as for midkine, hepatocyte growth factor, insulin-like growth factor-1 and angiopoietin-1. Signalling through NRP-1 accounted in part for the chemotactic activity for endothelial cells observed in arthritic paw homogenates. Importantly, therapeutic administration of anti-NRP1^B antibody significantly reduced disease severity and progression in CIA mice.

Conclusions

Our findings confirm that the arthritic synovium in murine CIA is a site of active angiogenesis, but an altered balance in the expression of angiogenic factors seems to favour the formation of non-functional and dilated capillaries. Furthermore, our results validate NRP-1 as a key player in the pathogenesis of CIA, and support the VEGF/VEGF receptor pathway as a potential therapeutic target in RA.     

Th1, Th2, and Th17 effector T cell-induced autoimmune gastritis differs in pathological pattern and in susceptibility to suppression by regulatory T cells

Th cells can be subdivided into IFN-gamma-secreting Th1, IL-4/IL-5-secreting Th2, and IL-17-secreting Th17 cells. We have evaluated the capacity of fully differentiated Th1, Th2, and Th17 cells derived from a mouse bearing a transgenic TCR specific for the gastric parietal cell antigen, H(+)K(+)-ATPase, to induce autoimmune gastritis after transfer to immunodeficient recipients. We have also determined the susceptibility of the disease induced by each of the effector T cell types to suppression by polyclonal regulatory T cells (Treg) in vivo. Each type of effector cell induced autoimmune gastritis with distinct histological patterns. Th17 cells induced the most destructive disease with cellular infiltrates composed primarily of eosinophils accompanied by high levels of serum IgE. Polyclonal Treg could suppress the capacity of Th1 cells, could moderately suppress Th2 cells, but could suppress Th17-induced disease only at early time points. The major effect of the Treg was to inhibit the expansion of the effector T cells. However, effector cells isolated from protected animals were not anergic and were fully competent to proliferate and produce effector cytokines ex vivo. The strong inhibitory effect of polyclonal Treg on the capacity of some types of differentiated effector cells to induce disease provides an experimental basis for the clinical use of polyclonal Treg in the treatment of autoimmune disease in humans.     

Limited ability of antigen-specific Th1 responses to inhibit Th2 cell development in vivo

Th1 and Th2 cells mutually antagonize each other's differentiation. Consequently, allergen-specific Th1 cells are believed to be able to suppress the development of Th2 cells and to prevent the development of atopic disorders. To determine whether a pre-existing Ag-specific Th1 response can affect the development of Th2 cells in vivo, we used an immunization model of Ag-pulsed murine dendritic cell (DC) transfer to induce distinct Th responses. When transferred into naive mice, Ag-pulsed CD8alpha(+) DCs induced a Th1 response and the production of IgG2a, whereas CD8alpha(-) DCs primed a Th2 response and the production of IgE. In the presence of a pre-existing Ag-specific Th2 environment due to Ag-pulsed CD8alpha(-) DC transfer, CD8alpha(+) DCs failed to prime Th1 cells. In contrast, CD8alpha(-) DCs could prime a Th2 response in the presence of a pre-existing Ag-specific Th1 environment. Moreover, exogenous IL-4 abolished the Th1-inducing potential of CD8alpha(+) DCs in vitro, but the addition of IFN-gamma did not effectively inhibit the potential of CD8alpha(-) DCs to prime IL-4-producing cells. Thus, Th1 and Th2 cells differ in their potential to inhibit the development of the other. This suggests that the early induction of allergen-specific Th1 cells before allergy sensitization will not prevent the development of atopic disorders.     

The role of B cells in the development of CD4 effector T cells during a polarized Th2 immune response

Previous studies have suggested that B cells promote Th2 cell development by inhibiting Th1 cell differentiation. To examine whether B cells are directly required for the development of IL-4-producing T cells in the lymph node during a highly polarized Th2 response, B cell-deficient and wild-type mice were inoculated with the nematode parasite, Nippostrongylus brasiliensis. On day 7, in the absence of increased IFN-gamma, IL-4 protein and gene expression from CD4 T cells in the draining lymph nodes were markedly reduced in B cell-deficient mice and could not be restored by multiple immunizations. Using a DO11.10 T cell adoptive transfer system, OVA-specific T cell IL-4 production and cell cycle progression, but not cell surface expression of early activation markers, were impaired in B cell-deficient recipient mice following immunization with N. brasiliensis plus OVA. Laser capture microdissection and immunofluorescent staining showed that pronounced IL-4 mRNA and protein secretion by donor DO11.10 T cells first occurred in the T cell:B cell zone of the lymph node shortly after inoculation of IL-4-/- recipients, suggesting that this microenvironment is critical for initial Th2 cell development. Reconstitution of B cell-deficient mice with wild-type naive B cells, or IL-4-/- B cells, substantially restored Ag-specific T cell IL-4 production. However, reconstitution with B7-1/B7-2-deficient B cells failed to rescue the IL-4-producing DO11.10 T cells. These results suggest that B cells, expressing B7 costimulatory molecules, are required in the absence of an underlying IFN-gamma-mediated response for the development of a polarized primary Ag-specific Th2 response in vivo.