Natural immunosurveillance against spontaneous, autochthonous breast cancers revealed and enhanced by blockade of IL-13-mediated negative regulation.

INTRODUCTION: We and others previously observed immunosurveillance against transplantable tumors in mice, and enhancement thereof by blockade of negative regulation by T reg cells or the NKT-IL-13-myeloid cell-TGF-beta regulatory circuit. However, it was unknown whether natural immunosurveillance inhibits growth of completely spontaneous autochthonous tumors, and whether it can be improved by inhibition of negative regulation. MATERIALS AND METHODS: To examine the existence of T cell-mediated immunosurveillance against spontaneous tumors, BALB-neuT mice were treated with anti-CD4 and/or anti-CD8. A role for IL-13 in the suppression of immunosurveillance was investigated by treating mice with IL-13 inhibitor. RESULTS: We show that even spontaneous autochthonous breast carcinomas arising in Her-2/neu transgenic mice appear more quickly when the mice are depleted of T cells, evidence for T-cell mediated immunosurveillance slowing tumor growth. This immunosurveillance could be further enhanced by blockade of IL-13 (but not IL-4) which slowed the appearance of these autologous tumors compared to control antibody-treated mice. CONCLUSION: Thus, even completely spontaneous, autochthonous breast cancers can be controlled in part by natural immunosurveillance, and blockade of negative regulation can improve this control.

IL-13 blockade reduces lung inflammation after Ascaris suum challenge in cynomolgus monkeys.

BACKGROUND: Airway inflammation is a hallmark feature of asthma and a driver of airway hyperresponsiveness. IL-13 is a key inducer of airway inflammation in rodent models of respiratory disease, but a role for IL-13 has not been demonstrated in primates. OBJECTIVE: We sought to test the efficacy of a neutralizing antibody to human IL-13 in a cynomolgus monkey model of lung inflammation. METHODS: Using cynomolgus monkeys (Macaca fascicularis) that are sensitized to Ascaris suum through natural exposure, we developed a reproducible model of acute airway inflammation after segmental A suum antigen challenge. This model was used to test the in vivo efficacy of mAb13.2, a mouse mAb directed against human IL-13, and IMA-638, the humanized counterpart of mAb13.2. Bronchoalveolar lavage (BAL) cells and BAL fluid were collected before and after antigen challenge and assayed for cellular content by means of differential count. RESULTS: Total BAL cell count, eosinophil number, and neutrophil number were all reduced in animals treated with mAb13.2 or IMA-638 compared with values in control animals that were untreated, given saline, or treated with human IgG of irrelevant specificity. In addition, levels of eotaxin and RANTES in BAL fluid were reduced in anti-IL-13-treated animals compared with levels seen in control animals. CONCLUSION: These findings support a role for IL-13 in maintaining lung inflammation in response to allergen challenge in nonhuman primates. CLINICAL IMPLICATIONS: IL-13 neutralization with a specific antibody could be a useful therapeutic strategy for asthma.

Efficacy of IL-13 neutralization in a sheep model of experimental asthma.

IL-13 contributes to airway hyperresponsiveness, mucus secretion, inflammation, and fibrosis, suggesting that it plays a central role in asthma pathogenesis. Neutralization of IL-13 with sIL-13Ralpha2-Fc (sIL-13R) reduces allergen-induced airway responses in rodent models of respiratory disease, but its efficacy in a large animal model has not been previously reported. In this study, we determined whether two different strategies for IL-13 neutralization modified experimental asthma in sheep. Sheep with natural airway hypersensitivity to Ascaris suum antigen were treated intravenously either with sIL-13R, a strong antagonist of sheep IL-13 bioactivity in vitro, or with IMA-638 (IgG1, kappa), a humanized antibody to human IL-13. Higher doses of IMA-638 were used because, although it is a potent antagonist of human IL-13, this antibody has 20 to 30 times lower binding and neutralization activity against sheep IL-13. Control animals received human IgG of irrelevant specificity. Sheep were treated 24 h before inhalation challenge with nebulized A. suum. The effects on antigen-induced early and late bronchial responses, and antigen-induced hyperresponsiveness, were assessed. Both sIL-13R and IMA-638 provided dose-dependent inhibition of the antigen-induced late responses and airway hyperresponsiveness. The highest dose of IMA-638 also reduced the early phase response. These findings suggest that IL-13 contributes to allergen-induced airway responses in this sheep model of asthma, and that neutralization of IL-13 is an effective strategy for blocking these A. suum-induced effects.

Intraepithelial NK cell-derived IL-13 induces intestinal pathology associated with nematode infection.

IL-13 is a Th2-derived cytokine associated with pathological changes in asthma and ulcerative colitis. Moreover, it plays a major role in the control of gut nematode infection and associated immunopathology. The current paradigm is that these effects are due to T cell-derived IL-13. We show in this study that an innate source of IL-13, the intraepithelial NK cell, is responsible for the disruption of intestinal tissue architecture and induction of goblet cell hyperplasia that characterizes infection with the intestinal helminth Trichinella spiralis. IL-13 or IL-4Ralpha (but not IL-4) null mice failed to induce intestinal pathology. Unexpectedly, SCID and athymic mice developed the same pathology found in immunocompetent mice following infection. Moreover, immunodeficient mice expressed IL-13 in the intestine, and abnormal mucosal pathology was reduced by in vivo administration of a soluble IL-13 antagonist. IL-13 expression was induced in non-T intraepithelial CD3- NK cells. Epithelial cells expressed the IL-13 signaling receptor, IL-13Ralpha1, and after infection, IL-4Ralpha. Furthermore, the soluble IL-13 decoy receptor IL-13Ralpha2, which regulates IL-13 responses, was also induced upon infection. These data provide the first evidence that intestinal tissue restructuring during helminth infection is an innate event dependent on IL-13 production by NK cells resident in the epithelium of the intestine.

Antagonizing deactivating cytokines to enhance host defense and chemotherapy in experimental visceral leishmaniasis.

In experimental visceral leishmaniasis, inhibition of interleukin 10 (IL-10) signaling enhances Th1-cell-associated responses, promoting gamma interferon (IFN-gamma) secretion, granuloma assembly, macrophage activation with substantial liver parasite killing, and synergy with pentavalent antimony (Sb) chemotherapy. To determine if inhibiting other suppressive cytokines has similar therapeutic potential, Leishmania donovani-infected BALB/c mice were injected with anti-IL-4 monoclonal antibody or receptor fusion antagonists of IL-13 or transforming growth factor beta (TGF-beta). Targeting IL-13 or TGF-beta enabled inhibition of L. donovani replication but little parasite killing; anti-IL-4 had no effect. None of the three antagonists promoted IFN-gamma production, granuloma maturation, or Sb efficacy. Excess IL-13 and TGF-beta exacerbated liver infection; however, effects were transient. Among IL-10, IL-4, IL-13, and TGF-beta, cytokines capable of disabling Th1-cell mechanisms (including those which support chemotherapy), IL-10 appears to be the appropriate target for therapeutic inhibition in visceral L. donovani infection.

IgE generation and mast cell effector function in mice deficient in IL-4 and IL-13.

IL-4 and IL-13 are potent cytokines that drive production of IgE, which is critical to the development of atopic disease. In this study, we directly compared IgE generation and IgE-dependent mast cell effector function in mouse strains lacking IL-4, IL-13, IL-4 + IL-13, or their common receptor component, IL-4Ralpha. Although serum IgE was undetectable under resting conditions in most animals deficient in one or both cytokines, peritoneal mast cells from mice lacking IL-4 or IL-13 had only partial reductions in surface IgE level. In contrast, peritoneal mast cells from IL-4/13(-/-) and IL-4Ralpha(-/-) animals were severely deficient in surface IgE, and showed no detectable degranulation following treatment with anti-IgE in vitro. Surprisingly, however, intradermal challenge with high concentrations of anti-IgE Ab induced an ear-swelling response in these strains, implying some capacity for IgE-mediated effector function in tissue mast cells. Furthermore, upon specific immunization with OVA, both IL-4/IL-13(-/-) and IL-4Ralpha(-/-) mice produced detectable levels of serum IgE and Ag-specific IgG1, and generated strong ear-swelling responses to intradermal administration of anti-IgE. These findings suggest that a mechanism for IgE production exists in vivo that is independent of IL-4 or IL-13.

IL-13 regulates the immune response to inhaled antigens.

The large inhibitory effect of IL-13 blockers on the asthma phenotype prompted us to ask whether IL-13 would play a role in regulating the allergic immune response in addition to its documented effects on structural pulmonary cells. Because IL-13 does not interact with murine T or B cells, but with monocytes, macrophages, and dendritic cells (DCs), we examined the role of IL-13 in the activation of pulmonary macrophages and DCs and in the priming of an immune response to a harmless, inhaled Ag. We found that a majority of cells called "alveolar or interstitial macrophages" express CD11c at high levels (CD11c(high)) and are a mixture of at least two cell types as follows: 1) cells of a mixed phenotype expressing DC and macrophage markers (CD11c, CD205, and F4/80) but little MHC class II (MHC II); and 2) DC-like cells expressing CD11c, CD205, MHC II, and costimulatory molecules. Endogenous IL-13 was necessary to induce and sustain the increase in MHC II and CD40 expression by pulmonary CD11c(high) cells, demonstrated by giving an IL-13 inhibitor as a measure of prevention or reversal to allergen-primed and -challenged mice. Conversely, IL-13 given by inhalation to naive mice increased the expression of MHC II and costimulatory molecules by CD11c(high) cells in an IL-4Ralpha-dependent manner. We found that exogenous IL-13 exaggerated the immune and inflammatory responses to an inhaled, harmless Ag, whereas endogenous IL-13 was necessary for the priming of naive mice with an inhaled, harmless Ag. These data indicate that blockade of IL-13 may have therapeutic potential for controlling the immune response to inhaled Ags.

Unmasking immunosurveillance against a syngeneic colon cancer by elimination of CD4+ NKT regulatory cells and IL-13.

We have previously observed a novel role of natural killer T (NKT) cells in negative regulation of antitumor immune responses against an immunogenic regressor tumor expressing a transfected viral antigen. Here, we investigated whether hidden spontaneous antitumor immunosurveillance, in the absence of a vaccine, could be revealed by disruption of this negative regulatory pathway involving CD4+ NKT cells and interleukin-13 (IL-13), in a murine pulmonary metastasis model of a nontransfected, nonregressor, syngeneic tumor, the CT26 colon carcinoma. Lung metastases of CT26 were decreased in CD4+ T cell-depleted BALB/c mice, suggesting that CD4+ T cells were involved in negative regulation of antitumor responses. CD1-knock out (CD1-KO) mice, which have conventional CD4+ T cells and CD4+CD25+ regulatory T cells but lack CD1-restricted CD4+ NKT cells, were significantly resistant to lung metastasis of CT26. The metastases were not further decreased in CD4+ T cell-depleted CD1-KO mice, implying that CD4+ NKT cells might be the primary negative regulator of antitumor immune responses in BALB/c mice. CD8+ T cells were found to act as effectors in antitumor immune responses, since the inhibition of lung metastases observed in naive CD1-KO or CD4+ T cell-depleted mice was abrogated by depletion of CD8+ T cells. Lung metastases were significantly decreased by treatment of mice with an IL-13 inhibitor, but not by deficiency or inhibition of IL-4. Thus, even for a nonregressor tumor, immunosurveillance exists but is negatively regulated via CD4+ NKT cells possibly mediated by IL-13, and can be unmasked by removal of these negative regulatory components.

Is interleukin-13 critical in maintaining airway hyperresponsiveness in allergen-challenged mice?

Interleukin (IL)-13 is regarded as being a central effector in the pathophysiology of airway hyperresponsiveness. We have described a mouse model in which chronic allergen exposure results in sustained airway hyperresponsiveness and aspects of airway remodeling, and here sought to demonstrate that this component of airway hyperresponsiveness is independent of biologically active IL-13. Sensitized mice were subjected to either brief or chronic periods of allergen exposure and studied 24 hours after brief or 4 weeks after chronic allergen inhalation. A soluble murine anti-IL-13 receptor fusion protein that specifically binds to and neutralizes IL-13 was given daily during the 4 days before the day of outcome measurements in both protocols. Outcome measurements included airway responses to intravenous methacholine, bronchoalveolar lavage fluid cell counts, and airway morphometry. Compared with the saline control, brief allergen challenge resulted in airway hyperresponsiveness, which was prevented by anti-IL-13 treatment. Chronic allergen challenge resulted in sustained airway hyperresponsiveness and indices of airway remodeling; IL-13 blockade failed to reverse this sustained airway hyperresponsiveness. These results confirm that IL-13 is critical for the development of airway hyperresponsiveness associated with brief allergen exposure, but is not necessary to maintain the sustained airway hyperresponsiveness associated with airway remodeling.

Mast cells, Fc epsilon RI, and IL-13 are required for development of airway hyperresponsiveness after aerosolized allergen exposure in the absence of adjuvant.

In certain models of allergic airway disease, mast cells facilitate the development of inflammation and airway hyper-responsiveness (AHR). To define the role of the high affinity IgE receptor (FcepsilonRI) in the development of AHR, mice with a disruption of the alpha subunit of the high affinity IgE receptor (FcepsilonRI(-/-)) were exposed on 10 consecutive days to nebulized OVA. Forty-eight hours after the last nebulization, airway responsiveness was monitored by the contractile response of tracheal smooth muscle to electrical field stimulation (EFS). After the 10-day OVA challenge protocol, wild-type mice demonstrated increased responsiveness to EFS, whereas similarly challenged FcepsilonRI(-/-) mice showed a low response to EFS, similar to nonexposed animals. Further, allergen-challenged FcepsilonRI(-/-) mice showed less airway inflammation, goblet cell hyperplasia, and lower levels of IL-13 in lung homogenates compared with the controls. IL-13-deficient mice failed to develop an increased response to EFS or goblet cell hyperplasia after the 10-day OVA challenge. We transferred bone marrow-derived mast cells from wild-type mice to FcepsilonRI(-/-) mice 1 day before initiating the challenge protocol. After the 10-day OVA challenge, recipient FcepsilonRI(-/-) mice demonstrated EFS-induced responses similar to those of challenged wild-type mice. Transferred mast cells could be detected in tracheal preparations. These results show that FcepsilonRI is important for the development of AHR after an aerosolized allergen sensitization protocol and that this effect is mediated through FcepsilonRI on mast cells and production of IL-13 in the lung.

IL-13 receptor alpha 2 down-modulates granulomatous inflammation and prolongs host survival in schistosomiasis.

An important feature of many chronic parasitic infections is the ability of the invading pathogen and host to establish a compromise, which ensures successful parasitism without killing the infected host. For many helminth infections, down-modulating the immune response is critical because persistent inflammation can become more damaging to the host than the invading pathogen itself. Such is the case with schistosomiasis mansoni, where chronic granulomatous inflammation in the liver causes portal hypertension, porto-pulmonary shunting, bleeding from collateral bypass vessels, and eventual death if not suppressed effectively. CD4(+) T helper type 2 cells (Th2) (secreting IL-4, IL-5, and IL-13) characterize the host response after Schistosoma mansoni infection, and recent studies have identified IL-13 as the principal mediator of hepatic fibrosis. Here, we show that the IL-13 receptor alpha 2 (IL-13R alpha 2) is a critical mediator of immune down-modulation, identifying the receptor as a life-sustaining off signal for chronic and pernicious inflammation in schistosomiasis.

IL-21 effects on human IgE production in response to IL-4 or IL-13.

In human atopic disease, IgE sensitizes the allergic response, while IgG4 is protective. Because IL-4 and IL-13 trigger switch recombination to both IgE and IgG4, additional agents must regulate the balance between these isotypes to influence susceptibility or tolerance to atopy. In this report, we define in vitro conditions leading to activation or inhibition of human IgE and IgG4 production by IL-21. IL-21 reduced IL-4-driven IgE synthesis by mitogen-stimulated human PBMC. IL-21 inhibition of human IgE production was not a direct effect on B cells, was not seen following B cell activation with IL-13, and was overcome by CD40 ligation. Neither IFN-gamma, IL-10, IL-12, CD40L expression, nor apoptosis was responsible for the inhibitory effect. In contrast, IL-21-stimulated secretion of IgG4 from PBMC. Our findings indicate that IL-21 may influence the production of both human IgE and IgG4, and thus contribute to the regulation of atopic reactions.

Transforming growth factor-beta production and myeloid cells are an effector mechanism through which CD1d-restricted T cells block cytotoxic T lymphocyte-mediated tumor immunosurveillance: abrogation prevents tumor recurrence.

Our previous work demonstrated that cytotoxic T lymphocyte (CTL)-mediated tumor immunosurveillance of the 15-12RM tumor could be suppressed by a CD1d-restricted lymphocyte, most likely a natural killer (NK) T cell, which produces interleukin (IL)-13. Here we present evidence for the effector elements in this suppressive pathway. T cell-reconstituted recombination activating gene (RAG)2 knockout (KO) and RAG2/IL-4 receptor alpha double KO mice showed that inhibition of immunosurveillance requires IL-13 responsiveness by a non-T non-B cell. Such nonlymphoid splenocytes from tumor-bearing mice produced more transforming growth factor (TGF)-beta, a potent inhibitor of CTL, ex vivo than such cells from naive mice, and this TGF-beta production was dependent on the presence in vivo of both IL-13 and CD1d-restricted T cells. Ex vivo TGF-beta production was also abrogated by depleting either CD11b+ or Gr-1+ cells from the nonlymphoid cells of tumor-bearing mice. Further, blocking TGF-beta or depleting Gr-1+ cells in vivo prevented the tumor recurrence, implying that TGF-beta made by a CD11b+ Gr-1+ myeloid cell, in an IL-13 and CD1d-restricted T cell-dependent mechanism, is necessary for down-regulation of tumor immunosurveillance. Identification of this stepwise regulation of immunosurveillance, involving CD1-restricted T cells, IL-13, myeloid cells, and TGF-beta, explains previous observations on myeloid suppressor cells or TGF-beta and provides insights for targeted approaches for cancer immunotherapy, including synergistic blockade of TGF-beta and IL-13.

Respiratory syncytial virus-induced airway hyperresponsiveness is independent of IL-13 compared with that induced by allergen.

BACKGROUND: IL-13 is a central mediator of allergen-induced airway hyperresponsiveness (AHR), but its role in respiratory syncytial virus (RSV)-induced AHR is not defined. The combination of allergen exposure and RSV infection is known to increase AHR and lung inflammation, but whether IL-13 regulates this increase is similarly not known. OBJECTIVE: Our objective was to determine the role of RSV infection and IL-13 on airway responsiveness and lung inflammation on sensitized and challenged mice. METHODS: Using a murine model of RSV infection and allergen exposure, we examined the role of IL-13 in the development of AHR and lung inflammation in IL-13 knockout mice, as well as using a potent IL-13 inhibitor (IL-13i). Mice were sensitized and challenged to allergen, and 6 days after the last challenge, they were infected with RSV. IL-13 was inhibited using an IL-13 receptor alpha(2)-human IgG fusion protein. AHR to inhaled methacholine was measured 6 days after infection, as was bronchoalveolar lavage fluid and lung inflammatory and cytokine responses. RESULTS: RSV-induced AHR was unaffected by the IL-13i, despite prevention of goblet cell hyperplasia. Similar results were seen in IL-13-deficient mice. In sensitized and challenged mice, RSV infection significantly increased AHR, and after IL-13i treatment, AHR was significantly reduced, but to the levels seen in RSV-infected mice alone. CONCLUSIONS: These results indicate that despite some similarities, the mechanisms leading to AHR induced by RSV are different from those that follow allergen sensitization and challenge. Because IL-13 inhibition is effective in preventing the increases in AHR and mucus production in sensitized and challenged mice infected with RSV, IL-13i could play an important role in preventing the consequences of viral infection in patients with allergic asthma.

Antigen-specific production of interleukin (IL)-13 and IL-5 cooperate to mediate IL-4Ralpha-independent airway hyperreactivity.

The pathogenesis of human asthma and the development of key features of pulmonary allergy in mouse models has been critically linked to IL-13. Analyses of the receptor components employed by IL-13 have shown that delivery of this cytokine to the airways of naive IL-4Ralpha gene targeted (IL-4Ralpha(-/-)) mice fails to induce disease, suggesting that this membrane protein is critical for transducing IL-13-mediated responses. The current study demonstrates that, in contrast to naive mice, T helper 2 bias, airways hyperreactivity (AHR) and tissue eosinophilia develop in Ovalbumin-sensitized IL-4Ralpha(-/-) mice and that these responses can be inhibited by the IL-13 antagonist sIL-13Ralpha2Fc. Therefore, antigen stimulation induces an IL-13-regulated response that is independent of IL-4Ralpha. To determine the role of IL-5 and eosinophils in the development of disease in antigen-exposed IL-4Ralpha(-/-) mice, pulmonary allergy was examined in mice deficient in both factors. IL-4Ralpha/IL-5(-/-) mice were significantly defective in their ability to produce IL-13 and failed to develop AHR, suggesting that IL-5 indirectly regulates AHR in allergic IL-4Ralpha(-/-) mice by an IL-13-dependent mechanism. Collectively, these results demonstrate that IL-13-dependent processes regulating the development of AHR and T helper bias persist in the in the lungs of allergic IL-4Ralpha(-/-) mice.

Targeted inactivation of the IL-4 receptor alpha chain I4R motif promotes allergic airway inflammation.

The insulin/interleukin-4 (IL-4) receptor (I4R) motif mediates the association of insulin receptor substrate (IRS)-2 with the interleukin-4 (IL-4)Ralpha chain and transduces mitogenic signals in response to IL-4. Its physiological functions were analyzed in mice with a germline point mutation that changed the motif's effector tyrosine residue into phenylalanine (Y500F). The Y500F mutation abrogated IRS-2 phosphorylation and impaired IL-4-induced CD4+ T lymphocyte proliferation but left unperturbed Stat6 activation, up-regulation of IL-4-responsive gene products, and Th cell differentiation under Th2 polarizing conditions. However, in vivo the Y500F mutation was associated with increased allergen-induced IgE production, airway responsiveness, tissue eosinophilia, and mucus production. These results define an important role for the I4R motif in regulating allergic inflammation.

Regulation and function of the interleukin 13 receptor alpha 2 during a T helper cell type 2-dominant immune response.

Highly polarized type 2 cytokine responses can be harmful and even lethal to the host if they are too vigorous or persist too long. Therefore, it is important to elucidate the mechanisms that down-regulate these reactions. Interleukin (IL)-13 has emerged as a central mediator of T helper cell (Th)2-dominant immune responses, exhibiting a diverse array of functional activities including regulation of airway hyperreactivity, resistance to nematode parasites, and tissue remodeling and fibrosis. Here, we show that IL-13 receptor (R)alpha2 is a critical down-regulatory factor of IL-13-mediated tissue fibrosis induced by the parasitic helminth Schistosoma mansoni. IL-13Ralpha2 expression was induced after the onset of the fibrotic response, IL-10, IL-13, and Stat6 dependent, and inhibited by the Th1-inducing adjuvant IL-12. Strikingly, schistosome-infected C57BL/6 and BALB/c IL-13Ralpha2-deficient mice showed a marked exacerbation in hepatic fibrosis, despite displaying no change in granuloma size, tissue eosinophilia, or mastocytosis. Fibrosis increased despite the fact that IL-13 levels decreased significantly in the liver and serum. Importantly, pathology was prevented when IL-13Ralpha2-deficient mice were treated with a soluble IL-13Ralpha2-Fc construct, formally demonstrating that their exacerbated fibrotic response was due to heightened IL-13 activity. Together, these studies illustrate the central role played by the IL-13Ralpha2 in the down-regulation of a chronic and pathogenic Th2-mediated immune response.

Enhanced interleukin (IL)-13 responses in mice lacking IL-13 receptor alpha 2.

Interleukin (IL)-13 has recently been shown to play important and unique roles in asthma, parasite immunity, and tumor recurrence. At least two distinct receptor components, IL-4 receptor (R)alpha and IL-13Ralpha1, mediate the diverse actions of IL-13. We have recently described an additional high affinity receptor for IL-13, IL-13Ralpha2, whose function in IL-13 signaling is unknown. To better appreciate the functional importance of IL-13Ralpha2, mice deficient in IL-13Ralpha2 were generated by gene targeting. Serum immunoglobulin E levels were increased in IL-13Ralpha2-/- mice despite the fact that serum IL-13 was absent and immune interferon gamma production increased compared with wild-type mice. IL-13Ralpha2-deficient mice display increased bone marrow macrophage progenitor frequency and decreased tissue macrophage nitric oxide and IL-12 production in response to lipopolysaccharide. These results are consistent with a phenotype of enhanced IL-13 responsiveness and demonstrate a role for endogenous IL-13 and IL-13Ralpha2 in regulating immune responses in wild-type mice.

The role of IL-13 in established allergic airway disease.

The effectiveness of targeting IL-13 in models where airway hyperresponsiveness (AHR) and airway inflammation have already been established is not well-described. We investigated the effects of blocking IL-13 on the early and late phase airway responses and the development of AHR in previously sensitized and challenged mice. BALB/cByJ mice were sensitized (days 1 and 14) and challenged (days 28-30) with OVA. Six weeks later (day 72), previously sensitized/challenged mice were challenged with a single OVA aerosol and the early and late phase response and development of AHR were determined. Specific in vivo blockade of IL-13 was attained after i.p. injection of a soluble IL-13Ralpha2-IgG fusion protein (sIL-13Ralpha2Fc) on days 71-72 for the early and late responses and on days 71-73 for the development of AHR. sIL-13Ralpha2Fc administration inhibited the late, but not early, phase response and the OVA challenge-induced changes in lung resistance and dynamic compliance; as well, sIL-13Ralpha2Fc administration decreased bronchoalveolar lavage eosinophilia and mucus hypersecretion following the secondary challenge protocols. These results demonstrate that targeting IL-13 alone regulates airway responses when administrated to mice with established allergic airway disease. These data identify the importance of IL-13 in the development of allergen-induced altered airway responsiveness following airway challenge, even when administered before rechallenge of mice in which allergic disease had been previously established.

A push-pull approach to maximize vaccine efficacy: abrogating suppression with an IL-13 inhibitor while augmenting help with granulocyte/macrophage colony-stimulating factor and CD40L.

Although a role for CD4(+) helper cells in CD8(+) cytotoxic T lymphocyte (CTL) induction by vaccines is widely recognized, much less is known about a counterbalancing role of CD4(+) T cells in down-modulating this response, or about ways to optimize vaccine responses through abrogation of this negative regulatory mechanism. Here, we discovered a synergistic enhancement of vaccine-mediated CTL induction and protection by the relief of suppression through depletion of regulatory CD4(+) cells, including CD4(+) NKT cells, or blockade of IL-13 made by these cells, combined with the cytokine granulocyte/macrophage colony-stimulating factor and the costimulatory molecule CD40L. Indeed, in the absence of helper epitopes, granulocyte/macrophage colony-stimulating factor and the helper-mimetic molecule CD40L are not sufficient to replace help to induce CTL without abrogation of CD4(+) T cell-mediated suppression, suggesting a role for T cell help in overcoming suppression. The increased CTL induction translated to striking protection against viral infection by a vaccine by using this synergistic combined approach. These results argue for a push-pull approach to maximize vaccine efficacy, especially for HIV and cancer.