Transcription factor IRF4 drives dendritic cells to promote Th2 differentiation.

Atopic asthma is an inflammatory pulmonary disease associated with Th2 adaptive immune responses triggered by innocuous antigens. While dendritic cells (DCs) are known to shape the adaptive immune response, the mechanisms by which DCs promote Th2 differentiation remain elusive. Herein we demonstrate that Th2-promoting stimuli induce DC expression of IRF4. Mice with conditional deletion of Irf4 in DCs show a dramatic defect in Th2-type lung inflammation, yet retain the ability to elicit pulmonary Th1 antiviral responses. Using loss- and gain-of-function analysis, we demonstrate that Th2 differentiation is dependent on IRF4 expression in DCs. Finally, IRF4 directly targets and activates the Il-10 and Il-33 genes in DCs. Reconstitution with exogenous IL-10 and IL-33 recovers the ability of Irf4-deficient DCs to promote Th2 differentiation. These findings reveal a regulatory module in DCs by which IRF4 modulates IL-10 and IL-33 cytokine production to specifically promote Th2 differentiation and inflammation.

ICOS-expressing lymphocytes promote resolution of CD8-mediated lung injury in a mouse model of lung rejection.

Acute rejection, a common complication of lung transplantation, may promote obliterative bronchiolitis leading to graft failure in lung transplant recipients. During acute rejection episodes, CD8(+) T cells can contribute to lung epithelial injury but the mechanisms promoting and controlling CD8-mediated injury in the lung are not well understood. To study the mechanisms regulating CD8(+) T cell-mediated lung rejection, we used a transgenic model in which adoptively transferred ovalbumin (OVA)-specific cytotoxic T lymphocytes (CTL) induce lung injury in mice expressing an ovalbumin transgene in the small airway epithelium of the lungs (CC10-OVA mice). The lung pathology is similar to findings in humans with acute lung transplant. In the presence of an intact immune response the inflammation resolves by day 30. Using CC10-OVA.RAG(-/-) mice, we found that CD4(+) T cells and ICOS(+/+) T cells were required for protection against lethal lung injury, while neutrophil depletion was not protective. In addition, CD4(+)Foxp3 (+) ICOS(+) T cells were enriched in the lungs of animals surviving lung injury and ICOS(+/+) Tregs promoted survival in animals that received ICOS(-/-) T cells. Direct comparison of ICOS(-/-) Tregs to ICOS(+/+) Tregs found defects in vitro but no differences in the ability of ICOS(-/-) Tregs to protect from lethal lung injury. These data suggest that ICOS affects Treg development but is not necessarily required for Treg effector function.

IL-33-dependent induction of allergic lung inflammation by FcγRIII signaling.

Atopic asthma is a chronic inflammatory disease of the lungs generally marked by excessive Th2 inflammation. The role of allergen-specific IgG in asthma is still controversial; however, a receptor of IgG-immune complexes (IgG-ICs), FcγRIII, has been shown to promote Th2 responses through an unknown mechanism. Herein, we demonstrate that allergen-specific IgG-ICs, formed upon reexposure to allergen, promoted Th2 responses in two different models of IC-mediated inflammation that were independent of a preformed T cell memory response. Development of Th2-type airway inflammation was shown to be both FcγRIII and TLR4 dependent, and T cells were necessary and sufficient for this process to occur, even in the absence of type 2 innate lymphoid cells. We sought to identify downstream targets of FcγRIII signaling that could contribute to this process and demonstrated that bone marrow-derived DCs, alveolar macrophages, and respiratory DCs significantly upregulated IL-33 when activated through FcγRIII and TLR4. Importantly, IC-induced Th2 inflammation was dependent on the ST2/IL-33 pathway. Our results suggest that allergen-specific IgG can enhance secondary responses by ligating FcγRIII on antigen-presenting cells to augment development of Th2-mediated responses in the lungs via an IL-33-dependent mechanism.

Dynamic migration of γδ intraepithelial lymphocytes requires occludin.

γδ intraepithelial lymphocytes (IELs) are located beneath or between adjacent intestinal epithelial cells and are thought to contribute to homeostasis and disease pathogenesis. Using in vivo microscopy to image jejunal mucosa of GFP γδ T-cell transgenic mice, we discovered that γδ IELs migrate actively within the intraepithelial compartment and into the lamina propria. As a result, each γδ IEL contacts multiple epithelial cells. Occludin is concentrated at sites of γδ IEL/epithelial interaction, where it forms a ring surrounding the γδ IEL. In vitro analyses showed that occludin is expressed by epithelial and γδ T cells and that occludin derived from both cell types contributes to these rings and to γδ IEL migration within epithelial monolayers. In vivo TNF administration, which results in epithelial occludin endocytosis, reduces γδ IEL migration. Further in vivo analyses demonstrated that occludin KO γδ T cells are defective in both initial accumulation and migration within the intraepithelial compartment. These data challenge the paradigm that γδ IELs are stationary in the intestinal epithelium and demonstrate that γδ IELs migrate dynamically to make extensive contacts with epithelial cells. The identification of occludin as an essential factor in γδ IEL migration provides insight into the molecular regulation of γδ IEL/epithelial interactions.

Protective effector memory CD4 T cells depend on ICOS for survival.

Memory CD4 T cells play a vital role in protection against re-infection by pathogens as diverse as helminthes or influenza viruses. Inducible costimulator (ICOS) is highly expressed on memory CD4 T cells and has been shown to augment proliferation and survival of activated CD4 T cells. However, the role of ICOS costimulation on the development and maintenance of memory CD4 T cells remains controversial. Herein, we describe a significant defect in the number of effector memory (EM) phenotype cells in ICOS(-/-) and ICOSL(-/-) mice that becomes progressively more dramatic as the mice age. This decrease was not due to a defect in the homeostatic proliferation of EM phenotype CD4 T cells in ICOS(-/-) or ICOSL(-/-) mice. To determine whether ICOS regulated the development or survival of EM CD4 T cells, we utilized an adoptive transfer model. We found no defect in development of EM CD4 T cells, but long-term survival of ICOS(-/-) EM CD4 T cells was significantly compromised compared to wild-type cells. The defect in survival was specific to EM cells as the central memory (CM) ICOS(-/-) CD4 T cells persisted as well as wild type cells. To determine the physiological consequences of a specific defect in EM CD4 T cells, wild-type and ICOS(-/-) mice were infected with influenza virus. ICOS(-/-) mice developed significantly fewer influenza-specific EM CD4 T cells and were more susceptible to re-infection than wild-type mice. Collectively, our findings demonstrate a role for ICOS costimulation in the maintenance of EM but not CM CD4 T cells.

Inducible costimulator controls migration of T cells to the lungs via down-regulation of CCR7 and CD62L.

We and others reported that inducible costimulator-deficient (ICOS(-/-)) mice manifest a defect in Th2-mediated airway inflammation, which was attributed to reduced Th2 differentiation in the absence of ICOS signaling. Interestingly, the number of CD4 T cells present in the airways and lungs after sensitization and challenge is significantly reduced in ICOS(-/-) mice. We now show that this reduction is not attributable simply to a reduced proliferation of ICOS(-/-) cells, because significantly more ICOS(-/-) than wild-type activated CD4 T cells are present in the lymph nodes, suggesting that more ICOS(-/-) CD4 T cells than wild-type CD4 T cells migrated into the lymph nodes. Further investigation revealed that activated ICOS(-/-) CD4 T cells express higher concentrations of the lymph node homing receptors, CCR7 and CD62L, than do wild-type CD4 T cells, leading to a preferential return of ICOS(-/-) cells to the nondraining lymph nodes rather than the lungs. Blocking reentry into the lymph nodes after the initiation of Th2-mediated airway inflammation equalized the levels of CD4 and granulocyte infiltration in the lungs of wild-type and ICOS(-/-) mice. Our results demonstrate that in wild-type CD4 T cells, co-stimulation with ICOS promotes the down-regulation of CCR7 and CD62L after activation, leading to a reduced return of activated CD4 T cells to the lymph nodes and a more efficient entry into the lungs.

Fas ligand expression on T cells is sufficient to prevent prolonged airway inflammation in a murine model of asthma.

Our previous studies revealed that, in a murine model of asthma, mice that received Fas-deficient T cells developed a prolonged phase of airway inflammation, mucus production, and airway hyperreactivity that failed to resolve even 6 weeks after the last challenge. To investigate how Fas-Fas ligand (FasL) interaction occurs between T cells and other cells in vivo, Gld mice with abnormalities of the FasL signaling pathway were used. The reconstituted mice were made by transferring T cells from B6 or Gld mice to Rag(-/-) or FasL-deficient Rag(-/-) mice. We found that Rag(-/-) mice that received B6 T cells resolved the airway inflammation, whereas FasL-deficient Rag(-/-) mice that received Gld T cells developed a prolonged airway inflammation at Day 28, with decreased IFN-gamma production. Both FasL-deficient Rag(-/-) mice that received B6 T cells and Rag(-/-) mice that received Gld T cells also had completely resolved their airway inflammation by Day 28 after challenge. Interestingly, FasL-deficient Rag(-/-) mice that received Gld T cells eventually resolved airway inflammation at Day 42, with a similar level of IFN-gamma production to that of control group. These results demonstrate that FasL expression on either T cells only or non-T cells only was sufficient for the eventual resolution of airway inflammation, and the prolonged airway inflammation in FasL-deficient Rag(-/-) mice that received Gld T cells was correlated with decreased IFN-gamma production by Gld T cells.

Inhibition of Pyk2 blocks airway inflammation and hyperresponsiveness in a mouse model of asthma.

The objective of this investigation was to determine the role of Pyk2, an intracellular nonreceptor protein tyrosine kinase for postadhesive inflammatory cell migration, on airway inflammation and hyperresponsiveness in immune-sensitized mice. Blockade of Pyk2 was effected by intraperitoneal administration of dominant-negative C-terminal Pyk2 fused to a TAT protein transduction domain (TAT-Pyk2-CT). Ovalbumin challenge elicited infiltration of both eosinophils and lymphocytes into airways, increased mucus-containing epithelial cells, and caused increased airway hyperresponsiveness to methacholine in immune-sensitized mice. Pretreatment with 10 mg/kg TAT-Pyk2-CT intraperitoneally blocked all of these effects and further decreased secretion of Th2 cytokine IL-4, IL-5, and IL-13 into the bronchoalveolar lavage fluid. Intranasal administration of IL-5 caused eosinophil migration into the airway lumen, which was attenuated by systemic pretreatment with TAT-Pyk2-CT. In each paradigm, treatment with control protein TAT-GFP had no blocking effect. We conclude that Pyk2, which is essential for inflammatory cell migration in vitro, regulates airway inflammation, Th2 cytokine secretion, and airway hyperresponsiveness in the ovalbumin-sensitized mice during antigen challenge in vivo.

Inducible costimulator expression regulates the magnitude of Th2-mediated airway inflammation by regulating the number of Th2 cells.

BACKGROUND: Inducible Costimulator (ICOS) is an important regulator of Th2 lymphocyte function and a potential immunotherapeutic target for allergy and asthma. A SNP in the ICOS 5' promoter in humans is associated with increased atopy and serum IgE in a founder population and increased ICOS surface expression and Th2 cytokine production from peripheral blood mononuclear cells. However, it is unknown if increased ICOS expression contributes to disease progression or is a result of disease pathology. METHODOLOGY/PRINCIPAL FINDINGS: We developed a mouse model in which ICOS surface expression levels are genetically predetermined to test our hypothesis that genetic regulation of ICOS expression controls the severity of Th2 responses in vivo. Using ICOS+/+ and ICOS+/- mice in a Th2 model of airway inflammation, we found that T cells from the ICOS+/- mice had reduced ICOS expression and decreased Th2-mediated inflammation in vivo. Although the activation status of the T cells did not differ, T cells isolated from the lungs and draining lymph nodes of ICOS+/- mice at the peak of inflammation produced less Th2 cytokines upon stimulation ex vivo. Using 4get mice, which express GFP upon IL-4 transcription, we determined that the decreased Th2 cytokines in ICOS+/- is due to reduced percentage of Th2 cells and not a defect in their ability to produce IL-4. CONCLUSION: These data suggest that in both mice and humans, the level of ICOS surface expression regulates the magnitude of the in vivo Th2 response, perhaps by influencing Th2 differentiation.

CD28 and ICOS play complementary non-overlapping roles in the development of Th2 immunity in vivo.

Previous work has shown ICOS can function independently of CD28, but whether either molecule can compensate for the other in vivo is not known. Since ICOS is a potent inducer of Th2 cytokines and linked to allergy and elevated serum IgE in humans, we hypothesized that augmenting ICOS costimulation in murine allergic airway disease may overcome CD28 deficiency. While ICOS was expressed on T cells from CD28(-/-) mice, Th2-mediated airway inflammation was not induced in CD28(-/-) mice by increased ICOS costimulation. Further, we determined if augmenting CD28 costimulation could compensate for ICOS deficiency. ICOS(-/-) mice had a defect in airway eosinophilia that was not overcome by augmenting CD28 costimulation. CD28 costimulation also did not fully compensate for ICOS for antibody responses, germinal center formation or the development of follicular B helper T cells. CD28 and ICOS play complementary non-overlapping roles in the development of Th2 immunity in vivo.

ICOS costimulation expands Th2 immunity by augmenting migration of lymphocytes to draining lymph nodes.

The T cell costimulatory molecule ICOS regulates Th2 effector function in allergic airway disease. Recently, several studies with ICOS(-/-) mice have also demonstrated a role for ICOS in Th2 differentiation. To determine the effects of ICOS on the early immune response, we investigated augmenting ICOS costimulation in a Th2-mediated immune response to Schistosoma mansoni Ags. We found that augmenting ICOS costimulation with B7RP-1-Fc increased the accumulation of T and B cells in the draining lymph nodes postimmunization. Interestingly, the increased numbers were due in part to increased migration of undivided Ag-specific TCR transgenic T cells and surprisingly B cells, as well as non-TCR transgenic T cells. B7RP-1-Fc also increased the levels of the chemokines CCL21 and CXCL13 in the draining lymph node, suggesting ICOS costimulation contributes to migration by direct or indirect effects on dendritic cells, stromal cells and high endothelial venules. Further, the effects of B7RP-1-Fc were not dependent on immunization. Our data support a model in which ICOS costimulation augments the pool of lymphocytes in the draining lymph nodes, leading to an increase in the frequency of potentially reactive T and B cells.

CD43 regulates Th2 differentiation and inflammation.

CD43 is a highly glycosylated transmembrane protein that regulates T cell activation. CD43(-/-) T cells are hyperproliferative and the cytoplasmic tail of CD43 has been found to be sufficient to reconstitute wild-type proliferation levels, suggesting an intracellular mechanism. In this study, we report that upon TCR ligation CD43(-/-) T cells demonstrated no increase in tyrosine phosphorylation but a decreased calcium flux. Interestingly, CD43(-/-) T cells preferentially differentiated into Th2 cells in vitro, and CD43(-/-) T cells show increased GATA-3 translocation into the nucleus. In vivo, CD43(-/-) mice exhibited increased inflammation in two separate models of Th2-mediated allergic airway disease. In contrast, in Th1-mediated diabetes, nonobese diabetic CD43(-/-) mice did not significantly differ from wild-type mice in disease onset or progression. Th1-induced experimental autoimmune encephalomyelitis to MOG(35-55) was also normal in the CD43(-/-) mice. Nonetheless, the CD43(-/-) mice produced more IL-5 when restimulated with MOG(35-55) in vitro and demonstrated decreased delayed-type hypersensitivity responses. Together, these data demonstrate that although CD43(-/-) T cells preferentially differentiate into Th2 cells, this response is not sufficient to protect against Th1-mediated autoimmune responses.

Signaling through Fc gamma RIII is required for optimal T helper type (Th)2 responses and Th2-mediated airway inflammation.

Although inhibitory Fc gamma receptors have been demonstrated to promote mucosal tolerance, the role of activating Fc gamma receptors in modulating T helper type (Th)2-dependent inflammatory responses characteristic of asthma and allergies remains unclear. Here, we demonstrate that signaling via activating Fc gamma receptors in conjunction with Toll-like receptor 4 stimulation modulated cytokine production from bone marrow-derived dendritic cells (DCs) and augmented their ability to promote Th2 responses. Ligation of the low affinity receptor Fc gamma RIII was specifically required for the enhanced Th2 responses, as Fc gamma RIII(-/-) DCs failed to augment Th2-mediated airway inflammation in vivo or induce Th2 differentiation in vitro. Further, Fc gamma RIII(-/-) mice had impaired Th2 cytokine production and exhibited reduced airway inflammation, whereas no defect was found in Fc gamma RI(-/-) mice. The augmentation of Th2 immunity was regulated by interleukin 10 production from the DCs but was distinct and independent of the well-established role of Fc gamma RIII in augmenting antigen presentation. Thus, our studies reveal a novel and specific role for Fc gamma RIII signaling in the regulation of Th cell responses and suggest that in addition to immunoglobulin (Ig)E, antigen-specific IgG also contributes to the pathogenesis of Th2-mediated diseases such as asthma and allergies.

T-cell costimulation blockade in immunologic diseases: role of CD28 family members.

The destruction of many immune-mediated diseases is a result of T-cell responses against usually harmless antigens. Extensive research has been conducted to discover new mechanisms to specifically modulate harmful effector T cells while leaving normal immune responses intact. Since proteins of the CD28 family members are expressed on T cells, blockade of these proteins has become a possible target for potential therapies. The CD28 family contains proteins that have the ability to both enhance and diminish T-cell responses. Therefore, blockade of targets that enhance T-cell signaling may reduce destructive autoimmune responses, while blockade of targets that diminish T-cell signaling may enhance antitumor responses. In this article, the function of these proteins will be reviewed and a sample of clinical trials highlighting the potential efficacy and drawbacks of their use in humans will be described briefly. Finally, inducible costimulator and programmed death-1, two future targets of T-cell therapies, will be highlighted.

Fas-positive T cells regulate the resolution of airway inflammation in a murine model of asthma.

Persistent airway inflammation, mucus production, and airway hyperreactivity are the major contributors to the frequency and severity of asthma. Why lung inflammation persists in asthmatics remains unclear. It has been proposed that Fas-mediated apoptosis of inflammatory cells is a fundamental mechanism involved in the resolution of eosinophilic airway inflammation. Because infiltrating eosinophils are highly sensitive to Fas-mediated apoptosis, it has been presumed that direct ligation of Fas on eosinophils is involved. Here, we utilize adoptive transfers of T cells to demonstrate that the delayed resolution of eosinophilia in Fas-deficient mice is a downstream effect of Fas deficiency on T cells, not eosinophils. Interestingly, the mice that received Fas-deficient T cells, but not the controls, developed a persistent phase of inflammation that failed to resolve even 6 wk after the last challenge. This persistent phase correlated with decreased interferon (IFN)gamma production by Fas-deficient T cells and could be reproduced with adoptive transfer of IFNgamma-deficient T cells. These data demonstrate that Fas deficiency on T cells is sufficient for the development of long-term allergic airway disease in mice and implies that deregulation of death receptors such as Fas on human T cells could be an important factor in the development and/or chronic nature of asthma.