Anna Tafuri

Costimulation through the inducible costimulator ligand is essential for both T helper and B cell functions in T cell-dependent B cell responses.

Costimulation through the inducible costimulator (ICOS) and its ligand (ICOSL) is essential for T cell–dependent B cell responses, but the cellular and temporal dynamics underlying its in vivo effects are poorly defined. Here we have shown that Icosl−/− and Icos−/− mice had similar phenotypes and that ICOS-ICOSL costimulation modulated the early but not late phases of IgG1 affinity maturation. Exploiting the adoptive transfer of T or B cells from primed Icosl−/− mice, we provided genetic evidence that costimulation through ICOSL was essential for primary but not secondary helper T cell responses and for the control of both T and B cell activities, resulting in T cell–dependent IgG1 production.

The Murine Nck SH2/SH3 Adaptors Are Important for the Development of Mesoderm-Derived Embryonic Structures and for Regulating the Cellular Actin Network

ABSTRACT Mammalian Nck1 and Nck2 are closely related adaptor proteins that possess three SH3 domains, followed by an SH2 domain, and are implicated in coupling phosphotyrosine signals to polypeptides that regulate the actin cytoskeleton. However, the in vivo functions of Nck1 and Nck2 have not been defined. We have mutated the murine Nck1 and Nck2 genes and incorporated β-galactosidase reporters into the mutant loci. In mouse embryos, the two Nck genes have broad and overlapping expression patterns. They are functionally redundant in the sense that mice deficient for either Nck1 or Nck2 are viable, whereas inactivation of both Nck1 and Nck2 results in profound defects in mesoderm-derived notochord and embryonic lethality at embryonic day 9.5. Fibroblast cell lines derived from Nck1−/− Nck2−/− embryos have defects in cell motility and in the organization of the lamellipodial actin network. These data suggest that the Nck SH2/SH3 adaptors have important functions in the development of mesodermal structures during embryogenesis, potentially linked to a role in cell movement and cytoskeletal organization.

Role of ICOS versus CD28 in antiviral immunity.

The costimulatory protein ICOS is inducibly expressed on activated T cells. Previous results have shown that ICOS–/– mice are defective in germinal center formation, antibody (Ab) production and class switch as well as Th1 and Th2 cytokine production in response to protein or parasite antigens. However, ICOS‐Ig failed to block antiviral Ab responses. To date the immune response to viruses has not been examined in ICOS–/– mice. In this report we compared antiviral Ab responses to LCMV, VSV and influenza virus in ICOS–/– versus wild‐type mice. Our results show that ICOS is important in the Ab response to all three viruses, with greater effects on primary as compared to secondary responses. Although ICOS–/– mice are impaired in some immune responses following influenza infection, the effects were less severe than for CD28–/– mice. There was no defect in initial influenza‐specific CD8 T cell expansion in ICOS–/– mice or in cytotoxic effector function. However, ICOS was important in maintaining CD4 cytokine production and CD8 T cell numbers late in the primary response. Upon secondary infection, ICOS–/– mice show wild‐type levels of influenza‐specific CD8 T cells, whereas CD28–/– mice show greatly impaired secondary CD8 T cell expansion. Overall, our results show that ICOS plays a clear role in the primary response to viruses at the level of Ab production, germinal center formation and Th cytokine production, but has diminished effects following secondary viral challenge.

Epidermolysis bullosa and embryonic lethality in mice lacking the multi-PDZ domain protein GRIP1

Glutamate receptor-interacting protein 1 (GRIP1) is an adaptor protein composed of seven PDZ (postsynaptic density-95/Discs large/zona occludens-1) domains, capable of mediating diverse protein–protein interactions. GRIP1 has been implicated in the regulation of neuronal synaptic function, but its physiologic roles have not been defined in vivo. We find that elimination of murine GRIP1 results in embryonic lethality. GRIP1−/− embryos develop abnormalities of the dermo-epidermal junction, resulting in extensive skin blistering around day 12 of embryonic life. Ultra-structural characterization of the blisters (or bullae) revealed cleavage of the dermo-epidermal junction below the lamina densa, an alteration reminiscent of the dystrophic form of human epidermolysis bullosa. Blisters were also observed in the lateral ventricle of the brain and in the meninges covering the cerebral cortex. These genetic data suggest that the GRIP1 scaffolding protein is required for the formation and integrity of the dermo-epidermal junction and reveal the importance of PDZ domains in the organization of supramolecular structures essential for mammalian embryonic development.

Nck-mediated recruitment of BCAP to the BCR regulates the PI(3)K-Akt pathway in B cells

The adaptor Nck links receptor signaling to cytoskeleton regulation. Here we found that Nck also controlled the phosphatidylinositol-3-OH kinase (PI(3)K)–kinase Akt pathway by recruiting the adaptor BCAP after activation of B cells. Nck bound directly to the B cell antigen receptor (BCR) via the non–immunoreceptor tyrosine-based activation motif (ITAM) phosphorylated tyrosine residue at position 204 in the tail of the immunoglobulin-α component. Genetic ablation of Nck resulted in defective BCR signaling, which led to hampered survival and proliferation of B cells in vivo. Indeed, antibody responses in Nck-deficient mice were also considerably impaired. Thus, we demonstrate a previously unknown adaptor function for Nck in recruiting BCAP to sites of BCR signaling and thereby modulating the PI(3)K-Akt pathway in B cells.

B7RP-1 Is Not Required for the Generation of Th2 Responses in a Model of Allergic Airway Inflammation but Is Essential for the Induction of Inhalation Tolerance

The recently described ICOS-B7RP-1 costimulatory pathway has been implicated in the generation of effector Th2 responses and, hence, has become an attractive therapeutic target for allergic diseases. In the present study, we used B7RP-1-deficient mice to investigate the role of B7RP-1 in the generation and maintenance of Th2 responses in a model of mucosal allergic airway inflammation. We found that exposure of B7RP-1 knockout mice to aerosolized OVA in the context of GM-CSF leads to airway eosinophilic inflammation. This response was long lasting because rechallenge of mice with the same Ag recapitulated airway eosinophilia. Moreover, significant expression of T1/ST2 on T cells and production of Th2-affiliated cytokines (IL-5, IL-4, and IL-13) and Igs (IgE and IgG1) conclusively demonstrate the generation of a Th2 response in the absence of B7RP-1. In addition, expression of two major Th2-associated costimulatory molecules—CD28 and ICOS—indicates T cell activation in the absence of B7RP-1 signaling. Finally, B7RP-1 knockout mice are resistant to the induction of inhalation tolerance as indicated by the sustained eosinophilia in the lung and IL-5 production. In summary, our results demonstrate that in a model of mucosal allergic sensitization, the ICOS-B7RP-1 pathway is redundant for the generation of Th2 responses but essential for the induction of inhalation tolerance.

Nck adaptor proteins modulate differentiation and effector function of T cells

Understanding the molecular mechanisms regulating T cell reactivity is required for successful reprogramming of immune responses in medical conditions, characterized by dysfunctions of the immune system. Nck proteins are cytoplasmic adaptors mediating diverse cellular functions, including TCR signaling. By enhancing TCR signal strength, Nck proteins influence thymic selection and regulate the size and sensitivity of the peripheral T cell repertoire. Here, we investigated the contribution of Nck proteins to CD4+ T cell differentiation and effector function using Nck.T−/− mice. Impaired GC formation and reduced Tfh were observed in Nck.T−/− mice after immunization with T cell‐dependent antigens. Th2/Tfh‐related cytokines, such as IL‐4, IL‐10, and IL‐21, were decreased in Nck.T−/− mice T cells. Moreover, an increased susceptibility to cell death of Tfh cells in Nck.T−/− mice was associated with decreased levels of Akt phosphorylation. As a result of this dysregulation in Tfh cells of Nck.T−/− mice, we found impaired production and affinity maturation of antibodies against T cell‐dependent antigens. Thus, Nck proteins not only participate in thymic selection and generation of the peripheral T cell repertoire but also are involved in the differentiation and effector functions of CD4+ T cells.