Although both CD28 and ICOS bind PI3K and provide stimulatory signal for T cell activation, unlike CD28, ICOS does not costimulate IL-2 secretion. CD28 binds both PI3K and Grb2, whereas ICOS binds only PI3K. We have generated an ICOS mutant, which can bind Grb2 by replacement of its PI3K binding motif YMFM with the CD28 YMNM motif, and shown that it induces significant activation of the IL-2 promoter. However, this mutant ICOS was insufficient to activate the NF-κB pathway. In this study, we show that Gads, but not Grb2, is essential for CD28-mediated NF-κB activation, and its binding to CD28 requires the whole CD28 cytoplasmic domain in addition to the YMNM motif. Mutagenesis experiments have indicated that mutations in the N-terminal and/or C-terminal PXXP motif(s) of CD28 significantly reduce their association with Gads, whereas their associations with Grb2 are maintained. They induced strong activity of the NFAT/AP-1 reporter comparable with the CD28 wild type, but weak activity of the NF-κB reporter. Grb2- and Gads-dominant-negative mutants had a strong effect on NFAT/AP-1 reporter, but only Gads-dominant-negative significantly inhibited NF-κB reporter. Our data suggest that, in addition to the PI3K binding motif, the PXXP motif in the CD28 cytoplasmic domain may also define a functional difference between the CD28- and ICOS-mediated costimulatory signals by binding to Gads.

Tissue factor (TF), the trigger protein of the extrinsic blood coagulation cascade, is abundantly expressed in various cancers including gastric cancer. Anti‐TF monoclonal antibodies (mAbs) capable of targeting cancers have been successfully applied to armed antibodies such as antibody‐drug conjugates (ADCs) and molecular imaging probes. We prepared an anti‐TF mAb, clone 1084, labeled with astatine‐211 (211At), as a promising alpha emitter for cancer treatment. Alpha particles are characterized by high linear energy transfer and a range of 50‐100 µm in tissue. Therefore, selective and efficient tumor accumulation of alpha emitters results in potent antitumor activities against cancer cells with minor effects on normal cells adjacent to the tumor. Although the 211At‐conjugated clone 1084 (211At‐anti‐TF mAb) was disrupted by an 211At‐induced radiochemical reaction, we demonstrated that astatinated anti‐TF mAbs eluted in 0.6% or 1.2% sodium ascorbate (SA) solution were protected from antibody denaturation, which contributed to the maintenance of cellular binding activities and cytocidal effects of this immunoconjugate. Although body weight loss was observed in mice administered a 1.2% SA solution, the loss was transient and the radioprotectant seemed to be tolerable in vivo. In a high TF–expressing gastric cancer xenograft model, 211At‐anti‐TF mAb in 1.2% SA exerted a significantly greater antitumor effect than nonprotected 211At‐anti‐TF mAb. Moreover, the antitumor activities of the protected immunoconjugate in gastric cancer xenograft models were dependent on the level of TF in cancer cells. These findings suggest the clinical availability of the radioprotectant and applicability of clone 1084 to 211At‐radioimmunotherapy.

It has been proposed that sustained ICOS expression in chronic inflammatory immune conditions, such as autoimmunity and allergy, contributes to symptom exacerbation. Therefore modulation of ICOS gene expression could be a potential therapeutic strategy for such immune diseases. However, the precise molecular mechanisms controlling ICOS gene expression remain poorly understood. In this study, we explored transcription factors involving in ICOS gene expression and examined their roles in a physiological situation. Microarray analysis revealed that one AP-1 molecule, Fos-related antigen-2 (Fra2), was highly correlated with ICOS expression. Ectopic expression of Fra2 and other AP-1 molecules upregulated ICOS expression on T cells. We identified an AP-1-responsive site (AP1-RE) within the ICOS promoter region and demonstrated AP-1 actually binds to AP1-RE upon TCR/CD28 stimulation. Meanwhile, we found several cytokines could upregulate ICOS expression on both naïve and effector T cells in a manner independent of TCR/CD28 stimulation. These cytokine stimuli induced AP-1 binding to AP1-RE. Together, our results indicate AP-1 transcription factors are involved in ICOS gene expression downstream of both TCR/CD28 signaling and cytokine receptor signaling, and suggest AP-1 activation via cytokine receptor signaling may be one of the mechanisms maintaining high level ICOS expression in chronic inflammatory immune responses. Keywords: Costimulatory molecules r Gene regulation r T cell Supporting Information available onlineCorrespondence: Prof. Ryo Abe e-mail: rabe@rs.noda.tus.ac.jp IntroductionAlthough engagement of the T-cell Ag-specific receptor by Ag/MHC products is essential for the initial stages of T-cell activation, a second signal termed a costimulatory signal is necessary for clonal expansion and functional differentiation of Agspecific T cells [1]. ICOS is a third member of CD28 family of C 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu Eur. J. Immunol. 2012. 42: 1850-1862 Molecular immunology 1851 costimulatory molecules [2]. ICOS has several unique features, which differ from CD28. For instance, ICOS is induced upon T-cell activation and has a minimal role in the induction of IL-2 production [2][3][4][5]. Until now, a large number of studies have demonstrated that ICOS serves to enhance T-helper cell differentiation and effector function, and plays a critical role in T-dependent B-cell antibody responses [4][5][6]. Furthermore, in humans, ICOS has been identified as one of the genes which when mutated is responsible for common variable immune deficiency (CVID) [7], indicating its critical role in immune responses. Meanwhile, it has been proposed that sustained ICOS signaling could exert a pathological role for certain chronic immune responses, such as autoimmunity and allergy. For example, Roquin mutant mice, that show deficiency in the degradation pathway of ICOS mRNA, express ICOS at a high level and develop an autoimmune phenotype in a manner dependent on ICOS [8]. We previously reported t...

Multimodal imaging can provide multidimensional information for understanding concealed microstructures or bioprocesses in biological objects. The combination of over–1000 nm near–infrared (OTN–NIR) fluorescence imaging and magnetic resonance (MR) imaging is...

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