Although it is well-known that the ICOS-ICOS ligand (ICOSL) costimulatory pathway is important for many immune responses, recent accumulated evidence suggests that dysregulation of this pathway may lead to and/or exaggerate autoimmune responses. ICOS is induced on the cell surface after T cell activation. Similarly, ICOSL is up-regulated on APCs by several mitogenic stimuli. However, the mechanism regulating expression of the ICOS-ICOSL pair, and the significance of controlling their expression for an appropriate immune response, is largely unknown. To gain a better understanding of the importance of fine control of the ICOS-ICOSL costimulatory pathway, we generated ICOS-transgenic (Tg) mice that have high constitutive expression of ICOS in all T cells. Using ICOS-Tg mice, we studied whether in vivo immune responses were affected. Unexpectedly, we first found that ICOS-Tg mice exhibited a phenotype resembling ICOS-deficient mice in their Ag-specific Ab response, such as a defect in class switch recombination. Further examination revealed that ICOSL expression of APCs was significantly suppressed in ICOS-Tg mice. Interestingly, suppression of ICOSL was induced by interaction of ICOSL with ICOS, and it seemed to be regulated at the posttranscriptional level. The suppressive effect of the ICOS-ICOSL interaction overcame the positive effect of CD40 or B cell activation factor of the TNF family (BAFF) stimulation on ICOSL expression. Together, our studies demonstrate a novel mechanism for the regulation of ICOSL expression in vivo and suggest that the ICOS costimulatory pathway is subject to negative feedback regulation by ICOSL down-regulation in response to ICOS expression.

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.

CD28 stimulation contributes to activation of the IL-2 promoter by up-regulating the activity of several transcription factors, including nuclear factor kappaB (NF-kappaB)/Rel family members. However, the signal-transducing cascades linking the CD28 molecule and activation of NF-kappaB remain unclear. Protein kinase C (PKC) , CARMA1 and Bcl10 have recently been reported to integrate TCR-mediated NF-kappaB activation. However, since the data in these studies were drawn from experiments in which T cells were usually stimulated with both TCR and CD28, the relative contributions of TCR- and CD28-mediated signals to initiation of the NF-kappaB pathway remain elusive. To examine the role of these molecules in NF-kappaB activation through CD28-mediated stimulation, Bcl10 was over-expressed in Jurkat cells and their NF-kappaB activation by CD28- or TCR-cross-linking was evaluated. We found that CD28 stimulation alone can induce NF-kappaB activation in Bcl10-over-expressing Jurkat cells, whereas TCR stimulation alone has only little effect. In addition, we found that Bcl10-induced NF-kappaB activation through CD28-mediated stimulation could be blocked by the dominant-negative form of PKC or CARMA1. Furthermore, genetic studies revealed that Grb2/Gads binding, but not phosphatidylinositol 3-kinase binding, is important in CD28-mediated NF-kappaB activation. These findings indicate that the PKC-CARMA1-Bcl10 signaling pathway participates in the CD28 co-stimulatory signal independently of the TCR-signaling pathway, which leads us to propose that the activation of the NF-kappaB-signaling pathway via PKC-CARMA1-Bcl10 may be markedly dependent on CD28 stimulation rather than TCR stimulation.

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Gene downregulation by antisense morpholino oligonucleotides (MOs) is achieved by either hybridization around the translation initiation codon or by targeting the splice donor site. In the present study, an antisense MO method is introduced that uses a 25-mer MO against a region at least 40-nt upstream from a poly(A) tail junction in the 3′-untranslated region (UTR) of maternal mRNA. The MO removed the poly(A) tail and blocked zebrafish cdk9 (zcdk9) mRNA translation, showing functional mimicry between miRNA and MO. A PCR-based assay revealed MO-mediated specific poly(A) tail removal of zebrafish mRNAs, including those for cyclin B1, cyclin B2 and tbp. The MO activity targeting cyclins A and B mRNAs was validated in unfertilized starfish oocytes and eggs. The MO removed the elongated poly(A) tail from maternal matured mRNA. This antisense method introduces a new application for the targeted downregulation of maternal mRNAs in animal oocytes, eggs and early embryos.