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The combination of anti-CD2 mAb 9.6 and 9-1, specific for distinct epitopes, induces proliferation of resting human T cells. The mitogenic activity of this mAb mixture depends upon accessory cells and the 9-1 mAb Fc domain. To further study the functional properties of these mAb, their variable regions were cloned and expressed as monospecific single-chain Fv (scFv) proteins fused to the human IgG1 Fc domain (scFvlg). A novel bispecific scFvlg was constructed by cloning the two monospecific scFv binding sites in tandem, with the 9.6 scFv placed N-terminal to the 9-1 scFvlg. Monospecific scFvlg binding to CD2 was comparable to that of the corresponding parental mAb, while the bispecific scFvlg exhibited binding activity similar to that of the 9-1 scFvlg. The combination of 9.6 scFvlg and 9-1 mAb was mitogenic, whereas mixtures including the 9-1 scFvlg were non-stimulatory, confirming the unique properties of the 9-1 IgG3 Fc. Without the IgG3 tail, the bispecific 9.6/9-1 scFvlg was directly mitogenic and was a more potent mitogen than the mAb mixture but was accessory cell dependent. Unlike the combination of mAb, the bispecific reagent did not directly mobilize calcium in T cells. In comparison to the mAb mixture, bispecific 9.6-9-1 scFvlg-mediated stimulation of a mixed lymphocyte reaction was significantly more resistant to inhibition of the CD28 co-stimulatory pathway by the inhibitor CTLA-4-Ig. These results show that expression of the 9.6 and 9-1 binding sites together on a bispecific scFvlg increased the mitogenic properties of the mAb and altered the degree of accessory cell signals required for T cell activation. [ABSTRACT FROM PUBLISHER]

We report a physical and functional association between the Tec-family tyrosine kinase Itk (Emt/Tsk) and the nuclear import chaperone karyopherin alpha (Rch1alpha) in human T cells. The Itk-SH3 domain and the Rch1alpha proline-rich (PR) motif were crucial for the Itk/Rch1alpha constitutive interaction as demonstrated by directed mutagenesis of the Rch1alpha PR motif (proline 242 to alanine, P242A). TCR-CD3 stimulation of Jurkat T cells resulted in increased Itk/Rch1alpha complex formation, recruitment of karyopherin beta to the protein complex and Rch1alpha tyrosine phosphorylation. Analysis of in vitro kinase reactions with a panel of recombinant glutathione-S-transferase (GST) fusion tyrosine kinases (Itk, Lck, ZAP-70 and Jak3) revealed that only GST-Itk efficiently phosphorylated a recombinant GST-Rch1alpha fusion. We observed constitutive nuclear localization of Itk that was up-regulated following either TCR-CD3 stimulation or over-expression of wild-type Rch1alpha in T cells. Further, nuclear localization of Itk and TCR-CD3-mediated IL-2 production were significantly down-regulated following expression of the Rch1alpha-P242A mutant, implicating a role for Rch1alpha in the nuclear translocation of Itk.