Recombinant immunoreceptors with specificity for the carcinoembryonic Ag (CEA) can redirect grafted T cells to a MHC/Ag-independent antitumor response. To analyze receptor-mediated cellular activation in the context of CD28 costimulation, we generated: 1) CEA+ colorectal tumor cells that express simultaneously B7-1 and B7-2, and 2) CEA-specific immunoreceptors that harbor intracellularly the signaling moities either of CD28 (BW431/26-scFv-Fc-CD28), CD3ζ (BW431/26-scFv-Fc-CD3ζ), or FcεRIγ (BW431/26-scFv-Fc-γ). By retroviral gene transfer, we grafted activated T cells from the peripheral blood with these immunoreceptors. T cells that express the FcεRIγ or CD3ζ signaling receptor lysed specifically CEA+ tumor cells and secreted high amounts of IFN-γ upon receptor cross-linking, whereas anti-CEA-CD28 receptor-grafted T cells did not, indicating that CD28 signaling alone is not sufficient for efficient T cell activation. CD28 costimulation did not affect cytolysis by T cells equipped with γ- or ζ-signaling receptors, but enhanced both IFN-γ secretion and proliferation. CD28 costimulation, however, was required for efficient IL-2 secretion of anti-CEA-γ receptor-grafted T cells. Both purified CD4+ and CD8+ T cells grafted with immunoreceptors required CD28 costimulation for complete T cell activation. We integrated both CD28 and CD3ζ signaling domains into one combined immunoreceptor molecule (BW431/26-scFv-Fc-CD28/CD3ζ) with dual signaling properties. T cells grafted with the combined CD28/CD3ζ signaling receptor secreted high amounts of IL-2 upon Ag binding without exogenous B7/CD28 costimulation, demonstrating that both MHC-independent cellular activation and CD28 costimulation for complete T cell activation can be delivered by one recombinant receptor molecule.

Plasmacytoid dendritic cells (pDCs) represent a DC subtype that exerts divergent functions in innate and adoptive immunity including the immediate reaction to microbial factors and the induction of immunoregulatory responses. It is thought that different DC subtypes may be critically involved in the pathogenesis of multiple sclerosis (MS). In our study we assessed the phenotype, maturation and functional properties of peripheral blood pDCs from 35 clinically stable, untreated multiple sclerosis patients, 30 healthy controls and 9 patients with pneumonia, which was used as a non-specific inflammatory condition (NIC). Ex vivo expression of CD86 and 4-1BBL was significantly lower on pDCs from multiple sclerosis patients than from controls and patients with NIC (22 versus 47 versus 41% and 12 versus 35 versus 32%, respectively). When stimulated with IL-3 and CD40L, pDCs of multiple sclerosis patients showed inefficient maturation as demonstrated by significantly lower or delayed upregulation of CD86, 4-1BBL, CD40 and CD83. Additionally, in multiple sclerosis, stimulation of pDCs by unmethylated cytosine-phosphate-guanosine oligodeoxynucleotides (CpG ODN) resulted in a significantly lower interferon (IFN) alpha secretion than in controls. In multiple sclerosis, but not in controls, pDCs failed to upregulate proliferative responses and IFN-gamma secretion of autologous peripheral blood mononuclear cells (PBMC) in a co-culture system. Moreover, depletion of pDCs in multiple sclerosis patients, but not in controls, had no effect on generation of CD4+Foxp3+ regulatory T cells. We also provide data showing that glatiramer acetate (GA) treatment partially restores phenotype and function of pDCs in multiple sclerosis patients. These findings suggest functional abnormalities of pDCs in these patients, which might be of importance in the understanding of the development of immune dysregulation in this disease.

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T cells engrafted by a recombinant immunoreceptor with predefined Ag specificity can efficiently lyse Ag-positive target cells in a MHC Ag-independent manner. It is yet unresolved how receptor-grafted CD4+ T cells contribute to MHC Ag-independent target cell lysis. To address this issue, we grafted isolated CD8+ and CD4+ T cells from the peripheral blood with recombinant anti-carcinoembryonic Ag and anti-CD30 receptors, respectively. Cytotoxicity analyses revealed that grafted CD4+ T cells exert cytolysis of Ag-positive target cells with an efficiency similar to that of grafted CD8+ T cells. Lysis by receptor-grafted CD4+ T cells is Ag specific and is inhibited by blocking the target Ag or the Ag binding site of the recombinant receptor. Both Fas-sensitive and Fas-resistant target cells are lysed with equal efficiency, and lysis of Fas-sensitive target cells is not blocked by an anti-Fas ligand Ab, indicating that cytolysis by receptor-grafted CD4+ T cells is independent of the Fas pathway. We conclude that cytolysis by CD4+ T cells equipped with a recombinant immunoreceptor is MHC Ag and Fas independent and likely to be mediated by perforin present in receptor-grafted CD4+ T cells.