Modulation of the immune response using immunoglobulin fusion proteins has shown great promise for clinical immunotherapy of autoimmune diseases. Alefacept is an immunoglobulin fusion protein composed of the first extracellular domain of human LFA-3 fused to the hinge, C H 2 and C H 3 domains of human IgG 1 . Alefacept has previously been reported to inhibit T cell proliferation. Here, we analyzed the effects of alefacept on lymphocytes in vitro and characterized the role of autologous NK cells in its mechanism of action. Alefacept, but not a C H 2 binding mutant of Alefacept, inhibited CD3-induced T cell proliferation only in the presence of live NK cells, consistent with an important role for Fc + R engagement. Alefacept caused preferential depletion of CD69+ T cell subsets. Cytotoxicity assays revealed that alefacept, but not the C H 2 binding mutant, induced NK cell-mediated death of activated T cells and sorting into CD45R0 + and CD45RA + subpopulations showed that lymphocyte deletion occurred preferentially in the CD45R0 + subset. Activated CD45R0 + cells expressed higher levels of CD2 than CD45R0 -cells, providing a possible explanation for the selective targeting of this subset. Our results suggest that selective targeting of CD45R0 + T cells by NK cells represents a potential therapeutic mechanism of action of alefacept.
The molecular mechanisms mediating the inhibitory effects of a humanized CD4 mAb YHB.46 on primary human CD4+ T cells were investigated. Preincubation of T cells with soluble YHB.46 caused a general inhibition of TCR-stimulated protein tyrosine phosphorylation events, including a reduction in phosphorylation of p95vav, linker for activation of T cells, and Src homology 2 domain-containing leukocyte protein of 76-kDa signaling molecules. A marked reduction in activation of the Ras/mitogen-activated protein kinase pathway was also observed. Examination of the earliest initiation events of TCR signal transduction showed that YHB.46 inhibited TCR-ζ chain phosphorylation together with recruitment and tyrosine phosphorylation of the ζ-associated protein of 70-kDa tyrosine kinase, particularly at Tyr319, as well as reduced recruitment of p56lck to the TCR-ζ and ζ-associated protein of 70-kDa complex. These inhibitory events were associated with inhibition of TCR endocytosis. Our results show that the YHB.46 mAb is a powerful inhibitor of the early initiating events of TCR signal transduction.
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