Targeting of alpha4beta1 Integrin and CD4/CD8 on Human T Cells, in Addition to CD3 and CD28, Induces Maximal Activation, as Assessed by Single-Cell Analysis of MAP Kinase Pathway Activation and Cytokine Production.

Optimal T cell activation requires the formation of immunological synapses between T cells and antigen-presenting cells (APC). Immunological synapse formation involves the binding of ligands on APC to co-stimulatory receptors on T cells, resulting in maximal activation. Most prior in vitro studies examining the process of T cell activation have utilized immobilized mAbs or ligands to assess the roles of co-stimulatory molecules in T cell activation. Micro-domain formation in such models may be inflexible, potentially yielding misleading results about the relative importance of individual costimulatory pathways. To more physiologically mimic immunologic synapse formation, we stimulated T cells with soluble primary mAbs that were then cross-linked by secondary antibodies. Specifically, we induced cross-linking of surface proteins on purified human T cells using a combination of mAbs recognizing four potential co-stimulatory receptors (CD3, CD4 or CD8, CD28 and VLA4/a4b1 integrin), and assessed phospho-Erk1/2 and cytokine production at a single cell level using flow cytometry. Consistent with the predictions of the Bretscher-Cohn two-signal model, CD28 induced a modest increase in Erk1/2 phosphorylation (in CD4) and IL-2 production (in CD4+ and CD8+ T cells) and IFNg (in CD8+ T cells), while low dose (0.1mcg/ml) anti-CD3 mAb alone failed to induce significant activation (P additional co-stimulations through a4b1/VLA4 and CD4 or CD8 may be necessary to induce optimal T cell signaling, targeting of a4b1 and CD4/CD8 by specific agonists or antagonists may have therapeutic benefit in immunomodulation (e.g., augmentation of vaccine responses and/or abrogation of pathogenic T cell activation).