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<0.05). However, CD28 stimulation consistently augmented activation in both CD4+ and CD8+ T cells in the presence of anti-CD4 and anti-CD8 mAbs (P<0.05). More interestingly, stimulation by CD4 or CD8 mAbs induced an additive effect on Erk1/2 phosphorylation and IL-2 and IFNg production regardless of CD28 stimulation (P<0.05). In addition, stimulation via a4b1/VLA4 increased the level of T cell activation in the presence of three distinct signals in both CD4+ T cells and also in CD8+ T cells (P<0.05). We then examined the dependence of T cell activation on all four types of stimuli (i.e., via CD3, CD28, CD4 or CD8, and VLA4). Following stimulation with low-dose anti-CD3 mAb (0.1mcg/ml), the extent of Erk1/2 phosphorylation and IL-2/IFNg production was found to be dependent on the numbers of stimulatory signals received through all four pathways in a statistically meaningful manner (see Figure). In conclusion, optimal T cell activation requires not only CD3 and CD28, but also signaling via CD4/CD8 and VLA4, supporting a “four-signal” model of T cell activation. These data suggest that

1. additional co-stimulations through a4b1/VLA4 and CD4 or CD8 may be necessary to induce optimal T cell signaling,

2. 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).

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