Abstract The two-signal model of T cell activation has been focused on what costimulatory signals are necessary to facilitate and enhance T cell response to a specific antigen. However, it is also intriguing to study the ability for costimulatory interactions to induce signaling in the absence of specific antigen recognition. We examine the signaling of CD4+ T cells engaging high densities of CD80 molecules in the presence of ICAM-1 in an antigen-independent T cell response. Examination of freshly isolated naive mouse T cells interacting with CD80 and ICAM-1 reveals a novel role for CD28:CD80 interactions in antigen independent ring junction formation in which CD80:CD28 interactions were located in the central region. The unengaged T-cell receptor is also localized to this central region. These cells exhibit increased cytoplasmic Ca++ compared to cells interacting with ICAM-1 alone. With as many cells fluxing Calcium as seen in antigen dependent stimulation. Mouse T cells interacting with ICAM-1 and CD80 also show an increased level of phosphorylated tyrosine residues. Although there is signaling in an antigen independent T cell response, it is not sufficient for inducing IL-2 secretion. The higher expression levels of CD80 achieved in the bilayer distinguish these results from earlier studies (Bromley et al, 2001; Markiewicz et al 2005) showing no interaction of mouse CD28 and CD80 in the absence of antigen. Thus, CD28, as well as NKG2D (Somersalo et al, 2004; Markiewicz, 2005), can stimulation formation of antigen independent ring junctions. These experiments also demonstrate that CD4+ T cells can be induced to form antigen independent adhesion rings under conditions of high CD80 density. The function of these structures is under investigation. NIH AI44931