MEM83 is an inserted domain (I-domain)-specific antibody that up-regulates the interaction of LFA-1 with ICAM-1 through an outside-in activation mechanism. We demonstrate here that there is no change in the affinity of the MEM83 antibody for the I-domain in either its low (wild-type) or high affinity form and that MEM83 does not enhance the binding of the wild-type I-domain to ICAM-1. Furthermore, we show that the antibody acts as an activating agent to induce LFA-1/ICAM-1-dependent homotypic cell aggregation only as an IgG, but not as a Fab fragment. On the basis of these data, we propose an avidity-based mechanism that requires no direct activation of the LFA-1 I-domain by the binding of the antibody; rather, activation is enhanced when there is an interaction with both arms of the IgG. A molecular model of the antibody interaction with LFA-1 illustrates the symmetry and accessibility of the two MEM83 epitopes across the LFA-1/ICAM-1 heterotetramer. We hypothesize that MEM83 stabilizes adjacent LFA-1 molecules in their active form by the free energy that is gained from the binding of the I-domains to each arm of the IgG. This leads to stabilization of the open state of the integrin and outside-in signaling. Our model supports a mechanism in which both affinity and avidity regulation are required in the activation of LFA-1.The interaction of leukocyte function-associated antigen-1 (LFA-1) 4 and intercellular adhesion molecules (ICAMs) plays a key role in the immune system and in the development of arteriosclerosis, autoimmune diseases, and inflammation (1-5). Further understanding of the LFA-1 activation mechanism is necessary for the development of novel therapeutics that could modulate the immune system (6, 7). Inhibitors that stabilize the inactive form or block the active form of LFA-1 suppress immune responses and may prevent inflammatory and autoimmune disease (8 -10). Similarly, up-regulation of LFA-1 can be used to enhance immune responses (11-13) and may have roles in restoring T cell function in human immunodeficiency virusimmunocompromised individuals (14) or in overcoming tumor-specific toleration (15).Activation of the integrin LFA-1 results in a rapid and reversible increase in affinity for its ligands, . ICAM-1-3 regulate T cell activation (13) and leukocyte homing (20, 21); ICAM-4 is a red blood cell-specific ligand that can interact with a several 2/CD18 integrins (22); and ICAM-5 plays a role in leukocyte recruitment to neurons in the central nervous system (19). In T cells, LFA-1 and ICAM-1 act as costimulatory molecules to the peptide/major histocompatibility complex (23,24). The role of LFA-1 and ICAM-1 interaction is critical to the formation of the immunological synapse, and aberrant activation or expression of LFA-1 may lead to selectively disabled T cell receptor clustering (25) or the development of autoimmune diseases (26,27).Recognition and binding of its ICAM-1 ligand by LFA-1 are mediated through the inserted domain (I-domain) in its ␣-subunit; this forms a heterotetrameric comple...