LFA-1 Affinity Regulation Is Necessary for the Activation and Proliferation of Naive T Cells

The activation of LFA-1 (lymphocyte function-associated antigen) is a critical event for T cell co-stimulation. The mechanism of LFA-1 activation involves both affinity and avidity regulation, but the role of each in T cell activation remains unclear. We have identified antibodies that recognize and block different affinity states of the mouse LFA-1 I-domain. Monoclonal antibody 2D7 preferentially binds to the low affinity conformation, and this specific binding is abolished when LFA-1 is locked in the high affinity conformation. In contrast, M17/4 can bind both the locked high and low affinity forms of LFA-1. Although both 2D7 and M17/4 are blocking antibodies, 2D7 is significantly less potent than M17/4 in blocking LFA-1-mediated adhesion; thus, blocking high affinity LFA-1 is critical for preventing LFA-1-mediated adhesion. Using these reagents, we investigated whether LFA-1 affinity regulation affects T cell activation. We found that blocking high affinity LFA-1 prevents interleukin-2 production and T cell proliferation, demonstrated by TCR cross-linking and antigen-specific stimulation. Furthermore, there is a differential requirement of high affinity LFA-1 in the activation of CD4⁺ and CD8⁺ T cells. Although CD4⁺ T cell activation depends on both high and low affinity LFA-1, only high affinity LFA-1 provides co-stimulation for CD8⁺ T cell activation. Together, our data demonstrated that the I-domain of LFA-1 changes to the high affinity state in primary T cells, and high affinity LFA-1 is critical for facilitating T cell activation. This implicates LFA-1 activation as a novel regulatory mechanism for the modulation of T cell activation and proliferation.LFA-1 (lymphocyte function-associated antigen), an integrin family member, is important in regulating leukocyte adhesion and T cell activation (1, 2). LFA-1 consists of the α_L (CD11a) and β₂ (CD18) heterodimer. The ligands for LFA-1, including intercellular adhesion molecule ICAM³-1, ICAM-2, and ICAM-3, are expressed on antigen-presenting cells (APCs), endothelial cells, and lymphocytes (1). Mice that are deficient in LFA-1 have defects in leukocyte adhesion, lymphocyte proliferation, and tumor rejection (3–5). Blocking LFA-1 with antibodies can prevent inflammation, autoimmunity, organ graft rejection, and graft versus host disease in human and murine models (6–10).LFA-1 is constitutively expressed on the surface of leukocytes in an inactive state. Activation of LFA-1 is mediated by inside-out signals from the cytoplasm (1, 11). Subsequently, activated LFA-1 binds to the ligands and transduces outside-in signals back into the cytoplasm that result in cell adhesion and activation (12, 13). The activation of LFA-1 is a critical event in the formation of the immunological synapse, which is important for T cell activation (2, 14, 15). The active state of LFA-1 is regulated by chemokines and the T cell receptor (TCR) through Rap1 signaling (16). LFA-1 ligation lowers the activation threshold and affects polarization in CD4⁺ T cells (17). Moreover, productive LFA-1 engagement facilitates efficient activation of cytotoxic T lymphocytes and initiates a distinct signal essential for the effector function (18–20). Thus, LFA-1 activation is essential for the optimal activation of T cells.The mechanism of LFA-1 activation involves both affinity (conformational changes within the molecule) and avidity (receptor clustering) regulation (21–23). The I-domain of the LFA-1 α_L subunit is the primary ligand-binding site and has been proposed to change conformation, leading to an increased affinity for ligands (24–26). The structural basis of the conformational changes in the I-domain of LFA-1 has been extensively characterized (27). Previously, we have demonstrated that the conformation of the LFA-1 I-domain changes from the low affinity to the high affinity state upon activation. By introducing disulfide bonds into the I-domain, LFA-1 can be locked in either the closed or open conformation, which represents the “low affinity” or “high affinity” state, respectively (28, 29). In addition, we identified antibodies that are sensitive to the affinity changes in the I-domain of human LFA-1 and showed that the activation-dependent epitopes are exposed upon activation (30). This study supports the presence of the high affinity conformation upon LFA-1 activation in cell lines. It has been demonstrated recently that therapeutic antagonists, such as statins, inhibit LFA-1 activation and immune responses by locking LFA-1 in the low affinity state (31–34). Furthermore, high affinity LFA-1 has been shown to be important for mediating the adhesion of human T cells (35, 36). Thus, the affinity regulation is a critical step in LFA-1 activation.LFA-1 is a molecule of great importance in the immune system, and its activation state influences the outcome of T cell activation. Our previous data using the activating LFA-1 I-domain-specific antibody MEM83 indicate that avidity and affinity of the integrin can be coupled during activation (37). However, whether affinity or avidity regulation of LFA-1 contributes to T cell activation remains controversial (23, 38, 39). Despite the recent progress suggesting that conformational changes represent a key step in the activation of LFA-1, there are considerable gaps to be filled. When LFA-1 is activated, the subsequent outside-in signaling contributes to T cell activation via immunological synapse and LFA-1-dependent signaling. It is critical to determine whether high affinity LFA-1 participates in the outside-in signaling and affects the cellular activation of T cells. Nevertheless, the rapid and dynamic process of LFA-1 activation has hampered further understanding of the role of high affinity LFA-1 in primary T cell activation. The affinity of LFA-1 for ICAM-1 increases up to 10,000-fold within seconds and involves multiple reversible steps (23). In addition, the activation of LFA-1 regulates both adhesion and activation of T cells, two separate yet closely associated cellular functions. When LFA-1 is constitutively expressed in the active state in mice, immune responses are broadly impaired rather than hyperactivated, suggesting the complexity of affinity regulation (40). Therefore, it is difficult to dissect the mechanisms by which high affinity LFA-1 regulates stepwise activation of T cells in the whole animal system.In the present study, we identified antibodies recognizing and blocking different affinity states of mouse LFA-1. These reagents allowed us to determine the role of affinity regulation in T cell activation. We found that blocking high affinity LFA-1 inhibited IL-2 production and proliferation in T cells. Furthermore, there is a differential requirement of high affinity LFA-1 in antigen-specific activation of CD4⁺ and CD8⁺ T cells. The activation of CD4⁺ T cells depends on both high and low affinity LFA-1. For CD8⁺ T cell activation, only high affinity LFA-1 provides co-stimulation. Thus, affinity regulation of LFA-1 is critical for the activation and proliferation of naive T cells.