No abstract

Abstract. The leukocyte function-associated molecule-1 (LFA-1) plays a key role in cell adhesion processes between cells of the immune system. We investigated the mechanism that may regulate LFA-1-1igand interactions, which result in cell-cell adhesion. To this end we employed an intriguing anti-LFA-1 ot mAb (NKI-L16), capable of inducing rather than inhibiting cell adhesion. Aggregation induced by NKI-L16 or Fab fragments thereof is not the result of signals transmitted through LFA-1. The antibody was found to recognize a unique Ca2+-dependent activation epitope of LFA-1, which is essentially absent on resting lymphocytes, but becomes induced upon in vitro culture. Expression of this epitope correlates well with the capacity of cells to rapidly aggregate upon stimulation by PMA or through the TCR/CD3 complex, indicating that expression of the NKI-L16 epitope is essential for LFA-1 to mediate adhesion. However, expression of the NKI-L16 epitope in itself is not sufficient for cell binding since cloned T lymphocytes express the NKI-L16 epitope constitutively at high levels, but do not aggregate spontaneously. Based on these observations we propose the existence of three distinct forms of LFA-I: (a) an inactive form, which does not, or only partially exposes the NKI-L16 epitope, found on resting cells; (b) an intermediate, NKI-L16 ÷ form, expressed by mature or previously activated cells; and (c) an active (NKI-L16 ÷) form of LFA-1, capable of high affinity ligand binding, obtained after specific triggering of a lymphocyte through the TCR/CD3 complex, by PMA, or by binding of NKI-L16 antibodies.

Abstract. Transition of leukocyte function-associated antigen-1 (LFA-1), from an inactive into an activate state depends on the presence of extracellular Mg 2+ and/or Ca 2+ ions. Although Mg 2+ is directly involved in ligand binding, the role of Ca 2+ in LFA-1 mediated adhesion remained obscure. We now demonstrate that binding of Ca 2+, but not Mg 2+, directly correlates with clustering of LFA-1 molecules at the cell surface of T cells, thereby facilitating LFA-l-ligand interaction. Using a reporter antibody (NKI-L16) that recognizes a Ca2+-dependent epitope on LFA-1, we found that Ca 2+ can be bound by LFA-1 with different strength. We noticed that weak binding of Ca 2+ is associated with a dispersed LFA-1 surface distribution on T cells and with non-responsiveness of these cells to stimuli known to activate LFA-1. In contrast, stable binding of Ca 2÷ by LFA-1 correlates with a patch-like surface distribution and vivid ligand binding after activation of LFA-1. Mg:+-dependent ligand binding does not affect binding of Ca 2+ by LFA-1 as measured by NKI-L16 expression, suggesting that Mg 2+ binds to a distinct site, and that both cations are important to mediate adhesion. Only Sr 2+ ions can replace Ca 2+ to express the L16 epitope, and to induce clustering of LFA-1 at the cell surface.We conclude that Ca 2+ is involved in avidity regulation of LFA-1 by clustering of LFA-1 molecules at the cell surface, whereas Mg 2+ is important in regulation of the affinity of LFA-1 for its ligands.

These data suggest that the presence of TAA-specific TILs forms a prognostic factor, predicting improved survival in advanced-stage melanoma patients. The absence of TAA-specific T cells in the circulation suggests that homing of the tumor-specific T cell population to the tumor site contributes to the effectiveness of antitumor immunity.