4-1BB T-CELL ANTIGEN BINDS TO MATURE B-CELLS AND MACROPHAGES, AND COSTIMULATES ANTI-MU-PRIMED SPLENIC B-CELLS

4-1BB is expressed on activated murine T cells and may function as an accessory signaling molecule during T-cell activation. To identify putative 4-1BB ligands, a fusion protein consisting of the extracellular domain of 4-1BB fused to human placental alkaline phosphatase (4-1BB-AP) was constructed. Alkaline phosphatase activity could then be used as an indicator of the relative amount of bound 4-1BB. These studies indicated that 4-1BB-AP specifically bound to the surface of various mature B and macrophage cell lines. 4-1BB-AP bound at low levels to T cell lines (non-activated and anti-CD3-activated), pre-B-cell lines, and an immature macrophage cell line. 4-1BB-AP did not bind to a glial tumor cell line, HeLa cells, or COS cells. In addition, 4-1BB-AP bound at higher levels to F(ab')(2) anti-mu-activated primary B cells compared to anti-CD3-activated primary T cells. Scatchard analysis indicated that the A20 B cell lymphoma expressed 3680 binding sites per cell with a K-d of 1.86 nM. Affinity cross-linking studies demonstrated that a major cell surface species of 120 kDa bound to 4-1BB-AP; 4-1BB-AP also bound to a minor species of approximately 60 kDa.The addition of paraformaldehyde-fixed SF21 cells expressing recombinant 4-1BB synergized with F(ab')(2) anti-mu in inducing splenic B cell proliferation suggesting that 4-1BB may function as a regulator of B cell growth.