Role of CD2 cross‐linking in cytoplasmic calcium responses and T cell activation

Abstract

The relationship between the increase of intracellular free Ca²⁺ concentration ([Ca²⁺],) in resting T cells after stimulation with monoclonal antibody (mAb) to CD2 (E rosette receptor) and the subsequent proliferation response was investigated. Although the Combination of 9.6 plus 9‐1 mAb to CD2 was both mitogenic and induced an increase in [Ca²⁺]i, cross‐linking of an individual CD2 mAb on the cell surface induced an increase in [Ca²⁺]_i without directly stimulating T cell proliferation. The [Ca²⁺]_i response from cross‐linking an individual mAb was not epitope dependent,since 21 of 21 mAb to CD2 were effective when cross‐linked with a polyclonal goat anti‐mouse Ig second step. The kinetics of calcium mobilization was highly dependent upon the procedure for cross‐linking, since the binding of biotinconjugated 9.6 mAb followed by avidin gave a large and rapid response, whereas cross‐linking of 9.6 with an anti‐x mAb, 187.1, caused a minimal response, and the cross‐linking of 9.6 followed by a polyclonal goat anti‐mouse Ig gave an intermediate response. In addition, ligation of CD2 by rosetting with sheep red blood cells alone was sufficient to cause increased [Ca²⁺]_i. In functional studies only the procedures associated with minimal CD2 cross‐linking induced proliferation of resting T cells in combination with interleukin (IL) 2. The proliferation also required IL1 or accessory cells. Cross‐linking 9.6 on the cell surface also enhanced proliferation in the presence of phorbol myristate acetate or a CD28 mAb, 9.3, under conditions that were accessory cell independent. In contrast to the proliferation following stimulation with 9.6 plus 9−1, the combination of 9.6 plus 9‐1 F(ab′)₂ fragments lost mitogenic activity. The 9.6 plus 9‐1 F(ab′)₂ combination was similar to 9.6 cross‐linking in that either could induce responsiveness to recombinant IL2 or CD28 mAb 9.3. The combination of 9.6 plus 9‐1 F(ab′)₂ fragments was still able to increase [Ca²⁺]_i in T cells with slow kinetics. Together these results suggest that the binding of mAb to CD2 under conditions that cause a slow rather than a rapid increase in [Ca²⁺]_i is associated with T cell activation. Furthermore, they suggest that in studies of T cell activation, sheep eryth‐rocyte rosette formation should not be used to isolate T cells since rosetting may effect [ca²⁺]_i.