Regulation of 4-1BB expression by cell-cell interactions and the cytokines,interleukin-2 and interleukin-4

4-1BB expression increased gradually following T cell activation, and by day 3 post-stimulation with immobilized anti-CD3 (anti-CD3i) or concanavalin A (Con A), splenic T cells were routinely 35–45% 4-1BB⁺ by flow cytometric analysis. 4-1BB was expressed on activated CD8⁺, CD4⁺, CD28⁺ and CD45RB⁺ T cells. Optimal 4-1BB expression was seen by day 6 post-stimulation and was cell density dependent. When T cells were cultured for 6 days at 1 × 10⁶/well in a 24-well plate with anti-CD3i, 82% of the cells were 4-1BB⁺. In contrast, at lower cell densities (4 × 10⁵, 2 × 10⁵ and 1 × 10⁵), optimal 4-1BB expression was observed only if the cultures were supplemented with recombinant interleukin-2 (IL-2) or recombinant IL-4 (IL-4). In agreement, with these results, modes of inducing endogenous IL-2 production such as cross-linking the costimulatory molecule, CD28, or the addition of syngeneic accessory cells to T cells activated with anti-CD3i, resulted in high levels of 4-1BB expression. The addition of interleukin-1α(IL-1α) or interferon-γ (IFN-γ) did not increase 4-1BB expression on anti-CD3i-activated T cells. In addition, if T cells were incubated with IL-2, IL-4, IL-1α, IFN-γ or anti-CD28 alone, no 4-1BB expression was induced. T cells activated with soluble anti-CD3 (anti-CD3s) in the presence of IL-2, IL-4, or accessory cells, did not express higher levels of 4-1BB on their cell surface. These data suggest that initial events crucial for efficient T cell activation, such as signals delivered through the T cell receptor/CD3 complex and the CD28 molecule, are instrumental in regulating subsequent 4-1BB expression.