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

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Abstract
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.