Abstract
Background: IL-7 is constantly available, most mature T cells express the IL-7R complex, and IL-7 signaling is required for mature T cell survival, thus implicating IL-7 as a trophic cytokine. However, since IL-7Rα (CD127) expression on T cells is dynamically regulated in response to activation and IL-7 exposure, increased IL-7 levels present during lymphopenia augment homeostatic expansion, and IL-7 therapy induces dramatic alterations in T cell homeostasis, it can be inferred that IL-7’s effects on T cells critically depends on concentration. We postulated therefore, that dose response effects may be central to IL-7’s capacity to modulate T cell homeostasis.
Methods: We evaluated dose response effects of IL-7 on naïve vs. memory CD4+ and CD8+ mature human T cells in vitro using five distinct biologic effects of IL-7 as endpoints: Stat5a phosphorylation, co-stimulation of anti-CD3 mediated proliferation, Bcl-2 up-regulation, CXCR4 up-regulation, and IL-7Rα down-regulation. Using CD45RO based immunomagnetic bead separation (Miltenyi), fresh human peripheral blood T cells were separated into naïve (CD45RO−) vs. memory (CD45RO+) subsets, then cultured for 5 days with increasing concentrations of IL-7 (0.1ng/ml – 10ng/ml). On day 5, cells were analyzed by flow cytometry for the endpoints noted. Intracellular pathways implicated in IL-7 signaling on T cells were probed using PI3K (LY294002) and mTOR (Rapamycin) inhibitors.
Results: The biologic effects of IL-7 on mature T cells can be grouped into two categories. The first category consists of Stat5a phosphorylation and co-stimulation for proliferation. These effects occur at very low doses (0.1ng/ml) with gradually increasing percentages of cells responding with increasing doses. These responses appear to reflect receptor occupancy by the IL-7 molecule since subsets with higher IL-7Rα receptor expression show proliferative effects at lower IL-7 doses. Further, the proliferative effects of IL-7 are fully inhibited by either LY294002 (10μM) or Rapamycin (10ng/ml). In contrast to IL-7’s low dose effects, Bcl-2 and CXCR4 up-regulation, and IL-7Rα down-regulation can be grouped into a second category of effects that occur only in response to high dose IL-7 (10ng/ml). High dose effects occur in an “all or nothing” pattern with T cell subsets bearing low levels of IL-7Rα expression demonstrating the same dose response as subsets with high IL7Rα expression. Furthermore, high dose effects of IL-7 utilize differential signaling pathways compared to the low dose effects, as they are not inhibited by either LY294002 or Rapamycin.
Conclusions: We have identified two categories of IL-7 effects on mature T cells. Low dose effects, which are primarily involved in co-stimulation for proliferation and PI3K/mTOR dependent, and are likely to be highly modulated by receptor regulation and small changes in IL-7 availability. Then in contrast, high dose effects including Bcl-2, CXCR4 and IL-7Rα modulation, which utilize separate signaling pathways as they are not PI3K/mTOR dependent. Whether high dose effects of IL-7 reflect signaling through a separate, low affinity IL-7R is currently under investigation. These results demonstrate previously unrecognized distinctions in IL-7 signaling pathways, and may help to explain why substantial alterations in T cell homeostasis occur when IL-7 is elevated during lymphopenia despite IL-7’s constant availability in a lymphoreplete environment.
A model for low dose effects of low-LET radiation delivered at high dose rates