Mechanism of synergistic proliferation in response to IL-2/15 and c-kit ligand involves both structural and functional cooperation: Insights into lymphohematopoietic stem cell expansion

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Lymphohematopoietic stem cells show synergy in growth when exposed to IL-2 (or IL-15) and c-kit ligand (KL), yet the molecular mechanisms whereby proliferative synergy occurs between IL-2 and KL are unknown. As compared to unstimulated cells in serum free media by MTS proliferation assay, KL (100ng/mL) stimulation alone for 72h yields 160% growth ( 8%), IL-2 (150IU/mL) stimulation yields 300% growth ( 27%) and the combination leads to 382% increased growth (26%). All pairwise comparisons are significantly different from one another (KL vs IL-2, p=0.001, KL vs IL-2 and KL, p=0.0001, and IL-2 vs IL-2 and KL, p=0.02.) Structural and functional mechanisms contribute to this effect. Structurally, the c-kit receptor physically associates with the beta and gamma chains of the IL-2/15 receptor on the cell membrane. Functionally, costimulation with IL-2 and KL upregulates MAPK signaling intermediaries to a greater degree than either cytokine alone, a phenomenon not observed in the PI3K pathway or in Jak/STAT pathways. Interestingly, MAPK inhibition with the MEK1/2 inhibitor UO126 (50uM) led to a 48% decrease in KL-dependent proliferation (p=0.003), an 89% decrease in proliferation in response to IL-2 (p=0.03), but no significant change in cells proliferating in response to IL-2 and KL, suggesting that alternative signaling systems are uniquely activated by IL-2 and KL in combination. AKT inhibition with the PI3K (10uM) inhibitor LY249002 dramatically suppressed growth in all conditions: 150% reduction in KL mediated growth, 163% reduction in IL-2 mediated growth, and 237% reduction in growth in response to IL-2 and KL together (all p values < 0.001). Finally, we show that costimulation with IL-2 and KL uniquely modulates control of cell cycle entry over either cytokine alone. After 24 h, 11% of cells stimulated with KL are in S-phase, 28% of cells stimulated with IL-2 alone are in S-phase, compared to 41% of cells stimulated with IL-2 and KL (p=0.01). Cyclins D2 and D3 are largely upregulated by the combination, likely through enhanced STAT5 binding to the D cyclin promoter. Furthermore, p27 is more rapidly lost when cells are stimulated with the combination of IL-2 and KL rather than either cytokine alone. Taken together, these findings provide early evidence that the proliferative synergy observed from the combination of IL-2/15 plus KL are the result of unique structural and functional molecular cooperation that results in lymphohematopoietic stem cell expansion.