Figure 1.
Th17 differentiation in vitro requires PIKFYVE activity. Primary murine CD4+ T cells isolated by negative selection were activated under Treg, Th17, Th1, and Th2 polarization conditions for 10 min. (A) Immunoblotting was performed for p-PIKFYVE(S307) and total PIKFYVE. (B) Densitometry was performed on immunoblots from panel A. p-PIKFYVE(S307) was normalized to total PIKFYVE levels. Data are shown as mean ± SEM; N = 3 biological replicates. (C) Imaging flow cytometry was utilized to measure the relative abundance of PtdIns(3,5)P2 generated in response to Treg, Th17, Th1, and Th2 polarization conditions 10 min after activation. Data are shown as mean ± SEM; N = 4 biological replicates. (D) Primary murine CD4+ T cells isolated by negative selection were activated under Th17 conditions in the presence of iAKT, apilimod, rapamycin, or vehicle control for 10 min. (E) Densitometry was performed on immunoblots from panel D. p-PIKFYVE(S307) was normalized to total PIKFYVE levels. Data are shown as mean ± SEM; N = 3 biological replicates. (F) Primary naive murine CD4+ T cells isolated by negative selection (CD4+CD25−CD44low) were polarized for 4 days under Th17 conditions with vehicle control or apilimod. Flow cytometry was performed on gated CD4+ T cells to measure IL-17 and FoxP3. Data are shown as mean ± SEM; N = 3 biological replicates. Primary murine CD4+ T cells were isolated by negative selection and cultured under Th17 conditions with vehicle or apilimod for 5 days. Multidimensional flow cytometry was utilized to monitor FoxP3, IL-17a, GITR, CD39, CTLA-4, PD-1, and LAG-3 expression on CD4+ T cells. (G and H) (G) A tSNE analysis on the multidimensional flow cytometry data and (H) clustering analysis resolved subgroups in that developed in the cultures. Data shown are representative examples from three biological replicates. Naive CD4+ T cells were isolated by negative selection from FoxP3 GFP reporter mice. Cells were polarized under Treg, Th17, and Th17+apilimod conditions for 4 days, and FoxP3-expressing cells were isolated from these cultures by FACS. The isolated FoxP3 cells were cocultured with naive cells polarized under Th17 conditions for 4 days. (I–K) Cell proliferation was measured by flow cytometry and (K) the generation of IL-17 was measured. Data shown as mean ± SEM; N = 3 biological replicates. P values were calculated with one-way ANOVA followed by the Tukey test for panels B, C, E, and K. Student’s t test was used to calculate P values in panel F. Source data are available for this figure: SourceData F1.

Th17 differentiation in vitro requires PIKFYVE activity. Primary murine CD4+ T cells isolated by negative selection were activated under Treg, Th17, Th1, and Th2 polarization conditions for 10 min. (A) Immunoblotting was performed for p-PIKFYVE(S307) and total PIKFYVE. (B) Densitometry was performed on immunoblots from panel A. p-PIKFYVE(S307) was normalized to total PIKFYVE levels. Data are shown as mean ± SEM; N = 3 biological replicates. (C) Imaging flow cytometry was utilized to measure the relative abundance of PtdIns(3,5)P2 generated in response to Treg, Th17, Th1, and Th2 polarization conditions 10 min after activation. Data are shown as mean ± SEM; N = 4 biological replicates. (D) Primary murine CD4+ T cells isolated by negative selection were activated under Th17 conditions in the presence of iAKT, apilimod, rapamycin, or vehicle control for 10 min. (E) Densitometry was performed on immunoblots from panel D. p-PIKFYVE(S307) was normalized to total PIKFYVE levels. Data are shown as mean ± SEM; N = 3 biological replicates. (F) Primary naive murine CD4+ T cells isolated by negative selection (CD4+CD25−CD44low) were polarized for 4 days under Th17 conditions with vehicle control or apilimod. Flow cytometry was performed on gated CD4+ T cells to measure IL-17 and FoxP3. Data are shown as mean ± SEM; N = 3 biological replicates. Primary murine CD4+ T cells were isolated by negative selection and cultured under Th17 conditions with vehicle or apilimod for 5 days. Multidimensional flow cytometry was utilized to monitor FoxP3, IL-17a, GITR, CD39, CTLA-4, PD-1, and LAG-3 expression on CD4+ T cells. (G and H) (G) A tSNE analysis on the multidimensional flow cytometry data and (H) clustering analysis resolved subgroups in that developed in the cultures. Data shown are representative examples from three biological replicates. Naive CD4+ T cells were isolated by negative selection from FoxP3 GFP reporter mice. Cells were polarized under Treg, Th17, and Th17+apilimod conditions for 4 days, and FoxP3-expressing cells were isolated from these cultures by FACS. The isolated FoxP3 cells were cocultured with naive cells polarized under Th17 conditions for 4 days. (I–K) Cell proliferation was measured by flow cytometry and (K) the generation of IL-17 was measured. Data shown as mean ± SEM; N = 3 biological replicates. P values were calculated with one-way ANOVA followed by the Tukey test for panels B, C, E, and K. Student’s t test was used to calculate P values in panel F. Source data are available for this figure: SourceData F1.

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