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. 2017 Aug 1;7:7018. doi: 10.1038/s41598-017-07365-8

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© The Author(s) 2017

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Runx1 is required for differentiation of ROR-γt expressing iNKT17 cells. (a) Representative frequency of iNKT subsets, iNKT1, iNKT2 and iNKT17 in WT (top) and PLZF-cre Runx1 cKO (bottom) mice. Functional subsets were defined by expression of PLZF, Tbet, ROR-γt. iNKT1 (Tbet⁺ PLZF^lo), iNKT2, (PLZF^hi ROR-γt⁻ Tbet⁻) and iNKT17 (ROR-γt⁺ PLZF^med Tbet⁻). (b) Absolute cell number of iNKT1, iNKT2 and iNKT17 cells in WT (black bars) and PLZF-cre Runx1 cKO (white bars) mice. (c) Percent of thymic iNKT1, iNKT2 and iNKT17 cells in WT (black bars) and PLZF-cre Runx1 cKO (white bars) mice. (a-c) Data is representative and calculated from 22 WT and 12 PLZF-cre Runx1 cKO mice. (d) Expression of Runx1 in thymic iNKT1, iNKT2 and iNKT17 in WT and PLZF-cre Runx1 cKO mice with isotype control for WT iNKT1. Data is representative of 7mice/genotype. (e) Production of IL-4 and IL-17A by iNKT2 and iNKT17 cells in WT and IL-4 in PLZF-cre Runx1 cKO mice. Production of IL-17A by Runx1-deficient iNKT cells is not determined (N.D.) due to absence of ROR-γt expressing iNKT17 cells. Thymocytes were stimulated for 6hr with PMA/ionomycin, and then examined for cytokine productions using flow cytometry. (f) Production of IFN-γ by iNKT1 cells in WT (top) and PLZF-cre Runx1 cKO (bottom) mice. Thymocytes were stimulated for 6hr with PMA/ionomycin, and then examined for cytokine production using flow cytometry. (g) Frequency of cells positive for cytokine production (IL-4, IL-17A and IFN-γ) in stimulated iNKT2, iNKT17, and iNKT1 respectively in WT (black bars) and PLZF-cre Runx1 cKO (white bars) mice. Production of IL-17A by Runx1-deficient iNKT cells is not determined (N.D.) due to absence of ROR-γt expressing iNKT17 cells. (h) Quantification of MFI of cytokine (IL-4, IL-17A and IFN-γ) produced by stimulated iNKT2, iNKT17, and iNKT1, respectively in WT (black bars) and PLZF-cre Runx1 cKO (white bars) mice. Production of IL-17A by Runx1-deficient iNKT cells is not determined (N.D.) due to absence of ROR-γt expressing iNKT17 cells. (e-h) Data is representative and calculated from 8 WT and 4 PLZF-cre Runx1 cKO mice. All statistical analysis was done using Student’s t-test. Means ± S.E.M.