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. 2019 Sep 30;9:13991. doi: 10.1038/s41598-019-50614-1

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© The Author(s) 2019

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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TFH1-like cells derived from TH1 cells uniquely co-express Bcl-6 and T-bet. (A) Schematic depicting culturing conditions utilized for the differentiation of the indicated cell populations. Briefly, naïve CD4⁺ T cells were cultured on plate-bound anti-CD3 and anti-CD28 as follows: T_H1 (5 ng/mL IL-12, 5 μg/mL anti-IL-4), T_FH0-like (50 ng/mL IL-6, 10 μg/mL anti-IL-4, 10 μg/mL anti-IFN-γ), or T_H0 (10 μg/mL anti-IL-4, 10 μg/mL anti-IFN-γ). After 3 days, cells were removed from stimulation and plated under the following conditions: T_H1 (5 ng/mL rmIL-12, 2.5 μg/mL anti-IL-4, 500 U/mL rhIL-2), T_FH1-like (expanded from T_H1 population; 5 ng/mL rmIL-12, 2.5 μg/mL anti-IL-4, 10 U/mL rhIL-2), T_FH0-like (10 μg/mL anti-IL-4, 10 μg/mL anti-IFN-γ, 50 ng/mL rmIL-6, 10 U/mL rhIL-2), or T_H0 (10 μg/mL anti-IL-4, 10 μg/mL anti-IFN-γ, 10 U/mL IL-2) for an additional 2 days. (B) qRT-PCR was used to assess expression of the indicated genes. The data were normalized to Rps18 and presented as fold change relative to the T_FH1-like sample (mean of n = 4–7 ± s.e.m.). **P < 0.01, ***P < 0.001; one-way ANOVA with Tukey multiple-comparison test. (C) Immunoblot analysis of Bcl-6 and T-bet protein expression in the indicated T helper cell populations. β-actin serves as a loading control. Shown is a representative blot of four independent experiments.