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. 2021 May 24;12:3073. doi: 10.1038/s41467-021-23220-x

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© The Author(s) 2021

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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Fig. 2 — a Comparison of serum leptin levels between healthy individuals with T_FH increase (fold changes of T_FH cells, the ratio of day 7/day 0 > 1) (n = 61, grey) and healthy individuals without T_FH increase (fold changes of T_FH cells, the ratio of day 7/day 0 ≤ 1) (n = 15, red) in healthy adults (18–60 years, n = 76) immunized with the influenza vaccine (Supplementary Table 4). Dotted line indicates healthy individuals with low leptin levels (lower 25%) and sufficient leptin levels (higher 75%). b Comparison of T_FH changes and vaccine response in low leptin group (n = 19) and sufficient leptin group (n = 57). c Real-time PCR analysis of T_FH-related genes in naïve CD4⁺ T cells from PBMC of healthy controls with anti-CD3/CD28 activation for 3 days, and further treated with leptin for 12 h (CXCR5: *P = 0.0176, Bcl6: **P = 0.0046, IL21: **P = 0.0085, PDCD1: **P = 0.0050, ICOS: **P = 0.0344) (n = 7). d, e Representative FACS plots and statistics showing the differentiation of CXCR5⁺PD-1⁺ T_FH cells (d) (100 ng/mL: *P = 0.0257, 200 ng/mL: **P = 0.0012, 400 ng/mL: **P = 0.0005, 800 ng/mL: **P < 0.0001), and the secretion of IL-21 (e) (100 ng/mL: **P = 0.0004, 200 ng/mL: **P < 0.0001, 400 ng/mL: **P < 0.0001, 800 ng/mL: **P < 0.0001) from purified human naïve CD4⁺ T cells stimulated with anti-CD3/CD28 and indicated concentrations of leptin for 4 days (dots, n = 5; bars, mean ± SEM). f, g Statistics showing the percentages of CXCR5⁺PD-1⁺ T_FH cells (f) (IL-12: *P = 0.0105, *P = 0.0107; IL-23: **P = 0.0063, **P = 0.0087; TGF-β: *P = 0.0187,*P = 0.0161; IL-12+TGF-β: **P = 0.0063,**P = 0.0052; IL-23+TGF-β: *P = 0.0452,*P = 0.0211; IL-12+IL-23+TGF-β: *P = 0.0206,**P = 0.0023) and IL-21⁺ cells (g) (IL-12: **P = 0.0068, **P = 0.0071; IL-23: **P < 0.0001, **P = 0.0012; TGF-β: *P = 0.0287,**P = 0.0096; IL-12+TGF-β: **P = 0.0023,*P = 0.0129; IL-23+TGF-β: *P = 0.0145,**P = 0.0044; IL-12+IL-23+TGF-β: **P = 0.0084,**P = 0.0082) differentiated from naïve CD4⁺ T cells with anti-CD3/CD28 stimulation and indicated cytokine combinations for 4 days (dots, n = 5; bars, mean ± SEM). Data from individuals (dots) and the median values (bars) are shown for (a); Mann–Whitney test was used for analysis (a). Percentages and numbers are shown for (b); Chi-Square test was used for analysis (b). Real-time PCR and FACS statistics are shown for individuals (dots, n = 3) and mean ± SEM (bars) values, and analysed by Mann–Whitney U-test (a, c), one-way ANOVA Dunnett’s multiple comparisons test (d–g). *P < 0.05, **P < 0.01. Results are representative of three independent experiments.