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. 2018 Feb 9;8:2729. doi: 10.1038/s41598-018-20740-3

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© The Author(s) 2018

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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Oral ETEC vaccination induced increased frequencies of activated Tfh-like cells in peripheral blood. (a–c) ICOS expression was analysed in PBMCs by FCM before (preVacc) and after (postVacc) primary and booster ETEC vaccinations. Strong ALS responders and weak/non-responders were randomly selected from each vaccination group (Group B; vaccine alone and groups C and D; vaccine with 10 or 25 µg dmLT, respectively, in the primary vaccination study; all booster vaccinations were given without adjuvant). (a and b) Frequencies of ICOS⁺ cells among CD4⁺CXCR5⁺ cTfh and CD4⁺CXCR5⁻ cells in the primary (a, n = 14) and booster (b, n = 10) vaccination studies (separate sets of individuals). (c) Frequencies of ICOS⁺ cells among cTfh in strong versus weak/non-responders (data pooled from the primary and booster vaccination study, n = 24). (d) PD-1 expression in ICOS⁺ and ICOS⁻ cTfh cells in post vaccination samples (symbols as in Fig. 1a and b, n = 19). (e) Representative PD-1 staining among ICOS⁺ and ICOS⁻ cTfh cells. (f) Frequencies of IL-21-producing cells among ICOS⁺ and ICOS⁻ cTfh cells isolated from 4 strong ALS responders after stimulation of PBMCs with PMA and ionomycin. (g) Representative staining of IL-21 expression in cTfh cells. (a–d, f) Each symbol represents one individual. Horizontal lines or bars indicate median values.