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. 2020 May 19;10:8218. doi: 10.1038/s41598-020-65129-3

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© The Author(s) 2020

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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CD28 blockade inhibits NKT cell cytokine production. NKT cells were sorted as α-GalCer:CD1d Tetramer+ TCRβInt/+ CD24− CD44 + and NK1.1−(Stage 2) or NK1.1 + (Stage 3). Sorted NKT cells were co-cultured with splenocytes pre-loaded with vehicle or 1 or 10 ng/mL α-GalCer in the presence of media or CTLA-4Ig for 72 hours. Culture supernatants were analyzed for production of (A) IFN-γ, (B) IL-4, (D) TNF-α, (E) IL-6, (F) IL-2, (G) IL-5, (H) IL-13, (I) IL-10, (J) IL-9, (K) IL-17A, (L) IL-17F, (M) IL-21, and (N) IL-22. (C) Ratio of IFN-γ to IL-4 production by stage 2 and stage 3 NKT cells. All data displayed in graphs correspond to mean +/− SEM of 3 biological replicates. Statistical significance was determined using one-way ANOVA followed by Bonferroni tests. P values as follows: *p < 0.05, **p < 0.001, and ***p < 0.0001. Flow cytometry gating strategy outlined in Materials and Methods.