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. 2019 Apr 5;9:5661. doi: 10.1038/s41598-019-41506-5

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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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Evaluation of Gag-specific rectal CD4⁺ and CD8⁺ T cells at biopsy and autopsy. Rectal biopsies (day 77) and autopsies were performed and single cell suspensions were prepared from tissue samples, stimulated with 15 mer overlapping SIV Gag-specific peptide pool and their cytokine expression profiles were measured by multi-colour intracellular cytokine staining. Proportion of cytokine expressing (a) CD4⁺ T cells and (b) CD8⁺ T cells from biopsy (top panel) and autopsy (bottom panel) expressing the three cytokines of interest, IFN-γ, TNF, IL-2 individually or in a combination are presented as stacked bar charts as in Fig. 6. Rectal sample could not be obtained from DA45 and CE71 at autopsy and are hence labelled as “*”. Please see Supplementary Tables 3, 4 for absolute T cell counts. (c) The total IL-2 (top panel), TNF (middle panel) and IFN-γ (bottom panel) was also measured at both biopsy (left) and autopsy (right) by CD4⁺ and CD8⁺ T cells in both unadjuvanted and IL-4R antagonist vaccine groups. (d) The extent of CD4⁺ T cell (left) and CD8⁺ T cell (right) poly-functionality was also determined between the two vaccinated groups. (e) IL-2 expression by CD4⁺ T cells (top panel) and CD8⁺ T cells (bottom panel) from the three protected animals (AAB5, 55A3 and 94D5) were also compared to the remaining macaques at both biopsy and autopsy. Unpaired student’s t tests were performed to assess statistical significance.