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. 2017 Nov 23;7:16179. doi: 10.1038/s41598-017-15532-0

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© The Author(s) 2017

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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Latently infected CD4+ T_CM cells exhibit altered phosphorylation signatures relative to uninfected cells following T cell activation. (a) Experimental timeline of in vitro primary CD4+ T_CM cell model generation. After DHIV-Nef +/ HIV-1 _LAI (X4) infection on day 5, uninfected and infected cells are cultured in parallel. (b) Scatter plots of Gag p24 expression in CD4+ T_CM cells before (left) and after stimulation with CD3/CD28 (right) measured by flow cytometry. Representative data from donor 2; n = 10,000 cells. (c) Range of latent infection levels for replicate infections across 3 donors as estimated by CD3/CD28 stimulation of viral reactivation. (d) Viral reactivation after 48 hours of stimulation across a panel of LRAs. Data presented as means ± SD (n = 3) for donor 2. (e,f) Heatmaps depict phospho-protein time courses following CD3/CD28 (e) or PMA/I stimulation (f) in uninfected and infected CD4+ T_CM cells for 3 donors over 48 hours measured by bead-based immunoassay. Phospho-signals were measured in biological triplicate and 90% of measurements had an SD < 18%. Data are normalized to the maximum signal for each paired stimulation time course (uninfected and infected cells). Raw data are provided in Supplementary Datasets S1–S6. (g,h) Flow cytometry analysis of p-ERK at 15 minutes (g) and p-p38 at 24 hours (h) in uninfected, latently infected (p24−) and infected/reactivaing (p24+) T_CM cells following CD3/CD28 (top) or PMA/I stimulation (bottom). Gating for p24 (left plots) and histogram intensities for each population (middle plots) are shown for a representative donor. Mean fluorescence intensity (MFI) of phospho-protein levels (right plots) presented as means ± SD for 3 donors.