Journal: Frontiers in Immunology

Article Title: Phosphoproteomics Reveals Regulatory T Cell-Mediated DEF6 Dephosphorylation That Affects Cytokine Expression in Human Conventional T Cells

doi: 10.3389/fimmu.2017.01163

Figure Lengend Snippet: Experimental setup and quality controls for phosphoproteomics in primary human T cells. (A) Conventional CD4 + CD25 – T cells (Tcons) were cocultured either with allogeneic Tcons or regulatory T cells (Tregs), and cocultures were stimulated for 5 min with cross-linked anti-CD3/anti-CD28 antibodies. Stimulation was stopped on ice. Tstim (blue) and Tsup (red) were obtained after separation of T cell receptor (TCR)-stimulated Tcon:Tcon or Tcon:Treg cocultures, respectively. Unstimulated Tcons (Trest; gray) from the same donor were processed in parallel. Proteins were digested, peptides dimethyl-labeled and mixed, before phosphopeptides were enriched and measured by mass spectrometry (MS). Relative abundance of phosphopeptides was quantified by calculating the intensity ratios between the different samples as indicated. (B) An aliquot of cells used for phosphoproteomics was stimulated for 3 h before coculture separation, and suppression of cytokine mRNA was measured in re-isolated responder Tcons [Trest, Tstim, and Tsup as in panel (A) ]. As additional control, responder Tcons were stimulated without allogeneic Tcons (control Tstim). IL2 and IFNG mRNA were measured by quantitative RT-PCR, normalized to GAPDH mRNA. Results are presented as fold change compared to Trest (set to 1). The upper panel shows a representative donor (mean ± SD of technical PCR duplicates). Percentage suppression of respective cytokines in Tsup as compared to Tstim was calculated and is summarized for the three phosphoproteomics donors (lower panel). T cells were processed in three independent experiments (one experiment/donor) and phosphopeptide enrichment was performed in two independent experiments. (C) The number of unique phosphopeptides detected in each donor was determined, and the overlap is depicted as Venn diagram.

Article Snippet: We first isolated CD25 high cells with CD25-specific MACS beads (2 μl/10 7 cells, Miltenyi Biotec; cat. no. 130-092-983) as described previously ( ).

Techniques: Phospho-proteomics, Labeling, Mass Spectrometry, Isolation, Control, Quantitative RT-PCR

Journal: Diabetes

Article Title: Dynamic Immune Phenotypes of B and T Helper Cells Mark Distinct Stages of T1D Progression

doi: 10.2337/db18-1081

Figure Lengend Snippet: Teff response to IL-2 and Teff suppression by Treg is decreased in autoAb+ subjects. A: IL-2–mediated STAT5 phosphorylation in autoAbneg FDRs was compared with autoAb+ nonprogressors (Non-Prog) and autoAb+ progressors (Prog) in CD4+CD25dimCD45RO+ memory T cells at time 1, ages ≤18 years. Cells were activated with 25 IU/mL IL-2 for 10 min and then fixed, permeabilized, and stained with antibodies against pSTAT5 (pY694), CD25, CD4, and CD45RO. Percentage positive pSTAT5 was determined based on media stimulation of the same sample as shown in Supplementary Fig. 1A. Significance was determined using a Mann-Whitney two-tailed test. B: IL-2/pSTAT5 response of Teff stratified by number of autoantibodies (numeric 0–4: includes autoAbneg FDRs, autoAb+ nonprogressors, and autoAb+ progressors) at time 1, ages ≤18 years. AutoAbs include microinsulin autoAb, GAD65 standard, IA-2, ICA, ICA512, and Zn transporter (if assayed). Significance was determined using a one-way ANOVA (Kruskal-Wallace test) with Benjamini-Hochberg procedure for controlling false discovery rate. None of the comparisons was statistically significant. C: Treg suppression of CD4+CD25dim Teff in autoAbneg FDRs was compared with autoAb+ nonprogressors and autoAb+ progressors in total CD4+ T cells at time 1. Percent suppression was determined as in Supplementary Fig. 1B. Significance was determined using Mann-Whitney two-tailed tests. None of the two-way comparisons were statistically significant. D (left panel): Paired analysis of percent suppression between time 1 and time 2 for all autoAb+ subjects (nonprogressors and progressors) seropositive for two or more autoAbs. Significance was determined using a two-tailed Wilcoxon matched-pairs signed rank test. D (right panel): Percent suppression of Teff stratified by number of autoantibodies (numeric 0–4: includes autoAbneg FDRs, autoAb+ nonprogressors, and autoAb+ progressors). AutoAbs include microinsulin autoAb, GAD65 standard, IA-2, ICA, ICA512, and Zn transporter (if assayed). Significance was determined using a one-way ANOVA (Kruskal-Wallace test) with Benjamini-Hochberg procedure for controlling false discovery rate. 0 vs. 3 autoAbs: P = 0.014; q = 0.06. All other comparisons were not statistically significant.

Article Snippet: In brief, CD4 + T cells depleted of CD25 hi cells were isolated from PBMCs using a no-touch Miltenyi CD4 T Cell Isolation Kit II and positive Miltenyi CD25 Microbeads II prior to staining with carboxyfluorescein succinimidyl ester (Sigma-Aldrich).

Techniques: Phospho-proteomics, Staining, MANN-WHITNEY, Two Tailed Test