PMC

Label-free quantitative analysis of conventional and regulatory T cell proteomes. General analytical workflow based on cell sorting by flow cytometry using the DEREG mouse model and parallel proteomic analysis of Tconv and Treg cell populations by nanoLC-MS/MS and label-free relative quantification.

Schematic representation of the Treg proteomic signature. A, Schematical representation of the proteins differentially regulated in CD4⁺Foxp3⁺ Treg compared with CD4⁺Foxp3⁻ Tconv. The proteins were organized based on their GO term annotations. B and C, Boxplots of the log2-transformed MS intensities (LFQ values) of the 5 most up- (B) and down- (C) regulated proteins in Treg cells. Each point corresponds to a biological replicate. Gene names are presented on top of each graph.

Comparison of transcriptomic and proteomics Treg data sets. Tile map of the log2-transformed relative quantification (Treg/Tconv) in our data set (1), the Treg and Tconv data from the transcriptomic data set Hill et al. 2007 mapped to the moe430–20 Affymetrix Mouse Genome 430 2.0 Array (Annotation: GPL1261) (2) and the proteomic data set from Barra et al. 2015 (3). The top 50% of proteins/genes that presented a 2-fold change between Treg and Tconv in 2 or 3 of the data sets are presented ordered with decreasing log2-transformed fold change (missing values are in gray). Known markers are highlighted in red. Symbols on the right indicate if the genes/proteins that were considered as significant hits by the authors according to their statistical thresholds. The numerical values of this figure can be found in the supplemental Table S3.

Overexpression of Themis1 in Treg does not affect their development or phenotype at steady state. A, Immunoblot analysis of Themis1 and Gapdh quantities in Treg and Tconv isolated from WT mice and Themis1-Tg mice (left) and the densitometric quantification of Themis1 expression normalized with Gapdh (right; from 2 independent experiments). B, Relative quantification of Themis1 mRNA levels in Treg and Tconv relative to β₂-microglobulin (from 4 independent experiments). C and D, Frequency and absolute numbers of CD4⁺CD25⁺ and CD4⁺Foxp3⁺ cells among CD4⁺ T cells from thymus (C) and spleen (D) collected from WT mice (n = 5) and Themis1-Tg mice (n = 7). The results are presented as mean ± S.E. E, Mean fluorescence intensity (MFI) of selective markers expressed by CD4⁺Foxp3⁺ T cells in the spleen from WT mice and Themis1-Tg mice (n = 4). ns, nonsignificant. In C and D, the data are representative of two independent experiments. Nonparametric test **p ≤ 0.01; ***p ≤ 0.001. WT mice (WT) and Themis1-Tg mice (Tg).

Proteomic analysis of Foxp3⁺ regulatory T cells. A, Volcano plot showing proteins differentially regulated in Treg compared with Tconv in 7 independent experiments. Proteins with a Benjamini-Hochberg adjusted p value ≤0.05 were considered significantly regulated if they presented a log2-fold change ≥1 (in orange) or ≤-1 (in cyan) (correspond to a 2-fold change of protein quantity). Proteins considered as well-characterized markers of Treg are shown in red (upregulated proteins) and dark blue (downregulated proteins) with their corresponding gene names. Themis1 is indicated in yellow. B, Heatmaps of Tconv (left) and Treg (right) log2-transformed LFQ intensities retrieved from MaxQuant for the 40 most up- or downregulated proteins in the Treg cells (top and bottom, respectively). The columns correspond to the mean of technical replicates for the 7 independent experiments and proteins are ranked by decreasing mean intensity in the Treg cells. Gene names are indicated on the left, with marker proteins historically used to differentiate Treg from Tconv in red. C, Barplot of the log2-transformed fold changes (Treg/Tconv) of the 40 most up- or downregulated proteins in the Treg cells (top and bottom, respectively). Historical marker proteins known to be more abundant in Treg compared with Tconv are in red, and those known to be less abundant in Treg are colored in dark blue. All the regulated proteins are indicated by their gene name.

Overexpression of Themis1 improves the suppressive functions of Treg. A, CD4⁺CD25⁻CD45RB^high colitogenic T cells were sorted from WT mice and injected into Rag2^−/− mice to induce colitis. In addition, CD4⁺CD25^bright Treg cells were sorted either from Themis1-Tg mice or littermates control and were cotransferred in a ratio of 1:4 (Treg:Tconv). B, Mice were monitored for their body weight for 8 weeks. C, The intensity of disease was assessed 8 weeks after T cell transfer by measuring macroscopic appearance and wall thickness of the colon. Bars show mean value of 3 independent experiments with 8 to 10 mice per group ± S.E. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001. D, Suppressive activity of Treg cells from WT and Themis1-Tg mice was assessed in coculture experiments using Cell Trace Violet (CTV)-labeled and naive Tconv from WT mice as effector cells stimulated with plate-bound anti-CD3 and irradiated syngeneic antigen-presenting cells (APC). After 3 days of coculture, Tconv proliferation was assessed by CTV dilution. E, Representative histograms of the proliferation of Tconv alone or in the presence of the indicated ratios of Treg from WT or Themis1-Tg mice. Percentages indicate the proportion of CTV low effector cells. Results are representative of the 7 independent experiments. F, Suppressive functions of Treg from WT or Themis1-Tg mice at different ratios Treg:Tconv. Data are expressed as percentage of inhibition and presented as mean values ± S.E. obtained from seven experiments. G, Boxplot of the number of Foxp3⁺ Treg at different ratio Treg/Tconv in coculture experiment in vitro after 3 days. Data are presented as mean values ± S.E. obtained from seven experiments. Nonparametric test *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001. WT mice (WT) and Themis1-Tg mice (Tg).