Additional file 1 of CD4+ and CD8+ regulatory T cell characterization in the rat using a unique transgenic Foxp3-EGFP model
Creators: name: Ménoret, Séverine
name: Tesson, Laurent
name: Remy, Séverine
name: Gourain, Victor
name: Sérazin, Céline
name: Usal, Claire
name: Guiffes, Aude
name: Chenouard, Vanessa
name: Ouisse, Laure-Hélène
name: Gantier, Malika
name: Heslan, Jean-Marie
name: Fourgeux, Cynthia
name: Poschmann, Jeremie
name: Guillonneau, Carole
name: Anegon, Ignacio
Publisher: name: figshare
Year: 2024
Subjects: subject: Immunology
subject: FOS: Clinical medicine

description: Additional file 1: Supplementary figure 1. Flow cytometry analyses of spleen and thymus cells from Foxp3-EGFP rats. A) Spleen, thymus and bone marrow were harvested from 12 weeks-old Foxp3-EGFP or wild-type (WT) rats and single cell suspensions were gated by SSC and FSC on lymphocytes followed by the identification with mAbs of major cell populations such as TCR+ cells (TCR+CD4+, TCR+CD8+), TCR− cells (TCR-CD4+ and TCR-CD8+) and CD161+ for NK cells. These populations were then analyzed for FOXP3 expression by EGFP expression and by using an anti-FOXP3 mAb. Contour plots from one animal representative of 6 analyzed in the same conditions. Right hand graphs are the mean and SEM of all animals analyzed. Student’s t test *P &lt; 0.05, **P &lt; 0.01, ***P &lt; 0.001, and ****P &lt; 0.0001. B) PBMCs from WT and Foxp3−EGFP+ rats were analyzed using untreated (left panels) or permeabilized and fixed (2 middle panels) cells with the conditions used for the analysis using anti-FOXP3 antibodies. Compared to untreated cells, EGFP signals were reduced in permeabilized cells, as previously described [59]. Co-labeling using anti-FOXP3 antibodies and EGFP in permeabilized cells (2 right panels) showed co-staining in CD4+ and CD8+ T cells from Foxp3-EGFP animals. C. EGFP+ cells were sorted from spleen, permeabilized and fixed followed by analysis using anti-FOXP3 antibodies. All sorted EGFP+ cells expressed FOXP3. Supplementary figure 2. Suppression assay using T CD4+EGFP+ and CD8+EGFP+ cells. Representative histograms of a suppressive assay using spleen cells from 12 weeks-old Foxp3-EGFP rats. TCR+CD4+CD25+CD127lowEGFP+ (labeled with CPD-450) responder cells and TCR+CD8+CD45RClow/−EGFP+ cells were sorted and added to an MLR in a 1:1 or 1:0.5 Tresponder to Tregs cells. The MLR was performed by co-culturing in a 1:1 ratio spleen CD4+CD25− Tconv cells from SPD rats (MHC haplotype u) labeled with CPD-670 along with enriched spleen APCs from Lewis 1A (MHC haplotype a) rats. Decrease in the percentage of CPD-670 bright cells and increase in the percentages of CPD-670 low cells denote dilution of CPD-670 with cell proliferation. The right histogram shows the CPD-670 dilution of proliferating T cells in the absence of Treg and the others show less proliferation in the presence of the EGFP+ Treg. Representative experiment out of 4 performed. Supplementary figure 3. RNAseq analyses of EGFP+ and EGFP− within CD4+ and CD8+ T cells. TCR+CD25highCD127lowCD4+ and TCR+CD45RClow/− CD8+ EGFP+ and EGFP− T cells were cell sorted and RNAseq analyses were performed comparing CD4+EGFP+ vs. CD4+EGFP− cells, and CD8+EGFP+ vs. CD8+EGFP− cells and CD8+EGFP+ vs. CD4+EGFP+ cells. A. R-Shiny representations of RNAseq levels for individual samples of the indicated genes with immune functions expressed in units per million reads. B. GO analyses of analysis of functional pathways, Cell component analysis showed that the proteins encoded by comparison of TCR+CD25highCD127lowCD4+EGFP+ vs. TCR+CD25highCD127lowCD4+EGFP−, TCR+CD45RClow/−CD8+EGFP+ vs. TCR+CD45RClow/−CD8+EGFP− and TCR+CD25highCD127lowCD4+EGFP+ vs. TCR+CD45RClow/−CD8+EGFP+ cells. (a) Comparison of 5 principal pathways in immune cell activation (b) Comparison of 3 principal pathway in immune cell proliferation. (c) Comparison of 4 principal pathway in immune cell adhesion. (d) Comparison of 11 pathways in miscellaneous process. C. Venn diagrams showing the overlap in genes that were significantly (adjusted P value, 0.05 and absolute log2 fold-change of genes differentially upregulated (left) or down regulated (right). D. Heatmap analysis of DEG grouped together for all CD4+EGFP+ vs. CD4+EGFP− cells, CD8+EGFP+ vs. CD8+EGFP− cells, as well as CD4+EGFP+ cells vs. CD8+EGFP+ cells. Supplementary figure 4. Confirmation of RNAseq results on CD4+EGFP+ and CD8+EGFP+ cells by FACS. A. Histograms of TCR+CD4+EGFP+, TCR+CD4+EGFP-, TCR+CD8+EGFP+and TCR+CD8+EGFP- cells analyzed with the indicate mAbs. Percentages above the traits correspond to the number of positive cells above staining with isotype control mAbs. One experiment representative of 4 performed in the same conditions. Lower graphs are the mean and SEM f all animals analyzed. Student’s t test *P &lt; 0.05, **P &lt; 0.01, ***P &lt; 0.001, and ****P &lt; 0.0001. B. R-Shiny representation of RNAseq expression levels expressed in individual sample in units per million reads for Il2ra (Cd25), Cd44 and Icos. Supplementary figure 5. Analysis using anti-FOXP3 antibodies of EGFP+ cells induced in vitro. Spleen cells from Foxp3-EGFP animals were used to sort Tconv CD4+CD25-EGFP− Treg and T CD8+CD45RClow/-EGFP- cells and cultured for 3 days in the presence of anti-CD3/CD28 mAbs, IL-2 and in the absence or presence of hTGFbeta. CD4+ or CD8+ EGFP+ cells were cell sorted, permeabilized, fixed and analyzed using anti-FOXP3 antibodies. Results show that all EGFP+ cells were also anti-FOXP3+ cells (n = 3). graphs are the mean and SEM of all animals analyzed. Student’s t test *P &lt; 0.05, **P &lt; 0.01, ***P &lt; 0.001, and ****P &lt; 0.0001. Supplementary figure 6. IL-2 induce expansion of EGFP+ Tregs in spleen. Foxp3-EGFP rats received or not hIL-2 at low doses and the percent of the indicated cell populations was analyzed in spleen. Percentages of cells in the indicated cell populations of IL-2-treated and untreated rats. Left graph shows mean ± SEM, n = 3, and right contour plots show a representative animal. Supplementary figure 7. CD4+ and CD8+ FOXP3+ Treg in Foxp3-EGFP mice. Lymphocytes (PBL) and spleen cells from Foxp3-EGFP mice were analyzed for the expression of EGFP+ cells as well as for FOXP3+ cells using antibodies among CD4+ and CD8+ T cells. Graphs show mean ± SEM and each point represents an individual animal. Table S1. Frequency of CD4+EGFP+ and CD8+EGFP+ in Foxp3-EGFP rats and Foxp3-EGFP mice. Table S2. Immune genes upregulated in TCD4+EGFP+ vs. TCD4+EGFP- cells. Table S3. Immune genes upregulated in TCD8+EGFP+ vs. TCD8+EGFP- cells. Table S4. Immune genes upregulated in CD4+EGFP+ vs. CD8+EGFP+ cells. Table S5. Genes that are upregulated in Venn diagrams. Table S6. Genes that are downregulated in Venn diagrams. Table S6. Unique genes (333) in CD8+EGFP+ vs. CD8+EGFP- Treg. Table S6. Common genes (24) between CD4+EGFP+ vs. CD4+EGFP- cells and CD8+EGFP+ vs. CD8+EGFP- cells. Table S6. Unique genes (212) between CD4+EGFP+ vs. CD8+EGFP+ Treg. Table S6. Common genes (24) between CD4+EGFP+ vs. CD8+EGFP+ cells. Table S6. Common genes (3) between CD4+EGFP+ vs. CD8+EGFP+ and CD8+EGFP+ vs. CD8+EGFP- cells and CD4+EGFP+ vs. CD4+EGFP- cells. Table S6. Common genes (3) between CD4+EGFP+ vs. CD8+EGFP+ and CD8+EGFP+ vs. CD8+EGFP- and CD4+EGFP+ vs. CD4+EGFP- cells. Table S7. Increase in different EGFP+ and EGFP- cell subsets in blood following IL-2 treatment. Table S8. Antibodies used in the study.