pe cy7 mouse anti human cd127  (Thermo Fisher)

Journal: Nature Communications

Article Title: A DAP5/eIF3d alternate mRNA translation mechanism promotes differentiation and immune suppression by human regulatory T cells

doi: 10.1038/s41467-021-27087-w

Figure Lengend Snippet: a Representative parental populations of CD24 + CD127 −/dim CD25 + cells (representative of three donors). Human naive CD4 + lymphocytes were activated at d 1 and d 7 by αCD3, soluble αCD28, and 150 U/ml IL-2 and cultured for 12 d. Treatments of 20 nM RAD001, 2 ng/ml activated TGF-beta, RAD001+TGF-beta or DMSO (untreated) were added during activation and subsequent expansions as needed. IL-2 was refreshed every 2–3 d. After 12 d, cells were harvested, treated cells enriched for Treg cells by magnetic-labeling recovery. Sorted cells were stained and analyzed by flow cytometry. Total lymphocytes were gated for doublet exclusion, live CD4 + T cells, and CD25 + CD127 − cells as shown. b TGF-beta or RAD001 treatments increase the percentage of CD25 + CD127 − in CD4 + population. Quantitation of CD4 + CD127 dim/− CD25 + population for each treatment shown from a representative data of three donors. c TGF-beta treatment increases the percentage of FOXP3 + CD25 + cells in CD4 + population, shown from representative data of three donors. FMO-CD127 and FMO-FOXP3 were used as references for gating. d Lymphocytes from six different donors were cultured, treated, and stained as described in a . Percentages of FOXP3 + CD25 + cells for each donor were normalized to its untreated control and plotted cumulatively. e Number of CD25 + on CD4 + cells is unchanged by treatments. Samples from d were analyzed for percentage of overall CD25 + cells and for CD25 intensity (MFI, median fluorescence intensity of CD25-labeling fluorophore). Six independent donors per group were tested. f TGF-beta and RAD001+TGF-beta treatments increase numbers and intensity of expression of FOXP3 + on CD4 + T cells (MFI of FOXP3-labeling fluorophore). g iTreg cells were generated as in a , enriched for CD4 + CD127 dim/ − CD25 + cells by sorting, responder cells (autologous PBMCs) labeled with CFSE and co-cultured with labeled responders at different ratios with overnight activation. Suppression ability of treated and untreated cells for all ratios shown, expressed as 1-[division index of tested cells/average division index of responders alone]. Division indices obtained from the FlowJo Proliferation Platform of tests on three independent donors per test condition. a – g P values determined by statistical analysis using two-way ANOVA tests with Dunnett post-ANOVA test determination with SEM shown. Source data are provided as a Source Data file.

Article Snippet: In all, 1–2 × 10 5 cells were incubated with Blue LIVE/DEAD Fixable Dead Cell Stain kit (Life Technologies, Cat. # L-23105) or Zombie Aqua Fixable Viability Kit (BioLegend, Cat. # 423102) in 1× PBS, and then stained with surface antibodies diluted in Stain Buffer (BD Pharmingen, Cat. #554656): FITC mouse anti-human CD4 (BD Pharmingen, Cat. #555346), APC mouse anti-human CD25 (BD Pharmingen, Cat. #555434), PE-Cy7 mouse anti-human CD127 (Invitrogen BD Pharmingen, Cat. #5 25-1278-42), PE mouse anti-human CD101 (BioLegend, Cat. #331012), Brilliant Violet 421 mouse anti-human CD103 (BioLegend, Cat. #350213) and PE mouse anti-human GITR (BioLegend, Cat. #371203).

Techniques: Cell Culture, Activation Assay, Labeling, Staining, Flow Cytometry, Quantitation Assay, Fluorescence, Expressing, Generated

Journal: Nature Communications

Article Title: A DAP5/eIF3d alternate mRNA translation mechanism promotes differentiation and immune suppression by human regulatory T cells

doi: 10.1038/s41467-021-27087-w

Figure Lengend Snippet: Human naive CD4 + T cells were cultured and induced to differentiate to iTreg cells, then analyzed by flow cytometry as described in the legend to Fig. . a RAD001+TGF-beta-induced CD4 + CD127 dim/− CD25 + FOXP3 + iTreg cells that express high levels of GITR, CD101, CD103, TGF-beta, and IL-10. Quantitation was derived from three independent donors. P values were determined by statistical analysis using unpaired t test, with mean and SEM shown. b Representative CD4 + CD127 dim/− CD25 + FOXP3 + cell population from three studies expressing CD101, +/− TGF-beta, +/− RAD001 treatment. Percentages normalized to untreated control for each donor. FMO-CD127 and FMO-FOXP3 were used as references for gating. c Fold increase in CD101 and percentage of the CD4 + CD127 dim/− CD25 + FOXP3 + population obtained from three independent donor iTreg cells in b , normalized to each donor untreated control. d Representative flow cytometry of three independent donors of the CD4 + CD127 dim/− CD25 + FOXP3 + cell population expressing CD103, +/− TGF-beta, +/− RAD001 treatment. Percentages normalized to untreated control for each donor. FMO-CD127 and FMO-FOXP3 were used as a reference for gating. e Fold increase in CD103 and percentage of the CD4 + CD127 dim/− CD25 + FOXP3 + population from three donors in d normalized to each donor untreated control and plotted. P values were determined for c – e by two-way ANOVA test with Dunnett post-ANOVA test determination, with mean values and SEM shown. Source data are provided as a Source Data file.

Article Snippet: In all, 1–2 × 10 5 cells were incubated with Blue LIVE/DEAD Fixable Dead Cell Stain kit (Life Technologies, Cat. # L-23105) or Zombie Aqua Fixable Viability Kit (BioLegend, Cat. # 423102) in 1× PBS, and then stained with surface antibodies diluted in Stain Buffer (BD Pharmingen, Cat. #554656): FITC mouse anti-human CD4 (BD Pharmingen, Cat. #555346), APC mouse anti-human CD25 (BD Pharmingen, Cat. #555434), PE-Cy7 mouse anti-human CD127 (Invitrogen BD Pharmingen, Cat. #5 25-1278-42), PE mouse anti-human CD101 (BioLegend, Cat. #331012), Brilliant Violet 421 mouse anti-human CD103 (BioLegend, Cat. #350213) and PE mouse anti-human GITR (BioLegend, Cat. #371203).

Techniques: Cell Culture, Flow Cytometry, Quantitation Assay, Derivative Assay, Expressing

Journal: Nature Communications

Article Title: A DAP5/eIF3d alternate mRNA translation mechanism promotes differentiation and immune suppression by human regulatory T cells

doi: 10.1038/s41467-021-27087-w

Figure Lengend Snippet: a Schema for silencing DAP5 in activated CD4 + T cells during differentiation. Human naive CD4 + T cells from two different donors were isolated from PBMCs as described in Fig. legend. Cells were treated with 1 μM of Accell SMARTpool siRNAs targeting DAP5 or a non-targeting siRNA pool in serum-free X-Vivo 15 media containing 1% GlutaMAX for 24 h, activated with IL-2 and differentiated with TGF-beta and RAD001 to induce iTreg cells and maintained as described in Methods. Accell SMARTpool siRNAs were re-added to culture medium on d 3 and 6 after activation and cells submitted to flow cytometry on d 13. Studies included a GFP-siRNA non-targeting control to measure uptake efficiency by flow cytometry. b Levels of GFP uptake measured by flow cytometry in negative control (no GFP), IL-2 activated but otherwise untreated, and RAD001+TGF-beta treated (R+T) CD4 + T cells. Representative plot of two independent studies. c Equal protein amounts of cell lysates from two donors obtained as in a were pooled and subjected to immunoblot analysis. Samples correspond to untreated and RAD001 + TGF-beta treated cells. DAP5 protein levels were reduced by 70–80% with silencing. Immunoblots are representative of two independent experiments. d Human CD4 + T cells treated as in a were subjected to flow cytometry for differentiated iTreg cells (CD4 + CD127 dim/– CD25 + FOXP3 + GITR + ) determined on d 13. Representative flow plots of two independent experiments shown indicating a 51% reduction in differentiated iTreg cells to baseline levels of Treg cells isolated from PBMCs prior to differentiation. e iTreg cells were tested for viability by Trypan Blue exclusion assay. P values determined using Fisher’s exact test with means and SEM shown. There is no statistically significant difference in viability between pairs of non-silenced and DAP5 silenced matched conditions. f Percentages of CD4 + CD127 dim/− CD25 + FOXP3 + GITR + cells for each of two donors were normalized to its untreated control and plotted cumulatively for IL-2 activated but otherwise untreated, and RAD001+TGF-beta treated (R+T) CD4 + T cells. Source data are provided as a Source Data file.

Article Snippet: In all, 1–2 × 10 5 cells were incubated with Blue LIVE/DEAD Fixable Dead Cell Stain kit (Life Technologies, Cat. # L-23105) or Zombie Aqua Fixable Viability Kit (BioLegend, Cat. # 423102) in 1× PBS, and then stained with surface antibodies diluted in Stain Buffer (BD Pharmingen, Cat. #554656): FITC mouse anti-human CD4 (BD Pharmingen, Cat. #555346), APC mouse anti-human CD25 (BD Pharmingen, Cat. #555434), PE-Cy7 mouse anti-human CD127 (Invitrogen BD Pharmingen, Cat. #5 25-1278-42), PE mouse anti-human CD101 (BioLegend, Cat. #331012), Brilliant Violet 421 mouse anti-human CD103 (BioLegend, Cat. #350213) and PE mouse anti-human GITR (BioLegend, Cat. #371203).

Techniques: Isolation, Activation Assay, Flow Cytometry, Negative Control, Western Blot, Trypan Blue Exclusion Assay