Journal: Frontiers in Immunology

Article Title: Dissecting the role of CSF2RB expression in human regulatory T cells

doi: 10.3389/fimmu.2022.1005965

Figure Lengend Snippet: Differential CD131 expression in human CD4 + CD25 - Tconvs and Tregs. (A) Relative CSF2RB mRNA levels in flow-sorted CD4 + CD25 - CD127 + Tconvs and CD4 + CD25 + CD127 - Tregs from freshly isolated human PBMCs (n= 10). The relative quantities of CSF2RB mRNA were normalized to the expression of B2M mRNA. (B) Granulocytes, monocytes and lymphocytes were gated based on forward and side scatter characteristic of cells from cells obtained from healthy human donors. Tconvs and Tregs were further gated based on CD4, CD25 and CD127 expression. Histograms show CD131 expression (red line) in comparison with matched isotype control (black line) for each cell type. One representative donor out of two. (C) CD131 surface and intracellular protein expression was studied in CD4 + CD25 - CD127 + Tconvs and CD4 + CD25 + CD127 - Tregs in 13-22 independent donors. (D, E) FOXP3 expression was analyzed within the total Treg population or within CD131 + Tregs. (D) Representative FACS plot for one donor. (E) FOXP3 fluorescence intensity in CD131 + Tregs normalized to the total Treg population (n=18 independent donors). Graphs display mean ± SD. Normal distribution was assessed by Shapiro-Wilk normality test with a significance level of 0.05. Significance was calculated by (A, C) Mann-Whitney test or (E) Wilcoxon matched-pairs test. ns for P > 0.05, ** for P ≤ 0.01, **** for P ≤ 0.0001.

Article Snippet: The following primers were used for PCR: CSF2RB - Hs01036514_m1, CSF2RA -Hs00531296_g1, IL3RA -Hs00608141_m1, IL5RA -Hs00602482_m1, IL10 -Hs00961622_m1, EPOR -Hs00959427_m1 (ThermoFisher Scientific) and B2M -4326319E-1402015 (Applied Biosystems).

Techniques: Expressing, Isolation, Comparison, Control, Fluorescence, MANN-WHITNEY

Journal: Frontiers in Immunology

Article Title: Dissecting the role of CSF2RB expression in human regulatory T cells

doi: 10.3389/fimmu.2022.1005965

Figure Lengend Snippet: CD131 expression on TCR-stimulated CD4 + CD25 - Tconvs and Tregs. (A) Relative CSF2RB mRNA levels in flow-sorted CD4 + CD25 - CD127 + Tconv and CD4 + CD25 + CD127 - Treg cells from human PBMCs upon in vitro stimulation with anti-CD3/CD28 mAbs and IL-2. Cells were stimulated with 1 or 10µg/ml anti-CD3 mAb, 1µg/ml anti-CD28 mAb and 25 or 300U/ml IL-2 (n= 6-10). The relative quantities of CSF2RB mRNA were normalized to the expression of B2M mRNA. Graph includes data from Figure 1A at day 0 for comparison (B) CD131 intracellular protein expression was studied in CD4 + CD25 - CD127 + Tconvs and CD4 + CD25 + CD127 - Tregs flow-sorted from human PBMCs, labelled with cell proliferation trackers CFSE or CTV and in vitro -stimulated with anti-CD3/CD28/CD2 coated beads for 4 days in the presence of 25U/ml IL-2. One representative donor out of two is shown. (C) CD131 surface and intracellular protein expression was studied in CD4 + CD25 - CD127 + Tconv and CD4 + CD25 + CD127 - Treg cells stimulated for 4 days using 1µg/ml anti-CD3 mAb, 1 µg/ml anti-CD28 mAb and 25U/ml IL-2 (n= 7-13 independent donors). Graphs display mean ± SD. Normal distribution was calculated by Shapiro-Wilk normality test with a significance level of 0.05. Significance was calculated by (A) Kruskal-Wallis test with Dunn’s multiple comparisons test and (C) unpaired two-tailed t test for normal distributed data. * for P ≤ 0.05 ** for P ≤ 0.01,*** for P ≤ 0.001, **** for P ≤ 0.0001.

Article Snippet: The following primers were used for PCR: CSF2RB - Hs01036514_m1, CSF2RA -Hs00531296_g1, IL3RA -Hs00608141_m1, IL5RA -Hs00602482_m1, IL10 -Hs00961622_m1, EPOR -Hs00959427_m1 (ThermoFisher Scientific) and B2M -4326319E-1402015 (Applied Biosystems).

Techniques: Expressing, In Vitro, Comparison, Two Tailed Test

Journal: Frontiers in Immunology

Article Title: Dissecting the role of CSF2RB expression in human regulatory T cells

doi: 10.3389/fimmu.2022.1005965

Figure Lengend Snippet: Characterization of CSF2RB -KO Tregs (A–E) CD4 + CD25 + CD127 - Tregs were flow-sorted from human PBMCs and cultured in vitro for 7 days with 10µg/ml anti-CD3 mAb, 1µg/ml anti-CD28 mAb and 300 U/ml IL-2 prior CSF2RB gene knock-out (KO) by Cas9/sgRNA ribonucleoprotein (RNP) transfection or controls (Mock). (A) CD131 surface and intracellular expression, (B) cell viability and (C) FOXP3 and CD25 expression were studied 3-4 days post-electroporation in 4-7 independent donors. Histograms show data from one representative donor. Isotype (if applicable) is shown as a grey shadow, mock as a black line and CSF2RB -KO as a red line. (D) In vitro suppressive capacity of MOCK or CSF2RB -KO Tregs was assessed by measuring their ability to inhibit proliferation of CFSE-labelled CD4 + CD25 - Tconv (Tresp) when stimulated with monocyte-derived dendritic cells (moDC) and anti-CD3 mAb. Top panel: CFSE expression for one representative donor. Dotted black line: proliferation of unstimulated Tresp; grey shadow: Tresp proliferation in the absence of Tregs; black line: Tresp proliferation when co-cultured with Mock Tregs; red line: Tresp proliferation when co-cultured with CSF2RB -KO Tregs. Bottom panel: percentage of suppression of CD4 + Tresp that diluted CFSE dye is represented for 3 independent donors. (E) CD131 expression was measured by flow cytometry in Tregs harvested from suppression assay cultures and gated as live, CD3 + CD4 + CFSE - cells. (F) 4 days after nucleofection, MOCK or CSF2RB -KO Tregs were re-stimulated with anti-CD3/CD28 mAbs and IL-2 for 4 days. Tregs were re-stimulated with PMA and ionomycin in the presence of Brefeldin A prior staining for cytokine expression (n=4 independent donors). Mean ± SD is shown for cumulative data. Normal distribution was calculated by Shapiro-Wilk normality test with a significance level of 0.05. Significance was calculated by (A, B, D, E) paired two-tailed t test for normal distributed data or (C) by Wilcoxon matched-pairs test (F) one-way ANOVA with Holm-Sidak’s multiple comparisons test. ns for P > 0.05, * for P ≤ 0.05, ** for P ≤0.01, *** for P ≤ 0.001.

Article Snippet: The following primers were used for PCR: CSF2RB - Hs01036514_m1, CSF2RA -Hs00531296_g1, IL3RA -Hs00608141_m1, IL5RA -Hs00602482_m1, IL10 -Hs00961622_m1, EPOR -Hs00959427_m1 (ThermoFisher Scientific) and B2M -4326319E-1402015 (Applied Biosystems).

Techniques: Cell Culture, In Vitro, Knock-Out, Transfection, Expressing, Electroporation, Derivative Assay, Flow Cytometry, Suppression Assay, Staining, Two Tailed Test

Journal: Frontiers in Immunology

Article Title: Dissecting the role of CSF2RB expression in human regulatory T cells

doi: 10.3389/fimmu.2022.1005965

Figure Lengend Snippet: CSF2RB expression of Tregs in patients with autoimmunity. (A) CSF2RB mRNA levels in Tregs isolated from healthy individuals (n= 14), and patients with MS (n= 11), RA (n= 9) and SLE (n= 8). Data obtained and reanalyzed from . (B) IL3RA, CSF2RA and IL5RA mRNA levels in Tregs isolated from healthy individuals (n= 14), and patients with MS (n= 11), RA (n= 9) and SLE (n= 8). Data obtained and reanalyzed from . Mean ± SD is shown. Normal distribution was assessed by Shapiro-Wilk normality test with a significance level of 0.05. Significance was calculated by Kruskal-Wallis test with Dunn’s multiple comparisons test. ns for P > 0.05; ** for P ≤ 0.01.

Article Snippet: The following primers were used for PCR: CSF2RB - Hs01036514_m1, CSF2RA -Hs00531296_g1, IL3RA -Hs00608141_m1, IL5RA -Hs00602482_m1, IL10 -Hs00961622_m1, EPOR -Hs00959427_m1 (ThermoFisher Scientific) and B2M -4326319E-1402015 (Applied Biosystems).

Techniques: Expressing, Isolation