Journal: bioRxiv

Article Title: TNFa and IL-6 promote ex-vivo proliferation of lineage-committed human regulatory T cells

doi: 10.1101/2021.08.09.455690

Figure Lengend Snippet: FACS purified human Tregs were stimulated with either aCD3/28 beads or CD28SA and cultured in the presence of 300 IU/ml rhIL-2 with or without TNFa and IL-6 as indicated. ( A and B ) Flow cytometric analysis of FOXP3 ( A ) and HELIOS ( B ) expression in Tregs on day 9 after stimulation. Representative histograms (left) and summaries of MFI from 3 independent experiments (right) are shown. Statistical significance of differences was assessed using one-way ANOVA with Geisser-Greenhouse’s correction and Dunnett’s multiple comparisons test using either Tconv or Treg treated with IL-2 alone as a baseline reference. p values are marked as ns=not significant, p>0.05, *=p<0.05, **=p<0.01, ***=p<0.001, ****=p<0.0001. ( C ) Heatmap summary of TSDR demethylation of Tregs expand-ed in various conditions. Results shown are averages of Treg cultures using 2 unrelated male donors in 2 independent experiments.

Article Snippet: The cellular suspensions were first stained with surface antibodies to CD4 (Per-CP; clone SK3), CD25 (PE-Cy7; clone 4E3), TNFR2 (APC; clone hTNFR-M1), and fixable viability dye (APC-e780, all from purchased from BD Biosciences) prior to fixation and permeabilization using kit according to manufacturer protocol (catalog number 88-8824-00; Thermofisher) and intracellular staining for FOXP3 (e450; clone PCH101) and HELIOS (FITC; clone 22F6, both from BD Biosciences).

Techniques: Purification, Cell Culture, Expressing

Journal: bioRxiv

Article Title: TNFa and IL-6 promote ex-vivo proliferation of lineage-committed human regulatory T cells

doi: 10.1101/2021.08.09.455690

Figure Lengend Snippet: FACS purified human Tregs were stimulated with either aCD3/28 beads or CD28SA and cultured in the presence of 300 IU/ml rhIL-2 with or without TNFa and IL-6 for 10 and 14 days, as indicated. ( A ) Representative aCD3/28 bead stimulated Treg expansion kinetics and summary of final fold expansion on day 10 of Treg expanded in the presence or absence of etanercept (5mcg/mL) (left). Similarly, CD28SA stimulated Tregs were expanded for 14 days (right). Results of 3 independent experiments using 3 unrelated donors are shown. ( B ) Tregs were gene edited to delete TNFR2 gene using CRISPR-Cas9 and then stimulated with either aCD3/28 beads (left) or CD28SA (right). Results shown are from 4 independent experiments using 4 unrelated donors. ( C ) Same as panel B, except the Tregs were cultured in the presence of IL-6 and TNFa. Results shown are from 4 independent donors in 4 independent experiments. ( D ) Tregs were expanded as shown in panel C and flow cytometric analysis of FOXP3, HELIOS and CD25 expression was performed on day 8 after stimulation. Results show summary of MFI of FOXP3, HELIOS and CD25 from 4 independent donors in 4 independent experiments. Paired t-test was used to determine statistical significance of the differences observed. p values are stated.

Article Snippet: The cellular suspensions were first stained with surface antibodies to CD4 (Per-CP; clone SK3), CD25 (PE-Cy7; clone 4E3), TNFR2 (APC; clone hTNFR-M1), and fixable viability dye (APC-e780, all from purchased from BD Biosciences) prior to fixation and permeabilization using kit according to manufacturer protocol (catalog number 88-8824-00; Thermofisher) and intracellular staining for FOXP3 (e450; clone PCH101) and HELIOS (FITC; clone 22F6, both from BD Biosciences).

Techniques: Purification, Cell Culture, CRISPR, Expressing