Journal: Frontiers in Immunology
Article Title: Allogeneic cord blood regulatory T cells decrease dsDNA antibody and improve albuminuria in systemic lupus erythematosus
doi: 10.3389/fimmu.2023.1217121
Figure Lengend Snippet: CD4 + CD25 + CD127 low UCB-Tregs suppress SLE-PBMC and shift their cell population distribution. (A) Functional analysis of ex vivo -expanded day 14 CD4 + CD25 + CD127 low UCB-Tregs on suppression of Tcon cells from healthy donor and SLE-PBMCs. Two-way ANOVA demonstrated that ratio ( p < 0.0001), group ( p = 0.0029), and interaction ( p = 0.0068) between UCB-Treg : Tcon and UCB-Treg : SLE-PBMC were statistically significant. Data are presented as mean ± SEM ( n = 3). p < 0.05 was considered statistically significant. ** p < 0.01; **** p < 0.0001 by two-way ANOVA with Tukey multiple comparison tests. (B) Expression levels of surface and intracellular markers on tSNE map. Unstimulated or stimulated CD4 + CD25 + CD127 low UCB-Tregs, SLE-PBMCs, or CD4 + CD25 + CD127 low UCB-Treg plus SLE-PBMC co-cultures (1:1) with CD3/CD28 plus IL-2 were stained with Live/Dead dye, CD45, CD14, CD3, CD4, CD25, CD127, CD8, CD19, IgD, CD27, CD62L, Helios, FoxP3, CD56, and HLA-DR antibodies. Stained live CD45 + cells from all six treatments were gated, down-sampled to 10,000 cells per sample which were concatenated. tSNE was run on six samples and the resulting tSNE plots were displayed expression intensities of surface and intracellular markers for all treatments in the concatenated file. (C) Subset analysis on tSNE map. Events in the tSNE embeddings were overlaid with manually gated lymphocytes, CD3 + CD19 - T, CD4 + T, CD4 + CD25 + T, CD4 + CD25 + CD127 low Treg, CD4 + CD8 + T, CD8 + T, CD3 - CD19 + B, CD27 - IgD - DN B, CD27 + IgD - Memory B, CD27 - IgD + Naïve B, CD27 + IgD + Plasma, CD56 + NK cells, and CD14 + monocytes and displayed for all treatments in the concatenated file. Stained cells were acquired on a Cytek Aurora flow cytometer and analyzed using FlowJo software. (D) Quantification analysis of subsets. Unstimulated or stimulated CD4 + CD25 + CD127 low UCB-Tregs, SLE-PBMCs, or CD4 + CD25 + CD127 low UCB-Treg plus SLE-PBMC co-cultures (1:1) with CD3/CD28 plus IL-2 were stained with Live/Dead dye, CD45, CD14, CD3, CD4, CD25, CD127, CD8, CD19, IgD, CD27, CD62L, Helios, FoxP3, CD56, and HLA-DR antibodies. CD4 + T, CD8 + T, CD4 + CD8 + T, CD4 + CD25 + CD127 low Treg, CD3 - CD19 + B, CD56 + NK cells, and CD14 + monocytes were quantified. Data are presented as mean ± SEM ( n = 6). p < 0.05 was considered statistically significant. p < 0.0001 by one-way ANOVA test.
Article Snippet: APC-eFluor 780-conjugated mouse anti-human CD45 antibody (Ab) (HI30), Alexa Fluor-532-conjugated mouse anti-human CD3 Ab (UCHT1), FITC-conjugated mouse anti-human CD3 Ab (UCHT1), PerCP-Cyanine5.5-conjugated mouse anti-human CD8a Ab (RTA-T8), Super Bright 600-conjugated mouse anti-human CD19 Ab (SJ25C1), PE-conjugated mouse anti-human CD25 Ab (BC96), PE-Cy5-conjugated mouse anti-human CD127 Ab (eBioRDR5), APC-conjugated mouse anti-human CD56 Ab (CMSSB), FITC-conjugated mouse anti-human CD16 Ab (eBioCB16(CB16)), PerCP-eFluor 710-conjugated mouse anti-human CD14 Ab (61D3), PE-Cy7-conjugated mouse anti-human HLA-DR Ab (LN3), and LIVE/DEAD™ fixable Blue dye were purchased from Thermo Fisher Scientific.
Techniques: Functional Assay, Ex Vivo, Comparison, Expressing, Staining, Flow Cytometry, Software
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