Journal: Stem cells translational medicine

Article Title: Mesenchymal Stromal Cells Suppress T-Cell-Mediated Delayed-Type Hypersensitivity via ALCAM-CD6 Interaction.

doi: 10.1093/stcltm/szad012

Figure Lengend Snippet: Figure 2. ALCAM or CD6 neutralization results in the abrogation of MSC-mediated suppression of T-cell activation CD4+CD25− T cells were magnetically sorted from human peripheral blood mononuclear cells and stimulated with anti-CD3/CD28-coated beads at a 1:1 ratio and cultured on the monolayer of human MSCs (hMSCs). hMSCs were pre-treated with or without ALCAM-neutralizing antibodies. (A) Representative flow cytometry histogram (left) and bar chart (right) demonstrating expression of CD69 (median fluorescence intensity; MFI) by CD4+ T cells in the indicated groups of cultures at 24 h. (B) Representative flow cytometry dot plots (left) and cumulative bar chart (right) showing frequencies of CD4+IFNγ+ Th1 cells (gated on CD4+ cells) in the indicated groups of cultures at 48 h of coculture. In another set of experiments, CD4+CD25− T cells were pre-treated with CD6 neutralizing antibody or control isotype prior to coculture. (C) Representative flow cytometry histogram (left) and bar chart (right) demonstrating expression of CD69 (MFI) by CD4+ T cells in the indicated groups of cultures at 24 h. (D) Representative flow cytometry dot plots (left) and cumulative bar chart (right) showing frequencies of CD4+IFNγ+ Th1 cells (gated on CD4+ cells) in the indicated groups of cultures at 48 h of coculture. Data from 3 independent experiments are shown. Data presented are mean ± SD. One-way ANOVA, *P < .05, **P < .01, ***P < .001, ****P < .0001.

Article Snippet: ELISPOT assay was used to measure the frequencies of directly primed IFNγ-producing T cells, as previously described.22 Briefly, a 96-well murine IFNγ ELISPOT plate was set up as per the manufacturer’s protocol (Immunospot, OH, USA).

Techniques: Neutralization, Activation Assay, Cell Culture, Flow Cytometry, Expressing, Fluorescence, Control

Journal: Stem cells translational medicine

Article Title: Mesenchymal Stromal Cells Suppress T-Cell-Mediated Delayed-Type Hypersensitivity via ALCAM-CD6 Interaction.

doi: 10.1093/stcltm/szad012

Figure Lengend Snippet: Figure 4. Silencing ALCAM expression on MSCs abrogates their capacity to suppress T-cell activation and proliferation. (A) Flow cytometry histogram (left) and bar graph (right) showing downregulation of ALCAM expression in mouse MSCs transfected with ALCAM-specific shRNA compared to control shRNA (Mock) on day 3 post-transfections to baseline levels (dotted line, isotype-stained control). Dotted line, isotype control; gray solid line, ALCAM-silenced MSCs, black solid line, Mock-silenced MSCs. CD4+CD25− T cells were magnetically sorted from C57BL/6 splenocytes and stimulated with anti-CD3/CD28-coated beads at a 1:2 ratio and cultured on the monolayer of ALCAM- or Mock-silenced MSCs. (B) Representative flow cytometry histogram (left) and bar chart (right) demonstrating expression of CD69 (MFI) by CD4+ T cells in the indicated groups of cultures at 24 h. (C) Representative flow cytometry dot plots (left) and cumulative bar chart (right) showing frequencies of CD4+IFNγ+ Th1 cells (gated on CD4+ cells) in the indicated groups of cultures at 48 h of coculture. (D) CD4+CD25− T cells were labeled with CellTrace CFSE prior to the coculture assays. Representative flow histogram showing CFSE dilution (left) and proliferation index (right) of indicated groups of cultures at 72 h. Data from 3 independent experiments are shown. Data presented are mean ± SD. One-way ANOVA, *P < .05, **P < .01, ****P < .0001.

Article Snippet: ELISPOT assay was used to measure the frequencies of directly primed IFNγ-producing T cells, as previously described.22 Briefly, a 96-well murine IFNγ ELISPOT plate was set up as per the manufacturer’s protocol (Immunospot, OH, USA).

Techniques: Expressing, Activation Assay, Flow Cytometry, Transfection, shRNA, Control, Staining, Cell Culture, Labeling