Journal: Open Life Sciences

Article Title: TGF-β1-overexpressing mesenchymal stem cells reciprocally regulate Th17/Treg cells by regulating the expression of IFN-γ

doi: 10.1515/biol-2021-0118

Figure Lengend Snippet: The effect of TGF-β1/MSCs on T cells. (a) T cells were co-cultured with MSCs or TGF-β1/MSCs, and the proportion of CD4 + CD25 + T cells in lymphocytes was detected by flow cytometry. (b) CD4 + T cells were co-cultured with MSCs or TGF-β1/MSCs for 3, 5, or 7 days, and proliferative responses were measured. (c) CD8 + T cells were co-cultured with MSCs or TGF-β1/MSCs in the presence of IL-2 after 3, 5, or 7 days. Live cells were collected, and the cytotoxic activity against MDCK cells was assessed. Data are expressed as the mean ± SD. Three independent experiments were analyzed using unpaired Student’s t -tests. * P < 0.05; ** P < 0.01; MSCs group vs control group. # P < 0.05; ## P < 0.01; TGF-β1/MSCs group vs MSCs group.

Article Snippet: Fluorescence-labeled monoclonal antibodies anti-CD4, anti-CD25, anti-CD34, anti-CD105, anti-CD44, anti-CD29, and anti-CD90 were, respectively, added and incubated in the refrigerator at 4°C for 30 min. All antibodies for flow cytometry were purchased from BD Biosciences.

Techniques: Cell Culture, Flow Cytometry, Activity Assay

Journal: Open Life Sciences

Article Title: TGF-β1-overexpressing mesenchymal stem cells reciprocally regulate Th17/Treg cells by regulating the expression of IFN-γ

doi: 10.1515/biol-2021-0118

Figure Lengend Snippet: The effect of TGF-β1/MSCs on Th17 differentiation of T cells. (a) After CD4 + CD25 − T cells were cultured with TGF-β1/MSCs for 3 days in the presence of IL-2, cytokines (IL-4, IL-10, IL-17A, IL-21, and IL-22) were measured by ELISA from the culture supernatant. (b) The expression of IL17, Foxp3, and RORγt in T cells was measured by Western blotting. (c) The mRNA levels of T-bet, IL-17, IL-17F, RORγt, Foxp3, and RORα were detected using RT-PCR. Data are expressed as the mean ± SD. Three independent experiments were analyzed using unpaired Student’s t -tests. * P < 0.05; ** P < 0.01; TGF-β1/MSCs group vs control group.

Article Snippet: Fluorescence-labeled monoclonal antibodies anti-CD4, anti-CD25, anti-CD34, anti-CD105, anti-CD44, anti-CD29, and anti-CD90 were, respectively, added and incubated in the refrigerator at 4°C for 30 min. All antibodies for flow cytometry were purchased from BD Biosciences.

Techniques: Cell Culture, Enzyme-linked Immunosorbent Assay, Expressing, Western Blot, Reverse Transcription Polymerase Chain Reaction

Journal: Stem Cell Research & Therapy

Article Title: Multiple intravenous injections of allogeneic equine mesenchymal stem cells do not induce a systemic inflammatory response but do alter lymphocyte subsets in healthy horses

doi: 10.1186/s13287-015-0050-0

Figure Lengend Snippet: Multiple allogeneic mesenchymal stem cell (MSC) injections result in changes in splenic regulatory T cell percentages. (A-D) There were no significant changes in splenic CD21 + B-cell (A) , CD4 + T-cell (B) , or CD8 + T-cell percentages (C) or CD4/CD8 ratios (D) following multiple MSC injections. (E) There were no significant changes in activated (CD25 + ) lymphocyte proportions. (F) There were significantly higher percentages of splenic FoxP3 + regulatory T cells in the horses injected with bone marrow (BM)-derived MSCs compared with horses injected with adipose tissue (AT)-derived MSCs. Data are presented as mean ± standard error of the mean. * P <0.05.

Article Snippet: The following antibodies were used: mouse-anti-equine CD3 (clone UC F6G 1:250; Jeffery Stott, University of California, Davis, CA, USA) [ ], mouse-anti-human CD21 (clone B-ly4 1:20; BD Pharmingen, San Jose, CA, USA) [ , ], polyclonal goat-anti-human CD25 (clone AF-223; R&D Systems) [ ], rat-anti-mouse/human FoxP3 (clone PCH101; ebioscience, San Diego, CA, USA) [ ], mouse-anti-CD4 (clone HB61A 1:133; VMRD, Pullman, WA, USA) [ ], mouse-anti-equine CD8 (clone F18P 1:500; J. Stott) [ ], and a donkey-anti-mouse secondary when necessary (1:50; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA).

Techniques: Injection, Derivative Assay

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: PBMC were stained with anti-CD4 FITC (clone OKT4), anti-CD25 APC (clone 4E3) and anti-CD127 PE (clone HIL-7R-M21, all from BD Biosciences, San Jose, CA).

Techniques: Purification, Cell Culture, CRISPR, Expressing

Journal: Cell Death & Disease

Article Title: HIV integrase inhibitor, Elvitegravir, impairs RAG functions and inhibits V(D)J recombination

doi: 10.1038/cddis.2017.237

Figure Lengend Snippet: Evaluation of effect of Elvitegravir on B cells progression in mice by FACS analysis. ( A ) Schematic representation of steps involved during the in vivo experiment. Balb/c mice (vehicle control and Elvitegravir-treated) were fed with Elvitegravir (8 days; 30 mg/kg). Mice were sacrificed and bone marrow cells were collected, stained with CD45, CD25 surface markers and FACS analysed. ( B ) Representative FACS dot plots of CD45 + CD25 + cells from vehicle control and Elvitegravir-treated mice are shown. Two mice each from independent batches of vehicle control (a,b batch I, c,d batch II) and Elvitegravir-treated groups (e,f batch I, g,h batch II) are presented. ( C ) Table showing percentage of CD45 + CD25 + cells obtained following flow cytometric analysis from control ( n =11) and Elvitegravir-treated ( n =16) mice from all three batches. Mice, from the Elvitegarvir treated group, that are affected ( n =11) and unaffected ( n =5) by Elvitegravir are shown. ( D ) Histogram showing CD45 + CD25 + B cells. Double-positive B cells detected following FACS analysis from bone marrow cells of vehicle control (white) are depicted in comparison to Elvitegravir-treated group which is divided into affected (black) and unaffected (grey). While vehicle control mice possessed average 78.03% of CD45 + CD25 + B cells in bone marrow it was reduced to average 62.5% following Elvitegravir treatment. ~30% mice were insensitive to treatment. P value <0.001

Article Snippet: Anti-CD3-FITC (Cat No. 555274), anti-CD8-APC-Cy7 (Cat No. 557654), anti-CD19-APC (Cat No. 550992), anti-CD25-PECy7 (Cat No. 552880) and anti-CD45-APC (Cat No. 559864) were from BD Biosciences.

Techniques: In Vivo, Staining