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: 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

Journal: Journal for Immunotherapy of Cancer

Article Title: Combined innate and adaptive immunotherapy overcomes resistance of immunologically cold syngeneic murine neuroblastoma to checkpoint inhibition

doi: 10.1186/s40425-019-0823-6

Figure Lengend Snippet: Treated 9464D-GD2 tumors have significantly fewer T regulatory cells, with a higher CD8+ T cell to Treg ratio, and more CD4+ T cells and monocytes/macrophages compared to untreated tumors. Untreated 9464D-GD2 tumors and tumors treated with 12 Gy and combined ½ dose IT-IC, anti-CTLA-4 (CTLA), CpG, and anti-CD40 (CD40) were harvested on treatment day 13, and tumor microenvironment was analyzed by flow cytometry ( a ). Representative dot plots of Treg populations (defined as CD25 + FoxP3+ of CD45 + CD4+ live cells) are shown for three representative untreated ( b ) and treated ( c ) tumors (numerical values shown are the % of CD45 + CD4+ live cells that are Tregs)

Article Snippet: For cell surface staining, cells were incubated with anti-GD2-APC (clone 14G2a; BioLegend), anti-CD45-eF450 (clone 30-F11; eBioscience), anti-CD3-Alexa700 (clone 17A2; BioLegend), anti-CD4-PE-Dazzle594 (clone GK1.5; BioLegend), anti-CD8a-APC-eFluor780 (clone 53–6.7; eBioscience), anti-CD11b-BB700 (clone M1/70; BD Horizon), anti-Ly6G-BV711 (clone 1A8; BioLegend), anti-CD25-BB515 (clone PC61; BD Horizon), anti-FoxP3-PE-Cy7 (clone FJK-16 s; eBioscience), and Ghost Dye Violet 510 (Tonbo Biosciences).

Techniques: Flow Cytometry

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Nanoparticles Containing an Insulin-ChgA Hybrid Peptide Protect from Transfer of Autoimmune Diabetes by Shifting the Balance between Effector T cells and Tregs

doi: 10.4049/jimmunol.1900127

Figure Lengend Snippet: A–C: Single-cell suspensions were prepared from the pancreas and spleen of control and 2.5HIP-PLG treated mice euthanized at 2 or 8 weeks post adoptive transfer, followed by staining with I-Ag7/2.5HIP tetramer and antibodies. Gates were set on live, CD45+, lineage-negative (lin−), CD4+, 2.5HIP tet+ cells. A: Representative example of Foxp3 and CD25/Foxp3 staining from the pancreas of a 2.5HIP-PLG treated mouse euthanized at 8 weeks. B: Summary of 2–3 independent experiments for control 2 wks (n = 6), 2.5HIP-PLG 2 wks (n = 6), and 2.5HIP-PLG 8 wks (n = 4). Each symbol represents an individual mouse. *P < 0.05, **P < 0.01. C: Data are representative of 3 independent experiments (n = 6) from mice euthanized at 2 weeks. **P < 0.01, ***P < 0.001.

Article Snippet: Other antibodies used included: anti-CD45:BUV395 (30-F11, BD), anti-CD4:BV711 (GK1.5, Biolegend), anti-Foxp3:PE and eFluor450 (FJK-16s, eBioscience), anti-CD25:BB515 (PC61, BD), anti-CTLA-4:PE-Cy7 (4C10–4B9, Biolegend), anti-GITR:BV510 (DTA-1, BD), anti-ICOS:BV605 (7E.17G9, BD), anti-CD127:PE-Cy7 (SB/199, eBioscience), anti-CD103:eFluor450 (2E7, eBioscience), anti-CD44:BV510 (IM7, BD), anti-CD73:BV605 (TY/11.8, Biolegend), anti-FR4:PE-Cy7 (12A5, eBioscience), anti-IFN-γ:APC (XMG1.2, BD), anti-TNF-α: FITC (MP6-XT22, eBioscience), and anti-T-bet: BV605 (4B10, Biolegend).

Techniques: Adoptive Transfer Assay, Staining

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Nanoparticles Containing an Insulin-ChgA Hybrid Peptide Protect from Transfer of Autoimmune Diabetes by Shifting the Balance between Effector T cells and Tregs

doi: 10.4049/jimmunol.1900127

Figure Lengend Snippet: A–C: Single-cell suspensions were prepared from the pancreas and spleen of control and 2.5HIP-PLG-treated mice euthanized at 2 or 8 weeks post adoptive transfer, followed by staining with I-Ag7/2.5HIP tetramer and antibodies. Gates were set on live, CD45+, lineage-negative (lin−), CD4+, 2.5HIP tet+ cells, CD25+ Foxp3+ cells. A. Representative example of CTLA-4, GITR, and ICOS staining on bulk 2.5HIP tet+ or tet+ Treg cells from the pancreas of a 2.5HIP-PLG-treated mouse euthanized at 8 weeks. B: Summary of 2 independent experiments for control 2 wks (n = 4), 2.5HIP-PLG 2 wks (n = 3–4), and 2.5HIP-PLG 8 wks (n = 3–4). Each symbol represents an individual mouse. C: Geometric mean fluorescence intensity (gMFI) data from mice euthanized at 2 weeks from 1 experiment is shown (n = 2).

Article Snippet: Other antibodies used included: anti-CD45:BUV395 (30-F11, BD), anti-CD4:BV711 (GK1.5, Biolegend), anti-Foxp3:PE and eFluor450 (FJK-16s, eBioscience), anti-CD25:BB515 (PC61, BD), anti-CTLA-4:PE-Cy7 (4C10–4B9, Biolegend), anti-GITR:BV510 (DTA-1, BD), anti-ICOS:BV605 (7E.17G9, BD), anti-CD127:PE-Cy7 (SB/199, eBioscience), anti-CD103:eFluor450 (2E7, eBioscience), anti-CD44:BV510 (IM7, BD), anti-CD73:BV605 (TY/11.8, Biolegend), anti-FR4:PE-Cy7 (12A5, eBioscience), anti-IFN-γ:APC (XMG1.2, BD), anti-TNF-α: FITC (MP6-XT22, eBioscience), and anti-T-bet: BV605 (4B10, Biolegend).

Techniques: Adoptive Transfer Assay, Staining, Fluorescence

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Nanoparticles Containing an Insulin-ChgA Hybrid Peptide Protect from Transfer of Autoimmune Diabetes by Shifting the Balance between Effector T cells and Tregs

doi: 10.4049/jimmunol.1900127

Figure Lengend Snippet: A–D: Single-cell suspensions were prepared from the pancreas and spleen of control and 2.5HIP-PLG-treated mice euthanized at 2 or 8 weeks post adoptive transfer, followed by staining with I-Ag7/2.5HIP tetramer and antibodies. Gates were set on live, CD45+, lineage-negative (lin−), CD4+, 2.5HIP tet+ cells, CD25+ Foxp3+ cells. A: Representative example of CD127 staining on tet+ Treg cells from the pancreas of a control (CTR) or 2.5HIP-PLG-treated (TOL) mouse euthanized at 2 weeks. C: Representative example of CD103 staining on bulk 2.5HIP tet+ or tet+ Treg cells from the pancreas of a 2.5HIP-PLG-treated mouse euthanized at 8 weeks. B&D: Summary of 2–3 independent experiments for control 2 wks (n = 4–6), 2.5HIP-PLG 2 wks (n = 4–6), and 2.5HIP-PLG 8 wks (n = 3–4). Each symbol represents an individual mouse. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Article Snippet: Other antibodies used included: anti-CD45:BUV395 (30-F11, BD), anti-CD4:BV711 (GK1.5, Biolegend), anti-Foxp3:PE and eFluor450 (FJK-16s, eBioscience), anti-CD25:BB515 (PC61, BD), anti-CTLA-4:PE-Cy7 (4C10–4B9, Biolegend), anti-GITR:BV510 (DTA-1, BD), anti-ICOS:BV605 (7E.17G9, BD), anti-CD127:PE-Cy7 (SB/199, eBioscience), anti-CD103:eFluor450 (2E7, eBioscience), anti-CD44:BV510 (IM7, BD), anti-CD73:BV605 (TY/11.8, Biolegend), anti-FR4:PE-Cy7 (12A5, eBioscience), anti-IFN-γ:APC (XMG1.2, BD), anti-TNF-α: FITC (MP6-XT22, eBioscience), and anti-T-bet: BV605 (4B10, Biolegend).

Techniques: Adoptive Transfer Assay, Staining