rosettesep human t cell isolation kit  (STEMCELL Technologies Inc)

Journal: Frontiers in Immunology

Article Title: Human Regulatory Dendritic Cells Develop From Monocytes in Response to Signals From Regulatory and Helper T Cells

doi: 10.3389/fimmu.2020.01982

Figure Lengend Snippet: Human monocytes cultured with defined mixtures of Tregs and Th differentiate into cells that induce immune suppressive CD4 + FoxP3 + Tregs. (A) Dotplots and histograms showing CFSE dilution of CD4 + T cells cultured with monocyte-derived cells from the CD4 + T cell-monocyte cultures. (B) Graphical representation of statistical analysis of CFSE – cell percentage (mean ± SEM, n = 3) obtained from (A) . Similar results were obtained in two additional experiments. (C) Proliferation index calculated by Flowjo. (D) Expression of CD25 vs. FoxP3 in CFSE – CD2 + CD4 + cells. Numbers adjacent to gated areas indicate percentage of gated cells. (E,F) Graphical representation of statistical analysis of the MFI of CD25 and the percentage of CD25 high FoxP3 high cells in CD2 + CD4 + CFSE – proliferated T cells. (G) CD14 + monocytes were cultured with Tregs and Th at a ratio of 10:1:1 in the presence of anti-IL-10, 2 μg/ml; anti-TGFβ, 2 μg/ml or isotype control antibody, 5 μg/ml. After 72 h, 1 μg/ml LPS was added, and 16 h later FACS-purified myeloid cells (DR + CD2 – ) were further cultured with MACS sorted CFSE-labeled naïve allogeneic CD4 + T cells. The percentage of FoxP3 high cells in CD2 + CD4 + CFSE – cells was determined 6 days later. Mean ± SEM, n = 5. Similar results were obtained in two additional experiments. * P < 0.05; ** P < 0.001; *** P < 0.0001; ns, not significant. Data were analyzed with one-way ANOVA, followed by Dunnett’s test for multiple comparisons. (H,I) 10 5 CFSE labeled CD45RA + CD4 + T cells were cultured with 5 × 10 4 allogeneic myeloid cells purified from 3-day cultures of monocytes, Tregs and Th at 10:1:1. After 6 days the cells were analyzed by flow cytometry. Responder CD4 + T cells were gated as CD2 + CD4 + . (H) CFSE – FoxP3 + DC Reg -induced Tregs were analyzed for CD25 expression. (I) CD25 expression was used as the basis for sorting CD2 + CD4 + CFSE – cells. Percentages of FoxP3 – and FoxP3 + cells in CD2 + CD4 + CFSE – CD25 + cells are shown. (J–M) CD2 + CD4 + CFSE – CD25 + cells induced by 1:1 (Th:Treg), Th or Treg-derived myeloid cells were purified by FACS and cultured in a new MLR containing 10 5 CFSE labeled allogeneic CD45RA + CD4 + cells and irradiated DCs (5 × 10 4 ) and anti-CD3 (0.5 ng/ml, plate-bound). After 84–90 h, proliferation of CFSE-labeled naïve responder CD4 + T cells was analyzed by flow cytometry. Cells were gated as DAPI – CD2 + CD4 + . Data are representative of 2 independent experiments. (J–L) Dose-dependent suppression of induced Tregs on responder CD4 + T cells. (L) Heatmap of statistical analysis of the responder CD4 + T cell proliferation as indicated by CFSE in each division defined by Flowjo. Mean ± SEM, n = 3. Similar results were obtained in both experiments.

Article Snippet: The CD4 + T cells for these assays were purified from PBMCs using a RosetteSep CD4 + Human T cell Isolation Kit (Stem Cell Technologies) followed by magnetic purification (>95% CD2 + CD4 + CD45RA + cells by flow cytometry) using a Naïve CD4 + T Cell Isolation Kit II (Miltenyi Biotec) and subsequently labeled with CFSE.

Techniques: Cell Culture, Derivative Assay, Expressing, Control, Purification, Labeling, Flow Cytometry, Irradiation

Journal: Frontiers in Immunology

Article Title: Human Regulatory Dendritic Cells Develop From Monocytes in Response to Signals From Regulatory and Helper T Cells

doi: 10.3389/fimmu.2020.01982

Figure Lengend Snippet: Blockade of CTLA-4, IL-10 and TGF-β prevents DC Reg differentiation. (A–D) CD14 + monocytes were cultured for 4 days with Tregs or Th alone, or with Tregs and together at a 1:1 ratio in the presence of anti-CTLA-4 (5 μg/ml), anti-IL-10 (2 μg/ml), anti-TGFβ (2 μg/ml), or a mixture of these antibodies or isotype control antibody (5 μg/ml). (A) Images were captured with a bright-field DIC microscope on day 4. Original magnification 200×. (B) MFIs of DC-associated molecules on monocyte-derived cells are shown after excluding CD2 + HLA-DR – T cells. (C) On day 4, moDCs (HLA-DR + CD2 – cells) from the cultures in (A,B) were purified by FACS, and 200,000 cells were cultured in 200 μl containing 1 μg/ml LPS. After 16h, IL-10, IL-1β, IL-6 and TNFα levels in the cell-free supernatants were determined by Luminex. (D) On day 3, 1 μg/ml LPS was added to selected wells, and 16h later FACS-purified DC subsets were further cultured with MACS sorted CFSE-labeled naïve allogeneic CD4 + T cells. After another 6 days, CD2 + CD4 + T cells were analyzed by flow cytometry. Mean ± SEM of 4 donors. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, ns, not significant. Data were analyzed with one-way ANOVA, followed by Dunnett’s test for multiple comparisons.

Article Snippet: The CD4 + T cells for these assays were purified from PBMCs using a RosetteSep CD4 + Human T cell Isolation Kit (Stem Cell Technologies) followed by magnetic purification (>95% CD2 + CD4 + CD45RA + cells by flow cytometry) using a Naïve CD4 + T Cell Isolation Kit II (Miltenyi Biotec) and subsequently labeled with CFSE.

Techniques: Cell Culture, Control, Microscopy, Derivative Assay, Purification, Luminex, Labeling, Flow Cytometry

Journal: Frontiers in Immunology

Article Title: Human Regulatory Dendritic Cells Develop From Monocytes in Response to Signals From Regulatory and Helper T Cells

doi: 10.3389/fimmu.2020.01982

Figure Lengend Snippet: CRC CD4 + T cells induce DC Reg formation. (A) Left: representative human colon cancer tissue stained with antibodies against FoxP3 (brown) and CD14 (blue). Black arrows represent potential monocyte-Treg interactions. Right: representative human colon cancer tissue stained with antibodies against FoxP3 (brown) and T-bet (blue). Magnification 400×. (B) Representative flow cytometry analysis of CD25 + CD127 low Tregs in CD45 + CD2 + CD4 + T cells from colon cancer sample. (C) Comparison of the CD25 + CD127 low Treg percentages from healthy donor peripheral blood and colon cancer samples. Mean ± SEM. **** P < 0.0001, unpaired T test. (D,E) Lin – CD45 + CD2 + CD4 + T cells were purified from human CRC and co-cultured with peripheral blood CD14 + monocytes at a 1:10 ratio in the presence of anti-CD3 mAb for 4 days. (D) Images were captured with a bright-field DIC microscope on day 4. Original magnification 200×. (E) Flow cytometry analysis of the resultant antigen presenting cells (HLA-DR + CD2 – ) (red line) compared to monocytes cultured alone (gray line) or with GM-CSF and IL-4 (black line). (F) CD11c + HLA – DR + CD2 – cells were purified via FACS and stimulated for 16 h with LPS. Cells were washed extensively and cultured with CFSE-labeled allogeneic naïve CD4 + T cells in a MLR. The percentage of FoxP3 + cells in CD2 + CD4 + CFSE – cells was determined 6 days later (red line). Naïve CD4 + T cells cultured alone (black line) are shown as controls. Shaded histograms represent isotype controls. Data are representative of 3 independent experiments. (G) In the presence of anti-CD3 mAb, CD14 + monocytes were cultured with bulk CD45 + CD2 + CD4 + T cells sorted from CRC, in the presence of a mixture of anti-CTLA-4 (5 μg/ml), anti-IL-10 (2 μg/ml), anti-TGFβ (2 μg/ml), or isotype control antibody (5 μg/ml). At day 4, cells in culture were analyzed by flow cytometry for the indicated surface markers. (H) CD14 + monocytes were cultured with bulk CD45 + CD2 + CD4 + T cells sorted from CRC (red) or healthy donor peripheral blood (black). CD25 + CD127 low Treg percentages were analyzed before coculture. The graph shows the correlation between the percentage of Tregs in CD2 + CD4 + T cells prior to their initial culture with monocytes ( X axis) and the percentage of CFSE – FoxP3 + T cells among the responder CD4 + T cells cultured with the DCs generated in the initial culture ( Y axis). Linear regression was determined by prism. R 2 = 0.9509. P = 0.0002. (I) Representative flow cytometry analysis of human CRC DCs. DAPI – CD45 + cells were further gated with CD11c, HLA-DR. (J) Cytospin of freshly FACS-sorted CD11c + HLA-DR + DCs with Grunwald-Giemsa staining from CRC. Scale bar as indicated. (K) Flow cytometry analysis of CD40, CD80, CD86 and CD274 expression on gated CD11c + HLA-DR + DCs. Red line: Ab staining. Shaded histograms represent isotype controls. (L) CD45 + CD11c + HLA – DR + DCs from CRC were sorted by FACS and cultured with CFSE-labeled naïve allogeneic CD4 + T cells. The percentage of FoxP3 + cells in CD2 + CD4 + CFSE – cells was determined 6 days later.

Article Snippet: The CD4 + T cells for these assays were purified from PBMCs using a RosetteSep CD4 + Human T cell Isolation Kit (Stem Cell Technologies) followed by magnetic purification (>95% CD2 + CD4 + CD45RA + cells by flow cytometry) using a Naïve CD4 + T Cell Isolation Kit II (Miltenyi Biotec) and subsequently labeled with CFSE.

Techniques: Staining, Flow Cytometry, Comparison, Purification, Cell Culture, Microscopy, Labeling, Control, Generated, Expressing