Journal: The Journal of Clinical Investigation

Article Title: ST8Sia6 overexpression protects pancreatic β cells from spontaneous autoimmune diabetes in nonobese diabetic mice

doi: 10.1172/JCI181207

Figure Lengend Snippet: ( A and B ) Normalized cell count of classical macrophages (CD11b + CD11c – F4/80 + Gr1 – ), hybrid macrophages (CD11b + CD11c + F4/80 + Gr1 – ), cDC1s (CD11b – CD11c + F4/80 – Gr1 – ), and cDC2s (CD11b + CD11c + F4/80 – Gr1 – ) per islet ( A ) or per pancreatic lymph node ( B ) from euglycemic 14-week-old NOD βST or littermate mice. ( C ) Representative flow cytometry histogram of IL-12p35 expression in cDC2s from islets of euglycemic 14-week-old NOD βST and NOD littermate mice, compared with isotype control. ( D ) Representative flow cytometry histogram of IL-12p35 expression in hybrid macrophages from islets of euglycemic NOD βST and littermate mice, compared with isotype control. Vertical line denotes the positive gate. ( E ) Quantification of IL-12p35 expression shown in C and D across 7 NOD βST mice, 16 NOD littermate mice, and 5 isotype controls. Error bars represent SD. Mann-Whitney U tests were used to examine statistical differences between groups.

Article Snippet: The following antibodies were used: CD45-BUV395 (BD 564279 at 1:1,000), L/D-UV450 (Tonbo 13-0868 at 1:1,000), CD11c-BUV615 (BD 751222 at 1:100), CD11b-PacBlue (BioLegend 562894 at 1:200), Ly6C-BV480 (BioLegend 569439 at 1:500), CD8b-BV785 (BioLegend 126631 at 1:1,000), Ly6G-BV785 (BioLegend 127645 at 1:200), CD4-SparkBlue (BioLegend 100494 at 1:1,000), T-bet–RB780 (BD 569089 at 1:100), F4/80-ef660 (Invitrogen 50-4801-82 at 1:200), IFN-γ–APC (BioLegend 505810 at 1:100), isotype control–APC (BD 554686 at 1:100), FoxP3-APC (Tonbo 20-5773 at 1:100), CD11c-BV421 (BioLegend 117330 at 1:500), RORγt-BV421 (BD 562894 at 1:100), MHCII-BV510 (BioLegend 107635 at 1:400), L/D-BV510 (Tonbo 13-0870 at 1:1,000), CD8a-BV510 (BioLegend 100752 at 1:500), TCRβ-BV605 (BioLegend 109241 at 1:200), CD45-BV785 (BioLegend 103149 at 1:500), CD8a-BV785 (BioLegend 100750 at 1:500), CD11b-FITC (BioLegend 101206 at 1:200), CD45-FITC (BioLegend 103108 at 1:500), L/D-GR780 (Tonbo 13-0865 at 1:1,000), IL-12p35–PE (R&D Systems IC2191P at 1:100), isotype control–PE (BioLegend 400408 at 1:100), Siglec-E–PE–Cy7 (BioLegend 677108 at 1:100), isotype control–PE–Cy7 (BioLegend 400522 at 1:100), T-bet–PE–Cy7 (BioLegend 644824 at 1:500), F4/80-PE-Dazzle (BioLegend 123146 at 1:400), Gr1-PerCP (BioLegend 108426 at 1:500), CD4-PerCP (BioLegend 100538 at 1:500), PD-1–APC–Fire750 (BioLegend 135240 at 1:200), and PD-L1–BV421 (BioLegend 124315 at 1:100).

Techniques: Cell Counting, Flow Cytometry, Expressing, Control, MANN-WHITNEY

Journal: The Journal of Clinical Investigation

Article Title: ST8Sia6 overexpression protects pancreatic β cells from spontaneous autoimmune diabetes in nonobese diabetic mice

doi: 10.1172/JCI181207

Figure Lengend Snippet: ( A ) Confirmation of ST8Sia6-myc shutoff after 5 weeks of doxycycline treatment by IHC. Schematic of doxycycline treatment for temporal control of ST8Sia6 expression. Scale bars: 100 μm. ( B ) Diabetes-free incidence after treatment of 15 euglycemic NOD βST and 29 euglycemic littermate mice with doxycycline starting at 8 weeks of age. ( C ) Comparison with disease kinetics of 8-week-old euglycemic non-doxycycline-treated NOD βST ( n = 50) or littermate ( n = 151) mice (subset from ). ( D ) Diabetes-free incidence after treatment of 12 euglycemic NOD βST and 16 euglycemic littermate mice with doxycycline starting at 20 weeks of age. ( E ) Comparison with disease kinetics of 20-week-old euglycemic non-doxycycline-treated NOD βST ( n = 50) or littermate ( n = 115) mice (subset from ). Log-rank Mantel-Cox test was used to analyze statistical differences in diabetes-free incidence of indicated groups. ( F ) Insulitis distribution in pancreas sections of NOD βST mice treated with doxycycline for 5 weeks from 8 weeks or 20 weeks of age, compared with 300-day-old pancreata from NOD βST mice never treated with doxycycline (subset from ). Scoring and analysis as in . n = 69 islets from 12 mice (8-week doxycycline) or 127 islets from 11 mice (20-week doxycycline). ( G and H ) Ratio of T-bet + Tregs (T-bet + FoxP3 + CD4 + ) to Th1 T cells (T-bet + FoxP3 – CD4 + ) ( G ) or to SLECs (T-bet + CD8 + ) ( H ) from islets of 20-week-old mice treated with doxycycline for greater than 5 weeks. ( I ) Quantification of IL-12p35 expression in hybrid macrophages (CD11b + CD11c + F4/80 + Gr1 – ) and cDC2s (CD11b + CD11c + F4/80 – Gr1 – ) from islets of 20-week-old mice treated with doxycycline for greater than 5 weeks. Same gating schemes as in and 5. Error bars represent SD. Mann-Whitney U test was used for statistical analysis between groups.

Article Snippet: The following antibodies were used: CD45-BUV395 (BD 564279 at 1:1,000), L/D-UV450 (Tonbo 13-0868 at 1:1,000), CD11c-BUV615 (BD 751222 at 1:100), CD11b-PacBlue (BioLegend 562894 at 1:200), Ly6C-BV480 (BioLegend 569439 at 1:500), CD8b-BV785 (BioLegend 126631 at 1:1,000), Ly6G-BV785 (BioLegend 127645 at 1:200), CD4-SparkBlue (BioLegend 100494 at 1:1,000), T-bet–RB780 (BD 569089 at 1:100), F4/80-ef660 (Invitrogen 50-4801-82 at 1:200), IFN-γ–APC (BioLegend 505810 at 1:100), isotype control–APC (BD 554686 at 1:100), FoxP3-APC (Tonbo 20-5773 at 1:100), CD11c-BV421 (BioLegend 117330 at 1:500), RORγt-BV421 (BD 562894 at 1:100), MHCII-BV510 (BioLegend 107635 at 1:400), L/D-BV510 (Tonbo 13-0870 at 1:1,000), CD8a-BV510 (BioLegend 100752 at 1:500), TCRβ-BV605 (BioLegend 109241 at 1:200), CD45-BV785 (BioLegend 103149 at 1:500), CD8a-BV785 (BioLegend 100750 at 1:500), CD11b-FITC (BioLegend 101206 at 1:200), CD45-FITC (BioLegend 103108 at 1:500), L/D-GR780 (Tonbo 13-0865 at 1:1,000), IL-12p35–PE (R&D Systems IC2191P at 1:100), isotype control–PE (BioLegend 400408 at 1:100), Siglec-E–PE–Cy7 (BioLegend 677108 at 1:100), isotype control–PE–Cy7 (BioLegend 400522 at 1:100), T-bet–PE–Cy7 (BioLegend 644824 at 1:500), F4/80-PE-Dazzle (BioLegend 123146 at 1:400), Gr1-PerCP (BioLegend 108426 at 1:500), CD4-PerCP (BioLegend 100538 at 1:500), PD-1–APC–Fire750 (BioLegend 135240 at 1:200), and PD-L1–BV421 (BioLegend 124315 at 1:100).

Techniques: Control, Expressing, Comparison, MANN-WHITNEY

Journal: Advanced Science

Article Title: Tumor‐Associated Glycan Exploits Adenosine Receptor 2A Signaling to Facilitate Immune Evasion

doi: 10.1002/advs.202416501

Figure Lengend Snippet: GHCer promotes the differentiation of Treg cells and enhances its immunosuppressive activity. a) GHCer increases Treg differentiation from T cells of healthy donors. Human CD4 + T cells were cultured in a Treg differentiation medium containing anti‐CD3 and anti‐CD28 monoclonal antibodies, IL‐2, and TGF‐β in the presence of 30 µM GHCer, SSEA3Cer, SSEA4Cer, or PBS for 6 days. CD4 + T cells cultured in PBS for 6 days without activation by anti‐CD3 and anti‐CD28 antibodies were used as a control for Treg differentiation. b) GHCer‐induced Treg cells exhibit greater suppression of the proliferation of Tconv cells. Suppressive activities of human CD4 + CD25 + Treg induced in the presence of GHCer or PBS after washing twice were assessed by incubating with CFSE‐labelled, anti‐CD3/CD28 activated CD4 + CD25 − T cells (Tconv) at the indicated ratios. At 72 h, the proliferation of Tconv was analyzed by FACS. The Tconv cells without Treg cells were used as a control for normalization, represented by 100% (a black circle). The percentage of proliferating cells was normalized against the Tconv cells only. c) IL‐35 in the supernatants from GHCer‐induced Treg cells was measured using an IL‐35 Sandwich ELISA. d) IL‐10 production in supernatants from GHCer‐induced Treg cells was measured using an IL‐10 Sandwich ELISA. e) Flow cytometry analysis of LAG3, CTLA‐4, PD‐L1, CD39, and CD73 expression on human Treg cells induced in PBS or GHCer. f) Adenosine concentration in the supernatants of differentiated Treg on day 6. Supernatants from Treg differentiation were filtered through a 0.22 µm filter. Adenosine in the supernatant was measured by colorimetric assay. Data represent three experiments, and values are expressed as means ± SD. Statistical significance was calculated using ANOVA with Tukey correction for multiple comparisons. ∗p < 0.05; ∗∗p < 0.01; ***p < 0.001.

Article Snippet: IL‐10 and IL‐35 concentrations were measured in the supernatants of Treg differentiation cultures after 6 days, as described above, using specific ELISA kits (human: CSB‐ E13126 h, CUSABIO; mouse: 440507, BioLegend) according to the manufacturer's instructions.

Techniques: Activity Assay, Cell Culture, Bioprocessing, Activation Assay, Control, Sandwich ELISA, Flow Cytometry, Expressing, Concentration Assay, Colorimetric Assay