Journal: eLife

Article Title: IL-2/JES6-1 mAb complexes dramatically increase sensitivity to LPS through IFN-γ production by CD25 + Foxp3 - T cells

doi: 10.7554/eLife.62432

Figure Lengend Snippet: Purified CD8 + and CD4 + T cells from OT-I/Ly5.1 and OT-II/Ly5.1 mice (2 and 4 × 10 6 /mouse respectively), were adoptively transferred (AT) to C56BL/6 mice. One day later, mice were i.p. injected with PBS (Control), with both OT-I and OT-II specific peptides plus polyI:C (75 µg/mice) or with the latter plus αIFN-γ mAb (250 µg). Expansion of AT T cells was determined 3 d post stimulation by flow cytometry. Dot plots showing one representative mouse ( A ) out of two mice analysed by flow cytometry and a bar graph showing mean ± SD ( B ) are presented. ( C ) Three groups of C56BL/6 mice described above and one control group (no AT) were challenged with LPS (50% of MNLD). ( D ) IFN-γ -/- and normal C56BL/6 mice (IFN-γ +/+ ) were treated with IL-2/JES6 and challenged with LPS (50% of MNLD) as shown in . IFN-γ -/- C56BL/6 mice challenged with the same dose of LPS were used as the control. ( E ) Data pooled from three independent experiments (n = 13–16 technical replicates) described in D showing body temperature of mice 8 h after LPS challenge. ( F ) Scheme showing the proposed mechanism of how IL-2/JES6 induces LPS hyperreactivity. Experiments A-D were done at least twice with similar results; n = 2–6 technical replicates. Data were analysed using unpaired two-tailed Student’s t-test. Significant differences to control are shown (* p ≤ 0.05; *** p ≤ 0.001). Figure 6—source data 1. Source data for , panels B-E.

Article Snippet: C57BL/6 mice were injected i.p. with PBS, OT-I plus OT-II peptides (10 and 50 μg/mouse, respectively; MBL International, Woburn, Massachusetts, USA; or Genscript, Piscataway, New Jersey, USA, respectively) plus polyI:C (75 μg/mouse; Sigma-Aldrich, St. Louis, Missouri, USA) or with the latter plus αIFN-γ mAb (250 μg/mouse; XMG1.2; BioXcell, Lebanon, New Hampshire, USA).

Techniques: Purification, Injection, Control, Flow Cytometry, Two Tailed Test

Journal: eLife

Article Title: IL-2/JES6-1 mAb complexes dramatically increase sensitivity to LPS through IFN-γ production by CD25 + Foxp3 - T cells

doi: 10.7554/eLife.62432

Figure Lengend Snippet: Purified CD8 + and CD4 + T cells from OT-I/Ly5.1 and OT-II/Ly5.1 mice (2 and 4 × 10 6 /mouse respectively), were adoptively transferred (AT) to C56BL/6 mice. One day later, mice were i.p. injected with PBS (Control), with both OT-I and OT-II specific peptides plus polyI:C (75 µg/mice) or with the latter plus αIFN-γ mAb (250 µg). Expansion of AT T cells was determined 3 d post stimulation by flow cytometry. Dot plots showing one representative mouse ( A ) out of two mice analysed by flow cytometry and a bar graph showing mean ± SD ( B ) are presented. ( C ) Three groups of C56BL/6 mice described above and one control group (no AT) were challenged with LPS (50% of MNLD). ( D ) IFN-γ -/- and normal C56BL/6 mice (IFN-γ +/+ ) were treated with IL-2/JES6 and challenged with LPS (50% of MNLD) as shown in . IFN-γ -/- C56BL/6 mice challenged with the same dose of LPS were used as the control. ( E ) Data pooled from three independent experiments (n = 13–16 technical replicates) described in D showing body temperature of mice 8 h after LPS challenge. ( F ) Scheme showing the proposed mechanism of how IL-2/JES6 induces LPS hyperreactivity. Experiments A-D were done at least twice with similar results; n = 2–6 technical replicates. Data were analysed using unpaired two-tailed Student’s t-test. Significant differences to control are shown (* p ≤ 0.05; *** p ≤ 0.001). Figure 6—source data 1. Source data for , panels B-E.

Article Snippet: C57BL/6 mice were injected i.p. with PBS, OT-I plus OT-II peptides (10 and 50 μg/mouse, respectively; MBL International, Woburn, Massachusetts, USA; or Genscript, Piscataway, New Jersey, USA, respectively) plus polyI:C (75 μg/mouse; Sigma-Aldrich, St. Louis, Missouri, USA) or with the latter plus αIFN-γ mAb (250 μg/mouse; XMG1.2; BioXcell, Lebanon, New Hampshire, USA).

Techniques: Purification, Injection, Control, Flow Cytometry, Two Tailed Test

Journal: Nature Communications

Article Title: Gut microbiota dependent anti-tumor immunity restricts melanoma growth in Rnf5 −/ − mice

doi: 10.1038/s41467-019-09525-y

Figure Lengend Snippet: IEC of Rnf5 −/− mice activate immune response and change anti-microbial peptide (AMP) expression. a Villi length and crypt depth calculated from H&E-stained sections of intestines from WT or Rnf5 −/− mice (WT, n = 30; Rnf5 −/− , n = 32). b qRT-PCR analysis of AMPs mRNA levels in IECs from small intestine of naive WT or Rnf5 −/− mice ( n = 6). c Representative images (left) and quantification (right) of cleaved caspase-3 immunostained small intestine organoids from tumor-bearing WT or Rnf5 −/− mice ( n = 3). Scale bar = 100μm. Graph shows percentage of cleaved caspase-3 + cells per immunostained organoid ( n = 12 fields). d Intracellular IFN-γ and TNF-α staining of p14 CD8 + T cells incubated for 72 h with 2 μg/ml GP33 peptide recognized by the TCR of P14 and bone marrow-derived dendritic cells (BMDCs) that were incubated with medium alone (no stimulation) or with conditioned medium (CM) from shControl or shRNF5 MODE-K cells. e Representative images (left) and quantification (right) of CD11c + cell immunostaining in the small intestine of WT or Rnf5 −/− mice on day 24 after injection of YUMM1.5 cells. Scale bar = 50μm ( n = 4). f Frequencies of total DCs and pDCs in Peyer’s patches from WT and Rnf5 −/− mice on day 10 after YUMM1.5 cell injection ( n = 6). g 10 days after tumor injection, DCs from GALT, dLN, and ndLN were isolated, pooled per group ( n = 10 mice/group), and were incubated with OT-1 CD8 + T cells stimulated with 2 μg/ml OVA peptide (SINFEKL). Intracellular IFN-γ and TNF-α of OT-1 CD8 + T cells were detected. Data are representative of three independent experiments ( b , d ) and two independent experiments ( a , c , e , f , g ) ≥3 mice per group. Graphs show the mean ± s.e.m. * P < 0.05, ** P < 0.005, *** P < 0.001, **** P < 0.0001 by two-tailed t test or Mann–Whitney U test ( a , b , c , e , f ) or one-way ANOVA with Tukey’s ( d ) correction for multiple comparisons

Article Snippet: Cells were then mixed 1:1 with the BMDCs and incubated for 72 h pulsed with 2 μg/ml of OVA peptide (SINFEKL) (AnaSpec) or GP33 peptide (AnaSpec).

Techniques: Expressing, Staining, Quantitative RT-PCR, Incubation, Derivative Assay, Immunostaining, Injection, Isolation, Two Tailed Test, MANN-WHITNEY