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

Journal: Nature communications

Article Title: p38 MAPK-inhibited dendritic cells induce superior antitumor immune responses and overcome regulatory T cell-mediated immunosuppression

doi: 10.1038/ncomms5229

Figure Lengend Snippet: Antitumor activity of DC immunization and Treg depletion using anti-CD25 antibody. (a–c) Comparison of antitumor activity against pre-established tumors. C57BL/6 mice (5 per group) were inoculated subcutaneously with B16-OVA (a,b) or B16 (c) tumor cells (3 × 105) and 3 days later were immunized with 1 × 106 OT-I peptide-loaded (a), OT-II peptide-loaded (b) or TRP2 peptide-loaded (c) mDCs or mSBDCs twice at a weekly interval. Arrows represent the timing of DC-therapy. Anti-CD25 antibodies (50 μg/mouse) were administered intraperitoneally into some groups of mice 3 days before immunization. Error bars represent s.d. Representative results from one of three repeated experiments are shown. (d) IFN-γ ICS of splenocytes from tumor-bearing mice 10 days after second immunization was performed after restimulation with the immunizing peptides overnight in vitro. Numbers represent percentages of IFN-γ secreting lymphocytes gated on CD4+ or CD8+ T cells from one representative experiment of three performed. (e) In vivo CTL assay. CFSElo (left population in gates, non-target) and CFSEhi (right population in gates, target) cells were pulsed with control peptide and OT-I peptide, respectively, and were injected into mice 7 days after OT-I peptide-pulsed DC immunization. Splenocytes were collected 6 hours later for detection of CFSE-labeled cells. Numbers represents percentages of specific killing. Results are representative of one of three independent experiments. (f) Pooled data (n = 3) for in vivo CTL assay. Splenocytes were collected 36 hours later for detection of CFSE-labeled cells in OT-II and TRP2 peptide-loaded DC immunization. Error bars represent s.d. P values were calculated with Student’s t-test.

Article Snippet: MHC class II–restricted TRP1 (SGHNCGTCRPGWRGAACNQKILTVR), H2-K b –restricted TRP2 (SVYDFFVWL), H2-K b –restricted OT-I (SIINFEKL), and MHC class II–restricted OT-II (ISQAVHAAHAEINEAGR) peptides were purchased from GenScript.

Techniques: Activity Assay, In Vitro, In Vivo, CTL Assay, Injection, Labeling