Fig 1: Effects of tumor-derived G-CSF on the PALN and 18F-FDG-PET/CT in animal models of cervical cancer.a Representative image of G-CSF mRNA expression in ME180-Control or ME180-GCSF cells as evaluated by RT-PCR. b Representative G-CSF staining in ME180-Control cells or ME180-GCSF cells-derived tumors. c WBC/granulocyte counts of ME180-Control-derived tumor-bearing rats and ME180-GCSF-derived tumor-bearing rats (three rats per group). Rats were subcutaneously inoculated with ME180-Control or ME180-GCSF cells. Three weeks after inoculation, their subcutaneous tumors or peripheral blood samples were collected for analyses. d 18F-FDG-PET/CT scan of ME180-Control-derived tumor- and ME180-GCSF-derived tumor-bearing rats. Rats were subcutaneously inoculated with ME180-Control or ME180-GCSF cells. Three weeks after inoculation, 18F-FDG-PET/CT was performed. ME180-GCSF-derived tumor-bearing rats showed significant 18F-FDG-uptake in the PALN (circle). e PALNs resected after 18F-FDG-PET/CT. f Representative pathological findings from the PALNs resected after 18F-FDG-PET/CT (hematoxylin and eosin). Both of the images contain no malignant cells. g Effects of tumor-derived G-CSF on the induction of MDSC in rat models of cervical cancer. CD11b/c+HIS48+ cell populations detected in the peripheral blood and lymph nodes. Rats were subcutaneously inoculated with ME180-Control or ME180-GCSF cells. Three weeks after inoculation, the number of MDSC was evaluated by flow cytometry (three rats per group). h Effects of tumor-derived G-CSF and anti-Gr-1 antibody on PALN in mice models of cervical cancer. i Effects of anti-Gr-1 antibody on MDSC induction in mice models of cervical cancer. CD11b+Gr1+ cell populations detected in the peripheral blood and lymph nodes. h, i ME180-Control- or ME180-GCSF-derived tumor-bearing mice were treated with either control IgG or anti-Gr-1 antibody for three weeks (five mice per group). Then, the number of MDSC was evaluated by flow cytometry (five mice per group). j Ability of CD11b+Gr-1+ cells to suppress CD8+ T cell assessed by T-cell proliferation assay. CD11b+ Gr-1+ cells (MDSC) were isolated from spleen of ME180-GCSF-derived tumor-bearing mouse. CD8+ T cells (2 × 105 cells/well) were isolated from spleen of Balb/c mice and co-cultured with MDSC at indicated ratios. Cells were incubated for 72 h, after which BrdU was added for an additional 24 h. T cell proliferation was determined by BrdU incorporation (n = 6). Error bars indicate mean ± SD. Statistical significance was assessed using two-sided Welch t test. bp, base pairs. Scale bar, 50 μm. Source data are provided as a Source Data file.
Fig 2: Tregs modulate pro−inflammatory cytokines. In vitro CTL assays were carried out as described previously in Figure 1 using a 1:5 ratio of CD8+:Tregs. After 18 h of co-culture, CD8+ T−cells were analyzed using flow cytometry. (A) Representative contour plots show production of the pro−inflammatory cytokines IFN-γ as well as TNF. (B) Bar graphs show the frequency of IFN-γ− and TNF−producing CD8+ T−cells. Data shown are representative of three experiments using 3 wells/group/experiment and expressed as mean ± SE. rAd-OVA-primed OT-1 mice (n = 2/experiment) and IL−2/anti−IL−2−injected C57BL/6 mice (n = 4/experiment). ** p < 0.01, *** p < 0.001.
Fig 3: Role of ICAP‐1 during SP CD8+ thymocyte positive selection. Analysis of the multistep maturation stages encompassing positive selection. Cells were first gated on CCR7+CD3+, and the SM, M1 and M2 subpopulations were defined according to CD69 and H2‐Kb expression. Each of these populations was subsequently analyzed for the CD4 and CD8 levels. A representative dot plot result is shown (A), and data quantification of CD4+ and CD8+ proportions in the SM, M1 and M2 subsets is displayed in (B). (C) Cells were gated on CD3 and CD69 to mark the fractions 1 to 4, and each fraction was analyzed for CD4 and CD8 expression. Representative dot plots are shown, and panel (D) displays quantification of the data of CD4+ and CD8+ frequencies in fractions 1 to 4. (E) Analysis of CD3 expression on SP CD4+ and CD8+ thymocytes from ICAP‐1‐null and control mice was assessed by flow cytometry. Panels B, D, and E (bottom) show pooled data from two to four independent experiments (*** p < 0.001; ** p < 0.01; * p < 0.05).
Fig 4: CD8+ Treg cells resulted in enhanced mortality and increased virus load in the lung. (A) Splenic CD8+CD25+ T cells were isolated on day 6 from Foxp3-GFPtg mice infected with H5N1 virus. The cells were transferred into BALB/c mice (1 × 105 or 5 × 105 CD8+CD25+ T cells per mouse) and the mice (n = 10 mice per group) were then immediately challenged with H5N1 virus. (B) Survival of mice was monitored from day 7 to 16 after virus infection. Log-rank test for comparisons of survival curves between control mice and CD8+ Treg cells recipient mice; **p < 0.01. ***p < 0.001, log-rank test. (C) Lung viral load was assayed on day 6 after virus infection (n = 5 mice per group). (D) Total RNA was extracted from lung for real-time PCR for IFN-β and Mx-1. Data are shown as mean + SEM and are pooled from three independent experiments. n.s., p > 0.05. *p < 0.05. **p < 0.01. ***p < 0.001, unpaired two-tailed t-test.
Fig 5: CD8+CD25− T-cell proliferation was inhibited by CD8+ Treg cells through IL-10 in vitro. (A) CD8+CD25+ T cells, CD8+CD25− T cells, CD11c+ cells were isolated on day 6 from spleens of H5N1-infected C57BL/6 mice. CFSE-stained CD8+CD25− T cells were stimulated to proliferate in vitro; 2 × 105 CD8+ CD25− T cells and 5 × 104 CD11c+ cells were stimulated with 10 μg/mL NP366–374 peptide in the presence of different numbers of CD8+ Treg cells for 5 days. (B) CD4+CD25+ T cells, CD8+CD25+ T cells, CD8+CD25− T cells, and CD11c+ cells were isolated from spleens of C57BL/6 mice on day 6 after infection with H5N1 virus. CFSE-stained CD8+CD25− T cells were stimulated to proliferate in vitro; 2 × 105 CD8+ CD25− T cells and 5 × 104 CD11c+ cells were stimulated with 10 μg/mL NP366–374 peptide in the presence of different numbers of CD4+ or CD8+ Treg cells for 5 days. (C) 2 × 105 CD8+ CD25− T cells and 5 × 104 CD11c+ cells were stimulated with 10 μg/mL NP366–374 peptide in the presence of 1 × 105 CD8+ Treg cells in transwell plates for 5 days. (D) T-cell proliferation was done with 50 μg/mL anti-IL-10 mAb or isotype control antibodies in the wells. The number of CD8+ Treg cells used in the system (C and D) was 1 × 105. (E) CD8+CD25+ T cells and CD11c+ cells from C57BL/6 mice and CD8+CD25− T cells from DNIL-10R mice were isolated 6 days after infection with H5N1 virus and T-cell proliferation assays were performed. (A–E) Data shown are representative of at least three independent experiments with four mice per group.
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