Fig 1: Impaired trafficking of dermal DCs to draining LNs in CD44-deficient mice.(A, B, C) Recovery of endogenous DCs from draining inguinal and axillary LNs, 24 h after topical application of oxazolone and FITC, as measured by flow cytometry. (A) Gating strategy for data in Fig 6 is shown, where live CD45+ CD11c+ FITC+ cells were further gated according to expression of MHC class II and either Langerin, EpCAM, CD11b or F4/80, with representative contour and dot plots, showing percentage of cells in each gate (A). (B, C) Bar and whiskers plots showing percentage of LN cells that are FITC+ CD11c+ (B) and absolute numbers of CD11c+ MHC class II+ FITC+ cells in LN expressing subset markers Langerin, EpCAM, CD11b, and F4/80 (C). *P < 0.05, **P < 0.01, Mann–Whitney U-test. Data represent the mean (center bar) ± s.e.m. (whiskers) (n = 5 mice), one representative experiment of three.
Fig 2: Expression of PD-L1 and CSF1R in mouse myeloid cells and human blood CD11c+ cells after Lalistat2 treatment.(A) LAL enzymatic activity in HD1A myeloid cells after incubation with 10 µM, 50 µM, 100 µM, and 200 µM Lalistat2 or DMSO (S) for 72 hours. (B) Murine HD1A myeloid cells were incubated with 10 µM, 50 µM, 100 µM, and 200 µM Lalistat2 or DMSO (S) for 72 hours. Percentages of CD11c+, PD-L1+, and CSF1R+ cells in HD1A myeloid cells were analyzed by flow cytometry. (C) Expression of PD-L1 in HD1A myeloid cells after Lalistat2 or DMSO treatment for 72 hours by Western blot analysis. Representative blots are shown. (D) Human white blood cells from healthy individuals were incubated with 10 µM Lalistat2 (L) or DMSO (S) for 24 hours. Percentages of CD11c+ cells in the whole white blood cells were analyzed by flow cytometry. (E) Percentages of PD-L1+ and CSF1R+ cells in blood CD11c+ cells of healthy individuals treated with Lalistat2 (L) versus DMSO (S). Data are expressed as mean ± SD. Experiments were independently repeated, n = 4 for A and B, n = 3 for C, n = 6 for D, n = 5 for E. *P < 0.05, **P < 0.01, 1-way ANOVA.
Fig 3: P2X7 in DCs favor the membrane transfer from ACs to MutuDC cells(A and B) CellMask transference from H-ACs to the surface of the BM-DCs (A) and P2X7-KO BM-DCs (B) was analyzed to assess the role of DC P2X7 in membrane transference. Graphs show the fluorescence intensity on the surface of BM-DCs associated with ACs (red) and BM-DCs not associated with ACs (control, open black).(C) Graph shows the slope derived from each fluorescence intensity curve.(D) P2X7 expressing or parental HEK293 cells were loaded with CellTracker Orange (CMRA), and H-ACs were obtained by nutrient deprivation. The orthogonal projection of H-ACs shows the whole-cell distribution of fluorescence. ACs are shown as white pseudocolor.(E and F) Representative histograms and (F) percentage of BM-DCs which have phagocytosed CMRA labeled ACs. H-ACs and H-ACs-P2X7 phagocytosed by CD11c+ 7-AAD+ WT DCs (Upper panel) or CD11c+ 7-AAD+ P2X7-KO DCs (Lower panel). Phagocytosis of fluorescence latex beads was the positive control. The quantification of three independent experiments was graphed and represented as the mean ± SEM. Results were analyzed using non-parametric Mann-Whitney test, ∗ represent significant differences p < 0.05 and ∗∗ p < 0.01, treatments compared against control, n/s; not significant.
Fig 4: CCL21 blockade abolishes the RT efficiency and anti-tumor immunity enhanced by VEGF-C overexpression.a Left panels, Lyve1 and CCL21 staining of MLVs in mice bearing vector-GL261 or VEGF-C-GL261 tumors in the striatum with RT. Right panels, quantification of the coverage percentage of Lyve1 or CCL21, and the LV diameter (n = 8). b Monitoring and treatment scheme. CCL21 was blocked on days 16, 18, and 20 after inoculation. c Survival of mice with striatal VEGF-C-GL261 tumor injection treated with RT in the presence or absence of anti-CCL21 antibody (n = 18). d Representative T2-weighted single brain slices from mice in IgG isotype or anti-CCL21 groups (triangles indicate tumors). e Tumor volume quantified from MRI images on day 22 after inoculation of mice from IgG isotype or anti-CCL21 groups (n = 12). f, g Representative flow cytometry plots of CD8+ Ki67+ T cells as percentages of the total CD8+ T cells (f), and CD4+ Foxp3+ T cells as percentages of the total CD4+ T cells (g) in CLNs (left) and quantification (right) in tumors and CLNs from IgG isotype or anti-CCL21 groups on day 22 after inoculation (n = 8). h Ratios of CD8+ Ki67+ T cells to CD4+ Foxp3+ T cells in tumors and CLNs from IgG isotype or anti-CCL21 groups (n = 8). i Left panel, representative flow cytometry dot plots of DC trafficking from GL261 tumors to CLNs of mice in IgG isotype or anti-CCL21 groups shown by the quantity of CD11c+ MHCII+ FITC+ cells in the CLNs 24 h after intratumoral injection of FITC-labeled latex beads. Right panel, quantification of FITC+ DCs as a fraction of all MHC II+ CD11c+ cells in the CLNs of mice from the IgG isotype or anti-CCL21 group (n = 6). Data are presented as means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001; log-rank (Mantel–Cox) test (b); Student’s t-test (a, c–i). Data are from at least three (a, c, f–i) or two (d, e) independent experiments.
Fig 5: TSC1 deficiency in T cells decreases the survival of cardiac allografts and enhances CD8+ T cell accumulation in allografts. (A) Fully major histocompatibility complex (MHC)-mismatched cardiac allografts from BALB/c mice were transplanted into WT or TSC1-/- mice, and the survival of cardiac allografts was analyzed. Log-rank test; ***P < 0.005, n = 5-7 per group. (B) We determined the allograft rejection using histology with hematoxylin and eosin staining (B). Scale bar: 200 µm. We harvested some cardiac allografts on day 4 post-transplantation and analyzed the cell number of the infiltrated CD4+ and CD8+ (C and D), CD4+Foxp3+ (E), CD45+Gr-1+ (F), CD11b+ CD11c+ (G), and CD11b+ F4/80+ (H) cells. Student’s t-test for (D, E, F, and H) and Mann-Whitney U test for (G); *P < 0.05, **P < 0.01, n = 4 per group.
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