Bone marrow derived CX₃CR1+ cells are drivers of tumor angiogenesis.

<p>(<b>A</b>) FACS analysis of <i>cx₃cr1</i>^gfp cells purified by FACSAria Cell-Sorting System from BM of CCR2+ CD45.1 donor mice before their transfer to CCR2−/− mice (<b>B</b>) Imaging (IVIS) of the primary tumor on day 60, as recorded by the IVIS camera using – luciferin filter (recording luciferase activity of the cancer cells) as follows: CCR2+/+ C57BL/6 mice (WT) (a), CCR2−/− mice (b), CCR2−/− transplanted with GFP+ cells from BM of CCR2+ donor mice (c). All photos show a representative mouse per group (1 of 6 mice). (<b>C</b>) Computerized CCCD analysis of six mice per group. Results of six mice per group are shown as total flux (p/s ×10⁴) ±SE. * Indicates p<0.001. (<b>D</b>) Representative primary tumor sections were then analyzed by to immunostaining using different colors for CD45.1 (red color, only transferred <i>cx₃cr1</i>^gfp cells) and CD11b+ (green). (<b>E</b>) Analysis of 60 sections from six mice per group for the relative number of CD11b+ cells at tumor sections from each group, and of CD45.1 cells following cell transfer * Indicates p<0.001.</p

Loss of Runx3 abrogates functional activities of CD4⁺ DC.

<p>(A) CD4⁺ T cell priming in DC-Runx3^Δ mice <i>in </i><i>vivo</i>. CFSE-labeled OVA-specific OT-I CD8⁺ T cells or OT-II CD4⁺ T cells were introduced to DC-Runx3^Δ or WT control littermate mice followed by immunization with OVA. Shown are flow cytometric analyses of transgenic T cell grafts recovered from immunized recipient mice. Proliferation of OT-I CD8⁺ T cells (top) and OT-II CD4⁺ T cells (bottom) cells in DC-Runx3^+/+ (red) or DC-Runx3^Δ mice (blue) compared to PBS immunized mice (green) and non-labeled cells (brown). Shown representative of two independent experiments with two to three mice per group. (B) Total cell number of OT-II CD4+ T cell Proliferation quantification. PBS-immunized mice T cell proliferation is set as one (n=5). ***P < 0.001 (Student’s two-tailed t test). (C) Runx3 binding-pattern on MHCII genomic loci. Shown are ChIP-Seq tracing wiggle files uploaded to UCSC Genome Browser mm9 genome assembly and the normalized read coverage of whole cell extract (top) and Runx3 ChIP (bottom). The position of MACS2-identified Runx3 peaks is indicated by rectangles. For each panel the scale bar and diagram of gene exon structure are presented on top or bottom, respectively. (D and E) The capacity of splenic DC subtypes to phagocytose latex beads was analyzed in DC-Runx3Δ (blue) and WT (red) littermate mice 18h after i.v. injection of 1010 FITC-labeled 0.5μm latex beads. Shown are histograms of FITC-fluorescence in CD4+, CD8+ and DN DC subsets (D) or in Esamhi and Esamlow DC subsets (E). Shown representative of two independent experiments with two mice per group with the same finding. (F) The capacity of splenic DC to phagocytose latex beads was further analyzed using mixed BM chimera mice 18h after i.v. injection of 1010 FITC-labeled 0.5μm latex beads. Shown are histograms of FITC-fluorescence in CD4+ and Esamhi DC subsets gated on WT CD45.1 (red) or DC-Runx3Δ CD45.2 (red). Shown representative of two independent experiments with two mice per group.</p

Immunization with PS4-BMDC induces short-term anti-PS4 IgM and long-term anti-PS4 IgG Ab.

<p>Mice were immunized with PS4-BMDC either IP or IFP. Five days and three weeks after immunization, the mice were bled, and serum samples were tested for the presence of anti-PS4 IgM and IgG Abs by ELISA. p<0.05 tested by Wilcoxon non-parametric test (n = 10–25).</p

Challenge bacteria are trapped in the peritoneum of vaccinated mice and do not enter the blood or kill the mice; the vaccinated mice kill the bacteria after 18–24 hours.

<p>Mice were immunized IFP with PS4-BMDC or with un-pulsed BMDC. Three weeks later, the mice were challenged with Pn4 bacteria (100,000×LD₅₀) IP. The mice were bled 0.5, 2, 4, 8, 18 and 24 hours post-challenge, and peritoneal fluids were collected. The peritoneal fluid (A) and the blood samples (B) were plated on agar overnight and CFU were then counted. Note: x means that the mice died and the samples could not be collected.</p

Analysis of Runx3 occupied genomic regions in splenic CD4⁺ DC.

<p>(A) Venn diagram summarizing the overlap between Runx3 ChIP-seq bound genes and differentially expressed genes in splenic CD4⁺ DC. (B) Runx3 binding-pattern on five genomic loci differentially expressed in CD4⁺ DC. Shown are ChIP-seq Runx3 peaks and the normalized read coverage of whole cell extract (top) and Runx3 ChIP (bottom) uploaded to UCSC Genome Browser mm9 genome assembly. The position of MACS2-identifird Runx3 peaks is indicated by rectangles. For each panel the scale bar and diagram of gene exon structure are presented on top or bottom, respectively. (C) Enriched RUNX and zDC TF motif among Runx3 bound regions. Motifs were identified <i>de-novo</i> using the MEME-ChIP software.</p

Vaccination with type-specific PS-pulsed BMDC induces type-specific resistance: Only intact BMDC are effective.

<p>(A) LPS-stimulated (LPS) and non-stimulated (−) BMDC were pulsed with PS4 and injected once IP or IFP to C57BL/6 mice (5×10⁶ cells per mouse). Three-weeks later, the mice were challenged with Pn4 (100,000×LD₅₀ CFU) IP: *p<0.05 +LPS+PS4 compared to −LPS+PS4, +LPS−PS4 and −LPS−PS4, n = 25. (B) LPS-stimulated or non-stimulated BMDC were pulsed with PS4 or PS3 and injected IFP to C57BL/6 mice. Three weeks later, the mice were challenged with Pn4 or Pn3 or with a mixture of both Pn4 and Pn3 IP: *p<0.05 +LPS+PS4, Pn4 compared to +LPS+PS3, Pn4, +LPS−PS, Pn4 and to all groups of Pn4+Pn3. **p<0.05 +LPS+PS3, Pn3 compared to +LPS+PS4, Pn3, −LPS−PS, Pn3 and to all groups of Pn4+Pn3, n = 15. (C) LPS-stimulated, PS4-pulsed BMDC were fixed with glutaraldehyde 1%, lysed with DDW, sonicated (sonic.) or were not treated (untreat.). The cells were then injected into recipient mice. Control LPS-stimulated and PS4-pulsed splenic (sple.) T and B cells or soluble LPS+PS4 were injected to other groups of mice. After three weeks, the mice were challenged with Pn4 (100,000×LD₅₀): *p<0.05 untreated BMDC compared to all groups, n = 10.</p

PS4 on the surface of BMSC is transferred to endogenous cells in the peritoneum.

<p>BMDC derived from CD45.2 mice (donors) were stimulated with LPS and pulsed with PS4-Alexa633. The cells were injected IP into CD45.1 mice (recipients). After 24 hours, the mice were sacrificed, cold PBS was injected IP and the peritoneal fluid was collected. The collected cells were stained with anti-CD45.1-PE and anti-CD45.2-FITC, and were analyzed by flow cytometry. The numbers indicate the percentage of labeled cells.</p