Figure 4

<p>DCs generated from MARCO^-/- mice respond to LPS. Cytokine production by DCs generated from the bone marrow of WT or MARCO^-/- mice. The production of (A) IL12p70, (B) IL-10 and (C) TNF-α was measured. Experiments were repeated two times.</p

Figure 1

<p>Development of a MARCO^-/- mouse. A) Schematic of the construction of MARCO ^-/- allele for the generation of MARCO^-/- mice. B) PCR analysis of MARCO gene expression in wild-type (WT) and knockout (K/O) mice.</p

Figure 5

<p>DCs generated from MARCO^-/- mice demonstrate enhanced capacity for <i>in vitro</i> migration. WT and MARCO^-/- DCs were unpulsed or pulsed with B16 tumor lysate. Cells were added to the upper chamber of a transwell plate. Recombinant CCL-21 (100 ng/ml) was added to the lower chamber. After five hours, cells in the lower chamber were collected and analyzed by flow cytometry. The percent migration of IA^b and CD11c double positive cells was calculated.</p

Figure 3

<p>No difference in immune cell subsets or expression markers in WT and MARCO^-/- mice. Comparison of cell subsets in the (A) spleens and (B) lymph nodes of WT and MARCO^-/- mice. Comparison of cell surface markers on (C) unpulsed bone marrow-derived DCs and (D) DCs pulsed with LPS. Data shown is the average of three mice.</p

Figure 8

<p>Mice were injected s.c. with B16 cells on day 0. Mice were immunized s.c. with 1x10⁶ DCs on days 3, 6, and 9 with B16 tumor lysate pulsed WT or MARCO^-/- DCs or PBS. (A) Tumor growth and (B) survival were measured. n=7-8 mice per group. Experiment was performed two times with similar results.</p

Figure 7

<p>WT mice were vaccinated with lysate pulsed-DCs derived from the bone marrow of WT or MARCO^-/- mice on days 0, 3, and 6. On day 13, spleens were collected and T cells were purified using a T cell enrichment column. T cells were incubated with irradiated WT or MARCO ^-/- DCs at a ratio of 10 T cells : 1 DC. After 48 hours, supernatants were collected and IFN-γ production was measured by ELISA. Each data shows the average of three mice.</p

Figure 2

<p>No MARCO expression is measured in DCs generated from the bone marrow of MARCO^-/- mice. A) RT-PCR analysis of MARCO expression in DCs generated from the bone marrow of wild-type or knockout mice. DCs were unpulsed, pulsed with B16 tumor lysate, or pulsed with 1 µg/ml LPS for 24 hours. Amounts of mRNA were adjusted to give comparable GAPDH signals. B) DCs from wild-type or knockout mice were pulsed for 24 hours with PKH26-red labeled tumor lysate (red). DCs were stained for MARCO expression (green) and DAPI (blue).</p

Figure 6

<p>DCs generated from MARCO^-/- mice demonstrate enhanced capacity for migration <i>in vivo</i>. WT and MARCO^-/- DCs pulsed with B16 tumor lysate were stained with PKH26 red and injected s.c. to WT mice. After 60 hours, the draining lymph node was collected. (A) PKH26-red⁺ cells were visualized by microscopy (n=4 per group). (B) Lymph nodes were collected and dissociated into a single cell suspension. PKH26-red⁺ cells were quantified by flow cytometry. Data show as mean ± SEM of three mice per group.</p

MC-38 and B16 TIL are tumor specific.

A& B) Bar graph represents mean+ SD of CD8+ and CD4+ T cells infiltrating tumors (n = 8); C& D) MC-38 or B16 TIL were co-cultured with specific or irrelevant tumor cells for 48 hours. IFN-γ production was measured in culture supernatants by ELISA.</p

Expression of immune checkpoint receptors on MC-38 and B16 TIL.

<p>A&B) Flow cytometry analysis of PD-1, CTLA-4, BTLA, and LAG-3 expression on MC-38 (A) and B16 (B) TIL; C) PD-L1 expression on MC-38 tumor cells; D) fresh or cultured MC-38 TIL were co-cultured with MC-38 or irrelevant B16 tumor cells and incubated for 48 hours at 37°C. The number of IFN-γ producing cells in response to stimulation was evaluated in an ELISPOT assay. The number of spots was counted in triplicates and calculated using an automatic ELISPOT counter; E) TIL was cultured with ⁵¹Cr labeled MC-38 or B16 tumor cells at 50:1 and 25:1 effector to target ratios. After 5 hours, the percentage of specific ⁵¹Cr release was determined by the following equation: (experimental cpm − spontaneous cpm)/ (total cpm incorporated − spontaneous cpm) × 100. All determinations were done in triplicate, and the SE of all assays was calculated and was typically 5% of the mean or less.</p