Melanoma Cell Expression of CD200 Inhibits Tumor Formation and Lung Metastasis via Inhibition of Myeloid Cell Functions

CD200 is a cell surface glycoprotein that functions through engaging CD200 receptor on cells of the myeloid lineage and inhibits their functions. Expression of CD200 has been implicated in a variety of human cancer cells including melanoma cells and has been thought to play a protumor role. To investigate the role of cancer cell expression of CD200 in tumor formation and metastasis, we generated CD200-positive and CD200-negative B16 melanoma cells. Subcutaneous injection of CD200-positive B16 melanoma cells inhibited tumor formation and growth in C57BL/6 mice but not in Rag1−/−C57BL/6 mice. However, i.v. injection of CD200-positive B16 melanoma cells dramatically inhibited tumor foci formation in the lungs of both C57BL/6 and Rag1−/−C57BL6 mice. Flow cytometry analysis revealed higher expression of CD200R in Gr1+ myeloid cells in the lung than in peripheral myeloid cells. Depletion of Gr1+ cells or stimulation of CD200R with an agonistic antibody in vivo dramatically inhibited tumor foci formation in the lungs. In addition, treatment with tumor antigen specific CD4 or CD8 T cells or their combination yielded a survival advantage for CD200 positive tumor bearing mice over mice bearing CD200-negative tumors. Taken together, we have revealed a novel role for CD200-CD200R interaction in inhibiting tumor formation and metastasis. Targeting CD200R may represent a novel approach for cancer immunotherapy

CD200-positive tumors are more susceptible to adoptive T cell therapy.

<p><b>A.</b> 5×10⁵ of tumor cells were injected into each Rag1^−/− C57BL6 mouse s.c. Tumor growth (left) and survival of mice (right) were monitored over time. N = 5 mice per group and data shown represents two experiments with similar results. <b>B.</b> 5×10⁵ of tumor cells were injected into each Rag1^−/− C57BL/6 mouse s.c. Mice were then given 5×10⁶/mouse of purified CD8⁺ T cells from OT1 mice i.v. 5 days after tumor cell injection. Five mice per group were used and data shown represents two experiments with similar results. <b>C.</b> 5×10⁵ of tumor cells were injected into each Rag1^−/− C57BL/6 mouse s.c. Mice were then given 5×10⁶/mouse of purified CD4⁺ T cells from OT2 mice i.v. 5 days after tumor cell injection. Five mice per group were used and data shown represents two experiments with similar results. <b>D.</b> 5×10⁵ of tumor cells were injected into each Rag1^−/− C57BL/6 mouse s.c. Mice were then given 5×10⁶/mouse of purified OT1 and 5×10⁶/mouse of purified OT2 T cells i.v. 5 days after tumor cell injection. Five mice per group were used.</p

Tumor expression of CD200 inhibits the functions of Gr1⁺ myeloid cells.

<p>Gr1⁺ cells were isolated from spleens and lungs of C57BL/6 mice. The cells were then co-cultured with tumor cells at a 1∶1 ratio for 48 hours in the presence of 100 ng/ml of LPS (left panel) and 10 µg/ml of anti-CD200 mAb or an IgG2a isotype control mAb (right panel). The supernatants were collected from the co-cultures and were examined for the presence of IL-6, IL-10 and TNF-α. Experiments were repeated at least 3 times with similar results. Data shown are mean ± SEM of 5 mice. Student's two-tailed t test was used for the statistical analysis.</p

CD200 expression on tumor cells inhibits tumor lung metastasis in Rag1^−/− C57BL6 mice.

<p><b>A.</b> 1×10⁵ of B16.OVA.Ctrl or B16.OVA.CD200 cells were injected into each Rag1^−/− C57BL6 mouse s.c. The tumor growth was observed over time. <b>B.</b> Kaplan-Meier survival analysis and log-rank test were used to analyze mice survival. Mice with a tumor burden of 1.5×1.5 cm were sacrificed and counted as dead. Data shown in <b>A</b> and <b>B</b> represent two experiments with similar results. <b>C.</b> Rag^−/− C57BL6 mice were given 1×10⁵ B16.OVA.Ctrl or B16.OVA.CD200 cells per mouse via their tail vein. 20 days later mice were sacrificed and tumor lung metastasis was shown. <b>D.</b> Number of tumor foci in the lungs from mice shown in <b>C</b> were quantified. Error bars represent ± SEM. Student's two-tailed t test was used for the statistical analysis. <b>E.</b> Average weight of lungs from each group of mice shown in <b>C</b>. Error bars represent ± SEM. Student's two-tailed t test was used for the statistical analysis. <b>F.</b> Kaplan-Meier survival curve of mice who received i.v. injection of B16.OVA.Ctrl or B16.OVA.CD200 cells. Data shown in <b>C-F</b> represents two experiments with similar results.</p

CD200 on melanoma cells inhibits tumor formation and lung metastasis.

<p><b>A.</b> Flow cytometery analysis of B16.OVA.Ctrl and B16.OVA.CD200 cells for CD200, MHC class I H2-K^b expression. qRT-PCR was used to examine OVA gene expression. <b>B.</b> 1×10⁵ of B16.OVA.Ctrl or B16.OVA.CD200 cells were injected into each mouse subcutaneously. The tumor growth was observed over time. <b>C.</b> Kaplan-Meier survival analysis and log-rank test were used to analyze mice survival. Mice with a tumor burden of 1.5×1.5 cm were sacrificed and counted as dead. Data shown in <b>B</b> and <b>C</b> represent two experiments with similar results. <b>D.</b> C57BL/6 mice were given 1×10⁵ B16.OVA.Ctrl or B16.OVA.CD200 cells per mouse via their tail vein. 20 days later mice were sacrificed and tumor growth in the lungs were shown. <b>E.</b> Average number of tumor foci in the lungs from each group of mice shown in <b>D</b>. Error bars represent Mean ± SEM. Student's two-tailed t test was used for the statistical analysis. <b>F.</b> Average weight of lungs from each group of mice shown in <b>D</b>. Data shown in <b>D–F</b> represent two experiments with similar results.</p

Gr1⁺ myeloid cells express CD200R and mediate lung metastasis.

<p><b>A.</b> Flow cytometry analysis of CD200R expression in mononuclear cells from spleens and lungs. Cells were prepared from spleen and lung of normal C57BL/6 mice and were stained for CD11b, Gr1 and CD200R. The experiment was confirmed in three independent experiments with similar results. <b>B.</b> Two groups of C57BL/6 mice were injected with B16.OVA.Ctrl cells (1×10⁵ cells/mouse) via their tail vein. Mice were treated with either 250 µg/mouse of anti-Gr-1 (RB6-8C5, BioXcell) or an isotype-matched control antibody (anti-KLH, BioXcell). An untreated group of mice that received 1×10⁵/mouse of B16.OVA.CD200 cells were used for the comparison. 21 days after tumor cell injection, tumor lung metastasis was examined. <b>C.</b> Lung weight in groups of mice shown in <b>B</b>. Error bars represent ± SEM. Student's two-tailed t test was used for the statistical analysis. <b>D.</b> Average number of tumor foci in the lungs from each group of mice shown in <b>B</b>. Error bars represent ± SEM. Student's two-tailed t test was used for the statistical analysis. <b>E.</b> Flow cytometry analysis of lung mononuclear cells from anti-Gr-1 treated and control antibody treated mice. C57BL6 mice received either anti-Gr-1 (250 µg×3 doses per mouse) or isotype-matched control antibody i.p. Data shown represent three experiments with similar results.</p