Loss of CXCR4 in Myeloid Cells Enhances Antitumor Immunity and Reduces Melanoma Growth through NK Cell and FASL Mechanisms

CXCR4^MyeΔ/Δ mice acquire antitumor immunity dependent upon NK cells. A, Depletion of NK cells attenuated antitumor immunity of CXCR4^MyeΔ/Δ mice. CXCR4^MyeΔ/Δ mice (10/group) were treated for 2 days with asialo-GM1 antibody or normal rabbit serum containing an equivalent amount of IgG. Over 93% NK cells were depleted in asialo-GM1 antibody-treated mice. Mice were intravenously injected with 1.2 × 10⁵ B16F0 melanoma cells. Two weeks after tumor cell implantation, the lung weight was determined and compared with that of mice not injected with B16F0 cells (Wilcoxon rank-sum test). B, Tumor NK cell infiltration. 2 × 10⁵ B16F0 melanoma cells were intravenously injected into CXCR4^MyeΔ/Δ mice and CXCR4^WT mice (8/group). Sixteen days after injection of tumor cells, the lung tumor-infiltrating NK cells were analyzed by flow cytometry (Wilcoxon rank-sum test). C, Intratumoral quantitation of IFNγ-expressing NK cells was determined by FACS (Wilcoxon rank-sum test). D, Depletion of interstitial and vascular NK cells. C57BL/6 mice (5/group) were treated with asialo-GM1 antibody or isotype IgG. Two days after treatment, mice were injected intravenously with B16F0 melanoma cells (1.2 × 10⁵). Five minutes before euthanasia, mice were intravenously injected with 2 μg allophycocyanin-conjugated anti-mouse CD45 mAb. The interstitial (IST, CD45⁻) and marginated vascular (MV, CD45⁺) populations in lung tumors were analyzed by flow cytometry. E, Simultaneously, the interstitial and marginated vascular populations type II NKT (E) or type I NKT (F) in lung tumors were determined by flow cytometry. For A–C, group comparisons were made using Wilcoxon rank-sum test.

CXCR4/CXCL12 negatively regulates NK cell cytotoxicity. A, NK-cell cytotoxicity in vivo. 1 × 10⁸ Yac1-Fluc cells were intravenously injected into CXCR4^MyeΔ/Δ or CXCR4^WT mice. Images were taken at 1, 4, or 24 hours after Yac1 cells injection. Animals without Yac1 cell injection served as negative imaging background control (Ctrl). Data were expressed as log mean ± SD (n = 8, Wilcoxon rank-sum test with BH P value correction). B, Representative photos of imaged mice. C, NK-cell cytotoxicity in vitro. Murine NK cells were negatively isolated from the spleen of the CXCR4^MyeΔ/Δ or CXCR4^WT mice. The NK cells were cocultured with Yac1-Fluc reporter cells for 4 hours. Luciferase activity was determined for the un-lysed Yac1 cells to reflect the reverse NK-cell killing activity. Data are expressed as mean ± SD (n = 3) and statistically analyzed by two-way ANOVA with least-squares means using (BH) P value adjustment for multiple comparisons. D, mFasL (CD178) expression on immune cells. Peripheral immune cells from CXCR4^MyeΔ/Δ mice (n = 5) or CXCR4^WT mice (n = 5) were stained with CD178-APC and F4/80-BV421, Ly6G-APC/Cy7, CD8-Alexa Flour700, CD4-pacific blue, B220-Alexa Flour700, Nk1.1-APC/Cy7, and CD11c-AF700. The cells were sorted and analyzed by flow cytometry for CD178 expression on the various cell populations. Two-way ANOVA with model-based mean comparisons and BH P value correction. E, Neutralization of FasL affects NK-cell cytotoxicity ex vivo. NK cells were negatively selected from the spleens of CXCR4^MyeΔ/Δ mice and cocultured with Yac1-luci reporter cells at a 20:1 ratio for 5 hours. Anti-FasL antibody (10 μg/mL) or isotype IgG was applied. The luciferase activity was determined in the surviving target cells (n = 5). P values were determined by Wilcoxon rank-sum test to evaluate differences between NK⁺IgG and NK⁺FasL mAb.

Neutrophils are responsible for NK cytotoxicity in vivo. A, NK cell cytotoxicity toward YAC1 cells in neutrophil depleted mice. The neutrophils in CXCR4^MyeΔ/Δ mice were depleted with 250 μg/Ly6G mAb as described in Materials and Methods. Subsequently 1 × 10⁸ Yac1-Fluc cells were intravenously injected into the mice. Imaging was performed at indicated time points after Yac1 cells injection. Mice without Yac1 cell injection served as the negative imaging background control (Ctrl). Data analysis was performed with Wilcoxon rank-sum test with BH P value correction. B, Representative photos of imaging of mice. C, Depletion of neutrophils abrogated NK activation. Mice were injected in the peritoneum with 250 μg of Ly6G mAb or IgG isotype control daily for 3 days, and then 1 × 10⁸ Yac1 cells were intravenously injected. Twenty-four hours after cell injection, the lung leukocytes were isolated, and NK cells were sorted using Percy/cy5.5 conjugated CD3 and APC-conjugated NK1.1⁺ cells by FACS. Subsequent cell-surface expression of CD69-APC, NKG2A-APC, NKG2D-APC, CD27-pacific blue, and intracellular IFNγ-Alex Fluor700 and Ki67-pacific blue was analyzed. Data were analyzed by two-way ANOVA with model-based mean comparisons and BH P value correction. D, MDSC population in metastatic tumor. 2 × 10⁵ B16F0 melanoma cells were intravenously injected into CXCR4^MyeΔ/Δ mice and CXCR4^WT mice (8/group). After 16 days of injection, the lung tumor infiltrated MDSCs were analyzed by flow cytometry. The Wilcoxon rank-sum test was used for data analysis.

Neutrophils activate NK cells through cytokines. A, Yac1 cells (1 × 10⁸) were intravenously injected into myeloid CXCR4^Δ/Δ or CXCR4^WT mice. Twenty-four hours after injection, intracellular IL18 expression in the lung myeloid cells was determined by FACS. Data were analyzed by the Wilcoxon rank-sum test with BH P value correction (n = 6). B, A representative graph is shown. C, IL18 expression. B16F0 melanoma cells (2 × 10⁵) were intravenously injected into CXCR4^MyeΔ/Δ or CXCR4^WT mice (n = 8). Sixteen days after injection, IL18 in lung tumors was determined with mouse IL18 ELISA. D, Simultaneously, IL18 level in serum was measured. For C and D, data were analyzed by Wilcoxon rank-sum test.

The CXCR4 antagonist LY2510924 inhibits tumor growth and induces NK activation. A, LY2510924 antitumor activity. C57BL/6 mice (9 mice/group) were intravenously injected with B16F0 melanoma cells (1 × 10⁵; ref. 14) and treated subcutaneously with 3 mg/kg of LY2510924, or PBS vehicle twice a day. After 2 weeks of treatment, mice were sacrificed and the weight of tumor in tumor-bearing lungs was determined by subtracting the weight of the tumor-free lung. Data were plotted and statistically analyzed by the Wilcoxon rank-sum test. B, Peripheral immune cell profile. Blood was collected 2 weeks after PBS or LY2510924 treatment. Erythrocytes were excluded with lysis buffer and leukocytes were stained with specific antibodies for flow cytometry analysis using two-way ANOVA with model-based mean comparisons and BH P value correction. C and D, NK-cell distribution and activation. Leukocytes were isolated from the tumor-bearing lungs, stained with CD45-APC/Cy7, NK1.1-PE, and CD69-APC and analyzed by flow cytometry (Wilcoxon rank-sum test). E, Intracellular IL18 expression in leukocytes that infiltrated into lung tumors was analyzed by flow cytometry. Data were analyzed by two-way ANOVA with model-based mean comparisons and BH P value correction.