STAT1 Signaling Regulates Tumor-Associated Macrophage-Mediated T Cell Deletion

Inhibition of T cell immune response by F4/80⁺ TAM. A, Both Gr-1⁺ and F4/80⁺ cells from spleens of tumor-bearing mice failed to alter Ag-induced T cell proliferation. Gr-1⁺ or F4/80⁺ cells freshly isolated from spleens of CT-26 tumor-bearing BALB/c mice were added at different ratios to splenocytes from HA transgenic BALB/c mice stimulated with 10 μg/ml MHC class II-restricted control or HA-derived peptides. Cells were cocultured for 72 h, and incorporation of [³H]thymidine was measured in triplicates. Results are shown as average ± SD. Thymidine uptake in cells stimulated with control peptide was <1000 cpm. B, F4/80⁺, but not Gr-1⁺ cells derived from tumor are inhibitory to T cell response. Gr-1⁺ or F4/80⁺ cells were purified from CT-26 tumors and immediately added to splenocytes from HA transgenic mice stimulated with control or specific peptides. Incorporation of [³H]thymidine was measured in triplicates 72 h later, as described above. Results are presented as average ± SD. C, F4/80⁺ macrophages were purified from tumor tissues or from spleens of C3 tumor-bearing mice, or from spleens of naive mice and added at different ratios to splenocytes from OT-1 transgenic mice stimulated with specific MHC class I-restricted OVA-derived peptide SIINFEKL. Incorporation of [³H]thymidine was measured 72 h later, as described above. Results are presented as average ± SD. D, F4/80⁺ TAM suppress CTL development. Splenocytes from OT-1 transgenic mice were mixed at 1:10 ratio together with syngeneic splenocytes from naive C57BL/6 mice and incubated in 24-well plates (4 × 10⁶/well) for 5 days (control group). Cells were stimulated with 10 μg/ml OVA-derived peptide SIINFEKL. To evaluate the effect of TAM on CTL activity, F4/80⁺ macrophages freshly isolated from C3 tumor were added to the culture on day 0. TAM:splenocytes ratio was 1:10 (F4/80 group). Cytotoxicity against specific peptide-loaded EL-4 tumor cells was measured using ⁵¹Cr release assay. The results are presented as average ± SD. E, F4/80⁺ cells were isolated from spleens of naive tumor-free C57BL/6 or BALB/c mice using magnetic beads separation technique and labeled with either isotype control IgG or allophycocyanin-conjugated anti-Gr-1 Ab and FITC-conjugated anti-CD11b Ab. Typical result of three experiments is shown. F4/80⁺ cells were also isolated from spleens or ascites of EL-4 tumor-bearing C57BL/6 mice or MethA sarcoma-bearing BALB/c mice using magnetic beads separation technique and labeled with allophycocyanin-conjugated anti-Gr-1 Ab and FITC-conjugated anti-CD11b Ab. Typical result of three experiments is shown. F, Gr-1⁺ cells were isolated from tumor of EL-4 tumor-bearing mice using magnetic beads separation and labeled with either isotype control or FITC-conjugated anti-F4/80 Ab. The result of typical experiment is shown.

Tumor-associated F4/80⁺ macrophages cause deletion of T cells through induction of apoptosis. A, Stimulation of splenocytes with CD3/CD28 mAb in the presence of F4/80⁺ TAM induces apoptosis of CD4 and CD8 T cells. F4/80⁺ macrophages were purified from tumors or spleens of C3 tumor-bearing mice and mixed at 1:4 cell ratio with naive syngeneic splenocytes stimulated with anti-CD3 and anti-CD28 mAbs. After 24 and 48 h of incubation, cells were collected, washed in PBS, and labeled with anti-CD4 allophycocyanin, anti-CD8 FITC Abs, PE-conjugated annexin V, and 7-AAD. The proportion of annexin V-positive 7-AAD-negative apoptotic cells within the CD4⁺ or CD8⁺ T cell populations was measured by flow cytometry. Combined results of three performed experiments are shown. B, Stimulation of splenic cells from OT-1 transgenic mice with specific OVA-derived peptide in presence of F4/80⁺ TAM induces apoptosis of Ag-specific CD8 T cells. F4/80⁺ macrophages were purified from ascites or spleens of EL-4 tumor-bearing mice (ascitic tumor), mixed at 1:4 cell ratio with OT-1 transgenic splenocytes stimulated with specific OVA-derived peptide SIINFEKL. After 24 and 48 h of incubation, cells were collected, washed in PBS, and labeled, as described above. The proportion of annexin V-positive 7-AAD-negative apoptotic cells within the population of CD8⁺ T cells was measured by flow cytometry. Combined results of three performed experiments are shown. C, F4/80⁺ TAM cause in vitro deletion of CD8 T cells. F4/80⁺ macrophages were purified from ascites or spleens of EL-4 tumor-bearing mice (ascitic tumor) and mixed at 1:4 cell ratio with OT-1 transgenic splenocytes stimulated with specific OVA-derived peptide SIINFEKL. After 24 and 48 h of incubation, cells were collected, washed in PBS, and labeled with anti-CD8 FITC Ab. The proportion of CD8⁺ cells within whole cell population was measured by flow cytometry. D, F4/80⁺ macrophages were purified from ascites or spleens of EL-4 tumor-bearing mice and mixed at 1:4 cell ratio with naive syngeneic splenocytes stimulated with anti-CD3 and anti-CD28 mAbs. Neutralizing anti-IFN-γ, anti-TNF-α Abs, or control rabbit IgG (all from R&D Systems) were added at concentration of 10 μg/ml. After 48 h of incubation, cells were collected and the percentage of viable cells was counted using trypan blue exclusion. Experiments were performed in triplicates. Results are presented as average ± SD. ∗, Statistically significant differences from cells cultured without F4/80⁺ cells (p < 0.05). E, F4/80⁺ macrophages were purified from ascites or spleens of EL-4 tumor-bearing mice and mixed at 1:4 cell ratio with naive syngeneic splenocytes stimulated with anti-CD3 and anti-CD28 mAbs. Neutralizing anti-IFN-γ, anti-TNF-α Abs, or control rabbit IgG (all from R&D Systems) were added at concentration of 10 μg/ml. Incorporation of [³H]thymidine was measured in triplicates 72 h later, as described above. Results are presented as average ± SD. ∗, Statistically significant differences from cells cultured without F4/80⁺ cells (p < 0.05).

Arginase and iNOS are dramatically up-regulated in TAM. A, TAM are able to produce high amounts of NO. F4/80⁺ or Gr-1⁺ cells were purified from tumors or spleens of tumor-bearing mice and mixed at 1:16 cell ratio with 2 × 10⁵ naive syngeneic splenocytes stimulated with anti-CD3 and anti-CD28 mAbs. After 48-h incubation, cell supernatants were collected, filtered, and assayed for nitrites, as described in Materials and Methods. B, Freshly isolated F4/80⁺ TAM, but not Gr-1⁺ splenic myeloid cells from tumor-bearing mice express iNOS. Whole cell lysates were prepared from purified TAM or Gr-1⁺ splenocytes (C57BL/6 mice). Samples (30 μg of protein) were subjected to electrophoresis in 10% SDS-polyacrylamide gels, blotted onto 0.45-μm nitrocellulose membranes, and probed with anti-iNOS Ab. Western blotting has been done, as described in Materials and Methods. C, Comparative analysis of iNOS expression by F4/80⁺ macrophages derived from C57BL/6 mice: lane 1, F4/80⁺ cells were purified from naïve spleen; lane 2, from peritoneum of naïve mice; lane 3, from spleens of EL-4 tumor-bearing mice; and lane 4, from ascites of EL-4 tumor-bearing mice. Cells were lysed, and expression of iNOS was determined by Western blotting. D, Arginase I expression and activity in freshly derived Gr-1⁺ cells. Gr-1⁺ cells were purified from naïve spleens, spleens of CT-26 tumor-bearing mice, or tumor tissues. Cells were immediately lysed, and expression of arginase I was determined by Western blotting. The same cell lysates were used to determine arginase activity, as described in Materials and Methods. Two experiments with the same results were performed. Positive control: cell lysates of murine hepatocytes are known to express high arginase I activity. E, Arginase I expression and activity in freshly derived F4/80⁺ cells. Experiments were performed exactly as described in E and F, with the exception that instead of Gr-1⁺ cells F4/80⁺ macrophages were isolated from naive spleens, spleens, or tumor tissues of CT-26-bearing mice. Two experiments with the same results were performed. F, Block of arginase and iNOS activity prevents T cell inhibition mediated by TAM. F4/80⁺ macrophage isolated from spleens or tumor tissues of C3 tumor-bearing mice was incubated at 1:4 ratio with naive syngeneic splenocytes stimulated with anti-CD3 (1 μg/ml) and anti-CD28 mAbs (5 μg/ml) in the presence of iNOS (0.5 mM l-NNMA) or arginase (0.5 mM nor-NOHA) inhibitors. Cells were cocultured in triplicates for 72 h, and incorporation of [³H]thymidine was measured in triplicates. Results are shown as average ± SD.

The role of STAT signaling in TAM-mediated suppression of T cell response. A, Intracellular expression of phospho-STAT6 in F4/80⁺ macrophages. F4/80⁺ macrophages were isolated from ascites of EL-4 tumor-bearing mice (TAM) or from peritoneum of naive control mice (PEM). Cells were stained with anti-phospho-STAT6 Ab, as described in Materials and Methods. Control Ab: cells stained with isotype control IgG and secondary Abs. TAM derived from tumor-bearing mice display high intracellular phospho-STAT-6 expression. B, Immunosuppressive activity of TAM from STAT-6 knockout (KO) tumor-bearing mice was not affected. EL-4 tumor was established i.p. in control wild-type (wt) and STAT-6 (KO) mice. F4/80⁺ cells were isolated from ascites and cultured in triplicates together with control syngeneic splenocytes stimulated with anti-CD3 (1 μg/ml) and anti-CD28 (5 μg/ml) Abs. Cell proliferation was measured in triplicates using [³H]thymidine uptake. Cumulative results of three performed experiments are shown. The level of [³H]thymidine uptake in cells not stimulated with Abs was <1000 cpm. C, Arginase activity in TAM from STAT-6 KO mice was not impaired. F4/80⁺ TAM from EL-4 tumor-bearing wild-type (wt) and STAT-6 KO mice were cultured at 1:16 ratio with syngeneic splenocytes activated with anti-CD3 and anti-CD28 Abs, as described above. NO production was measured, as described in Materials and Methods. D, Expression of STAT3 and STAT1 in F4/80⁺ macrophages. F4/80⁺ cells were isolated from spleens (Spleen) or peritoneum (Periton.) of naive C57BL/6 mice (Naive), or from spleen or ascites (Ascites) of EL-4 tumor-bearing mice (Tumor). Whole cell lysates were prepared and subjected to SDS-PAGE electrophoresis and Western blotting. Membranes were probed with Abs, as indicated. E, Experiments were performed exactly as described in B. F4/80⁺ TAM from wild-type (wt) and STAT1 KO EL-4 tumor-bearing mice were used. F, F4/80⁺ TAM were isolated from ascites of EL-4 tumor-bearing wild-type (WT) and STAT1 knockout (KO) mice. Whole cell lysates were prepared, and the level of arginase and iNOS was evaluated using Western blotting, as described in Materials and Methods. G, NO production by wild-type or STAT1 KO TAM was measured, as described in C. Two experiments with the same results were performed. H, Arginase activity by TAM from wild-type or STAT1 KO EL-4 tumor-bearing mice was evaluated, as described in Materials and Methods. Two experiments with the same results were performed.