Tumor infiltrating CTL preferentially accumulate in CD200-positive tumors and are capable of producing more IFN-γ. Purified P1CTL were injected into RAG-2^−/−BALB/c mice with established J558-CD200 or J558-Ctrl tumors. At various times after P1CTL injection, single cell suspensions were prepared from tumors and were stained for Vα8.3, CD8 and/or IFN-γ. For IFN-γ staining, cells were stimulated with 50 ng/ml of PMA/Ionomycin for 5 h followed by a standard intracellular staining procedure. a. Flow cytometry analysis of frequencies of tumor antigen-specific T cells in CD200-positive and CD200-negative tumors. b. Numbers of tumor-infiltrating P1CTL at different times after injection. Data show mean + SD (n= 3–7 mice). c. Flow cytometry analysis of IFN-γ production by tumor infiltrating P1CTL. d. Quantitation of tumor-infiltrating P1CTL that were IFN-γ positive. Data show mean + SD (n=3–7 mice). Data shown in panels c and d were at day 5 after P1CTL transfer. Paired student’s t test was used for the statistical analysis.

CD200R expression on tumor-associated myeloid cells (TAMC). J558-Ctrl or J558-CD200 cells were injected into RAG-2^−/−BALB/c mice subcutaneously. When tumors were fully established, single cell suspensions were prepared from tumors and were stained for various cell surface markers followed by flow cytometry analysis. a. CD200R expression on CD11b⁺ myeloid cells. b. CD200R expression on three subsets of TAMC, i.e. CD11b⁺Gr-1^hi, CD11b⁺Gr-1^lo and CD11b⁺Gr-1⁻ cells. Shaded histograms represent isotype controls. Data are representative of at least 10 independent experiments.

Role of IL-10 produced by TAMC. a. Analysis of TAMC-associated factors by real-time PCR. RNA samples were prepared from established J558-CD200 and J558-ctrl tumors (the hosts were RAG-2^−/−BALB/c mice). Expression of genes associated with TAMC (IL-10 and TNF-α) was quantitated. b. CD11b⁺ cells from J558-CD200 or J558-Ctrl tumors were stimulated with100 ng/ml of PMA and ionomycin for 24 h, IL-10 and TNF-α releases in the culture supernatants were measured by ELISA. c. CD11b⁺ cells in spleens and tumors from mice bearing J558-Ctrl tumors were co-cultured with either J558-Ctrl or J558-CD200 cells at a 1:1 ratio for 24 h in the presence of 100 ng/ml of PMA and ionomycin. IL-10 concentrations in the culture supernatants were then detected by ELISA. Data are normalized to 1 × 10⁶/ml of TAMC, show mean + SD (n=3) and are representative of three independent experiments. Student’s t test was used for the statistical analysis.

Expression of CD200 on tumor cells prevents tumor recurrence after P1CTL therapy. a. Full-length CD200 cDNA was cloned from a variant of J558 cells and an expression vector was generated to transfect CD200-negative J558 cells and mastocytoma P815 cells. We also generated cells transfected with the empty expression vector. The solid lines represent CD200 staining while the dotted lines represent staining with an isotype control antibody. b. Tumor growth following P1CTL therapy. Tumor cells were injected into RAG-2^−/−BALB/c mice. When tumors were established, mice were treated with purified P1CTL. Numbers of mice with recurrent tumors were significantly different between the two groups (P<0.05, X² test). Data shown are representative of three independent experiments. c. Tumor free survival of J558 tumor-bearing mice that received P1CTL therapy. Data from the same group of mice shown in b is presented. d. Tumor cells were injected into RAG-2^−/−BALB/c mice at a dose of 1 × 10⁶/mouse s.c., 7 days after tumor cell injection, mice were treated with purified P1CTL (5 × 10⁶/mouse). Data are representative of two independent experiments.

Tumor expression of CD200 does not inhibit effector functions of CTL. a. Splenocytes from P1CTL-treated tumor-bearing mice were stained for CD8, Vα8.3 and CD200R. CD8⁺Vα8.3⁺ and CD8⁻Vα8.3⁻ cells were analyzed for CD200R expression. Shaded histograms represent isotype controls, solid lines represent CD200R staining. b. In vivo proliferation of P1CTL. 20 × 10⁶ irradiated (1000 rad) tumors cells were injected into RAG-2^−/−BALB/c mice intravenously (i.v.), 24 h later, mice received 5 × 10⁶ of CFSE-labeled P1CTL. 72 h after P1CTL injection, mice were sacrificed and splenocytes were stained for CD8 and Vα8.3. Data show CFSE dilution of CD8⁺Vα8.3⁺ cells and are representative of three independent experiments. c. IFN-γ production by activated P1CTL. Splenocytes from RAG-2^−/−P1CTL mice were activated in vitro with P1A 35–43, 72 h later, activated P1CTL were co-cultured with J558-CD200 or J558-Ctrl cells. 72 h after co-culture, IFN-γ release in the culture supernatants were measured by ELISA. Data are representative of two independent experiments. d. Effect of cancer cell CD200 expression on CTL-mediated destruction of target cells. A standard ⁵¹Cr-release assay, using activated P1CTL as effectors, ⁵¹Cr-labeled J558-Ctrl and J558-CD200 cells as targets, was performed. Data are representative of three independent experiments.

TAMC suppressive activity. Single-cell suspensions were prepared from tumors and were stained for CD11b or Gr-1 markers followed by anti-FITC or anti-PE MACS beads. The labeled cells went through MACS columns and CD11b⁺ or Gr-1⁺ cells (about 90% purity) were prepared. a. Flow cytometry analysis of CD11b⁺ cells after purification with MACS beads. b. Suppressive activity of CD11b⁺ cells. Spleen and lymph node cells from RAG-2^−/−P1CTL mice were co-cultured with CD11b⁺ cells from J558-CD200 or J558-Ctrl tumors at different ratios in the presence of P1A35-43 in U-bottomed 96-well plates. ³H-thymidine incorporation was performed 48 h later. % inhibition of P1CTL proliferation relative to controls was calculated. c. Flow cytometry analysis of Gr-1⁺ cells after purification with MACS beads. d. Suppressive activity of Gr-1⁺ cells. Spleen and lymph node cells from RAG-2^−/−P1CTL mice were co-cultured with Gr-1⁺ cells from J558-CD200 or J558-Ctrl tumors in the presence of P1A35-43. Data are representative of five independent experiments.

Role of TAMC-derived IL-10 in suppression of CTL activity. Spleen and lymph node cells from RAG2^−/−P1CTL mice were co-cultured with CD11b⁺ cells from J558-Ctrl tumors in the presence of P1A35-43. 5 µg/ml of anti-IL-10 antibody or an isotype matched control antibody were added in the culture (left panel). CD11b⁺ cells from BALB/c or IL-10^−/−BALB/c mice with J558-Ctrl tumors were compared for their suppressive activity using thymidine incorporation assay (right panel). Suppressor: effector =1:1. b. 0.3 × 10⁶/ml CD11b⁺ cells from WT or IL-10−/− mice with J558-Ctrl tumors were stimulated with 100 ng/ml of PMA and 100 ng/ml of ionomycin for 24 h. TNF-α production in the culture supernatants were detected by ELISA. c. Effect of P1CTL treatment of mice with established J558-Ctrl tumors. 5 × 10⁶ of CD8⁺ T cells from P1CTL transgenic mice were injected into each mouse with established J558-ctrl tumors. Numbers of mice with recurrent tumors were significantly different between the two groups (P<0.05, X² test). d. CD11b⁺ cells from J558-Ctrl tumors grown in wild type and IL-10^−/− mice were co-cultured with ⁵¹Cr-labeled J558 cells and J558T2S cells (with a P6R mutation in P1A epitope) for 12 h. ⁵¹Cr-releases into culture supernatants were measured in a scintillation counter. Specific lysis was calculated based on the following formula: specific lysis % = 100 × (cpm_coculture−cpm_{target minimal})/(cpm_{target max}−cpm_{target minimal}). Data are representative of 3–5 independent (a, b) and two (c, d). Paired student’s t test was used for statistical analysis.