CXC Chemokine Ligand 9/Monokine Induced by IFN-γ Production by Tumor Cells Is Critical for T Cell-Mediated Suppression of Cutaneous Tumors

Mig production by Mig-deficient and Mig-expressing tumor clones growing in vivo. Solid tumors were induced by intradermal transplantation of Mig-deficient MCA205-4 cells (A and B) or Mig-expressing MCA205-10 cells (C and D) into wild-type (A and C) or Mig^−/− (B and D) recipient mice. Tumors were excised on day +15 posttransplantation and prepared paraffin sections were stained with Mig-specific goat polyclonal Ab plus secondary rabbit anti-goat peroxidase-labeled Ab. Areas of intensive Mig production are indicated by arrows. Magnification, ×50 with ×200 inserts in upper right corner.

Tumor growth and activation of tumor-specific T cells induced by Mig-expressing vs Mig-deficient tumor cells. A, Mig-expressing MCA205-10 (⋄) or Mig-deficient MCA205-4 (▪) tumor cells were injected intradermally into groups of five C57BL/6 mice and tumor size was measured on the indicated days. The results indicate the mean tumor size for each recipient group. ∗, p < 0.05. B, Mig-expressing MCA205-10 tumor cells were injected intradermally into groups of five C57BL/6 mice and the recipients were treated by i.p. injections of 500 μl of control rabbit sera (□) or Mig-specific antisera (▪) every 48 h after transplantation. Tumor size was measured on the indicated days. The results indicate the mean tumor size for each recipient group. ∗, p < 0.05. C, Mig-expressing MCA205-10 or Mig-deficient MCA205-4 tumor cells were injected intradermally into C57BL/6 mice. On days 8 and 14 posttransplant, CD4⁺ and CD8⁺ T cell-enriched cell suspensions were prepared from the lymph nodes of the tumor recipients or from naive mice using negative selection and ELISPOT assay was used to enumerate tumor-specific IFN-γ-producing cells. The mean number ± SEM of IFN-γ-producing CD4⁺ (□) and CD8⁺ T cells (▪) per 5 × 10⁵ responder cells in triplicate cultures minus the number of spots from control cultures with syngeneic spleen cell stimulators for two individual mice are shown.

Recruitment of CXCR3-expressing T cells into Mig-deficient and Mig-producing tumors. Tumors were excised on day 14 posttransplant and digested in a collagenase, DNase and hyaluronidase mixture. Cell suspensions obtained were washed and stained with anti-CD45, anti-CD4 or anti-CD8, and anti-CXCR3 mAb. The tumor-infiltrating leukocyte cell population was gated as CD45⁺ cells (A) and then analyzed for the frequencies of CD4⁺ and CD8⁺ T cells that were positively stained by anti-CXCR3 mAb (B, gate R3) and were negative when stained with isotype control IgG (data not shown). The numbers in the dot plots indicate the mean percentage ± SEM of CXCR3⁺ T cells in 50,000 cell aliquots analyzed for three tumors in each group.

Proliferation and class I MHC expression of Mig-deficient and Mig-expressing tumor cells in vitro. A, Aliquots of Mig-deficient MCA205-4 (▪) and Mig-expressing MCA205-10 (□) tumor cells were cultured in triplicate for 48, 72, and 96 h. Cells were detached by 0.5% EDTA/trypsin and viable cells counted using trypan blue exclusion. The results are expressed as mean total cell number after the indicated time of culture. B, Mig-deficient MCA205-4 and Mig-producing MCA205-10 tumor cells were left untreated (solid line) or cultured with rIFN-γ (filled histogram). After 24 h, the cells were washed and stained with anti-class I MHC K^b or D^b mAb. Tumor cells incubated with isotype control Ab is shown as a negative control (dotted line).

In vivo growth of Mig-expressing, but not Mig-deficient, tumor cells is suppressed by NK and T cells. Wild-type syngeneic mice were treated with control rat IgG (□) or with anti-NK1.1 mAb (▪) before Mig-expressing MCA205-10 (A) or Mig-deficient MCA205-4 (B) tumor cells were injected intradermally into groups of C57BL/6 mice. Mig-expressing MCA205-10 (C) or Mig-deficient MCA205-4 (D) tumor cells were injected intradermally into groups of syngeneic wild-type mice (□) or B6.RAG1^−/− mice (▪). Results indicate the mean tumor size on the indicated days in each recipient group of five mice. ∗, p < 0.05.

Tumors constitutively producing Mig induce protective T cell-mediated immune responses. A, Mig-deficient MCA205-4 tumor cells were transduced with a control viral vector (•) or with a vector encoding Mig cDNA (Υ) and puromycin-selected transductants for each vector group were injected intradermally into groups of five syngeneic mice. The growth of solid tumors was monitored through day 25 posttransplantation. B, C57BL/6 mice were treated with control rat IgG (control), with anti-NK1.1 mAb (NK cell depleted), or with anti-CD4 plus anti-CD8 mAb (T cell depleted) and then received Mig-transduced MCA205-4 cells. The mean tumor size in each recipient group of five mice at day 14 posttransplantation is shown. ∗, p < 0.01. C, Mig-deficient tumor cells transduced with a control vector (control) or with a vector encoding Mig (Mig⁺) were injected intradermally into C57BL/6 mice. On day 8, CD4⁺ and CD8⁺ T cell-enriched cell suspensions were prepared from the draining lymph nodes of the tumor recipients or from naive mice using negative selection and tumor-specific IFN-γ-producing cells were enumerated by ELISPOT. The mean number ± SEM of IFN-γ-producing CD4⁺ (□) and CD8⁺ T cells (▪) per 5 × 10⁵ responder cells in triplicate cultures minus the number of spots from control cultures with syngeneic spleen cell stimulators for two individual mice is shown.