Perforin and interferon-γ activities independently control tumor initiation, growth, and metastasis

Shayna E. A. Street, Erika Cretney and Mark J. Smyth

Article Figures & Data


  • Fig. 1.

    NK-cell–mediated cytotoxicity of pfp and IFN-γ gene-targeted mice.

    Resting splenocytes and IL-2–activated adherent spleen NK cells were assessed by 4-hour 51Cr release assays using labeled targets as indicated for (A) B6 or (B) BALB/c strains of mice. An effector–target ratio of 100:1 is shown (4 were examined). The spontaneous release of 51Cr was always less than 15% (subtracted from test), and each experiment was performed twice using triplicate samples.

  • Fig. 2.

    NK-cell–mediated control of spontaneous metastasis by independent pfp and IFN-γ activities.

    BALB/c, BALB/c.SCID, BALB/c.IFN-γ0, BALB/c.pfp0, and BALB/c pfp0.IFN-γ0 mice or BALB/c mice treated with anti-asialo GM1 antibody (100 μg/injection) were inoculated subcutaneously with DA3 tumor cells (105 or 5 × 105) as indicated. Mice depleted of subsets in vivo were treated on days −4, 0 (day of subcutaneous tumor inoculation), and weekly thereafter. Forty-two days after tumor inoculation, the lungs of these mice were harvested and fixed, and colonies were counted and recorded as the mean number of colonies ± SE. Asterisks indicate the groups that are significantly different from BALB/c-untreated mice (*P < .0001; **P < .005; Mann-Whitney U test). A significant difference was also detected between the BALB/c.P0 mice or BALB/c.IFN-γ0 mice and BALB/cP0.IFN-γ0 mice (P < .0001). The number of mice per group is shown in parentheses. Note that for groups of 5 mice receiving 105DA3 tumor cells, the numbers of DA3 lung colonies were as follows: BALB/c.B6Cmv1 r = 8.0 ± 2.6; BALB/c.B6Cmv1 r + anti-asialo GM1 antibody = 62.4 ± 6.6.

  • Fig. 3.

    NK- and NKT-cell–mediated control of experimental tumor metastasis by independent pfp and IFN-γ activities.

    (A) B6, B6.pfp0, B6.RAG-10, B6.TNF0, B6.IFN-γ0, and B6.gldmice or B6 mice treated with anti-NK1.1 were inoculated subcutaneously between the shoulder blades with RM-1 tumor cells (2 × 106), and tumors were allowed to establish for 9 days. Subcutaneous tumors were then resected and a dose range of RM-1 cells (as indicated) injected through the tail vein. Mice were killed 14 days later, the lungs were removed and fixed, and colonies were counted and recorded as the mean number of colonies ± SE. A group of B6 mice was depleted of NK1.1+ cells in vivo by mAb treatment (100 μg/injection) on days −2, 0 (day of intravenous tumor inoculation), 2, and 9. (B) B6, B6.pfp0, B6.IFN-γ0, and B6.pfp0.IFN-γ0mice or B6 and B6.pfp0 mice treated with anti–mIFN-γ mAb (500 μg/injection) or control mAb (500 μg/injection) were treated as above. Mice received anti–IFN-γ mAb on days −2, 0 (day of intravenous tumor inoculation), 2, 7, and 10, with some groups receiving anti–mIFN-γ mAb early (days −2, 0) or late (days 7, 10). (A) Significant differences from the B6 group were determined by a Mann-Whitney U test (*P < .0001). Additionally, mice treated with anti-NK1.1 had significantly more metastases than any other group, including B6.pfp0 and B6.IFN-γ0 mice (P < .0001). (B) Significant differences from the B6.pfp0 group were determined by a Mann-Whitney U test and denoted (*P < .0001). The number of mice per group is shown in parentheses.

  • Fig. 4.

    NKT-cell–mediated control of MCA-induced fibrosarcoma by independent pfp and IFN-γ activities.

    (A) Groups of 10 to 43 B6, B6.Jα2810, B6.pfp0, B6.IFN-γ0, or B6 pfp0.IFN-γ0 mice were injected subcutaneously in the hind flank with 100 μg or 25 μg MCA diluted in 0.1 mL corn oil. Mice were observed weekly for tumor development over the course of 50 to 180 days. Tumors larger than 4 mm in diameter and demonstrating progressive growth over 3 weeks were counted as positive. Groups with statistically higher incidence than B6 mice were noted (*P < .01; **P < .05; Fisher exact test). (B) Representative individual fibrosarcomas from groups of 10 mice (above) were compared with a group of B6 mice receiving 400 μg MCA. Tumor size was measured daily with a caliper square as the product of 2 diameters, and results were recorded as the tumor size (cm2). The mean growth rate of tumors was determined from the gradients of individual plots and was normalized against the mean of the B6 (400 μg) group = 1.0. Data are plotted as the mean ± SE, and significant differences to the B6 (400 μg) control were determined by an unpaired t test with Welch correction (*P < .01)


  • Table 1.

    Depletion of NK cells using anti-asialo GM1 antibody in BALB/c.B6Cmv1 rmice

    Tissue*nNo. leukocytes
    % NK1.1+
    NK cells
    % TCRβ+
    % NK1.1+
    Spleen5105 ± 51-153 3.0 ± 0.425 ± 20.4 ± 0.1
    Spleen + Ab390 ± 7< 0.226 ± 30.5 ± 0.2
    Liver52.4 ± 0.419.7 ± 1.443 ± 47.7 ± 1.3
    Liver + Ab32.8 ± 0.10.3 ± 0.144 ± 47.5 ± 3.4
    • Cell suspensions were prepared as described and labeled with mAb specific for αβTCR and NK1.1.

    • * Mice were untreated or treated with 100 μg anti-asialo GM1 one day before tissue harvest.

    • n, number of mice tested.

    • Total number of leukocytes isolated from spleen or liver.21

    • F1-153 Results represent mean ± SE.