Necessary for IL-12-Mediated Regression of the The CXC Chemokines IP-10 and Mig Are

The role of the non-ELR-containing CXC chemokines IP-10 and Mig in antitumor activity induced by systemic treatment with IL-12 was examined in mice bearing the murine renal adenocarcinoma RENCA. IL-12 treatment produces a potent antitumor effect that is associated with tumor infiltration by CD8 1 T lymphocytes. The regression of tumor is associated with the elevated expression of the IFN- g -inducible chemokines IP-10 and Mig within the tumor tissue. IP-10 and Mig have been shown to function as chemoattractants for activated T lymphocytes. In animals treated with rabbit polyclonal Abs specific for IP-10 and for Mig, the IL-12-induced regression of RENCA tumors was partially abrogated. This effect was associated with a dramatic inhibition of T cell infiltration. Thus, it appears that IL-12-dependent, T cell-mediated antitumor activity requires the intermediate expression of IP-10 and Mig to recruit antitumor effector T cells to the tumor site. The Journal of Immunology, 1998, 161: 927–932. shown agarose-formaldehyde gel, and blotted by capillary transfer onto nylon membranes. The blots prehybridized 6 to 18 h at 42°C in 50% formamide, 1% SDS, 5 3 SSC, 1 3 Denhardt’s solution (0.02% Ficoll, 0.02% BSA, and 0.02% polyvinylpyr-rolidine), 0.25 mg/ml denatured salmon sperm DNA, and 50 mM sodium phosphate buffer, pH 6.5. Hybridization was conducted at 42°C for 12 to 18 h with 10 7 cpm of denatured probe. The filters were washed twice for 15 min each time at 55°C in 0.1% SDS-0.5 3 SSC. The blots were then exposed using XAR-5 x-ray film (Eastman Kodak, Rochester, NY) with DuPont (Wilmington, DE) Cronex Lightening Plus intensifying screens at 2 70°C. Expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA was used as an internal control and was applied in all experiments.Semiquantitative RT-PCR analysis of perforin mRNA was conducted as reported previously (6, 25, 32). One microgram of total RNA was amplified using an oligo(dT) antisense primer and AMV reverse transcriptase at 42°C for 1 h. The RT reaction products were used undiluted or at a 1/10 dilution for PCR amplification using 20 mM sense and antisense primers (see be- low) and Taq polymerase. PCR reactions were conducted in a Perkin-Elmer/Cetus DNA Thermal Cycler for 15 cycles (denaturation, 1 min, 94°C; annealing, 1 min, 60°C; amplification, 2 min, 72°C). The primer sequences used were as follows: perforin antisense primer, GGTGGAGT GGAGGTTTTTGTACC; and perforin sense primer, CAGAATGCAAG CAGAAGCACAAG (perforin product size, 486 bp). These primers were chosen from separate exons to ensure