These insights into the biology of IL-10 should allow a more rational approach to the design of clinical trials using recombinant vaccines in the treatment of human cancers. Acknowledgments The authors thank Dr. (a) IL-10 also enhanced the therapeutic effectiveness of a recombinant fowlpox virus, Sema6d which cannot replicate in mammalian cells; (b) Titers of rVV in immunized mice were NVP-QAV-572 lower, not higher; and (c) Although IL-10 did not alter levels of anti-vaccinia antibodies or natural killer cell activity, rVV-primed mice treated with IL-10 had enhanced vaccinia-specific cytotoxic T-lymphocyte activity. Thus, IL-10 enhanced the function of a recombinant poxvirus-based anti-cancer vaccine and may represent a potential NVP-QAV-572 adjuvant in the vaccination against human cancers using recombinant poxvirus-based vaccines. gene under the vaccinia early/late p7.5 promoter (provided by B. Moss, National Institute of Allergy and Infectious Diseases, Bethesda, MD, U.S.A.) and is inserted into the viral thymidine kinase (TK) gene by homologous recombination as previously described (23). The TK-disrupted control vaccinia virus designated V69 was constructed by generating the recombinant plasmid pGS69, which contains the influenza A/PR/8/34 nucleoprotein in flanking TK gene segments and lacks the gene (24). Viral stocks were propagated on BSC-1 cells and purified by ultracentrifugation on a 36% sucrose cushion. Recombinants were selected by expression of -gal and for the TK? pheno-type. Virus concentration was determined by the plaque titration method using BSC-1 cells. The fowlpox virus constructs included the wild-type strain, FPV.wt, originating from the POXVAC-TC strain (Schering Corp.. Kenilworth, NJ, U.S.A.) and the recombinant fowlpox virus, FPV.bg40k, containing the gene under the direction of the vaccinia virus 40-kDa promoter (provided by L. Gritz, Therion, Inc., Cambridge, MA, U.S.A.). The foreign sequences were inserted by homologous recombination into the test with p 0.05 used to determine significance. Quantitation of Vaccinia Viral Titers Two groups of mice were injected with 1 107 PFU of the VJS6 recombinant virus by tail vein injection. One group also received murine IL-10 (1 g) by i.p. injection starting 12 hours after virus administration and then daily for 5 days. Two mice from each group were killed on alternating days for 8 days and the lungs, spleen, kidneys, liver, and ovaries were removed and placed in PBS. The organs were homogenized in Tris, pH 8.0, subjected to three rounds of freeze-thawing, sonicated, and diluted in minimal essential medium culture media supplemented with 2% FCS. Vaccinia titers were determined by the plaque assay method on nearly confluent BSC-1 cells, as previously described (27). All samples were run in duplicate, and titers are reported as the number of PFU per milliliter. Direct Effect of IL-10 on Murine Tumor Cells In Vitro To determine whether IL-10 had any direct inhibitory properties on the CT26.WT or CT26.CL25 tumor cell line in vitro, a proliferative assay was performed. Tumor cells (5 103) were plated into 96-well plates and incubated at 37C for 24 hours. Murine IL-10 was added to the plates at the following concentrations in triplicate 0, 0.008 g, 0.04 g, 0.2 g, 1.0 g, NVP-QAV-572 and 3.0 g. 3H-Thymidine (1 Ci/well) was also added to each well and the plates incubated at 37C for 5 hours. Counts were obtained on a beta counter, and the amount of 3H-thymidine release was calculated. Enzyme-Linked Immunosorbent Assay Three groups of mice were vaccinated with either an i.v. injection of 1 1 107 PFU of VJS6 alone, 1 107 PFU of VJS6 followed by 5 days of recombinant murine IL-10 administration starting 12 hours after immunization (1 g, i.p., Q.D.), or recombinant murine IL-10 administration alone (PeproTech, Inc.). Pooled sera from two immunized mice were obtained 2, 4, 6, 14, and 21 days after treatment and were analyzed by enzyme-linked immunosorbent assay for the presence of antibodies (Abs) against -gal protein or wild-type vaccinia virus. Briefly, microtiter plates were dried down overnight at 37C in a nonhumidified incubator with 200 ng/well/50 l of purified -gal protein (Sigma Chemical Co., St. Louis, MO, U.S.A.). Alternatively, microtiter plates were coated with wild-type vaccinia virus (WT-VV) (5 105/well/50l) at 4C, overnight. The plates were incubated with 5% bovine serum albumin (BSA) in PBS on each well for 1 hour to prevent nonspecific Abs NVP-QAV-572 from binding. This was followed by a second 1-hour incubation with 50 1 of fivefold dilutions (starting at.