Figure 9. Effect of immunization on B cell control of tumor in subcutaneous and lung metastasis models.

Rag2^−/− SW_HEL+ and Rag2^−/− SW_HEL- mice were immunized s.c. with 5×10⁶ irradiated tumor cells emulsified in CFA, followed by i.p. anti-CD40 on days 3 and 6. Two weeks later mice were challenged with 1×10⁶ live tumor cells i.v. or s.c. Mice were euthanized on day 24 after i.v. challenge, or when s.c. tumors reached 100mm². A. Serum antibody isotypes. MFI of CBA signals for each isotype before and after immunization, and on day of death (mean±SEM, n≥7/group). Grey bar, naïve Rag2^−/− SW_HEL+ mice. White bar, Rag2^−/− SW_HEL+ mice at time of i.v. or s.c. challenge (day 14 after immunization). Black bar, Rag2^−/− SW_HEL+ mice on day of death). Top: i.v. challenge. Bottom: s.c. challenge. * = P < 0.05, ** = P < 0.01, *** = P < 0.001 by unpaired Student T test B. Left panel: Representative photos of lungs from Rag2^−/− SW_HEL+ and Rag2^−/− SW_HEL- mice after i.v. tumor challenge. Right panel: Number of metastases. Each dot represents an individual animal, with the bar representing mean±SEM (SW_HEL+, n = 17, SW_HEL-, n = 19). Data are pooled from 2 independent experiments. *** = P < 0.001 by nonparametric Mann-Whitney test. C. Individual tumor growth curves after s.c. tumor challenge. Left panel: Rag2^−/− SW_HEL+ mice. Right panel: Rag2^−/− SW_HEL- mice. D. Kaplan-Meier survival analysis after s.c. tumor challenge. Mice were euthanized when tumors reached 100mm². Black line, Rag2^−/− SW_HEL+ mice (n = 10). Red line, Rag2^−/− SW_HEL- mice (n = 9).