(A) Survival of BALB/c mice challenged with 5 × 10³ isogenic lymphoma cells expressing (n = 31) or not expressing (n = 24) free murine C3d. (B) Lymphoma tumor volumes 10 days after injection of 1 × 10⁷ C3d⁺ or C3d^– cells. On average, C3d⁺ tumors were 176 mm³ and C3d^– tumors 523 mm³. Data represent mean ± SEM analyzed by Mann Whitney 2-tailed test. (C) H&E (upper panels) and immunofluorescence staining (lower panels) of lymphoma tissue. C3d⁺ and C3d^– tumors have similar morphology (above). Both are recognized by antibodies against B220 (red; below), but only the transgenic C3d⁺ lymphoma stains with antibodies against an ENV fusion construct used to detect expression of C3d (green; below). Images are representative of tissues from 5 mice analyzed per condition and a minimum of 3 images/mouse. (D) Melanoma tumor volumes at various times after s.c. injection of 4 × 10⁴ C3d⁺ B16 (n = 10), or 4 × 10⁴ C3d^– (n = 9) melanoma cells in syngeneic mice. (E) Survival of recombinase activating gene 2–deficient (RAG-2–deficient) mice injected with 5 × 10³ isogenic C3d⁺ (n = 15) or C3d^– (n = 15) lymphoma cells. (F) Survival of mice injected with 5 × 10³ lymphoma cells of which 1%–50% were C3d⁺. (G) Survival of B cell–deficient (JH^–/– κ^–/–)mice challenged with 5 × 10³ C3d^– (n = 8) or C3d⁺ (n = 9) tumor cells. (H) Immunofluorescence analysis of frozen sections of C3d⁺ or C3d^– lymphoid tumors stained with anti-CD8 antibodies (green). Each panel is representative of at least 3 different tumors. CD8⁺ T cells were enumerated by flow cytometry, and results are shown on Figure 3H. All survival curves are Kaplan-Meier plots, and differences between curves were analyzed by the log rank Mantel-Cox test.