Immunization with MDA-LDL induces a specific Th2 response. C57BL/6 mice were immunized with homologous MDA-LDL in Freund’s adjuvant or remained naive. One week after the third injection, cellular and humoral immune responses were assessed. Three independent immunization studies were performed. (A) Splenocyte proliferation assay. Splenocytes of immunized (n = 6) or naive (n = 6) mice were cultured with titrated amounts of murine native LDL (open circles, immunized; open squares, naive) or murine MDA-LDL (filled circles, immunized; filled squares, naive), and antigen-specific proliferation was measured by ³[H]-thymidine uptake. (B) Increased IgG1 titers in plasma of immunized mice. Data represent titers from all mice studied (n = 14; P < 0.01, Student’s paired t test). (C) Increased frequency of MDA-LDL–specific IL-5 secreting cells in spleens of immunized mice as assessed by ELISpot assay. Splenocytes of immunized (n = 8; filled circles) and naive (n = 8; open circles) mice were incubated overnight with and without murine MDA-LDL, and the frequencies of MDA-LDL–specific IFN-γ or IL-5 spot-forming cells (SFCs) were assessed. Shown are the mean SFCs per 2 × 10⁶ cells for IFN-γ and IL-5 of individual mice from two experiments (P < 0.01, Student’s paired t test; IFN-γ vs. IL-5 SFC of immunized mice). (D) MDA-LDL–specific Th2 cytokine secretion in cultures of splenocytes from four mice incubated for 72 hours with 25 μg/ml murine native LDL (white bars) or murine MDA-LDL (black bars). Control cultures stimulated with anti-CD3 and anti-CD28 produced 11.7 ± 2.1 ng/ml IFN-γ, 156 ± 39 pg/ml IL-5, 470 ± 43 pg/ml IL-10, and 1.2 ± 0.4 ng/ml IL-13. *P < 0.05; MANOVA followed by Newman-Keuls test, comparing the stimulation with increasing amounts (0.25 [not shown], 2.5 [not shown], and 25 μg/ml) of native and MDA-LDL. All values shown in Figure 1 are mean ± SEM.