Fig. 2.

Immunization with P28-9, P28-12, and P28-19 induces a strong antigen-specific type 1 immune response. The frequencies of antigen-specific IFN-γ-producing CD4⁺ Th1 cells in the spleens of different groups of immunized mice were measured before and after E. muris challenge by flow cytometry. Splenocytes from 3 mice per group were pooled and stimulated in vitro for 18 h with the whole-cell lysate antigen prepared from E. muris cultured in DH82 cells. The frequencies of antigen-specific T cells producing IFN-γ were determined after the background staining of nonstimulated cells in wells containing medium only was subtracted. Immunization with P28-9, P28-12, or P28-19, or the P28 mixture resulted in the expansion of antigen-specific IFN-γ-producing CD4⁺ Th1 cells on day 7 following challenge with E. muris. The data are representative of two independent experiments (the experiments gave similar results), and each bar in the graph represents the frequency of Ehrlichia-specific IFN-γ-producing CD4⁺ T cells per million splenocytes pooled from 3 mice from each group.