Figure 1. CFA administration induces myeloid cell expansion and accumulation in the splenic red pulp.

(A) General scheme of single and repetitive immunizations. Mice received a single s.c. dose of CFA at day 0 alone or second dose of IFA or CFA at day 12. Then spleens were analyzed at day 15. Optionally, mice were challenged with LPS/IFN-γ at day 15 for 6 hours or 24 hours before spleens were further analyzed. (B) C57BL/6 mice were injected s.c. with CFA or CFA/CFA or remained untreated before spleens were analyzed according the scheme in A. Spleen cellularity was counted at day 15 (9 independent experiments with replicates: Ctrl = 14, CFA = 19, CFA/CFA = 21). (C) Mice were injected as described above, and spleen cells were analyzed by FACS for Ly6C⁺ and Ly6G⁺ cell subsets. Numbers above the dot plots represent the percentages of the red gated Ly6C^hi gated cells or the top right quadrant for Ly6G⁺ cells (4 independent experiments with replicates: Ctrl = 9, CFA = 14, CFA/CFA = 11). (D) Spleens from the indicated mice were collected at day 15 and cryosections were stained for Gr-1 for myeloid cells and B220 to indicate B cell zones as borders of the white pulp. Scale bars: 500 μm. Microscopy data are representative of at least 3 independent experiments with (n = 3 mice) for each group. (B and C) Statistical significance was assessed by comparison of control versus treated mice. Statistics by 1-way ANOVA with multiple comparisons and Tukey’s post test. **P < 0.01, ***P <0.005, ****P < 0.0001. Values correspond to the mean ± SD.