Interaction of Spl-PCs with BAFF-producing neutrophils. (A) Interactions of EYFP⁺ PCs (green) and Gr1⁺ cells (red) in the spleen of a representative anti-CD20–treated mouse (day 50 after the first anti-CD20 injection) observed by 3-color confocal microscopy with an LSM 700 (Zeiss) and 40× objective. Spleen sections were stained with anti-EYFP, anti-CD3 antibodies, and DAPI. Scale bar, 150 µm. PCs can be easily discriminated from memory B cells through their EYFP intensity (see supplemental Figure 11 and supplemental Methods). (B) Same analysis for control mice 3 months after 2 SRBC immunizations. Spleen sections were stained with anti-EYFP, anti-CD3 antibodies, and DAPI. Scale bar, 150 µm. (C) Number of Gr1⁺Ly6G⁺ and Gr1⁺Ly6G⁻ cells in spleens from controls 3 months after 2 SRBC immunizations and anti-CD20–treated mice 50 days after the first anti-CD20 injection. (D) Quantification of EYFP⁺ PC-Gr1⁺ cell interactions in the spleen of control and B-cell–depleted mice. Spleens from 3 mice per group were analyzed corresponding to about 600 EYFP⁺ PCs in the control group and 300 EYFP⁺ PCs in the anti-CD20–treated group. Interactions between EYFP⁺ PCs and Gr1⁺ cells were calculated by using ImageJ software. (E) Neutrophils (Gr1⁺Cd11b⁺Ly6G⁺ cells) were the predominant source of BAFF production in the spleen compared with other Gr1⁺ cells and CD4⁺ T cells (P < .05). CD4⁺ T cells, Gr1⁺Cd11b⁺Ly6G⁻ cells and Gr1⁺Cd11b⁺Ly6G⁺ cells were collected from the spleen of 3 immunized mice. About 10 000 cells per population for each mouse were sorted. BAFF cDNA expression was quantified by RT-PCR and normalized to B2m expression (2^−ΔCt). Significant differences are estimated by Mann-Whitney U test or 1-way analysis of variance, Dunn test for multiple comparisons (*P < .05; **P < .01). Mean values are indicated.