CD4⁺T-cells contribute to the persistence of splenic LLPCs in anti-CD20–treated AID-Cre-EYFP mice. (A-B) Analysis of interactions between EYFP⁺ PCs (green) and CD3⁺ T cells (blue) in the spleen on B-cell depletion. We analyzed total spleen sections from 3 mice by confocal microscopy corresponding to ∼400 EYFP⁺ PCs in controls and 300 EYFP⁺ PCs in anti-CD20–treated mice. Images were acquired by confocal microscopy with LSM 700 (Zeiss) and 40× objective. A threshold of confocal resolution allows the unambiguous detection of PCs. (A-B) Spleen sections were stained with anti-EYFP, anti-CD3 antibodies, and DAPI. Scale bars, 150 µm (A), 18 µm (B, left panel), 10 µm (B, right panel). (C) Number of EYFP⁺ B220⁻ PCs per spleen in controls, anti-CD20–, anti-CD20/anti-CD4–, and anti-CD4–treated mice (see supplemental Figure 2C for protocol). Analysis is from 2 independent tamoxifen-labeling experiments except for anti-CD4. (D) Survival of splenic EYFP⁺ PCs cocultured for 5 days with CD4⁺ T cells from anti-CD20–treated mice or CD4⁺ T cells from controls. Splenic EYFP⁺ PCs were cultured after sorting from 5 immunized mice, with a CD4⁺ T-cell–to-PC ratio of 5:1. CD4⁺ T cells were collected from untreated or anti-CD20–treated immunized mice (5 days after the second anti-CD20 injection). Assays were performed in duplicate per condition and per mouse. Significant differences are estimated by paired Student t test or 1-way analysis of variance, Dunn test for multiple comparisons (* P < .05; *** P < .001). Mean values are indicated.