CD138 expression promotes ASC accumulation and maturation. (A) Diagrammatic representation of the cell transfer and immunization protocol. CD45.1/2 WT cells and CD45.2 sdc1^−/− cells were injected at a ratio of 50:50. (B) Gating strategy and representative dot plots of B220⁺GL7⁺Fas⁺ GC B cells from WT (blue; co-stained for CD45.1 and CD45.2) and sdc1^−/− (red; CD45.2⁺) cells 7 days after secondary immunization. Graph represents the percentage of GC B cells generated on day 7 following immunization. (C) Gating strategy showing the detection by flow cytometry of NP-specific YFP⁺ ASCs on day 7 following immunization. WT (blue) ASCs co-stain for CD45.1 and CD45.2, whereas ASCs lacking CD138 expression (red) stain for CD45.2 only. CD138 expression was used to delineate WT gating. (D-E) Percentage (left) and frequency (right) of WT and CD138-deficient ASCs in the LN (D) and spleen (E) of host mice on day 7 after immunization. Results are expressed relative to total transferred B cells. Mice were immunized with NP-OVA either by a subcutaneous route in the case of LN data or by IP immunization for spleen data. (F) Percentage and frequency of WT and CD138-deficient ASCs in the BM of host mice 28 days after primary immunization with NP-KLH emulsified in alum. (G) ASC maturation is characterized by increased YFP expression. Maturation was measured by examining the mean fluorescence intensity (MFI) of YFP expression in sdc1-deficient ASCs relative to WT ASCs (sdc1^−/−:WT). (H) YFP⁺ ASCs from WT or CD138-deficient ASCs in the medulla of surgically exposed LNs were examined by two photon microscopy on day 7 following immunization. YFP intensity of individual cells was measured and the mean was calculated. Graph represents the mean (± SEM) of YFP intensity from 3 independent experiments. *P < .05; **P < .01; ***P < .001. ns, not significant; SSC, side scatter.