Fig 2. LTβR expression in B cells is not required for PC responses to a T-dependent antigen.

(A) Diagram of experimental scheme. Chimeras were made by mixing CD45.2⁺ WT or Ltbr^-/- BM cells with CD45.1⁺ wild-type cells at a 30:70 ratio and injecting into irradiated CD45.1⁺ recipients. After 8 weeks of reconstitution, mice were immunized with NP-CGG/Alum two times 21 days apart and analyzed 28 days after the booster immunization. (B) FACS plots of singlet cells with a gate on live Dump^-B220⁺ cells (top row, left). The B220⁺ cells were further gated on IgD⁺GL7^- FO B cells (top row, middle), with further gates on CD45.2⁺ or CD45.1⁺ cells (top row, right). Alternatively, B220⁺ cells were gated based on CD38^-GL7⁺ GC B cells (middle row, left) with further gates on CD45.2⁺ or CD45.1⁺ cells (middle row, middle). NP⁺ GC B cells were identified as NP16-PE⁺ and decoy SA-PE-AF647^- (middle row, right). Memory B cells were identified as CD38⁺GL7^-CD95⁺IgD^- (bottom row, left) with gates on CD45.2⁺ or CD45.1⁺ cells (bottom row, middle) or NP⁺ memory B cells (bottom row, right). (C) Chimerism based on the ratio of CD45.2⁺ WT or Ltbr^-/- to CD45.1⁺ WT FO B cells (top). GC B cell ratio of CD45.2⁺ cells to CD45.1⁺ cells normalized to the FO B cell ratio (middle). Memory B cell ratio of CD45.2⁺ cells to CD45.1⁺ cells normalized to the FO B cell ratio (bottom). (D) Representative FACS plots of CD138-enriched BM cells from a CD45.2⁺ WT recipient chimera pre-gated on live Dump^-B220^loCD138⁺ cells. PCs were identified as B220^- (left), TACI⁺CD98⁺ (top row, middle) with gates on CD45.2⁺ or CD45.1⁺ cells (top row, right). NP-CGG⁺ PCs were identified as NP-CGG-FITC⁺ (bottom row, left) with further gates on CD45.2⁺ or CD45.1⁺ cells (bottom row, right). (E) Ratio of CD45.2⁺ cells to CD45.1⁺ cells normalized to the FO B cell ratio for spleen PCs (top) or BM PCs (bottom). Data shown in all panels are representative of two independent experiments (n = 4–5 mice per group).