Fig 4. OE of LTβR increases PC numbers in vitro in response to LPS or anti-CD40 stimulation in the absence of ligand.

(A) Diagram of experimental scheme. Chimeras were made by retroviral transduction of CD45.2⁺ CD19-Cre^+/- BM with Empty-MSCV-Thy1.1 or loxp-EGFP-loxp-LTβR-MSCV-Thy1.1 retrovirus and transfer into irradiated CD45.1⁺ recipients. After reconstitution, B cells were isolated from the spleen for in vitro culture. (B) Representative FACS plots of CD45.2⁺B220^- splenocytes in chimeras stimulated with or anti-CD40 with IL-4, IL-5, and IL-21 for 3 days. Transduction efficiency based on Thy1.1 expression on FO B cells was 43–93% among the mice analyzed. Blocking of the LTβR ligands was assessed through the addition of human LTβR-Fc blocking antibody. (C) FACS plots of live Dump^-B220⁺CD45.2⁺ B cells with gates on Thy1.1⁺ cells and further gates on CD98⁺CD138⁺ PCs. (D) Percentages of CD98⁺CD138⁺ PCs among Thy1.1⁺ (left) and Thy1.1^- (right) B cells as gated in B. Numbers of CD98⁺CD138⁺ Thy1.1⁺ PCs (middle). (E) LTβR protein expression by flow cytometry of WT B cells stimulated with LPS for 3 days and retrovirally transduced with Empty-MSCV-Thy1.1 or LTβR-MSCV-Thy1.1 vectors. (F) Frequency of CD98⁺CD138⁺ PCs among Thy1.1⁺ transduced WT or Ltb^-/- B cells stimulated with LPS media for 3 days. Data shown in all panels are representative of two independent experiments (n = 2–3 mice per group). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.