Fig 2: TCF4 and IRF4 function downstream of p38a to regulate Blimp1 transcription.a Flow cytometry analysis of iPCs differentiated from cultured Cas9tg/+ B cells transduced with retroviruses encoding p38a or Tcf4 sgRNA (CD19+GFP+BFP+CD138+). Immunoblot analysis of BLIMP1, TCF4, and Actin expression in Tcf4 sgRNA expressing B cells at indicated time points after being transferred onto IL-21 feeder cells. b Schematic diagram of a putative TCF4-binding site in the Prdm1 locus. CUT&Tag analysis of TCF4 binding genomic fragments including the reported TCF4 binding site in Blimp1 enhancer region in cultured p38a KO and WT B cells at day 2 after transferred onto IL-21 feeder cells (n = 3 per group). c Flow cytometry analysis of iPCs among cultured p38a KO B cells transduced with retroviruses encoding TCF4 (CD19+GFP+CD138+) (n = 6 per group), protein levels of TCF4, IgG H-chain, BLIMP1, p38a and Actin were analyzed by immunoblot. d–f Same as described in (a–c) except that Irf4 sgRNAs were used or IRF4 was overexpressed in cultured p38a KO B cells. g Cultured p38afl/flVavcreCas9tg/+ and p38afl/flCas9tg/+ B cells were transduced with retroviruses encoding sgRNA targeting p38a, Tcf3, Tcf4 and Irf4 at day 2 of culture (n = 3 per group). Percentages of iPCs in sgRNA transduced B cells (CD19+GFP+BFP+CD138+) were analyzed by flow cytometry at day 8. NTC, non-targeting control. Data were representative of at least three independent experiments. Each symbol represents a representative sample. Small horizontal lines indicate the mean (±s.d.). Data were analyzed by two-tailed unpaired t-tests. Source data are provided as a Source Data file.

Fig 3: The p38a-BLIMP1 axis controls PC generation differentiated from in vivo generated GCB cells.a Sort GCB cells (CD3-B220+Fas+GL7+) from the spleen of OVA/Alum/LPS immunized mice (n = 10 per group) at day 7.5 were mixed and cultured on IL-21 feeder cells, and iPCs differentiated from p38a KO and WT GCB cells were analyzed at day 4. b Sorted p38a KO GCB cells from the spleen of OVA/Alum/LPS immunized mice (n = 10 per group) were mixed and cultured on IL-21 feeder cells and transduced with retroviruses encoding p38a or p38a kinase-dead mutants (K53M or T180A/Y182F) 1 day later. The percentage of iPCs in retrovirus-transduced B cells (CD19+GFP+CD138+) was analyzed by flow cytometry 3 days after transduction. c Flow cytometry analysis of iPCs among cultured p38a KO GCB cells from the spleen of OVA/Alum/LPS immunized mice (n = 10 per group) transduced with retroviruses encoding p38a, BLIMP1, TCF3 (E12 or E47), TCF4, or IRF4 (CD19+GFP+CD138+). Data were representative of at least three independent experiments. Each symbol represents a representative sample. Small horizontal lines indicate the mean (±s.d.). Data were analyzed by two-tailed unpaired t-tests. Source data are provided as a Source Data file.

Fig 4: The p38a-BLIMP1 signal axis is required for LPS-induced PC generation.a Naive B cells from the spleen of p38afl/flVavcre or p38afl/fl mice were stimulated with LPS for 4 days (n = 6 per group). The percentage of iPCs (CD19+CD138+) was analyzed by flow cytometry. b Cultured p38a KO and WT B cells were transduced with retroviruses encoding p38a or p38a kinase-dead mutants (K53M or T180A/Y182F) at day 1 of LPS-induced iPCs generation. iPCs among retrovirus-infected B cells (CD19+GFP+CD138+) were analyzed by flow cytometry 3 days after retroviral transduction (n = 6 per group). c Flow cytometry analysis of BLIMP1-expressing cells (CD19+GFP+), BLIMP1-expressing CD138- B cells (CD19+GFP+CD138-) and BLIMP1-expressing iPCs (CD19+GFP+CD138+) differentiated from cultured p38afl/flVavcreBlimp1gfp/+ and p38afl/flBlimp1gfp/+ B cells stimulated with LPS and MFI of GFP (indicating Blimp1 expression) in BLIMP1-expressing cells, GFP+CD138- B cells and iPCs (n = 4 per group). d Cultured p38a KO and WT B cells were transduced with retroviruses encoding BLIMP1, TCF3 (E12 or E47), TCF4, or IRF4 at day 1 of LPS-induced iPCs generation. iPCs among retrovirus-infected B cells (CD19+GFP+CD138+) were analyzed by flow cytometry 3 days after retroviral transduction (n = 6 per group). Data were representative of at least three independent experiments. Each symbol represents a representative sample. Small horizontal lines indicate the mean (±s.d.). Data were analyzed by two-tailed unpaired t-tests. Source data are provided as a Source Data file.

Fig 5: The activation of TCF3, TCF4 and IRF4 downstream of p38a in PC differentiation.a Cultured p38a-/- B cells were transduced with retroviruses encoding p38a or p38a kinase-dead mutations (K53M or T180A/Y182F) (GFP+) and luciferase reporter harboring TCF3, TCF4 or IRF4 binding sites in Prdm1 enhance region at day 2, and luciferase activity in retrovirus-infected B cells (CD19+GFP+) was analyzed at day 8 in iPC culture (n = 3 per group). b p38-/- 293 A cells were transfected with indicated plasmids, and the luciferase activity of IRF4 mutants was analyzed 24 h after transfection. Immunoblot analysis of p38a, p-p38a, IRF4 and Actin in p38-/- 293 A cells transfected with indicated plasmids (n = 3 per group). c Cultured Irf4-/- B cells were transduced with retroviruses encoding IRF4 or IRF4 mutants (T267A, T268A, T269A) at day 2, iPCs in retrovirally transduced B cells (CD19+GFP+CD138+) 6 days after transduction were analyzed by flow cytometry (n = 5 per group), and protein levels of IRF4, IgG H-chain, BLIMP1 and Actin were analyzed by immunoblot. Data were representative of at least three independent experiments. Each symbol represents a representative sample. Small horizontal lines indicate the mean (±s.d.). Data were analyzed by two-tailed unpaired t-tests. Source data are provided as a Source Data file.