Dysregulation of redox control by Trsp KO disrupts B-cell differentiation and transcriptional program. (A) Frequency of pre–pro-B (CD3^–CD11b^–Gr-1^–Ter-119^–IgM^–CD19^–CD43⁺), pro-B (CD3^–CD11b^–Gr-1^–Ter-119^–IgM^–CD19⁺CD43⁺), pre-B (CD3^–CD11b^–Gr-1^–Ter-119^–IgM^–CD19⁺CD43^–), and immature B (CD3^–CD11b^–Gr-1^–Ter-119^–IgM⁺CD19⁺CD43^–) cells in the BM of control and Trsp KO mice (n = 3 per group). P values were calculated by a 2-sided Student t test. (B) Representative image of polymerase chain reaction amplification. pre-B cells were isolated from BM by cell sorting, and Igh V(D)J gene rearrangement was determined by polymerase chain reaction amplification with Gapdh as a loading control (n = 2 per group). (C) Frequency of pre-B cells in the BM of each group categorized by age (young [age <20 weeks], n = 3 mice per group; aged [81 weeks], n = 5 mice per group). P values were calculated by a 2-sided Student t test. (D) MFI of C11-BODIPY 581/591 staining in pre-B and immature B cells in the BM cells of aged control mice and aged Trsp KO mice (n = 5 mice per group; age 81 weeks). P values were calculated by a 2-sided Student t test. (E) Heat map showing genes that regulate neutrophil and B-cell differentiation evaluated by RNA-seq in Trsp KO BM pre-B cells. The listed genes are from gene ontology biological process. Neutrophil includes neutrophil degranulation (GO:0043312) and Cebpa; B lymphocyte includes lymphocyte differentiation (GO:0030098), lymphocyte differentiation (GO: 0030098), regulation of B-cell receptor signaling pathway (GO:0050855), B-cell receptor signaling pathway (GO:0050853), regulation of B-cell proliferation (GO:0030888), and Ebf1, Irf4, and Irf8 (n = 4 per group). (F) GSEA enrichment plot for upregulate genes in RNA-seq of Trsp KO vs control in pre-B cells (n = 4 per group). (G) Schematic representation of the experimental procedure. LSK cells were sorted from the BM of Trsp^fl/fl or Trsp^fl/fl: R26-CreERT2 mice, expanded until day 6, and cultured under B-cell differentiation conditions with OP-9 stromal cells in the presence of IL-7 and FLT3-L. Cells were treated with 4-OHT for 24 hours on day 5. On day 20, they were analyzed by flow cytometry. (H) The frequency of B220⁺, B220⁺CD19⁺, and CD11b⁺ cells in each group is shown. P values were calculated by 1-way ANOVA test. (I) Schematic of BM transplantation assays. CD3^–CD11b^–Gr-1^–Ter-119^–CD19⁺IgM^– (pro-B/pre-B) BM cells sorted from the BM of control (Trsp^fl/fl) or Trsp KO (Trsp^fl/fl: Mx1-Cre) mice at 8 weeks after pI-pC injection, with rescue cells (CD45.1⁺ whole BM cells) injected into lethally irradiated CD45.1 mice. (J) Flow cytometric profiles for B220⁺ and CD11b⁺ in respective conditions and frequency of CD45.2⁺B220⁺ and CD45.2⁺CD11b⁺ cells in the BM of each group were assessed 1 month after transplantation (n = 5 per group). P values were calculated by a 2-sided Student t test. (K) The appearance of donor pro-B/pre-B–derived CD45.2⁺CD11b⁺ fractions in recipient PB is shown in each group (defined as >0.3% in live PB cells). P values were calculated using Fisher exact test. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001. EtOH, ethanol; FDR, false discovery rate; FLT3-L, FMS-related tyrosine kinase 3 ligand; Igh V(D)J, immunoglobulin heavy chain V(D)J; IL-7, interleukin-7; NES, normalized enrichment score; ns, not significant.