CD22 expression precedes CD19 expression during normal human B-cell differentiation. (A) Cartoon depicting the immunophenotypes used to flow sort the indicated HSPC populations across human fetal B-cell differentiation. Lineage markers: Lin: CD2/3/14/16/19/56/235a; Lin2: CD2/3/14/16/56/235a. (B) Bulk RNA-seq–normalized gene expression (transcripts per million [TPM]) of CD19 (left) and CD22 (right) in flow-sorted hematopoietic stem cells (HSCs), multipotent progenitors (MPPs), lymphoid-primed multipotent progenitors (LMPPs), early lymphoid progenitors (ELPs), Pre-ProB progenitors, ProB progenitors, and PreB cells from human FBM (n = 3). (C) Percentage of CD22⁺ cells within these FBM-derived HSPC populations analyzed by flow cytometry. (D) scRNA-seq UMAP plots showing CD22 expression within flow-sorted Lin⁻CD19⁻CD34⁺ cells from matched human FL (left) and human FBM (right) (n = 2; total, 30 000 cells). (E) Violin plots showing the log normalized median expression levels of CD22 in CD19⁻Lin⁻CD34⁺ cells from human FL and FBM. (F) Frequency of CD34⁺CD19⁻CD22⁺ cells in healthy FL (n = 8), FBM (n = 9), neonatal CB (n = 10), and adult BM (n = 13). Red horizontal lines represent the median. Inset: a representative FACS plot of CD22 and CD19 within the gated CD34⁺ cells. Blue cells are non-B-progenitor cells (mainly myeloid). Red cells are CD19⁺CD22⁺ bona fide B-cell progenitors. Green cells are early CD22⁺CD19⁻ B-cell progenitors. CB, cord blood; FACS, fluorescence activated cell sorting; UMAP, Uniform Manifold Approximation and Projection.