Relative increase of pre–B-1 B cells in the bone marrow of ICOS-deficient patients. Eight different precursor B-cell stages are defined and 1 mature IgM⁺/IgD⁺ B-cell stage. The pro-B and pre–B-I cell subsets (stages 1-5) of ICOS-deficient patients 3 and 4 show a relative increase as compared with the healthy control subject. (A) Analysis of expression of TdT and CD19 within a purified cytoplasmic (cy) CD79a⁺ lymphogate discriminates 4 different B-cell populations: a TdT^–/CD19^– (stage 2) and a TdT⁺/CD19^– (stage 3) pro–B-cell subset, a TdT⁺/CD19⁺ B-cell population (pre–B-I/pre–B-II stage 6), and a TdT^–/CD19⁺ (pre–B-II stage 7, immature and mature B cells). (B) Analysis of CyIgμ and surface membrane (Sm) IgM within a purified CD19⁺ lymphogate discriminates pre–B-I (CyIgμ^–/SmIgM^–), pre–B-II (CyIgμ⁺/SmIgM^–), and immature/mature (CyIgμ⁺/SmIgM⁺) B-cell subsets. (C) Analysis of SmIgM and SmIgD within a purified CD19⁺ lymphogate discriminates the pre–B-I and pre–B-II cell subsets together (SmIgM^–/SmIgD^–), the immature B cells (SmIgM⁺/SmIgD^–), and the mature B cells (SmIgM⁺/SmIgD⁺). (D) Schematic representation of the precursor B-cell compartment in a healthy donor and ICOS-deficient patients 3 and 4. The size of the 8 precursor B-cell stages is set to 100%.