Relationship between memory B-cell subsets and pneumococcal humoral response. PBMCs were incubated with PE-cyanin7–conjugated anti-CD19 (Coulter Immunotech High Wycombe), PE-conjugated anti-human IgD (Southern Biotechnology Associates), APC-conjugated anti-human IgM (The Jackson Laboratory), and FITC-conjugated anti-CD27 (DakoCytomation). Cells were then washed and acquired on FACSCalibur (BD Biosciences, Oxford, United Kingdom). A minimum of 5000 events were acquired on the B-cell gate, and the results are expressed as a percentage of CD19 events. FlowJo software (TreeStar) was used for data analysis. Because IgM memory B cells express both IgD and IgM, coexpression of either IgD or IgM together with CD27 was used to define this subset. IgM memory B cells (CD19⁺ CD27⁺ IgM^high IgD^+/lo) and switched memory B cells (CD19⁺ CD27⁺ IgM^– IgD^–) were calculated using a modified Piqueras classification.²⁸  (A) Patients who fail to mount a pneumococcal IgM response have significantly lower frequencies of IgM memory B cells compared with responders and healthy controls. (B) Scatter plot evaluating the association between pneumococcal IgM titers and IgM memory B-cell frequencies in CML patients. Samples were correlated using the Spearman rank correlation test. (C) Frequencies of class-switched memory B cells in the 33 patients who achieved a postimmunization IgG >200 U/mL compared with the 6 patients who failed to mount a positive pneumococcal IgM and IgG response; bars represent medians with interquartile range. A positive IgM Pneumovax II response was defined as a fourfold rise in serum IgM titers or an IgM titer >200 U/mL 4 weeks postimmunization irrespective of the preimmunization titer. A positive IgG response was defined as a twofold rise in serum IgG titer or an IgG titer >200 U/mL at 1 or 3 months.²⁶  (D) IgM memory B-cell frequencies at diagnosis (prior to initiation of imatinib) and once CCyR was achieved on imatinib. (E) Class-switched memory B-cell frequencies at diagnosis (prior to initiation of imatinib) and once CCyR was achieved on imatinib. (F) B-cell phenotype of a CML patient who developed a positive pneumococcal IgM response (patient A) compared with a nonresponder (patient B).