Table 1.
Antibodies used for ex vivo flow cytometry analysis.
Table 2.
Antibodies used for post-culture flow cytometry analysis.
Figure 1.
Expansion of total and IgG+ B-cells following mitogen-stimulation.
Panel (A) schematically illustrates the cellular composition of PBMCs directly ex vivo and after mitogen culture. The grey circle represents total PBMCs, the blue circle all CD19+ B-cells and the orange triangle IgG+ B-cells. (B) The composition of the IgG+ B-cell compartment ex vivo was analyzed by flow cytometry and is depicted as median proportions of individual B-cell subsets within total CD19+ B-cells for baseline samples of 62 donors. The individual B-cell subsets were subdivided based on IgD, CD38, CD10, CD21 and CD27 expression and include five memory B-cell subsets: classical MBCs (cMBCs, red), CD27− MBCs (yellow), activated MBCs (actMBC, black), atypical MBCs (atypMBC, green) and non-switched MBCs (nsMBC, white). Depicted in shades of grey are plasma blasts (PB), activated naïve B-cells (actN), classical naïve B-cells (cN) and double-negative naïve B-cells (dnN). Panels C–D show ex vivo and post-culture proportions of total CD19+ B-cells (C, blue dots) and IgG+CD19+ B-cells (D, orange dots) within viable PBMCs. Black lines indicate the median. Dots show all 269 cultures.
Table 3.
Definition of B-cell subsets ex vivo.
Figure 2.
Size and composition of the B-cell compartment ex vivo influences B-cell expansion during culture.
Flow cytometry analysis was performed to determine proportions and subsequently calculate absolute numbers of (A) total CD19+ B-cells (blue dots) and (B) IgG+CD19+ B-cells (orange dots) ex vivo and post-culture. Ex vivo proportions of (C) total CD19+ B-cells (blue dots) within viable PBMCs were plotted against the fold change in their absolute numbers, and (D) IgG+ B-cells (orange dots) within total CD19+ B-cells were plotted against the fold change in their proportion within the B-cell compartment. (E) Proportions of IgG+CD19+ B-cells (orange dots) and (F) IgG−CD19+ B-cells (green dots) within viable PBMCs were plotted against the fold increase in their respective absolute numbers post-culture compared to ex vivo. Colored dots show cultures from all 269 stimulated samples (3–7 time points per volunteer), while black dots show the cultures from only the 62 baseline samples (1 for each individual volunteer). The black dashed line indicates the median fold change (with value), grey dotted lines represent the upper and lower limit of the interquartile range. Spearman r and p values are shown for analysis of baseline samples (black dots) from the 62 donors assessed.
Figure 3.
Relationship between post-culture IgG+ ASC numbers and ex vivo or post-culture IgG+ B-cell proportions.
After 5 days of mitogen culture, proportions of IgG+ B-cells were analyzed by flow cytometry (orange dots) and IgG+ ASCs were quantified by ELISpot (red dots) and expressed as per million post-culture PBMCs (A). Black lines indicate the median. Post-culture (B) and ex vivo proportions (C) of IgG+ cells within PBMCs, and ex vivo proportions of IgG+ cells within CD19+ B-cells (D) were plotted against the number of IgG+ ASCs per million PBMCs. Light red dots show cultures from all 269 stimulated samples (3–7 time points per volunteer), while black dots show the cultures from only the 62 baseline samples (1 for each individual volunteer).