Figure 1.
Segregation of B cell phenotypes from T2 through naive B cell subsets.(A) SPADE on viSNE plots generated using the following markers that were used to identify B cell subsets: CD10, CD24, CD27, CD38, CD45RB, IgD, IgM, IgA, and IgG. The plots are from a female HCD and are representative of 10 HCDs. Nodes represent a cluster of phenotypically similar cells, the size of a node is proportional to the number of cells represented by it, and the color indicates the median expression of a given marker. Nodes representing B cell subsets were grouped into bubbles as indicated by the schema to the left. (B) SPADE on viSNE plots of TS B cells exported from the TS SPADE bubble in A and generated by rerunning viSNE using all expressed B cell markers. The SPADE plots depicted are from a female HCD and are representative of 10 HCDs. TS B cell populations were defined as T1 (CD10+CD24+++CD38+++CD21lo), T2 (CD10+CD24++CD38++CD21hi), and T3 (CD10−CD24+CD38+CD21hi). (C) SPADE trees demonstrating that T2 and T3 TS B cells have prominent IgMhi and IgMlo subpopulations. (D) SPADE trees demonstrating that IgMhi T2 B cells have higher expression of β7 integrin and lower expression of CCR7 than IgMlo T2 B cells. (E) qPCR quantification of CCR7, ITGB7 (β7 integrin), IGHM, and IGHD transcripts from sorted subsets expressed as 2−ΔCT values relative to 18S endogenous control (paired t test). *, P < 0.05; **, P < 0.01. (F) SPADE demonstrating that IgMhi T2 B cells also have high surface IgD expression. (G) SPADE aligns IgMhi T2 cells with IgMhi T3 cells with relatively high expression of CD45RB. (H) A SPADE on viSNE plot from A demonstrating the identification of IgMhi TS and naive B cell populations. (I) A PCA plot generated using all expressed markers on IgMhi and IgMlo subsets identified in H. Data points represent individual HCD (n = 10) and are surrounded by 95% confidence ellipses with a larger central mean data marker. (J) Variable contribution bar graphs demonstrate that homing receptors are major contributors to PCA1 and PCA2 in I. The dashed red reference line represents the value where the contribution was uniform.

Segregation of B cell phenotypes from T2 through naive B cell subsets. (A) SPADE on viSNE plots generated using the following markers that were used to identify B cell subsets: CD10, CD24, CD27, CD38, CD45RB, IgD, IgM, IgA, and IgG. The plots are from a female HCD and are representative of 10 HCDs. Nodes represent a cluster of phenotypically similar cells, the size of a node is proportional to the number of cells represented by it, and the color indicates the median expression of a given marker. Nodes representing B cell subsets were grouped into bubbles as indicated by the schema to the left. (B) SPADE on viSNE plots of TS B cells exported from the TS SPADE bubble in A and generated by rerunning viSNE using all expressed B cell markers. The SPADE plots depicted are from a female HCD and are representative of 10 HCDs. TS B cell populations were defined as T1 (CD10+CD24+++CD38+++CD21lo), T2 (CD10+CD24++CD38++CD21hi), and T3 (CD10−CD24+CD38+CD21hi). (C) SPADE trees demonstrating that T2 and T3 TS B cells have prominent IgMhi and IgMlo subpopulations. (D) SPADE trees demonstrating that IgMhi T2 B cells have higher expression of β7 integrin and lower expression of CCR7 than IgMlo T2 B cells. (E) qPCR quantification of CCR7, ITGB7 (β7 integrin), IGHM, and IGHD transcripts from sorted subsets expressed as 2−ΔCT values relative to 18S endogenous control (paired t test). *, P < 0.05; **, P < 0.01. (F) SPADE demonstrating that IgMhi T2 B cells also have high surface IgD expression. (G) SPADE aligns IgMhi T2 cells with IgMhi T3 cells with relatively high expression of CD45RB. (H) A SPADE on viSNE plot from A demonstrating the identification of IgMhi TS and naive B cell populations. (I) A PCA plot generated using all expressed markers on IgMhi and IgMlo subsets identified in H. Data points represent individual HCD (n = 10) and are surrounded by 95% confidence ellipses with a larger central mean data marker. (J) Variable contribution bar graphs demonstrate that homing receptors are major contributors to PCA1 and PCA2 in I. The dashed red reference line represents the value where the contribution was uniform.

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