Figure 2.
GALT is enriched in IgMhi T2 cells.(A) SPADE on viSNE plots depicting the expression of B cell lineage markers used to identify T1 and T2 cells as CD27−IgD+CD10+ in a concatenated (n = 7) GALT sample. GC, IgD−CD10+; M only, IgM-only memory (CD27+IgD−IgM+); PB, plasmablast (IgD−CD38hi; see also Fig. S1 E). (B) A minimal spanning tree generated by FlowSOM run on exported events (n = 4,520 from each tissue) from the TS bubble in A using CD10, CD24, CD38, and IgM as clustering parameters. The tree displayed shows aggregated events from both concatenated GALT and PBMC samples. Automatic metaclustering of the FlowSOM nodes identified six metaclusters; the identity of each can be inferred by the relative expression of CD21, CD24, CD38, and IgM (see also C). (C) Minimal spanning trees showing expression of CD21, CD24, CD38, and IgM on a concatenated (n = 7) PBMC sample. Clusters represent phenotypically similar cells, their size is proportional to the number of cells contained within them, and their color indicates the median expression of a given marker. (D) Pie charts demonstrating the proportion of TS B cell subsets inferred from metaclusters in B confirm that GALT is enriched in IgMhi T2 cells. (E) Minimal spanning trees demonstrating higher expression of CD69 and CD80 on GALT TS B cells. (F) Flow cytometry contour plots of concatenated (n = 3) liver perfusate samples and concatenated HCD PBMC (n = 3). TS B cells were gated as CD27−IgD+CD10+ as illustrated in Fig. S1 D. A reduced proportion of CD24++CD38++ T2 cells was observed in liver perfusates. (G) Flow cytometry plots of concatenated liver perfusate and PBMC samples demonstrating T2 cells as gated in F and naive (CD27−IgD+CD10−) B cells as gated in Fig. S1 D. A reduced frequency of IgMhiCD24hi T2 and naive cells was observed in liver perfusate samples compared with HCD PBMCs. (H) Flow cytometry dot plots with IgM mean fluorescence intensity (MFI) overlay of concatenated PBMC (n = 3) and liver perfusate (n = 3) samples demonstrating reduced frequency of IgMhiCD24hi TS and naive B cells in liver perfusate samples. (I) Scatter plots of flow cytometry data from individual samples gated as in G demonstrating reduced frequency of IgMhiCD24hi TS and naive B cells (CD27−IgD+CD10−) in liver perfusate samples compared with PBMCs (median values).

GALT is enriched in IgMhi T2 cells. (A) SPADE on viSNE plots depicting the expression of B cell lineage markers used to identify T1 and T2 cells as CD27−IgD+CD10+ in a concatenated (n = 7) GALT sample. GC, IgD−CD10+; M only, IgM-only memory (CD27+IgD−IgM+); PB, plasmablast (IgD−CD38hi; see also Fig. S1 E). (B) A minimal spanning tree generated by FlowSOM run on exported events (n = 4,520 from each tissue) from the TS bubble in A using CD10, CD24, CD38, and IgM as clustering parameters. The tree displayed shows aggregated events from both concatenated GALT and PBMC samples. Automatic metaclustering of the FlowSOM nodes identified six metaclusters; the identity of each can be inferred by the relative expression of CD21, CD24, CD38, and IgM (see also C). (C) Minimal spanning trees showing expression of CD21, CD24, CD38, and IgM on a concatenated (n = 7) PBMC sample. Clusters represent phenotypically similar cells, their size is proportional to the number of cells contained within them, and their color indicates the median expression of a given marker. (D) Pie charts demonstrating the proportion of TS B cell subsets inferred from metaclusters in B confirm that GALT is enriched in IgMhi T2 cells. (E) Minimal spanning trees demonstrating higher expression of CD69 and CD80 on GALT TS B cells. (F) Flow cytometry contour plots of concatenated (n = 3) liver perfusate samples and concatenated HCD PBMC (n = 3). TS B cells were gated as CD27−IgD+CD10+ as illustrated in Fig. S1 D. A reduced proportion of CD24++CD38++ T2 cells was observed in liver perfusates. (G) Flow cytometry plots of concatenated liver perfusate and PBMC samples demonstrating T2 cells as gated in F and naive (CD27−IgD+CD10−) B cells as gated in Fig. S1 D. A reduced frequency of IgMhiCD24hi T2 and naive cells was observed in liver perfusate samples compared with HCD PBMCs. (H) Flow cytometry dot plots with IgM mean fluorescence intensity (MFI) overlay of concatenated PBMC (n = 3) and liver perfusate (n = 3) samples demonstrating reduced frequency of IgMhiCD24hi TS and naive B cells in liver perfusate samples. (I) Scatter plots of flow cytometry data from individual samples gated as in G demonstrating reduced frequency of IgMhiCD24hi TS and naive B cells (CD27−IgD+CD10−) in liver perfusate samples compared with PBMCs (median values).

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