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. 2018 Nov 2;7:e39800. doi: 10.7554/eLife.39800

Figure 6. FCRL5hi identifies MSP121-specific Bmem after resolution of P.chabaudi infection.

(A) Flow cytometry showing expression of FCRL5 (right) on different subsets of splenic MSP121-specific B cells from NIMP23→Rag2-/- chimeric mice defined based on CD273 and CD80 expression (left) at 155dpi. (B) t-SNE analysis of splenic MSP121-specific B cells based on FCRL5, CD38, IgD, CD273 and CD80 expression measured by flow cytometry (n = 5). Clusters identified by PhenoGraph are colored and numbered. (C) PhenoGraph heat map showing median expression of FCRL5, CD38, IgD, CD273 and CD80 on the different clusters of MSP121-specific B cells. Arrows point at the different clusters displaying a memory B cell phenotype. (D) Expression profiles of FCRL5, CD38, CD273 and CD80 for the different PhenoGraph clusters visualized on the first component of ISOMAP. The regression line estimated using the generalized linear model (GLM) is added for each marker. Data representative of three independent experiments with 4–7 mice per group. (E) Heat map showing expression levels of different genes on splenic FCRL5— and FCRL5hi MSP121-specific B cells sorted at 155dpi, and MSP121-specific B cells sorted before infection (naïve), determined by RNAseq analysis. Each column corresponds to data from an individual mouse (n = 5 155 dpi, n = 5 0 dpi).

Figure 6.

Figure 6—figure supplement 1. Gating strategy for the sorting of splenic MSP121-specific FCRL5hi Bmem and FCRL5— B cells at 155dpi.

Figure 6—figure supplement 1.

Figure 6—figure supplement 2. High expression of FCRL5 identifies Bmem.

Figure 6—figure supplement 2.

(A) Flow cytometry gating strategy to identify splenic MSP121-specific FCRL5neg (red) and FCRL5hi (blue) B cells, and additional surface markers expressed on these cells, in NIMP23→ Rag2-/- mixed bone marrow chimeras infected with P. chabaudi (155dpi, top row) and immunized with recombinant MSP121 in Titermax Gold emulsion (day 29 post-immunization, bottom row). (B) Cumulative data showing geometric MFI for CD80, CD273, CD73, and CD38 memory markers on splenic MSP121-specific FCRL5neg and FCRL5hi B cells obtained from immunized mice. Mann Whitney U test. **, p<0.01. Error bars are SEM. Data representative of two independent experiments with 3–5 mice per group.