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. 2021 Sep 28;12:738123. doi: 10.3389/fimmu.2021.738123

Figure 2.

Figure 2

B cell TTd tetramer-binding assay. (A) A diagram depicting the theoretical basis of the gating strategy. TTd binding events will bind with equal fluorescence intensities to the tetramer and thus will align proportionally in the double positive quadrant of a TTd-AF647 vs. TTd-BV421 biaxial plot. In contrast, single-positive events represent fluorochrome-reactive B cells, as these are events that bind one tetramer and not the other. (B) Representative scatterplot showing the effect of unlabelled TTd versus 1%BSA/PBS pre-treatment on dual TTd tetramer staining of day 14 post-ADT PBMC sample. (C) Pre-treatment with 100M excess of unlabelled TTd prior to addition of TTd tetramers to PBMC of 5 individuals 14 days following ADT vaccination demonstrates a near complete abolishment of TTd-AF647+BV421+ events across all MBC subsets. (D) Median (range) of %TTd binding inhibition upon pre-treatment with unlabelled TTd, relative to 1%BSA/PBS pre-treated control, are shown for each subset.