Fig 1: Development of a multispectral panel for the assessment of tissue-specific T-cell regulation. (A) Representative example of antibody staining patterns for Helios (pink), CD20 (cyan), CD25 (red), IL-10 (yellow), Ki67 (magenta), CD4 (green) and FoxP3 (purple) in a tonsillar tissue section (left and right panels) and distribution of each marker with respect to the MZ, LZ and DZ (middle panels). Circles (pink/purple) in lower middle panels denote individual Helios (pink) and FoxP3 (purple) events. (B) Immunophenotyping gating strategy used for the identification of Tregs and Tfrs in lymphoid tissues of interest based on the expression and coexpression of the transcription factors FoxP3, Helios, the protein marker CD25 and IL10 in histocytometry. (C) Bar graphs (upper row) and violin plots (lower row) showing the frequencies of Tregs and Tfrs in follicular and extrafollicular areas and frequencies of Tfr subsets in a tonsillar tissue section. Circles in violin plots represent individual follicles whereas colors represent different phenotypic subgroups. Images were acquired at 40x (NA 1.3) with 1% magnification. Scale bars are 50um, 10um (zoomed in details) and 5um (Helios and FoxP3 staining patterns) respectively.