FIGURE 3 | Tissue map analysis of OVA-specific transgenic T cells in vivo.

Transgenic T-cell antigen receptor (TCR) T-cell (KJ1-26⁺; red), B-cell (B220⁺; green) and activated, dually phosphorylated extracellular signal-regulated kinase (pERK; blue) staining of a lymph-node section from a mouse that received adoptively transferred transgenic T cells and was immunized with ovalbumin (OVA) in complete Freund's adjuvant in vivo. Individual OVA-specific T cells were identified by setting standard integration contours (yellow) based on the staining (red) of the transgenic TCR by the clonotypic antibody KJ1-26 (a). By contrast, the B-cell follicles were located by a lattice of phantom contours set to detect green fluorescence (c) throughout the tissue. Such phantom contours (radius of 6 m and with a minimal distance between phantom centers of 20 m) generate fluorescence values that represent the B-cell follicles as a whole rather than the individual densely packed B cells. Plotting of the x and y coordinates of fluorescence allows generation of tissue maps identifying the localization of transgenic TCR T cells (red, b) and B cells (green, d) within the lymph node while the expression levels of KJ1.26 and B220 are quantified by histograms (a and c respectively). Further analysis of the transgenic TCR (KJ1-26⁺) gate allows analysis of the levels of activated, dually phosphorylated pERK expression (integral values) in individual antigen-specific cells (e) and the localization of pERK⁺ transgenic T cells within the tissue (f). Merging of the individual tissue maps (g) and selection of regional gates (h; that is, follicular (green) and paracortical (white) gates) allows quantification of both the number of transgenic T cells and also the levels of pERK expression in such T cells in these two microenvironments within the lymph node.

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