Extended Data Fig. 2: Spatiotemporal dynamics and activation of Ag-specific CD4+ T cells within dLNs upon VSV or LCMV infection.
(a, b) Track speed mean (a) and meandering index (b) of GFP+ Ag-specific (Tg7 when VSV-Ind was used, SMARTA in all other cases) CD4+ T cells in the mice described in Fig. 1d–g and Supplementary Movie 1, 3 days after PBS, VSV-Ind, rVSV or rLCMV injection. Data are pooled from 2 independent experiments. PBS, n = 395; VSV-Ind n = 11219; rVSV n = 6692; rLCMV n = 3537. One-way Anova test was applied. **** p value < 0.0001; (c, d) Mean fluorescent intensity of CD69 (c) and CD25 (d) within Ag-specific (SMARTA) CD4+ T cells in dLNs, 2 days after PBS, rVSV or rLCMV injection. Data are representative of 2 independent experiments. Mean + /- SEM is shown. n = 2. One-way Anova test was applied. ** p value < 0.01; *** p value < 0.001 (e, f) Methods used to determine the normalized distance from T area centre (e) and percentages of clustered T cells / section (f). e, T cell area volume was defined based on polyclonal B cell positioning and its centre was geometrically identified in Imaris. Ag-specific CD4+ T cells were localized using Imaris built-in spot detection function and distance from T cell area centre was calculated and normalized for T cell are volumes. f, A T cell cluster was defined as a minimum of 3 T cells aggregating within closest distance of 15 μm measured from cell centroids (see Materials and Methods). Cell clusters of less than 3 cells were manually removed.
