Antiviral CD8⁺ and CD4⁺ T cell interactions with CD11c⁺ microglia after immunotherapy. (A) Two-photon imaging experiments were performed in CD11c-YFP⁺ carrier mice 12 d after adoptive transfer of memory splenocytes containing OFP⁺ CD8⁺ P14 (red) and TFP⁺ CD4⁺ SMARTA (blue) memory T cells. Representative maximal projections extracted from 3D time lapses show interactions between CD11c-YFP⁺ microglia (green) and P14 (top) or SMARTA (bottom) cells over a 20–25-min time period. Arrowheads denote the position of the antiviral T cells at the different time points. White arrowheads indicate that the T cell was in motion at the denoted time point, whereas yellow indicates stable T cell arrest while in contact with the CD11c-YFP⁺ cell. The gray line depicted on the final CD4 T cell image shows the entire path taken by the cell while interacting with the CD11c-YFP⁺ target. (B) Instantaneous velocities were plotted versus time for individual CD8⁺ P14 and CD4⁺ SMARTA T cells interacting with CD11c-YFP⁺ microglia in the brain parenchyma at day 12 after immunotherapy. Each track represents a single T cell. Instantaneous velocities of <2 µm/min signify stable arrest. (C) The scatter plot shows the mean track velocities for individual CD8⁺ P14 and CD4⁺ SMARTA T cells interacting with CD11c-YFP⁺ microglia. Each dot represents an individual T cell. Horizontal black lines denote the mean ± SD of the groups. *, P < 0.05. (D) The bar graph depicts the proportional duration of time (none, <8 min, and >8 min) individual CD8⁺ P14 and CD4⁺ SMARTA T cells spent interacting with CD11c-YFP⁺ cells. All data in this figure are representative of two independent experiments (n = 3 mice per group).