Tcm conjugation with antigen-specific HTC leads to decelerated and confined HTC movement in the LNs. Lethally (10Gy) and sublethally (6.5Gy) irradiated C57BL/6 mice received 0.5 to 2 × 10⁶ 2c CD8⁺ cells (dyed with SNARF; red for lethal irradiation and magenta for sublethal irradiation [RIC]), and 1.5 to 10 × 10⁶ CB6 (“specific”) or C57BL/6 (“nonspecific”) derived Tcms (green, each dyed with CMTMR). Videos, using 2-photon microscopy were recorded at the indicated time points (see supplemental Methods). (A-B) 2c-CD8⁺-Tcm conjugates detected 4 to 16 hours after injection of 2 × 10⁶ 2c CD8⁺ and 10 × 10⁶ CB6 cells (arrows mark conjugates). (Picture presented is a single frame from supplemental Videos 1 and 2). A video was recorded 16 hours after injection of 0.5 × 10⁶ 2c CD8⁺, and 1.5 × 10⁶ CB6 or C57BL/6 cells. (C-D) Dot plot depicting velocity of individual cells at the LN (2c HTCs in the presence of “nonspecific” or “specific” Tcms). (E-F) Dot plot depicting displacement rate of individual 2c HTCs at the LN (in the presence of “nonspecific” or “specific” Tcms). Red horizontal bars indicate mean. (G-J) 3-D paths of tracked cells normalized to their starting coordinates in the presence of “nonspecific” (G-I), or “specific” (H-J) Tcms. Axes represent a distance of ± 100 μm for lethal irradiation and ± 30 μm for sublethal irradiation (RIC). Results presented represent 1 of 3 independent experiments (***P < .001).