Intracellular LFA-1 redistribution leads to unequal partitioning during division. (A) Representative image of real-time cell division on ICAM-1–coated plates 30 h after co-culture of naive CD11a-mYFP/OT-I CD8⁺ T cells (yellow) with N4-pulsed BMDCs. H, LFA-1^high; L, LFA-1^low. Bar, 5 µm. (B) Quantification of relative LFA-1 expression levels (mYFP intensity) of daughter T cells with and without APC contact during division. Data were analyzed based on real time imaging under N4-conditions. Each circle represents the ratio of total YFP intensity in each daughter cell by proximity to APC (proximity vs. distal). For divisions occurring outside of APC contact (no APC contact), proximity was assigned arbitrarily and both proximal and distal cells from the same parent cell showed similar YFP intensity (fold change = 1). Circles represent individual cells from five independent experiments with mean shown as a red line. Data represent mean ± SEM; n = 5. *, P < 0.0003. (C) Naive CD11a-mYFP/OT-I CD8⁺ T cells (1–3 × 10⁶) were labeled with Cell Proliferation Dye eFluor670 and i.v. transferred 24 h before infection with influenza virus x31-OVA. 56 h after infection, transferred cells were sorted and identified as the first division (Div1) and undivided (Undiv) cells based on proliferation dye dilution. Cell surface CD8 and CD11a-mYFP showed asymmetric expression on the first-divide cells (Div 1), but not the cell activation marker (CD69). Results are representative of 10 independent experiments (one mouse per experiment). (D) Flow cytometry analysis of cell surface receptors of the first division (Div1) and undivided (Undiv) CD8⁺ T cells generated from OT-I mice 56 h after infection with influenza virus x31-OVA (as described in C). Transferred cells were sorted on proliferation dye expression and stained for cell surface receptors, Con, isotype control. Results are representative of three independent experiments (one mouse per experiment).