Unequal LFA-1 expression leads to disparate migration and differentiation of first-division CD8⁺ T cells. (A) Frequency distribution of migration indices measured from sorted first-division LFA-1^high (YFP^high) versus LFA-1^low (YFP^low) CD8⁺ T cells migrating on ICAM-1+CCL21–coated plates were significantly different. Data collected from four independent experiments (one mouse per experiment; 44–72 cells per mouse) were fit to nonlinear regression, and multimodality was assessed with the Kolmogorov–Smirnov test. Asterisk indicates significance between LFA-1^high versus LFA-1^low (*, P < 0.01). (B) Flow cytometry analysis and homing index of first-division LFA-1^high versus LFA-1^low CD8⁺ T cells generated in vivo in x31-OVA–infected mice treated with 100 µg anti-CD62L i.v. ± 1 µg/g FTY720 i.p. 44 hpi. Lymph node and spleen were harvested at 56 hpi, and single-cell suspension was recorded on a flow cytometer (left). The homing index (right) of Div1 YFP^high or Div1 YFP^low cells was calculated as the ratio between anti-CD62L–treated mice and (anti-CD62L + FTY720)–treated mice. Circles represent individual mice from three independent experiments (one or two mice per experiment) with mean shown as a line. Data represent mean ± SEM. *, P < 0.0001. (C) mRNA levels of indicated genes from sorted first-division LFA-1^high versus LFA-1^low CD8⁺ T cells from x31-OVA–infected mice 56 hpi. Data represent mean ± SEM; n = 3 mice per group. *, P < 0.05. (D and E) First-division LFA-1^high and LFA-1^low CD8⁺ T cells were harvested at 56 hpi from influenza-infected mice and sorted based on YFP expression. Equal numbers (2 × 10³) of sorted first-division LFA-1^high versus LFA-1^low CD8⁺ T cells were injected into WT recipients, and mice were then inoculated with X31-OVA. The number of CD11a-mYFP⁺ CD8⁺ T cells found in the draining lymph node and lung 8 dpi (D; n = 3 mice per group) or 60 dpi (E) is shown. Data represent mean ± SEM; n = 3–6 mice per group. *, P < 0.001) as measured by flow cytometry. (F) Number of tissue-resident memory (TRM; CD103⁺ [integrin α_E], CD62L^low [L-selectin], CD44^high), central memory (TCM; CD62L⁺, CD44^high), and effector memory (TEM; CD62L^neg, CD44^high) CD11a-mYFP⁺ CD8⁺ T cells found in the lung 60 dpi. Data represent mean ± SEM; n = 3 mice/group. *, P < 0.001.