Importance of ICOS for memory-driven asthma. (A–E) ICOS^−/−:Rag1^−/− (I:R^−/−) and Rag1^−/− (R^−/−) mice were subjected to the Alt-induced memory-driven asthma protocol as per Fig. 1 A. Airway hyperreactivity (A) was measured by flexiVent, two-way ANOVA. Lung eosinophils (CD45⁺SiglecF⁺CCR3⁺ cells; B), ST2⁺, IL5⁺, and IL13⁺ ILC2s (CD45⁺NK1.1⁻CD25⁺ICOS⁺ST2⁺) cells were measured by FCM (C–E). *, P < 0.01; **, P < 0.001; ***, P < 0.0001, unpaired t test, n = 4/group. (F–J) ICOS^−/− and littermate ICOS^+/+ mice were subjected to the Alt-induced memory-driven asthma protocol as per Fig. 1 A. Airway hyperreactivity was measured by flexiVent (F). Lung eosinophils, ILCs (Lin⁻CD45⁺CD25⁺), ILC2s (Lin⁻CD45⁺CD25⁺PLZF⁺KLRG1⁺), and IL13⁺ ILC2s (CD45⁺Lin⁻CD25⁺PLZF⁺KLRG1⁺) cells were measured by FCM (G–J). *, P < 0.01; **, P < 0.001; ***, P < 0.0001; ns, not significant; two-way ANOVA. (K–P) Comparison of memory (ST2⁺) and nonmemory (ST2⁻) ILCs between ICOS^+/+ and ICOS^−/− mice. The frequency of ST2 ⁺ ILC2s (K) and KLRG1⁺ IL13⁺ and IL5⁺ ILC2s in ST2⁺ and ST2⁻ ILCs (L–P). *, P < 0.01; **, P < 0.001; ***, P < 0.0001, unpaired t test, n = 4–5/group. (Q–U) Rag1^−/− mice were intranasally pretreated with an anti-ICOSL antibody or an isotype control antibody before recall challenge. Airway hyperreactivity (Q) was measured by flexiVent, two-way ANOVA. Lung eosinophils (CD45⁺SiglecF⁺CCR3⁺ cells; R), ICOS⁺ST2⁺ ILCs (CD45⁺CD25⁺; S), and IL5⁺, IL13⁺ ILC2s (CD45⁺CD25⁺ICOS⁺ST2⁺; T and U) in the lung digest were measured by FCM. ***, P < 0.001, unpaired t test, n = 4–5/group.