Figure 7.

Virus specific CTLs poorly accumulate in infected ICAM-1- and ICAM-2- deficient lungs but give rise to normal T_RM CD8⁺ cells that protect from homo- and hetero- subtypic infections. (A) Experimental design and timeline of the accumulation of OT-I CD8⁺ effector T cells in the lungs of either WT or ICAM-1/2^-/- infected mice. A total of 2x10⁶ CD45.1 OT-I splenocytes were I.V. transferred into WT or ICAM-1/2^-/- mice and 18 hours later, mice were I.N. infected with a sublethal dose of PR8-SIINFEKL. (B) 7 days post infection, lungs were harvested and the number of CD45.1 OT-I CD8⁺CD62L^-CD44⁺ effector T cells recovered from the MedLNs were determined by flow cytometry of total lung cell suspensions. (C) The totals of endogenous Vβ8.3⁺/CD8⁺ T cells recovered from the PR8-SIINFEKL infected lungs of either WT or ICAM-1/2^-/- mice challenged in (B) were determined 7 days post PR8-SIINFEKL infection. I.N. PBS administration was performed in a control experiment. (D) Experimental design and timeline protocol for primary infection with X31 influenza virus. WT and ICAM-1/2^-/- mice were I.N. infected with 10⁴ PFU of X31 influenza or PBS treated. 40 days later, the numbers of T_RM CD8⁺ cells (CD62L^loCD44^highCD69⁺CD49a⁺CD103^-/+) recovered from the lungs of both mice groups were determined (top panel). Results shown (bottom panel) are the mean +- SEM. (E) Experimental design and timeline of primary X31 influenza infection (1ry) followed by secondary PR8 influenza challenge (2ry). WT and ICAM-1/2^-/- mice were I.N. infected with 10⁴ PFU of X31 influenza. 40 days later, all 4 groups were challenged I.T. with a high dose of PR8 influenza virus (600 PFU) and their weight (bottom panel) and survival (right panel) were monitored for 30 days post-secondary challenge. Mice that lost over 25% of their initial weight were humanly euthanized. (F) Experimental design and timeline protocol for primary PR8 infections (1ry) followed by a second challenge (2ry). WT and ICAM-1/2^-/- mice were initially infected with 30 PFU (I.N.) or 15 PFU (I.T.) of PR8. 40 days later, both groups were challenged I.T. with a lethal dose (3x10³ PFU) of PR8. 7 days later mice were sacrificed and the numbers of endogenous TCR Vβ8.3⁺/CD8⁺ CD62L^-CD44⁺ T cells were determined in the lungs of both mice groups. (G) Results shown from homosubtypic I.N. infection followed by I.T. challenge. (H) Results shown from homosubtypic I.T. infection and challenge. PBS administration was performed in control experiments. (I) Experimental design for detection of T_RM CD8⁺ and CD4⁺ cells generated following primary PR8 infection. WT and ICAM-1/2^-/- mice were I.N. infected with 30 PFU of PR8 influenza. 40 days later, the numbers of T_RM CD8⁺ cells recovered from total lung cell suspensions were determined and are shown in (J). The numbers of recovered T_RM CD4⁺ cells (CD62L^loCD44^highCD69⁺CD49b⁺CD103^-/+) are shown in (K). (B–D, G–K) Statistical significance was determined by two-tailed, unpaired Student’s t tests. (E) Statistical analysis was performed using a one-way ANOVA test comparing % body weight loss between groups. *P < 0.05, **P < 0.01, ns, not significant. The numbers of each experimental group are indicated in the graphs. The error bars indicate the SEM of each measurement.