Reduced antiviral response and impaired NK-cell function associated with reduced ζ-chain expression in LPS-treated mice. (A) Mice were infected by intranasal inoculation with an LD₅₀ dose of influenza virus (A/PR/8/34) 2 days before the third LPS treatment and monitored daily for mortality. The figure shows a representative survival curve, of 2 independent experiments performed, of control and LPS-treated mice after infection with influenza virus (n = 10). (B) Splenocytes from untreated and anti-NK1.1-treated mice (NK1.1-depleted mice) were stained for DX-5 and NK1.1 markers and analyzed by FACS. (C) In vivo cytotoxicity assay of CFSE-labeled allogeneic splenocytes was performed as described in “Cytotoxicity assays.” CFSE-labeled allogeneic (CFSE^low) and syngeneic (CFSE^high) cells were injected to untreated and NK1.1-depleted naive mice. After 24 hours, PBLs, lymph nodes (LNs), and splenocytes were analyzed to determine the ratio between allogeneic and syngeneic cells and the index of specific allogeneic cell clearance was calculated as described in “Cytotoxicity assays.” The mean value of 3 independent experiments with standard deviations is shown.* P < .005 (Student t test). (D) Representative plot of CFSE-labeled allogeneic (CFSE^low) and syngeneic (CFSE^high) cells within the spleen of control, LPS-treated or anti-NK1.1–treated mice 24 hours after administration (top panel). In the bottom panel, the results of 3 independent experiments are presented as the mean value plus or minus standard deviations (bottom panel). *P < .005 (Student t test). (E) Splenocytes were harvested from control and LPS-treated mice and subjected to in vitro NK-cytotoxicity assay with YAC-1 target cells in different effector to target ratios. Percentage of specific lysis was calculated as described in “Cytotoxicity assays.” *P < .01; **P < .001 (Student t test). (F) Before harvesting (24 hours), control and LPS-treated mice were injected intraperitoneally with poly (I:C); splenocytes were then harvested and subjected to in vitro NK-cytotoxicity assay as in E. (G) Total ζ-chain expression in splenic NK1.1⁺CD3⁻ cells from LPS-treated and control C57BL/6 mice.