Figure 1.
Virus replication kinetics in lungs of mice infected with H5N1 viruses HK483 or HK486.
At indicated days after I/N infection with 18 PFU of HK483 or HK486 virus, mice were euthanized and lung virus titers were determined by plaque assay on MDCK cells. The data are the average titers from 3 mice ± SD at each time point. Data is representative of 2 independent experiments. * P = 0.01; ** P = 0.0005; *** P = 0.0005; **** P = 0.02; ***** P = 0.004; ****** P = 0.002.
Figure 2.
CD8 T cell responses in the lung airways of mice infected with H5N1 viruses.
Panel A, Groups of BALB/C mice were infected I/N with 18 PFU of HK483 or HK486 virus. At the indicated days after infection, pooled cells from broncoalveolar lavage of 3 mice were collected. Cells were stained with anti-CD8, anti-LFA-1, and Kd/NP147 MHC I pentamers, and pentamer-binding LFA-1Hi CD8 T cells were quantified by flow cytometry. The data are representative of two independent experiments. Panel B, CFSE-labeled Thy1.1+ve TCR transgenic CL-4 CD8 T cells were adoptively transferred in to congenic Thy1.2/BALB/c mice, and infected I/N with 18 PFU of HK483 or HK486 virus 24 hours later. At day 5 after infection, cells from deep cervical lymph nodes were stained with anti-CD8, anti-Thy1.1, and Kd/HA518 tetramers. The histograms are gated on CD8+/Thy1.1+ MHC-I tetramer-binding cells, and the numbers are the percentage of divided cells of total gated cells. Data are representative of analysis of pooled cells from 3 mice/group. Panel C, As in panel B, CL-4 CD8 T cells were adoptively transferred into BALB/c mice, and infected with HK483 or HK486 virus. At the indicated time points after infection, cells from the deep cervical lymph nodes were stained with anti-CD8, anti-Thy1.1, Kd/HA518 tetramers, anti-LFA-1, anti-CD43, anti-CD62L, or anti-granzyme B. The flow cytometry plots are gated on CD8+/Thy1.1+ MHC-I tetramer-binding cells, and the numbers are the percentage of LFA-1LO/HI, CD43LO/HI, or CD62LO/HI, or granzyme BLO/HI of total gated cells.
Figure 3.
Premature contraction of CD8 T cells in mice infected with lethal dose of highly pathogenic H5N1 influenza viruses.
BALB/c mice were I/N inoculated with 1 MLD50 or 10 MLD50 of HK486 or 10 MLD50 of HK483 virus. At indicated days after infection, pooled cells from the BAL of 3 mice were stained with anti-CD8, anti-LFA-1, and Kd/NP147 pentamers. The numbers of activated LFA-1Hi CD8 T cells (Panel A) or NP147-specific LFA-1Hi CD8 T cells (Panel B) were quantified by flow cytometry. Data are from one of two independent experiments.
Figure 4.
Apoptosis of CD8 T cells in mice infected with H5N1 viruses.
A and B, BALB/c mice were infected with 18 PFU of HK483 or HK486. At the indicated days after infection, the percentages of total CD8 T cells (Panel A) and Kd/NP147-specific CD8 T cells (Panel B) expressing active form of caspase-3 were determined by flow cytometry; cells in the BAL were stained with anti-CD8, Kd/NP147 pentamers, and anti-active caspase-3. Panel C, C57BL/6 (+/+), Fas-deficient (Fas KO) or BIM-deficient (BIM KO) mice (n = 3) were infected with 18 PFU of HK483. On the eighth day after infection, the percentages of total CD8 T cells and of Db/PA224-specific CD8 T cells expressing active form of caspase-3 in the BAL were determined as in panels A and B above.
Figure 5.
Survival of C57BL/6, Fas KO, and BIM KO mice after inoculation with highly pathogenic HK483 virus.
C57BL/6 (+/+; n = 17), Fas KO (n = 12), or BIM KO (n = 5) mice were I/N inoculated with 18 PFU of HK483 virus and mouse survival was monitored for 16 days (Panel A). Panel B, At day 8 after infection, lungs from infected mice were examined for histopathological changes. Arrows indicate apoptotic cells in lung section from +/+ mouse or lymphocytic infiltrates adjacent to the bronchioles in lung section from BIM−/− mice.
Figure 6.
Oseltamivir therapy protects against accelerated activation and contraction of CD8 T cells following infection of mice with HK483 virus.
C57BL/6 mice were treated daily with indicated doses of oseltamivir (mg/Kg body weight) from −1 day to 3 day relative to infection with 100 PFU of HK483. Panel A, At days 5, 7, and 9 post-infection, BAL samples from 3 mice/dose were pooled and the number of CD8 T cells specific to CTL epitope PA224 were determined by flow cytometry. Panel B, At days 1, 2, 3, 5, 7, and 9 PI, 3 mice were euthanized at each time point/dose and lung virus titers were determined; error bars indicate SD. * P = 0.03 between 0 mg and 5 mg doses and P = 0.06 between 0 mg and 10 mg doses; ** P = 0.0003 between 0 mg and 5 mg doses, P = 0.08 between 0 mg and 10 mg doses, and P = 0.005 between 0 mg and 20 mg doses; *** P = 0.02 between 0 mg and 5 mg doses, P = 0.006 between 0 mg and 20 mg doses; **** P = 0.12 between 0 mg and 5 mg doses and P = 0.07 between 0 mg and 10 mg doses, and P = 0.004 between 0 mg and 20 mg doses. Panel C, Effect of oseltamivir treatment on survival of HK483-infected mice (n = 5/group). Data in panels B and C is representative of two independent experiments.
Figure 7.
Effect of CD8 T cell deficiency on survival of mice infected with H5N1 viruses.
C57BL/6 (+/+) or CD8-deficient (CD8 KO) mice were infected intranasally with the indicated doses of HK483 virus (Panels A and B). Three to six mice were infected at each dose and mouse survival was observed for 21 days.