FIG. 1.
T cell epitope identification and…
FIG. 1.
T cell epitope identification and cytokine production in MA15 virus-infected BALB/c and B6…
FIG. 1.T cell epitope identification and cytokine production in MA15 virus-infected BALB/c and B6 mice. (A) B6 mice (6 to 8 weeks old) were infected intranasally with 3 × 105 PFU MA15 virus in 50 μl DMEM. BALB/c mice were treated with clodronate, because in its absence, most BALB/c mice do not mount a virus-specific T cell response after infection with this virus dosage (35). At day 8, single cell suspensions were prepared from lungs and stimulated with 1 μM SARS-CoV-specific CD8 or CD4 T cell peptides for 5 h in the presence of brefeldin A. Frequencies of MA15 virus-specific T cells (determined by IFN-γ intracellular staining) are shown. Data are representative of three independent experiments, with 5 to 8 mice/group. (B) BALB/c or B6 mice (6 to 8 weeks old) were infected intranasally with 3 × 103 PFU or 3 × 105 PFU MA15 virus, respectively, in 50 μl DMEM. At day 7, single cell suspensions were prepared from lungs, LNs, and spleens and stimulated with SARS-CoV CD8 or CD4 T cell peptides for 5 h in the presence of brefeldin A. IL-2, TNF-α, and IFN-γ expression levels are shown. Data are summarized in Fig. 2A.
FIG. 2.
Characterization of T cell response…
FIG. 2.
Characterization of T cell response in MA15 virus-infected BALB/c and B6 mice. (A)…
FIG. 2.Characterization of T cell response in MA15 virus-infected BALB/c and B6 mice. (A) Numbers of MA15 virus-specific T cells are shown. Data are representative of two independent experiments, with 4 mice/group. (B) In vivo cytotoxicity assays were performed on day 6 p.i. as described in Materials and Methods. Data are representative of three independent experiments, with 3 or 4 mice/group.
FIG. 3.
SARS-CoV-specific T cell transfer cleared…
FIG. 3.
SARS-CoV-specific T cell transfer cleared virus and ameliorated clinical disease in both immunocompromised…
FIG. 3.SARS-CoV-specific T cell transfer cleared virus and ameliorated clinical disease in both immunocompromised and immunocompetent mice. Donor BALB/c mice were treated with clodronate 1 day prior to infection with 3 × 104 PFU MA15 virus. Donor B6 mice were infected with 3 × 105 PFU MA15. Mice were sacrificed 8 days p.i. and cells harvested from the infected lungs and spleens. Recipient SCID (A and B), B6 and RAG1−/− (C and D), or BALB/c (E and F) mice (6 to 8 weeks old) received 2.5 × 107, 5 × 107, or 10 × 107 splenocytes, 2 × 107 total lung cells, or 5 × 106 lung-derived CD4 or CD8 T cells at day −1 or no cells and were then infected intranasally with 3 × 104 PFU (SCID and BALB/c mice) or 3 × 105 PFU (B6 and RAG1−/− mice) MA15 virus in 50 μl DMEM at day 0. Mortality and weight were monitored daily. (A) Recipient SCID mice. Eleven mice were in the control group, 9 mice received 5 × 107 total splenocytes, and 10 mice received lung-derived CD4 or CD8 T cells. Survival was significantly greater in recipients of CD4 (P < 0.05) or CD8 (P < 0.01) T cell transfer and nearly significantly greater after total splenocyte transfer (P = 0.06). (C) B6 and RAG1−/− mice. Eight mice were in each group. B6 and RAG1−/− mice showed mild weight loss but remained asymptomatic after MA15 virus infection even in the absence of adoptively transferred cells. (E) BALB/c mice. Eight mice were in the control group; 8, 16, or 8 mice received 2.5 × 107, 5 × 107, or 10 × 107 splenocytes, respectively; and 8 mice received total lung cells. Survival was significantly greater after transfer of 5 × 107 or 10 × 107 splenocytes (P < 0.007 for both) or lung cells (P < 0.007). For virus titers, infected SCID (B), RAG1−/− (D), or BALB/c (F) mice received adoptively transferred cells or no cells and were sacrificed at the indicated time points. Lung homogenates were prepared, and titers were determined on Vero E6 cells. Viral titers are expressed as PFU/g tissue. There were 4 mice/group. AT, adoptive transfer. The horizontal line denotes the limit of detection.
FIG. 4.
Transfer of SARS-CoV-specific CD8 T…
FIG. 4.
Transfer of SARS-CoV-specific CD8 T cell lines decreased virus titers and ameliorated clinical…
FIG. 4.Transfer of SARS-CoV-specific CD8 T cell lines decreased virus titers and ameliorated clinical disease in BALB/c mice. Epitope S366-specific CD8 or epitope N353-specific CD4 T cell lines (TCLs) were propagated in vitro as described in Materials and Methods. (A) TCLs were stimulated with SARS-CoV CD8 peptide for 5 h in the presence of brefeldin A and examined for expression of IFN-γ. (B) In vivo cytotoxicity assays were performed 1 day after transfer of CD8 T cells as described in Materials and Methods. Data are representative of two independent experiments, with 3 or 4 mice/group. (C) CD8 or CD4 T cells (1 × 107) were transferred to 6- to 8-week-old BALB/c mice 1 day prior to i.n. infection with 3 × 104 PFU MA15 virus. Mortality was monitored daily. Eight mice were in the control group, 14 mice received CD8 T cells (P = 0.002), and 11 mice received CD4 T cells (P = 0.07). (D) For virus titers, lungs were homogenized at the indicated time points and titers were determined on Vero E6 cells. Viral titers are expressed as PFU/g tissue. There were 4 mice/group. The horizontal line indicates the limit of detection. *, P < 0.05. (E) Single cell suspensions were prepared from the lungs and spleens of infected BALB/c mice that had received 1 × 107 CD8 T cells. Cells were stimulated with SARS-CoV-specific CD8 T cell peptide S366 for 5 h in the presence of brefeldin A. IFN-γ expression frequencies of transferred T cells are shown. (F) Endogenous CD8 T cell responses at day 6 p.i. in recipients of transferred CD8 T cells were determined by IFN-γ intracellular staining. Data are representative of two independent experiments, with 4 mice/group.
FIG. 5.
Immunization with DC/peptide S366 complexes…
FIG. 5.
Immunization with DC/peptide S366 complexes induced protective CD8 T cell responses. BALB/c mice…
FIG. 5.Immunization with DC/peptide S366 complexes induced protective CD8 T cell responses. BALB/c mice (6 to 8 weeks old) were immunized with BMDCs pulsed with peptide S366. Seven days later, mice were infected intranasally with 3 × 104 PFU MA15 virus. Single cell suspensions were prepared from lungs and spleens at the indicated time points and stimulated with SARS-CoV S366 peptide for 5 h in the presence of brefeldin A. TNF-α and IFN-γ expression frequencies (A) and numbers (B) of SARS-CoV-specific T cells are shown. (C) Mortality was monitored daily. Ten mice were in the control group, and 10 and 19 mice were immunized with uncoated BMDCs (empty) and peptide S366-coated BMDCs, respectively. Survival was significantly greater in recipients of S366-coated BMDCs than in those receiving no (control) or empty BMDCs (P < 0.02). (D) For virus titers, lungs were homogenized at the indicated time points and titers were determined on Vero E6 cells. Viral titers are expressed as PFU/g tissue. There were 4 mice/group. The horizontal line denotes the limit of detection.
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