Figure 2
Figure 2. Production of IFNγ and granzyme B and detection of exocytosis in adoptively transferred CD8+ T cells. Chronically FV-infected (B10 × A.BY) F1 mice were treated with 4 × 106 naive, virus-specific CD8+ T cells from B6 mice carrying a TCRtg specific for the H-2Db–restricted gagL epitope of FV. The transferred CD8+ T cells were genetically labeled (CD45.1) to distinguish the host cell population from the donor cell population. B-type CpG-ODN 1668 (CpG) or control ODN without CpG motif (Control) (15 nmol) were injected twice. At 1 week after transfer, spleen cells of the recipient were harvested, and host (CD45.1−) and donor (CD45.1+) CD8+ T cells were stained for granzyme B (gzmB), IFNγ, and CD107a.9 Accumulated results are shown in the left column, with representative flow data in the right column. (A) The difference in the percentages of CD8+ donor T cells (CD45.1+) was statistically significant between the mice receiving CpG-ODNs or control ODNs. (B) Harvested cells were restimulated for 5 hours in vitro with α-CD3 and α-CD28 (closed symbols) or with 5 μg/mL FV DbGagL peptide and α-CD28 (open symbols) to detect intracellular IFNγ. Nonstimulated CD45.1+ CD8+ cells showed a background of less than 0.05% IFNγ-positive cells. The difference in absolute numbers of CD8+ donor (CD45+) T cells (CD45.1–) producing IFNγ was statistically significant between the mice receiving CpG-ODNs or control ODNs. (C) Harvested cells were analyzed directly ex vivo to detect intracellular granzyme B. The difference in absolute numbers of CD8+ donor and host T cells (CD45.1+) producing granzyme B was statistically significant between the mice receiving CpG-ODNs or control ODNs. Similar results were obtained for granzyme A production by CD8+ T cells (data not shown). (D) Harvested cells were analyzed directly ex vivo to detect CD107a expression. The difference in absolute numbers of CD8+ donor T cells (CD45.1+) expressing CD107a was statistically significant between the mice receiving CpG-ODNs or control ODNs. Percentages of positive donor (top right) and host (bottom right) cells are given in the respective quadrants. Mean values for each group are indicated by bars. Statistical analyses were done by unpaired t test.

Production of IFNγ and granzyme B and detection of exocytosis in adoptively transferred CD8+ T cells. Chronically FV-infected (B10 × A.BY) F1 mice were treated with 4 × 106 naive, virus-specific CD8+ T cells from B6 mice carrying a TCRtg specific for the H-2Db–restricted gagL epitope of FV. The transferred CD8+ T cells were genetically labeled (CD45.1) to distinguish the host cell population from the donor cell population. B-type CpG-ODN 1668 (CpG) or control ODN without CpG motif (Control) (15 nmol) were injected twice. At 1 week after transfer, spleen cells of the recipient were harvested, and host (CD45.1−) and donor (CD45.1+) CD8+ T cells were stained for granzyme B (gzmB), IFNγ, and CD107a.9  Accumulated results are shown in the left column, with representative flow data in the right column. (A) The difference in the percentages of CD8+ donor T cells (CD45.1+) was statistically significant between the mice receiving CpG-ODNs or control ODNs. (B) Harvested cells were restimulated for 5 hours in vitro with α-CD3 and α-CD28 (closed symbols) or with 5 μg/mL FV DbGagL peptide and α-CD28 (open symbols) to detect intracellular IFNγ. Nonstimulated CD45.1+ CD8+ cells showed a background of less than 0.05% IFNγ-positive cells. The difference in absolute numbers of CD8+ donor (CD45+) T cells (CD45.1–) producing IFNγ was statistically significant between the mice receiving CpG-ODNs or control ODNs. (C) Harvested cells were analyzed directly ex vivo to detect intracellular granzyme B. The difference in absolute numbers of CD8+ donor and host T cells (CD45.1+) producing granzyme B was statistically significant between the mice receiving CpG-ODNs or control ODNs. Similar results were obtained for granzyme A production by CD8+ T cells (data not shown). (D) Harvested cells were analyzed directly ex vivo to detect CD107a expression. The difference in absolute numbers of CD8+ donor T cells (CD45.1+) expressing CD107a was statistically significant between the mice receiving CpG-ODNs or control ODNs. Percentages of positive donor (top right) and host (bottom right) cells are given in the respective quadrants. Mean values for each group are indicated by bars. Statistical analyses were done by unpaired t test.

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