Fig 4.

Impaired WNV-specific CD8⁺ T cell responses in the spleens of infected Cd22^−/− mice. Splenocytes were harvested from naive or infected WT or Cd22^−/− mice at various time points after infection. (A) Tetramer staining. Total splenocytes were stained with surface markers for CD8⁺ T cells, NS4B-specific MHC I tetramer, and CD44. Tetramer⁺ CD44⁺ CD8⁺ T cells are quantified in representative dot plots as frequency of CD8⁺ cells (left dot plots) or in cell numbers (right bar graph). (B) CD8⁺ T cell ICS. Total splenocytes were restimulated ex vivo with 1 μM NS4B peptide as described in Materials and Methods. Data are shown as IFN-γ⁺ TNF-α⁺ cells as a frequency of total CD8⁺ T cells (left dot plots) and total cell numbers of CD8⁺ IFN-γ⁺ TNF-α⁺ populations (right bar graphs). (C) In vivo cytotoxicity assays were performed at described in Materials and Methods. CFSE-labeled unpulsed (CFSE-low) and NS4B-pulsed (CFSE-high) Ly5.1⁺ splenocytes were transferred into naive WT or Cd22^−/− mice or into WT or Cd22^−/− mice infected 5 days earlier with WNV; 2 h later, splenocytes were isolated and stained for surface Ly5.1 expression. (Left histograms) Representative histograms show CFSE-low and -high populations as a frequency of total Ly5.1⁺ cells. (Right graph) Each symbol represents percent killing of NS4B-pulsed Ly5.1⁺ target cells in an infected WT or Cd22^−/− mouse. (D) In vivo BrdU incorporation assay. Naive and day 5 and 7 infected WT and Cd22^−/− mice were injected with BrdU i.p. as described in Materials and Methods. Data are shown as BrdU⁺ tetramer⁺ cells as a frequency of total CD8⁺ T cells (left dot plots) and frequency of CD8⁺ tetramer⁺ cells that are BrdU⁺ (right bar graphs). (E) CD4⁺ T cell ICS. Total splenocytes were restimulated ex vivo with overnight with 1 μM NS3 peptide prior to ICS to detect IFN-γ in CD4⁺ T cells. Data are shown as IFN-γ⁺ cells as a frequency of total CD4⁺ T cells (left dot plots) and cell numbers of CD4⁺ IFN-γ⁺ populations (right bar graphs). Data show one representative of three independent experiments with at least three mice per group per time point. Statistics were performed using Student's t test. *, P < 0.05. n.s., not significant.