Figure 3

Frequency of GP33 antigen specific CD8+ T cells with PD-1 expression in the brains of Arm vs. Cl13 mice. (A) FACS plots showing the frequencies of intracellular IFN-γ and surface PD-1 expressing CD3⁺CD8⁺ T cells at five hours after ex vivo co-culture of splenic lymphocytes harvested from mice infected with LCMV Arm (left) or Cl13 (right), with CT2A-gp33 (top) or CT2A (bottom). Plot represents one of two replicates. (B) Histograms of PD-1 expression of GP33⁺lymphocytes (square in the left panel) after gating on Live⁺CD45⁺CD8⁺ T cells. Histograms (right) of four samples are overlapped while FACS plot (left) shows one; (C) Percentages of gp33-tetramer positive staining for CD8⁺CD45⁺ cells isolated from brains, PBMC or spleens of Arm- (upper graph) or Cl13-preinfected mice at seven days after orthotopic challenge with CT2A-gp33 (circles) or CT2A (squares). GP33⁺lymphocytes are selected as shown in the square in the left panel B after gating on Live⁺CD45⁺CD8⁺ T cells. (D) Percentages of brain-infiltrating IFNγ -expressing CD8⁺ T cells, harvested from 7-day old CT2A-gp33 (left), CT2A (center) or no-tumor control (right) challenged naive (non LCMV-infected) mice, 5 hours after in vitro incubation with GP33 peptide. Whiskers in box plots indicate maximum and minimum values measured (n = 4; each group), while line indicates the median. Statistical analyses by Student’s t-test, where *p < 0.05.