Analysis of CTL function during clone 13 persistence. A) Mononuclear cells were extracted from the spleen, liver and CNS of Armstrong (black bars) or clone 13 (white bars) infected mice (n = 3 mice per group) at the denoted time points. Following a 5 hr in vitro stimulation with GP33–41 peptide, P14 cells were examined flow cytometrically for the production of IFN-γ (top row), TNF-α (middle row) and IL-2 (lower row). Note that when compared to the P14 cells in the spleen and liver, an intermediate state of P14 functional exhaustion was observed in the CNS. This was most prominent at day 20 p.i. P14 cells in all compartments regained complete functionality by day 90 p.i. Each bar represents the mean ± SD. Statistical differences between Armstrong and clone 13 infected mice are denoted by asterisks (p < 0.05). B) Representative dot plots used to generate the bar graphs in panel A are shown for CNS P14 cytokine production at day 20 p.i. This time point was selected to show the relative preservation of CNS P14 function at a time point when functional exhaustion was most severe in the spleen and liver. Dot plots are gated on CD45+CD8+Thy1.1+ P14 cells, and the numbers indicate the frequency of P14 cells that produce the denoted cytokines. C) The relative loss in P14 function was calculated by dividing the frequency of TNF-α producing P14 cells (as shown in panel A) from the CNS, spleen, and liver of clone 13 infected mice by the frequency observed in Armstrong infected mice. This number was multiplied by 100 to generate percentages. Note the relative preservation of P14 function in the CNS when compared to peripheral tissues. Double asterisks (**) denote a statistically significant difference (p < 0.05) between the CNS and spleen as well as the CNS and liver. A single asterisk (*) denotes a statistically significant difference (p < 0.05) between the CNS and spleen only.