Kinetics of memory CD8 T-cell differentiation. (A-C) Mice were either infected with LCMV Armstrong (2 × 105 pfu intraperitoneally), or VV-GP33 (2 × 106 pfu intraperitoneally), or immunized with 1010 vp of AdHu5-GP33 intramuscularly, intravenously, or intraperitoneally (similar results were observed by all routes). Two groups of mice were used. In the first group, a small number of naive splenocytes from P14 mice (≈5 × 104 P14 cells) was adoptively transferred to naive B6 mice. The next day these P14 chimeras were infected or immunized. These are designated T-cell receptor Tg (P14). A second group of wild-type B6 mice was also used for confirmation of the P14 results (B6 non-Tg). Db/GP33-specific CD8 T-cell responses were monitored in the peripheral blood mononuclear cells of individual mice over time by MHC tetramer staining in conjunction with staining for CD62L and CD127. (A,B) The kinetics of CD62L and CD127 reversion on the population of Db/GP33 tetramer-positive CD8 T cells is plotted for each condition including P14 chimeras and B6 mice. For each group, n is 2 to 9. Error bars in panels A and B show standard deviations between samples from individual nice. (C) Examples of individual Db/GP33 tetramer staining and CD62L expression on Db/GP33 tetramer+ CD8 T cells are shown for each condition at 2 time points. The numbers show percent double positive cells over all cells positive for a given marker for the dot blots and percent of cells that were expressed high levels of a given marker as indicated by the brackets in the histograms. (D) Groups of BALB/c mice were immunized intramuscularly with 1010 vp of AdC68HIVgag37 for the acute cohort and 5 × 1011 vp for the memory cohort; they were killed at day 10 (acute) or 15 months (memory) after immunization. Groups of C57BL/6 mice were immunized intraperitoneally with 2 × 105 pfu LCMV Armstrong and killed on day 8 (acute) and 7 months (memory) after immunization. Lymphocytes isolated from indicated tissues were stained with a HIVgag tetramer for the AdC68HIVgag37–immunized mice and a GP33 tetramer for the LCMV mice. Cells were also stained with anti-CD8 and the indicated surface markers, and analyzed by flow cytometry. Results shown are gated on tetramer-positive cells (black line) or naive cells (filled curve).
