FIG 2.

Unhelped CD8_M expand and generate secondary CD8_M cells when maintained and boosted in an MHC-II-deficient environment. B6 mice and MHC-II^0/0 mice were primed i.p. with 5 × 10⁶ PFU VACV and, when applicable, similarly boosted 5 weeks later. (A) CD8_M in spleen were determined at 60 dpp or dpb; the cells were gated on CD8⁺ T cells. Representative flow cytometry plots on the left show IFN-γ and GzB expression after in vitro restimulation with VACV-infected DC2.4 cells (top row) and staining with K^b-TSYKFESV⁺ dimers (bottom row). The summary graphs on the right show the frequency of IFN-γ⁺ and K^b-TSYKFESV⁺ CD8_M. (B) Frequency of K^b-TSYKFESV⁺ CD8_M expressing the indicated cell surface molecules at 60 dpp or dpb. (C) In vivo killing of target cells by CD8_M. Splenocytes from naive B6 mice were labeled with CFSE at 3.0 μM (CFSE^High) or 0.8 μM (CFSE^Low). CFSE^High cells were pulsed with TSYKFESV, and CFSE^Low were not. Mixtures (1/1) of CFSE^High and CFSE^Low cells were injected i.v. into primed or primed/boosted B6 and MHC-II^0/0 mice at 60 dpp or dpb. Killing was determined 6 h later in spleen. Representative flow cytometry plots are shown on the left. Plots in the upper row are gated on CD8⁺ cells. The transferred cells can be distinguished by their CFSE expression. The histograms in the lower row show the peaks of CFSE^Low and CFSE^High cells. The number inside the plot indicates the percentage of specific killing of CFSE^High (TSYKFESV-pulsed) cells. Data correspond to groups of three to five mice and are representative of results from two or three experiments.