FIGURE 1.

Immunization with powerful immunogens induces a profound loss of cytolytic capacity upon restimulation with peptide. IL-2-rVV immunization augments primary cytolytic responses. Two BALB/c mice were immunized i.v. with 5 × 10⁶ PFU/mouse of different rVV. After 6 days, the spleens were removed, pooled, and tested in a 6-h ⁵¹Cr release assay against CT26.WT tumor cells infected with VV-WT (A) or pulsed with the β-gal peptide (B). Secondary responses are lost in mice immunized with IL-2-rVV. The splenocytes used for the previous experiment were further incubated in vitro with 1 μg/ml of the synthetic β-gal peptide for 6 days and then assayed in a 6-h ⁵¹Cr release assay against the CT26.CL25 β-gal-positive clone (C) or the CT26.WT cells pulsed with the β-gal peptide (D). Purification of CD8⁺ cells restores specific reactivity in mice immunized with IL-2-rVV. CD8⁺ cells were enriched through affinity columns from splenocytes used in Fig. 1C and D. This enriched population was admixed with naive, CD8-depleted splenocytes from syngeneic mice to constitute about 10% of total cells in culture. After a 6-day in vitro restimulation with β-gal peptide, the mixture was tested in a 6-h ⁵¹Cr release assay against CT26.WT cells pulsed with the β-gal peptide (E). Cytotoxicity toward the CT26.WT cells or the irrelevant E22 target cells was always <5% even at the highest E:T ratio (not shown). The E:T ratio was 33:1, then diluted threefold (11:1, 4:1, 1:1). The experiment was repeated four times with similar results.