Fig. 4.

L3-specific CTLs are not boosted by multiple sporozoite injections but can be boosted by heterologous immunization. (A) ELISPOT from BALB/c mice homologously immunized one or three times at 30-d intervals with 1 × 10⁴ CPS. ELISPOT was performed on the day listed by using 1 × 10⁻⁷ M CSP (open squares) or L3 (filled squares) peptides. *P = 0.002 (unpaired t test, 4–6 mice per group). (B) Flow cytometry for MACS tetramer-purified splenocytes from naïve mice or mice immunized once or three times with 2 × 10⁴ GAP. Gates and values denote the percentage of tetramer-positive CD8⁺CD4⁻B220⁻ single cells. (C) In vivo killing of CSP- or L3-coated target cells in naïve mice (open squares) or mice immunized with 3× RAS (filled squares), CSP peptide-pulsed DCs (open triangles), 3× RAS then CSP peptide-pulsed DCs (filled triangles), L3 peptide-pulsed DCs (open circles) or 3× RAS then L3 peptide-pulsed DCs (filled circles). *P < 0.05, **P < 0.01, ***P < 0.001 (unpaired t test; 2–3 mice per group). (D) Flow cytometry for MACS tetramer-purified splenocytes as in B from mice immunized 6 d earlier with mock-, CSP- or L3-peptide treated DCs. Gates and values as in B. (E) In vivo killing of CSP- or L3-coated target cells as in C in mice receiving CSP-pulsed DC priming and no booster immunization (open triangles), CSP DC priming and single RAS booster (filled triangles), L3 peptide-pulsed DC priming and no booster (open circles), or L3 peptide-pulsed DC priming and single RAS booster (filled circles). *P < 0.05, **P < 0.01 (unpaired t test); 4–5 mice per group. (F and G) Flow cytometry of total splenocytes showing CD11a^HIL3 tetramer-stained CD8⁺ T cells in mice immunized with mock- or L3 peptide-pulsed DCs or 2 × 10⁴ GAP parasites followed by L3-expressing Listeria infection 30 d later. The number of activated L3-specific cells per spleen was calculated and plotted in G. *P < 0.05, **P < 0.01, ***P < 0.001 (unpaired t test; 5 mice per group).