Suppressor assays.
(A) Presence of peptide-specific TRs cells in blood 7 days after injection. Blood mononuclear cells (2 × 105cells/well for subject Im1 and 3 × 105 cells/well for subject Im2) from preimmunization samples or samples obtained 7 days after immunization were cultured overnight, either separately or together (ratio, 1:1), in the presence of mature DCs pulsed with HLA-A*0201–restricted peptides from MP, LMP-2, and HIV gag at a DC/PBMC ratio of 1:60. Antigen-specific, INF-γ–producing cells were quantified by an ELISPOT assay. The asterisk indicatesP < .05 for comparison with baseline reactivity on Student t test. (B) Dose-dependent inhibition of T-cell function. PBMCs (3 × 105 cells/well) from recovery specimens (day 180) from subject Im2 were mixed with preimmunization specimens (nonsuppressor) or various doses of specimens obtained on day 7 (suppressor; ratio of day 7 to day 180 samples, 1:1 or 1:10). Antigen-specific, INF-γ–producing cells were quantified by an ELISPOT assay after overnight culture in the presence of DCs pulsed with influenza MP, EBV-LMP2, or HIV gag at a DC/PBMC ratio of 1:60. Data are representative of results from 2 similar experiments. The asterisk indicates P < .05 for comparison with baseline reactivity on Student t test. (C) Characterization of peptide-specific TRs. PBMCs (3 × 105cells/well) from recovery specimens (day 180) from subject Im2 were mixed (ratio, 1:1) with specimens from day 7, either unseparated, after CD8+ T cell depletion, or cultured physically separated in transwell cultures or in the presence of rIL-2 (100 U/mL). Antigen-specific, INF-γ–producing cells were quantified by an ELISPOT assay after overnight culture in the presence of DCs pulsed with MP, EBV-LMP2, or HIV-gag at a DC/PBMC ratio of 1:60. Data are representative of results from 2 similar experiments. One asterisk indicates P < .05 for comparison with baseline reactivity on Student t test, and 2 asterisks indicateP < .05 for comparison with the suppressed reactivity.
