KLH-dependent MHC class II–restricted T-cell proliferation in vitro correlates with DTH reactivity to KLH in vivo. (A) In vitro quantification of the KLH-specific immune response by PBL proliferation to KLH. 10⁵ PBLs (week 0, 4, and 10) were plated in 96 round-bottom culture plates with 200 μL CM and stimulated with 20 μg/mL KLH for 4 days. Proliferative activity was assessed after a final 6 hours' pulsing with 1 μCi (0.037 MBq)/well [³H]TdR. Negative control using FCS-supplemented complete medium showed only background proliferation (< 2000 cpm). Data shown represent the mean of triplicate wells. Standard deviations (error bars) were less than 15% for all data points. ▪ indicates patient 1; •, patient 2; ▴, patient 3; □, patient 4; and ○, patient 5. (B) KLH-dependent T-cell proliferation is MHC class II restricted. PBLs (week 0, 4, and 10) were allowed to proliferate to KLH in the absence or presence of anti–HLA-DR mAb (L243 hybridoma culture supernatant 1/1 vol/vol) and its respective isotype control. Proliferation was gradually restored by serial dilution (1/1, 1/10, or 1/100 vol/vol) of the supernatant. Data shown (patient 2) are representative for all patients (n = 5). ▦ indicates CM; ▪, KLH + isotype control; and □, KLH + anti–HLA-DR. (C) KLH-dependent T-cell proliferation correlates with the size of DTH to KLH (coefficient of regression r = 0.97, P < .01).