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Table I.

Effect of in vitro cultivation and antigen challenge in vitro/in vivoa

AProtocolResponse: SCID Sera
CultureSCID
FerritinCultivation timeFerritinFresh cell transferHuman IgG (μg/ml)Ferritin binding (μg/ml)*
NA − 1.8 ± 1 <0.1 
− − − 33 ± 10 <0.1 
− 10 − 15 ± 5 0.3 ± 0.1 
− − 62 ± 19 <0.1 
− 70 ± 31 3.8 ± 1.3 
10 − 10 ± 1 2.9 ± 0.4 
84 ± 29 11.6 ± 2.3 
10 21 ± 3 5.1 ± 0.6 
AProtocolResponse: SCID Sera
CultureSCID
FerritinCultivation timeFerritinFresh cell transferHuman IgG (μg/ml)Ferritin binding (μg/ml)*
NA − 1.8 ± 1 <0.1 
− − − 33 ± 10 <0.1 
− 10 − 15 ± 5 0.3 ± 0.1 
− − 62 ± 19 <0.1 
− 70 ± 31 3.8 ± 1.3 
10 − 10 ± 1 2.9 ± 0.4 
84 ± 29 11.6 ± 2.3 
10 21 ± 3 5.1 ± 0.6 
BCulture TTSCID TTHuman IgG titer/log3TT binding titer/log3
NA 7.988 ± 1.26 <4.192 
− − 11.963 ± 0.832 <4.192 
− 11.904 ± 1.112 10.376 ± 1.065 
− 11.234 ± 0.430 8.120 ± 1.759 
12.076 ± 0.766 12.672 ± 0.737 
BCulture TTSCID TTHuman IgG titer/log3TT binding titer/log3
NA 7.988 ± 1.26 <4.192 
− − 11.963 ± 0.832 <4.192 
− 11.904 ± 1.112 10.376 ± 1.065 
− 11.234 ± 0.430 8.120 ± 1.759 
12.076 ± 0.766 12.672 ± 0.737 
a

Responses by human splenocytes subjected to Ag challenge either in vitro, in vivo, or both were measured after transfer to SCID mice. Responses to both a neoantigen (A), horse ferritin, and a recall Ag (B), TT, were measured. A. Five SCID mice in each experiment were engrafted each with 5 × 106 human spleen cells from spleen NN, that had been subjected to in vitro cultivation for 0, 3, or 10 days with (%) or without (−) 1 μg/ml horse ferritin. The hu-SPL-SCID mice were boosted at day 11 with 10 μg of ferritin (+) in IFA or IFA alone (−). In experiments 7 and 8, 2.5 × 106 in vitro primed cells were transferred at the indicated day, followed by a similar number of fresh, autologous cells at day 10. *, Ferritin-specific responses were measured parallel to a human anti-ferritin Ab, 21-1B-9, and are recorded as micrograms per milliliter of this antibody, resulting in similar responses (OD490 readings). Data are given as means ± SD. Sera were taken 29 days after the start of the experiment. B. A compilation of five experiments, with four different spleens. A minimum of four mice was used in any group in all experiments. Each SCID mouse was engrafted with ∼2 × 107 cells that had been cultivated in vitro with (+) or without (−) optimal amounts of TT (see Materials and Methods). Seven days later, the resulting hu-SPL-SCID mice were then immunized with TT emulsified in alum (+) or with PBS emulsified in alum (−). TT-specific responses are reported in log3 titers (3 was the dilution factor) as geometric means ± SD. p values were calculated based on the geometric means ± SD. †, Dilutions were started at 1:100; therefore, nonresponders have titers below 100 = 4.192 log3. NA, not applicable.

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