Screening of TLR ligands
| TLR ligand . | Conc. tested, ng/mL . | Conc. at max. stimulation, ng/mL . | FVIII . | No. of experiments . | |||
|---|---|---|---|---|---|---|---|
| 0 ng/mL . | 10 ng/mL . | 1000 ng/mL . | 20 000 ng/mL . | ||||
| Control | 0 (0-1) | 100 | 7 (3-10) | 0 (0-2) | 10 | ||
| Zymosan/TLR2 | 0.1-10 000 | 10 000 | 1 (1) | 71 (21-121) | 9 (5-13) | 3 (1-5) | 2 |
| Poly (I:C)/TLR3 | 1-50 000 | 1000-10 000 | 1 (1) | 263 (227-298) | 31 (28-34) | 2 (1-2) | 2 |
| LPS/TLR4 | 0.1-10 000 | 1-100 | 2 (1-3) | 199 (144-292) | 87 (42-131) | 2 (1-3) | 2-3 |
| Flagellin/TLR5 | 0.01-1000 | 1-1000 | 1 (1) | 91 (35-146) | 53 (11-94) | 2 (1-3) | 2 |
| Loxoribine/TLR7 | 1-50 000 | 10 000-50 000 | 9 (2-12) | 478 (196-1023) | 212 (187-260) | 57 (12-112) | 3-5 |
| Imiquimod/TLR7 | 1-50 000 | 100-1000 | 2 (1-3) | 794 (205-1118) | 174 (126-220) | 38 (19-59) | 3-4 |
| CpG-ODN/TLR9 | 0.1-10 000 | 100 | 3 (0-6) | 676 (132-1884) | 198 (44-320) | 135 (24-194) | 4-5 |
| TLR ligand . | Conc. tested, ng/mL . | Conc. at max. stimulation, ng/mL . | FVIII . | No. of experiments . | |||
|---|---|---|---|---|---|---|---|
| 0 ng/mL . | 10 ng/mL . | 1000 ng/mL . | 20 000 ng/mL . | ||||
| Control | 0 (0-1) | 100 | 7 (3-10) | 0 (0-2) | 10 | ||
| Zymosan/TLR2 | 0.1-10 000 | 10 000 | 1 (1) | 71 (21-121) | 9 (5-13) | 3 (1-5) | 2 |
| Poly (I:C)/TLR3 | 1-50 000 | 1000-10 000 | 1 (1) | 263 (227-298) | 31 (28-34) | 2 (1-2) | 2 |
| LPS/TLR4 | 0.1-10 000 | 1-100 | 2 (1-3) | 199 (144-292) | 87 (42-131) | 2 (1-3) | 2-3 |
| Flagellin/TLR5 | 0.01-1000 | 1-1000 | 1 (1) | 91 (35-146) | 53 (11-94) | 2 (1-3) | 2 |
| Loxoribine/TLR7 | 1-50 000 | 10 000-50 000 | 9 (2-12) | 478 (196-1023) | 212 (187-260) | 57 (12-112) | 3-5 |
| Imiquimod/TLR7 | 1-50 000 | 100-1000 | 2 (1-3) | 794 (205-1118) | 174 (126-220) | 38 (19-59) | 3-4 |
| CpG-ODN/TLR9 | 0.1-10 000 | 100 | 3 (0-6) | 676 (132-1884) | 198 (44-320) | 135 (24-194) | 4-5 |
CD138− spleen cells were obtained from hemophilic mice treated with 4 weekly doses of 200 ng of FVIII and restimulated in vitro with FVIII at the concentrations indicated in the presence of TLR ligands in the range of concentrations shown. Newly formed anti-FVIII ASCs were detected by ELISPOT assay after 6 days of culture.
All results were normalized to compare experiments done on different days. Results obtained in differentiation cultures containing 10 ng/mL FVIII only were always set as 100%. The results shown represent the highest response obtained for each ligand tested. This highest response is expressed as the mean value and range of response, in percent, compared with the response after stimulation with 10 ng/mL FVIII.
Poly I:C indicates polyinosinic:polycytidylic acid; Conc., concentration; and max., maximum.