![Figure 1. Anti-VEGF and anti–IL-6 Abs reduce the inhibition of induction and maturation of DCs by MM patients' BM sera. (A) Immature DCs were induced from adherent healthy donor PBMCs by culturing in the presence of GM-CSF (1000 U/mL), IL-4 (10 ng/mL), and 20% MM patients' BM sera with control Ab (□), neutralizing anti–IL-6 Abs (5 μg/mL, ▨), anti-VEGF Abs (5 μg/mL, ▦), or both anti–IL-6 and VEGF Abs (▪). Cell surface phenotype was analyzed by flow cytometry using FITC- or PE-conjugated Abs for CD14 and CD1a. Percentage positivity is relative to an isotype-matched control Ab, and values indicate the mean ± SD of experiments with 6 MM patients' BM sera. Statistically significant decreased expression of CD14 (*, P = .01; **, P = .03) and increased expression of CD1a (*, P = .01) were observed with addition of MM patients' BM sera in the presence of anti–IL-6 and/or VEGF Abs. (B) Immature DCs induced in the presence of GM-CSF, IL-4, and 10% AB serum were then cultured for 3 days in the presence of 20% MM patients' BM sera, TNF-α (10 μg/mL), and either control Abs (□), neutralizing anti–IL-6 Abs (▨), anti-VEGF Abs (▦), or both anti–IL-6 and VEGF Abs (▪). Flow cytometric analysis was done using FITC-conjugated anti-CD83 Abs to assess maturation of DCs. Values indicate the mean ± SD of results from 6 MM patients. The expression of CD83 was significantly increased (*, P = .01) with addition of anti-VEGF Abs or both anti-VEGF and IL-6 Abs. (C) T cells (> 95% CD3+) obtained from the same PBMCs used for induction of DCs were incubated in 96-well round-bottom plates (2 × 105 cells/well) for 5 days with irradiated (15 Gy) autologous DCs, generated in media containing GM-CSF, IL-4, and 20% AB sera or MM patients' BM sera, with (▪) or without (□) neutralizing anti-VEGF Abs (5 μg/mL) and anti–IL-6 Abs (5 μg/mL) at indicated T-DC ratios. 3[H]thymidine (1.0 μCi [0.037 MBq]) was added to each well for the last 12 hours of 5-day cultures. Cells were then harvested and radioactivity was counted. Values indicate the mean ± SD of results from 5 triplicate experiments. T-cell proliferation was significantly inhibited with MM patients' BM sera compared with AB serum (*, P = .01), and this effect was neutralized by anti-VEGF and anti–IL-6 Abs (**, P = .01). Mean counts per minute (cpm) of T cells without stimulators was 454 and of DCs only was less than 100.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/102/4/10.1182_blood-2002-09-2828/6/h81634766001.jpeg?Expires=1724233767&Signature=F92ggU7IEAOJWd4~uSKHJTEahs2XNLSLZOw9twNl3eyiXw1aqn3Ebr8ynCpha84FhrdiKd-SYNPVfJTfpNrbjHcZS0asgMwmy9y8HwDPPppYg3lsFLb0coGaAsQ8ZOTYpba3m2LbDaawL8qtnfeZSVjEQxdJelVuXLMqh3tjz0AioQw1AB68bqWGBdYAua-6UCjoTuDrXd78-K4QjlU2rPZvr5aetU1jHK8bktfjcYLbBrs4Ftxor3V71Ig0VDc6NIdgyeAKFu6ImmOIlASHWP6t0HKOEdBI4R3gfmG7xOkwNRAseu7iJg9aFCG~KVx0j1XJmd57pTcx96cyIRk5eQ__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Anti-VEGF and anti–IL-6 Abs reduce the inhibition of induction and maturation of DCs by MM patients' BM sera. (A) Immature DCs were induced from adherent healthy donor PBMCs by culturing in the presence of GM-CSF (1000 U/mL), IL-4 (10 ng/mL), and 20% MM patients' BM sera with control Ab (□), neutralizing anti–IL-6 Abs (5 μg/mL, ▨), anti-VEGF Abs (5 μg/mL, ▦), or both anti–IL-6 and VEGF Abs (▪). Cell surface phenotype was analyzed by flow cytometry using FITC- or PE-conjugated Abs for CD14 and CD1a. Percentage positivity is relative to an isotype-matched control Ab, and values indicate the mean ± SD of experiments with 6 MM patients' BM sera. Statistically significant decreased expression of CD14 (*, P = .01; **, P = .03) and increased expression of CD1a (*, P = .01) were observed with addition of MM patients' BM sera in the presence of anti–IL-6 and/or VEGF Abs. (B) Immature DCs induced in the presence of GM-CSF, IL-4, and 10% AB serum were then cultured for 3 days in the presence of 20% MM patients' BM sera, TNF-α (10 μg/mL), and either control Abs (□), neutralizing anti–IL-6 Abs (▨), anti-VEGF Abs (▦), or both anti–IL-6 and VEGF Abs (▪). Flow cytometric analysis was done using FITC-conjugated anti-CD83 Abs to assess maturation of DCs. Values indicate the mean ± SD of results from 6 MM patients. The expression of CD83 was significantly increased (*, P = .01) with addition of anti-VEGF Abs or both anti-VEGF and IL-6 Abs. (C) T cells (> 95% CD3+) obtained from the same PBMCs used for induction of DCs were incubated in 96-well round-bottom plates (2 × 105 cells/well) for 5 days with irradiated (15 Gy) autologous DCs, generated in media containing GM-CSF, IL-4, and 20% AB sera or MM patients' BM sera, with (▪) or without (□) neutralizing anti-VEGF Abs (5 μg/mL) and anti–IL-6 Abs (5 μg/mL) at indicated T-DC ratios. 3[H]thymidine (1.0 μCi [0.037 MBq]) was added to each well for the last 12 hours of 5-day cultures. Cells were then harvested and radioactivity was counted. Values indicate the mean ± SD of results from 5 triplicate experiments. T-cell proliferation was significantly inhibited with MM patients' BM sera compared with AB serum (*, P = .01), and this effect was neutralized by anti-VEGF and anti–IL-6 Abs (**, P = .01). Mean counts per minute (cpm) of T cells without stimulators was 454 and of DCs only was less than 100.
