Ex vivo induction of multiple myeloma–specific cytotoxic T lymphocytes by Toshiaki Hayashi, Teru Hideshima, Masaharu Akiyama, Noopur Raje, Paul Richardson, Dharminder Chauhan, and Kenneth C. Anderson Blood Volume 102(4):1435-1442 August 15, 2003 ©2003 by American Society of Hematology

Anti-VEGF and anti–IL-6 Abs reduce the inhibition of induction and maturation of DCs by MM patients' BM sera. 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. Toshiaki Hayashi et al. Blood 2003;102:1435-1442 ©2003 by American Society of Hematology

Exogenous VEGF and IL-6 inhibit induction and maturation of DCs Exogenous VEGF and IL-6 inhibit induction and maturation of DCs. (A) 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 10% AB serum (□), with VEGF (20 ng/mL, ▦) or IL-6 (20 ng/mL, ... Exogenous VEGF and IL-6 inhibit induction and maturation of DCs. (A) 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 10% AB serum (□), with VEGF (20 ng/mL, ▦) or IL-6 (20 ng/mL, ▪), and TNF-α (10 μg/mL) was then added for 3 days. Cell surface expression of CD14, CD1a, and CD83 on DCs was analyzed by flow cytometry. Results indicate the mean ± SD of percent-positive cells in 4 experiments. Statistically significant increased expression of CD14 (*,P = .02) and decreased expression of CD1a (**, P = .02) and CD83 (***, P = .046) were observed with addition of IL-6 or VEGF. (B) T cells (2 × 105 cells/well) from healthy donor PBMCs were incubated for 5 days with irradiated (15 Gy) autologous DCs generated in media containing 10% AB serum, GM-CSF, and IL-4 (□) with VEGF (20 ng/mL, ▦) or IL-6 (20 ng/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. Results are representative of 3 experiments, and values indicate mean ± SD of triplicate wells. Statistically significant decreased stimulation of T cells was observed with addition of VEGF or IL-6 (*, P = .02). Mean cpm of T cells without stimulators was 424 and of DCs only was less than 100. Toshiaki Hayashi et al. Blood 2003;102:1435-1442 ©2003 by American Society of Hematology

Immature DCs phagocytose apoptotic MM bodies. Immature DCs phagocytose apoptotic MM bodies. (A) U266 cells were irradiated and stained with annexin-V–FITC and PI after 0, 2, 4, 6, 8, and 12 hours incubation at 37°C. Early apoptotic cells were defined as annexin-V–FITC+ and PI– using flow cytometry. Results are representative of experiments with 3 MM cell lines. (B) U266 cells labeled red with Vybrant Dil Cell-Labeling Solution were incubated 4 hours at 37°C after 30-Gy irradiation to allow apoptosis to occur and then were cocultured with immature DCs stained green with Vybrant DiO Cell-Labeling Solution at a ratio of 1:1 for 0, 2, 4, 6, and 8 hours at 37°C or 4°C. Cells were analyzed by flow cytometry and double-positive cells indicate uptake of apoptotic cells by immature DCs. Culturing at 4°C blocked phagocytosis of apoptotic bodies by immature DCs. Toshiaki Hayashi et al. Blood 2003;102:1435-1442 ©2003 by American Society of Hematology

Stimulation and induction of CTLs by DCs pulsed with apoptotic MM bodies versus cell lysates. Stimulation and induction of CTLs by DCs pulsed with apoptotic MM bodies versus cell lysates. (A) T cells (2 × 105 cells) from healthy donor PBMCs were incubated for 5 days with irradiated (15 Gy) autologous DCs cocultured with apoptotic U266 bodies (▪), U266 lysate (□), DCs alone (▨), or apoptotic U266 bodies alone (▦) at indicated T cell–to-DC (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. Mean cpm of T cells without stimulator was 409 and of DCs only and apoptotic U266 cells only were less than 50. Data shown indicate mean ± SD of triplicate wells and are representative of 3 experiments. Significantly greater proliferation was observed in T cells stimulated with apoptotic U266–pulsed DCs than other stimuli by Mann-Whitney U test (*, P = .02). (B) Cytotoxicity of CTLs against U266 cells was assessed with 51Cr-release assay. U266 cells (5 × 103 cells) labeled with 51Cr were cultured with CTLs induced by stimulation with DCs cocultured with apoptotic U266 bodies (▪), U266 lysate (▴), DCs alone (□), or apoptotic U266 cells alone (▵) at indicated effector-to-target (E/T) ratios in triplicate for 4 hours at 37°C. Supernatants were then harvested and radioactivity was counted. CTLs stimulated with apoptotic U266 bodies showed significantly higher specific lysis than other stimuli. Spontaneous release of target cells was less than 10%. Specific lysis of K562 cells by CTLs stimulated with DCs cocultured with apoptotic U266 bodies was maximum (14.8%) at an E/T ratio of 20:1. Results shown are mean ± SD of triplicate wells and representative of 3 experiments. Toshiaki Hayashi et al. Blood 2003;102:1435-1442 ©2003 by American Society of Hematology

Characterization of CTLs induced by stimulation with mature DCs cocultured with apoptotic MM cells. Characterization of CTLs induced by stimulation with mature DCs cocultured with apoptotic MM cells. (A) CTLs induced by stimulations with DCs cocultured with apoptotic U266 bodies showed significantly higher cytotoxicity (*, P = .02) against U266 cells (▪) than against RPMI 8226 cells (□) or K562 cells (▦), assessed with 51Cr-release assay at indicated effector-to-target (E/T) ratios for 4 hours at 37°C. Spontaneous release of target cells was less than 15%. Results shown are mean ± SD of triplicate wells and representative of 3 experiments. (B) HLA restriction of CTLs was examined using target blocking 51Cr-release assay. CTLs stimulated with apoptotic U266 body–pulsed DCs were cocultured with 51Cr-labeled U266 after incubation with blocking antibody against HLA class I (□), HLA class II (▨), control antibody (▦), or without antibody (▪). Cytotoxicity was significantly inhibited by anti–HLA class I Abs (*, P = .02). Spontaneous release of target cells was less than 15%. Results shown are mean ± SD of triplicate wells and representative of 2 experiments. (C) Cell surface phenotype of CTLs was analyzed by flow cytometry using FITC- or PE-conjugated Abs against CD3, CD8, CD4, and CD56 (filled histogram). Percentages positive are calculated relative to an isotype-matched control Ab (open histogram). Toshiaki Hayashi et al. Blood 2003;102:1435-1442 ©2003 by American Society of Hematology

Induction and characterization of CTLs against autologous MM cells. Induction and characterization of CTLs against autologous MM cells. (A) Immature DCs were induced from adherent cells in BMMCs of patients with MM (Pt 1 [λ-type] and 2 [IgA, κ]) by culturing with GM-CSF (1000 U/mL), IL-4 (10 ng/mL), and 10% AB serum, followed by coculture for 12 to 18 hours at a ratio of 5:1 with autologous MM cells incubated for 4 hours after irradiation (30 Gy) and then cultured for 3 days in the presence of TNF-α (20 ng/mL) to induce maturation. MNCs from the same MM patients were then cocultured with mature DCs at a ratio of 10:1 to 20:1 in AIM-V medium and stimulated with the apoptotic body–pulsed DCs weekly. Seven days after the second stimulation, cytotoxicity of the CTL against autologous MM cells (▪), RPMI 8226 cells (□), or K562 cells (▦) was examined using a lactate dehydrogenase assay at indicated effector-to-target (E/T) ratios. Significantly greater specific lysis of autologous MM cells was observed than of RPMI 8226 cells or K562 cells (*, P = .02). Spontaneous release of target cells was less than 20%. Results shown are mean ± SD of triplicate wells. (B) Cell surface phenotype of patient-derived CTLs was analyzed by flow cytometry using specific Abs against CD3, CD8, CD4, and CD56 (filled histogram). Percentages positive cells are calculated relative to an isotype-matched control Ab (open histogram). (C) HLA restriction of CTLs derived from patients with MM (Pt 3, nonsecretory; Pt 4, κ-type MM) was examined using target-blocking LDH assay. CTLs stimulated with apoptotic MM body–pulsed DCs were cocultured with autologous MM cells after incubation with neutralizing antibody against HLA class I (□), HLA class II (▨), control antibody (▦), or without antibody (▪). Cytotoxicity against autologous MM cells was significantly inhibited by anti–HLAclass IAbs (*, P = .02). Spontaneous release of target cells was less than 20%. Results shown are mean ± SD of triplicate wells from 2 representative patient-derived CTLs. (D) Cytotoxicity of CTLs induced by stimulation with DCs cocultured with apoptotic primary MM bodies of HLA-A2–positive patient against autologous MM cells (▪), U266 cells (▴), K562 cells (▵), or autologous PBMCs (□) was assessed with LDH assay at indicated effector-to-target (E/T) ratios for 4 hours at 37°C. CTLs induced by stimulations with DCs cocultured with primary MM bodies showed significantly greater cytotoxicity against autologous MM cells than against U226 cells, K562 cells, or autologous PBMCs. Spontaneous release of target cells was less than 20%. Results shown are mean ± SD of triplicate wells. Toshiaki Hayashi et al. Blood 2003;102:1435-1442 ©2003 by American Society of Hematology