| Objective:To investigate the cytotoxic effect of γδ T cells on multiple myeloma(MM) cells and the inhibitory effect on immature dendritic cells(im DCs) transdifferentiation into osteoclasts(OCs).Methods:(1) Peripheral blood mononuclear cells(PBMNCs) were isolated from healthy volunteers, and stimulated with zoledronic acid(Zol) at 1 μM, in combination with 200 IU/m L rh IL-2. After 1 week culture, γδ T cells were harvested and purified with immunomagnetic bead sorting system.(2) Well-grown MM RPMI 8226 cells collected and co-cultured with γδ T cells for 4 hours at different effector target ratios. Then the cytotoxic effect of γδ T cells was determined using lactate dehydrogenase(LDH) release assay kit. After the γδ T cells were treated with 10 μg/ml anti-human γδ TCR monoclonal antibody(m Ab), anti-human NKG2 D m Ab, anti-human γδ TCR m Ab+anti-human NKG2 D m Ab and anti-mouse Ig G m Ab, LDH release assay kit was repeated to analyze the cytotoxic effect of γδ T cells.(3) CD14+ PBMNCs were purified from PBMNCs in immunomagnetic bead sorting system and cultured in the medium containing interleukin-4(IL-4) and granulocyte-macrophage colony stimulating factor(GM-CSF), which induced cells differentiation toward im DCs. The cells were further cultured with macrophage colony stimulating factor(M-CSF) and soluble nuclear factor κB receptor activation factor ligands(s RANKL) for differentiation toward OCs.(4) The γδ T cells were co-cultured with cells at different stage from PBMNCs via im DCs transdifferentiation into OCs. Tartrate resistant acid phosphatase(TRAP) staining was used to observe the potential impact of γδ T cells on cells transdifferentiation into OCs.(5) After the γδ T cells were treated with 10 μg/ml anti-human γδ TCR m Ab, anti-human NKG2 D m Ab, anti-human γδ TCR m Ab+anti-human NKG2 D m Ab and anti-mouse Ig G m Ab, TRAP staining was repeated to observe the potential impact of γδ T cells on im DCs transdifferentiationinto OCs.(6) ELISA assay was used to determine interferon-γ(IFN-γ) and Type I collagen end Carboxy- terminal peptide(CTX-I) levels in supernatant of each culture groups.Results:(1) γδ T cells can be expanded with Zol and IL-2 in vitro.(2) Co-cultured γδ T cells with MM 8226 cells at 10:1、1:1、1:10 effector target ratios after 4 hours, the corresponding cytotoxic rates were 41.69%, 26.88% and 9.97%, respectively, showing γδ T cells‘s cytotoxicity against MM cells in the manner of effector target ratio dependence. After γδ T cells were treated with 10 μg/ml anti-human γδ TCR m Ab, anti-human NKG2 D m Ab, anti-human γδ TCR m Ab+anti-human NKG2 D and anti-mouse Ig G m Ab, co-cultured with MM 8226 cells at 10:1 ratio, the corresponding cytotoxic rates were 21.56%, 26.86%, 14.93% and 42.03%, respectively, indicating that blocking either γδ TCR or NKG2 D would decrease the cytotoxic rate and blocking γδ TCR give the lower rate.(3) When γδ T cells were directly co-cultured with CD14+ PBMNCs, rare TRAP positive giant cells were found, while the OCs number was 8.33±2.08( x ±S) per ten fields when γδ T cells were indirectly co-cultured with CD14+ PBMNCs at 1:5 ratio, significantly fewer than that in pure CD14+ PBMNCs culture(37.67±2.05 per ten fields)(p<0.05); When γδ T cells were co-cultured with im DCs at different ratios in direct or indirect ways, we found that γδ T cells inhibited the differentiation of the OCs. In direct way, the OC number in effector-target ratio of 1:10, 1:1 and 10:1 groups were 14.33±2.08, 9.67±2.5 and 3.33±1.53 per ten fields, respectively, significantly fewer than those in indirect way(24.67±2.08, 15.53±1.3 and 7.67±0.58 per ten fields, respectively)(p<0.05); Both direct and indirect way showed inhibitory effect depending on effector-target ratio. On the other hand, when γδ T cells were co-cultured with OCs, lysed and shrunk TRAP+ OCs were found. This phenomenon was more apparent at higher effector-target ratio.(4) After the γδ T cells were treated with anti-human γδ TCR m Ab, anti-human NKG2 D m Ab, anti-human γδ TCR m Ab+anti-human NKG2 D and anti-mouse Ig G m Ab, co-culture of the γδ T cells with im DCs at 10:1 ratio produced 2.33±0.47 TRAP+ giant cells per ten fields, similar with those withoutantibody treatment,(3.33±1.53 per ten fields)(p>0.05), showing that γδ TCR or NKG2 D did not change the inhibitory effect of γδ T cells on OC differentiation.(5) After co-culture of γδ T cells with im DCs at 1:1, 5:1 and 10:1 ratios in direct way for 24 hours, IFN-γ in the supernatant were 617.09 pg/ml, 900.1 pg/ml and 1277.34 pg/ml, respectively. In indirect way, they were 509.68 pg/ml, 786.49 pg/ml and 1045.5 pg/ml, respectively, showing that im DCs stimulated γδ T cells to secrete IFN-γ. IFN-γ level was higher in direct than in indirect co-culture and the higher the effector-target ratio, the higher IFN-γ level. CTX-I was undetectable in co-culture supernatant of γδ T cells and CD14+ PBMNCs. In co-culture of γδ T cells and im DCs at the effector-target ratio of 1:1, the supernatant CTX-I 397.01 pg/ml and 459.47 pg/ml(P<0.05), respectively in direct way and indirect way. When γδ T cells were directly co-cultured with OCs at the ratio of 1:1 for 24 h, supernatant CTX-1 was undetectable. CTX-1 in indirect culture was 83.24 pg/ml, lower than that in pure OC culture(889.54 pg/ml)(P<0.05).Conclusions:(1) γδ T cells might kill MM cells in vitro with the mechanism associated with γδ TCR and NKG2 D mediated signal pathways.(2) γδ T cells might inhibit the differentiation of OCs from im DCs in different stages and might directly damage the mature OCs. These effects might be irrelevant to γδ TCR and NKG2 D.(3) IFN-γ may play a role in γδ T cells’ inhibiting OC differentiation. Therefore, the γδ T cells are promising to be a good choice in immunotherapy for MM. |
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