| Objective:To investigate the effect of Vγ9Vδ2 T cells regulating immature dendritic cells’(imDCs’)transdifferentiation into osteoclasts(OCs),to screen out the relevant critical genes and signaling pathways during the transdifferentiation and to evaluate regulation of Vγ9Vδ2 T cells on the imDCs’transdifferentiation into OCs in the myeloma microenvironment.Methods:1.Investigate the effect of Vγ9Vδ2 T cells on osteoclastogenesis from imDCs:(1)Cell culture:?Vγ9Vδ2 T cells cultured in vitro:Peripheral blood mononuclear cells(PBMNCs)were cultured with zoledronate(ZOL)and recombinant human interleukin-2(rhIL-2)to amplify Vγ9Vδ2 T cells.?ImDC cells cultured in vitro:Immunomagnetic bead sorting CD14~+cells were cultured with recombinant human granulocyte-macrophage colony-stimulating factor(rhGM-CSF)and recombinant human interleukin 4(rhIL-4)to differentiate to imDCs.?Co-culture system established:ImDCs were transdifferentiated into OCs in the present of recombinant human macrophage colony-stimulating factor(rhM-CSF)and recombinant human receptor activator nuclear factor?B ligand(rhRANKL).ImDCs were cultured alone as control group.Using Transwell inserts with Vγ9Vδ2 T cells in the upper compartment and imDC in the lower compartment(asγδT group).(2)Means of intervention:?Different radios of co-culture cells:the ratio of Vγ9Vδ2 T cells and imDCs 10:1,1:1 and 1:10,respectively.?Different stage of intervention:co-culture was performed initially when imDCs were cultured(d 0-1γδT group),at the later stage of culture(d 3-4γδT group)and from the beginning of culture till day 4(d 0-4γδT group),with Vγ9Vδ2 T cells and imDCs ratio of 1:1.(3)Detection of number,activity and realated genes of osteoclasts:the cells in the lower compartment were collected for the tartrate resistant acid phosphatase(TRAP)staining and toluidine blue staining.The number of TRAP~+OC was counted and lacunar resorption area was measured.The mRNA expression of CTR and MMP9 was detected by real-time RT-PCR and Western-blot.2.Study on the OC mRNA expression change after co-cultured with Vγ9Vδ2 T cells from imDCs ImDCs were cultured alone with rhM-CSF and rhRANKL as control group.γδT cells in the upper department were co-cultured with the same number of imDCs in the lower compartment for 24 hours(asγδT group).On day 9,mRNA microarray was used to screen out expression differences.Bioinformatics analyses were used to find the possible gene regulatory network and signal pathway.Real-time RT-PCR was used to detect critical genes relevant to Vγ9Vδ2 T cells’regulation on imDCs’transdifferentiation into OC.3.Evaluate regulation of Vγ9Vδ2 T cells on the imDCs’transdifferentiation into OCs in the myeloma microenvironment:ImDCs were cultured alone with rhM-CSF and rhRANKL as control group.ImDCs were cultured with the condition medium(CM)of multiple myeloma(MM)cell line RPMI8226(MM-CM group).γδT cells in the upper compartment were co-cultured with lower compartment imDCs in the CM of MM(asγδT-MM-CM group).Real-time PCR,Western blot and immunofluorescence were used to detect the expression of genes associated with the osteoclast differentiation,including RANK,Cathepsin K,ATP6V0D2 and c-Fos.The level of type I collagen carboxy-terminal telopeptide(CTX-I)in the culture supernatant was measured by ELISA.Results:1.(1)TRAP staining showed that TRAP~+OCs inγδT group were significantly fewer than in control group(P<0.01).The higher theγδT:imDCs,the fewer TRAP staining positive OCs.There was a significant difference between the adjacent ratio groups(P<0.05).The number of TRAP~+OCs in d 0-1γδT group was significantly lower than that in the control group(P<0.05)and also significantly lower than that d3-4γδT group(P<0.05).No significant difference was found between d 3-4γδT group and control(P>0.05).Toluidine blue staining showed that lacunar resorption area on dentine slices inγδT group was significantly smaller than that of control group(P<0.01).The higher cell ratio ofγδT:imDCs,the smaller resorption area.There was significantly difference between the adjacent ratio groups(P<0.01).Real-time RT-PCR and Western blot results showed that mRNA and protein expression of CTR and MMP9 was significantly lower inγδT groups(Vγ9Vδ2 T cells:imDCs were 1:1and 1:10,repectively)than that in control group(P<0.01 vs control).2.The mRNA microarray and bioinformatics analysis showed that there were significant differences of gene expression in a total of 293 genes.Among them,123were upregulated and 170 downregulated.GO and KEGG functional enrichment analysis found that genes with different expression were relevant to some biological processes such as osteoclast differentiation,immunoregulatory,cytokines and cytokine receptor interactions,etc.FCGR3A,Fcgr3B,BTK and FOSL2,ATP6V0D2,BTUK and Cathepsin K had significantly different expression.They were involved in osteoclast differentiation.Real-time RT-PCR results showed that the mRNA level of BTK,RANK,Cathepsin K,c-Fos,ATP6V0D2,MMP9 and CTR was significantly lower inγδT group than that of control(P<0.05).3.(1)TRAP staining showed the number of TRAP~+OCs was significantly increased in MM-CM group(38.81±3.78 vs 30.52±1.89,P<0.01)and significantly decreased inγδT-MM-CM group(10.93±2.08 vs 30.52±1.89,P<0.01),compared with the control group.Toluidine blue staining showed that lacunar resorption size was significantly increased in MM-CM group(0.45±0.13 vs 0.31±0.08,P<0.01)and significantly decreased inγδT-MM-CM group(0.04±0.01 vs 0.31±0.08,P<0.01),compared with the control group.Compared with control group,mRNA and protein expression level of c-Fos,ATP6V0D2 and Cathepsin K was increased in MM-CM group,but decreased inγδT-MM-CM group(P<0.05).The fluorescence intensity of c-Fos was reduced inγδT-MM-CM group(P<0.05).The level of CTX-I in culture supernatant was significant higher(639.56±58.59 vs 326.73±29.67,P<0.05)in MM-CM group and significantly lower inγδT-MM-CM group(101.24±19.31 vs326.73±29.67,P<0.01),compared with the control group.Conclusions:1.Co-culture transwell system of Vγ9Vδ2 T cells and imDCs was established in the present study.Vγ9Vδ2 T cells from amplification of zol and rhIL-2 in vitro culture suppressed imDC transdifferentiating into OC,indicating the co-culture system was valid.Vγ9Vδ2 T cells inhibited osteoclast differentiation from imDC and their resorption activity.The inhibitory effect was Vγ9Vδ2 T cells number-dependent.ImDC at the early stage of osteoclast differentiation were vulnerable to Vγ9Vδ2 T cells inhibition.2.The mRNA expression microarray screened out that BTK,c-Fos,ATP6V0D2 and Cathepsin K as the relevant genes of Vγ9Vδ2 T cells’inhibition on imDCs transdifferentiation into OC and their resorptive activity.3.Myeloma condition medium facilitated imDCs’transdifferentiation into OCs and enhanced their osteolytic effect,stimulating myeloma microenvironment.Vγ9Vδ2 T cells inhibited imDCs’transdifferentiation and OCs’function,possible via down-regulation of RANK,c-Fos,ATP6V0D2 and cathepsin K in myeloma microenvironment. |
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