γδT Cells Inhibiting Immature Dendritic Cell Differentiation Into Osteoclast In Vitro

Object: To observe the amplification of γδ T cells from peripheral blood of healthyvolunteers or patients with multiple myeloma (MM) and to explore the role of γδ Tcell in the progress of immature dendritic cell (imDC) differentiation into osteoclast(OC).Methods:(1) Peripheral blood mononuclear cell (PBMNC) from healthy volunteersor patients with MM were cultured with1μM zoledronate (Zol) and100IU/mlrecombinant human interleukin-2(IL-2). The production capacity was assessed byflow cytometry and cell count after7days of culture.(2) PBMNC from healthyvolunteers were cultured with granulocyte macrophage colony-stimulating factor(GM-CSF) and recombinant human interleukin-4(IL-4) and induced to differentiateto imDC. On day6, expression of CD14, CD1a and CD51/61were investigated withflow cytometry. ImDC were then cultured with receptor activator nuclear factor Bligand (RANKL) and macrophage colony-stimulating factor (M-CSF) and induced todifferentiate into OC.(3) γδ T cells were isolated with immune magnetic bead fromPBMNC which had been cultured with Zol and IL-2for7days. PBMNC at day6ofculture with GM-CSF and IL-4were regarded as imDC. Coculture system wasestablished using millicell inserts, with γδ T cells in the upper compartment and imDCin the lower compartment in the ratio of10:1. After7or14days of coculture, the cellsin the lower compartment were collected for the detection of CD51/61expression byflow cytometry, tartrate resistant acid phosphatase (TRAP) staining using the TRAPkit and bone resorption observation staining to understand the potential impact of γδ Tcells on imDC transdifferentiation into OC.(4) Immune magnetic bead isolated γδ Tcells and cultured imDC mentioned above were cocultured directly with ratio of10:1and the impact of γδ T cells on imDC transdifferentiation into OC was followed in thesame way.(5) Supernatant liquid of γδ T cell-imDC indirect coculture system wascollected and level of tumor necrosis factor-alpha (TNF-α) and type I collagen carboxy-terminal peptide (CTX-1) were measured using ELISA kit to explain thepossible mechanism of the impact of γδ T cells on imDC.Results:(1) γδ T cells can be expanded from PBMNC of MM patients, and theproduction capacity was similar to that of healthy volunteers (P>0.05).(2) ImDCinduced from human PBMNC proved to differentiate into OC when cultured withRANKL and M-CSF.(3) The rate of CD51/61expression, the number of TRAP+multinuclear cell/low power field and the absorption area of dentine weresignificantly lower in imDC indirectly cocultured with γδ T cells, compared withcontrol imDC, indicating fewer OC production.(4) When directly cocultured withimDC, γδ T cell might also inhibit OC production.(5) Under the circumstance of γδ Tcell-imDC indirect coculture, CTX-1level remained stable but TNF-α gotsignificantly higher.Conclusions: γδ T cells can be selectively expanded from PBMNC of MM patientsand healthy volunteers cultured with Zol and IL-2. γδ T cells might inhibit imDCdifferentiation into OC. γδ T cells-based immunotherapy is expected to be a newtherapy for myeloma bone disease.