Deficient invariant natural killer T cells had impaired regulation on osteoclastogenesis in myeloma bone disease

Abstract Recent research showed that invariant natural killer T (iNKT) cells take part in the regulation of osteoclastogenesis. While the role of iNKT cells in myeloma bone disease (MBD) remains unclear. In our study, the quantity of iNKT cells and the levels of cytokines produced by them were measured by flow cytometry. iNKT cells and osteoclasts were induced from peripheral blood mononuclear cells after activation by α‐GalCer or RANKL in vitro. Then, gene expressions and the levels of cytokines were determined by RT‐PCR and ELISA, respectively. The results showed that the quantity of iNKT and production of IFN‐γ by iNKT cells were significantly decreased in newly diagnosed MM (NDMM), and both negatively related with severity of bone disease. Then, the osteoclasts from healthy controls were cultured in vitro and were found to be down‐regulated after α‐GalCer‐stimulated, while there was no significant change with or without α‐GalCer in NDMM patients, indicating that the regulation of osteoclastogenesis by iNKT cells was impaired. Furthermore, the inhibition of osteoclastogenesis by iNKT cells was regulated by IFN‐γ production, which down‐regulated osteoclast‐associated genes. In conclusion, the role of α‐GalCer‐stimulated iNKT cells in regulation of osteoclastogenesis was impaired in MBD, as a result of iNKT cell dysfunction.

Invariant NKT (iNKT) cells are a distinct subset of T cells that express a semi-invariant TCR a-chain encoded by Va24-Ja18 paired with a limited repertoire of Vb chains encoded by Vb11 in human beings and recognize glycolipid ligands presented by the non-polymorphic MHC class I-like molecule CD1d. 12,13 iNKT cells can be activated by a synthetic glycolipid isolated from a marine sponge, a-galactosylceramide (a-GalCer or KRN7000), leading to rapid production of several cytokines such as IL-4 and IFN-c. 14,15 NKT cells exhibit bridges connecting the innate immune system to the adaptive immune system. Furthermore, they are able to interact with immune cells, such as monocytes, T cells, B cells, dendritic cells and NK cells, by producing Th1 and Th2 cytokines. 16 Many studies have confirmed that NKT cell level and function are reversibly defective in MM. [17][18][19][20] In addition, a recent study showed that the function of iNKT is defective which is related to osteoclastogenesis and inflammatory bone destruction in RA patients. 21 Furthermore, a previous study illustrated that iNKT cells have the unique characteristic of enhancing osteoclast progenitor and precursor development. 22 However, the effect of iNKT on MBD remains unknown. The aim of this study was to investigate the role of iNKT cells during osteoclastogenesis and the underlying mechanism of osteoclastogenesis dysregulation in MBD.

| Patients and sample
The study cohort included 37  Myeloma Working Group uniform response criteria. 20 According to body X-ray scanning data obtained before treatment within 1 week after diagnosis, bone morbidity was graded into three stages (Stage A included patients with no osteolytic lesions or osteoporosis alone; Stage B included patients with one to three osteolytic lesions, and Stage C included patients with more than three osteolytic lesions and/or a pathological fracture). 23,24 We have used the <3/>3 as cutoff for bone lesions as advanced bone disease (BD; Stage C) includes more than three lytic lesions in the Durie-Salmon staging system. CD34 + OCN + cells were routinely regarded as osteoblast precursors (OBPs), 25 and CD14 + CD16 + cells were routinely as osteoclast precursors (OCPs). 26

| Quantitative real-time PCR
Quantitative real-time PCR was performed as described previously. 28 RNA was extracted from OCs using the TRIzol reagent (Invitrogen, USA), and mRNA expression was quantified using the Bio-Rad iQ 5 Real-time system (Bio-Rad, Hercules, CA, USA). The primer sequences of osteoclast-associated genes are shown in Table 2.

| Statistical analyses
Among 37 NDMM patients, the percentage of iNKT cells was significantly lower in the patients with Stage C bone disease than those with Stage A/B bone disease and HCs (median 0.04% vs 0.07% and 0.09%, P = .042 and P < .001, respectively; Figure 1D).
We ously. 26 The results indicated that the percentage of iNKT cells was significantly correlated with the population of OCPs (r = À.413, P = .011; Figure 1F). However, there were no significant differences between the level of iNKT cells percentage and the level of osteoblast precursors (OBPs), bone formation markers (OCN and PINP).
According to the percentages of CD4 + CD8 À , CD4 À CD8 + and DN iNKT cell subsets of T cells in the MM patients with Stage A/B or C bone disease (Table 4), the percentage of CD4 À CD8 + iNKT cells was significantly lower in the patients with Stage C bone disease than those with Stage A/B bone disease and HCs (P = .029 and P < .001, respectively; Figure 1G). These results suggested that the quantity of iNKT cells which may be mainly CD4 À CD8 + iNKT cells related to the state of bone destruction, especially osteoclastogenesis.

| Deficiency in proliferative responses of iNKT cells on a-GalCer and impaired regulation of osteoclastogenesis by iNKT cells in NDMM patients
We examined the proliferative responses of iNKT cells on a-GalCer

| Inhibition of osteoclastogenesis by iNKT cells via IFN-c production
To examine whether inhibition of osteoclastogenesis is regulated by cytokines produced by iNKT cells, we measured the level of IFN-c,  Figure 4A). However, no significant dif- Our study first investigated the role of iNKT cells in osteoclastogenesis and the underlying mechanism in MBD.
It was previously reported that iNKT cells are a small population of T lymphocytes derived from the thymus at the double positive selection (CD4 + CD8 + ) stage of T cell development [33,34]. While a significant proportion of iNKT cells remain in the thymus, where they become long-lived residents, other part of iNKT cells are often migrated to different tissues, like liver, spleen, bone marrow, peripheral blood and lymph nodes [35,36]. In healthy humans, the relative frequency of iNKT cells of BM is similar to that of PB [37].