Elotuzumab-mediated ADCC with Th1-like Vγ9Vδ2 T cells to disrupt myeloma–osteoclast interaction

2.1 Human γδ T cell expansion

Th1-like γδ T cells were expanded from peripheral blood mononuclear cells (PBMCs) as previously reported.^{4, 7} In brief, PBMCs were cultured for 1 week with ZA at 1 μM in combination with IL-2 at 100 IU/mL. γδ T cell expansion was analyzed with the Gallios flow cytometer (Beckman Coulter).

2.2 Human OC differentiation

Monocytes were isolated from PBMCs using the Pan Monocyte Isolation Kit (Miltenyi Biotec) and cultured at 1 × 10⁶ cells/mL in 24-well culture plates for 14 days in the presence of M-CSF (10 ng/mL) and receptor activator of nuclear factor-κB ligand (RANKL) (50 ng/mL) as previously described.¹⁰ Culture media were changed every 2 days.

2.3 Cytotoxicity assay by flow cytometry

Target MM cells were stained by PKH26 fluorescent dye and cultured alone or with effector cells; then, dead cells were stained with 7 amino-actinomycin D (7-AAD). The cells were analyzed by flow cytometry, and the distributions of 7-AAD-stained cells were calculated within PKH26-labeled MM cells.

Methods are further described as supporting information including Supplementary Materials and Methods—Data S1 and Table S1.

3.1 γδ T cells exert ADCC with ELO

Zoledronic acid and IL-2 expanded Th1-like γδ T cells from PBMCs, which expressed intracellular IFN-γ (Figure S1) and increased CD16 on their surface (Figure 1A). We next examined cytotoxic activity by Th1-like γδ T cells in combination with clinically available therapeutic mAbs. SLAMF7 was expressed on MM.1S and OPM-2 cells but not on RPMI 8226 cells, while CD38 was expressed on all these MM cell lines (Figure S2A). ELO, the anti-CD38 mAbs daratumumab (DARA), or isatuximab (ISA) alone marginally induced cell death in these MM cell lines (Figure S2B). The expanded Th1-like γδ T cells induced cytotoxic activity for these MM cell lines; interestingly, the addition of ELO further enhanced the cytotoxic activity of the Th1-like γδ T cells against SLAMF7-expressing MM.1S and OPM-2 cells but not against the SLAMF7-lacking RPMI 8226 cells (Figure 1B). Furthermore, addition of anti-CD16-blocking Ab mitigated the ELO-mediated cytotoxicity (Figure S3A), indicating ELO-mediated ADCC with the γδ T cells. Although the expanded γδ T cells expressed SLAMF7 on their surface, ELO pretreatment for the γδ T cells only marginally affected their cytotoxic activity against MM cells (Figure S3B, C). ELO-mediated ADCC was further confirmed using γδ T cells expanded from different healthy donors (Figure 1C) and primary CD138⁺ MM cells isolated from patients with MM (Figure 1D). However, neither DARA nor ISA enhanced the cytotoxic activity of Th1-like γδ T cells in our experimental conditions (Figure S4), indicating good compatibility of ELO with Th1-like γδ T cells. These results suggest that Th1-like γδ T cells can be utilized as an effector for ELO-mediated ADCC.

3.2 Osteoclasts highly express SLAMF7 and are susceptible to ELO's ADCC

Intriguingly, the addition of ELO also enhanced the cytotoxic activity of Th1-like γδ T cells against OCs (Figure 2A,B), although ELO alone did not induce any cytotoxic activity against OCs (Figure 2B). Blockade of CD16 significantly reduced the ELO-mediated OC death with the γδ T cells (Figure S5A), indicating ELO-mediated ADCC against OCs with the γδ T cells. CD38 was reported to be expressed in monocytes but downregulated in osteoclastic lineage cells differentiated from monocytes.¹¹ In sharp contrast to CD38, SLAMF7 expression was upregulated during osteoclastic differentiation from monocytes, although monocytes only marginally expressed SLAMF7 (Figure 2C and Figure S5B), and confirmed in mature multi-nucleated OCs (Figure S5C). SLAMF7 was robustly upregulated in pre-osteoclasts before expressing cathepsin K, a marker of mature OCs, as well as cathepsin K-expressing mature OCs (Figure 2C). The SLAMF7 expression was induced in parallel with NFATc1 upon RANKL treatment (Figure S5D,E), suggesting RANKL-mediated SLAMF7 induction in osteoclastic lineage cells. Furthermore, ELO was able to enhance γδ T cell-induced cell death against both MM cells and OCs even in their coculture settings (Figure 2D). These results collectively demonstrate that SLAMF7 is highly expressed in both MM cells and OCs, and that OCs as well as MM cells are susceptible to ELO-mediated ADCC with Th1-like γδ T cells. ELO and Th1-like γδ T cells in combination are suggested to target and disrupt the MM cell–OC vicious cycle.

SLAMF7, a therapeutic target of MM cells, is also robustly upregulated in osteoclastic lineage cells. Th1-like γδ T cells expanded by ZA and IL-2 can exert cytotoxic activity against OCs as well as MM cells, which is substantially enhanced through ADCC with ELO. The unique function of Th1-like γδ T cells warrants their further utilization for innovative immunotherapies.