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

Abstract

Multiple myeloma (MM) cells and osteoclasts (OCs) activate with each other to cause drug resistance. Human Th1-like Vγ9Vδ2 (γδ) T cells, important effectors against tumors, can be expanded and activated ex vivo by the aminobisphosphonate zoledronic acid in combination with IL-2. We previously reported that the expanded γδ T cells effectively targeted and killed OCs as well as MM cells. Because the expanded γδ T cells expressed CD16 on their surface, we investigated the utilization of the expanded γδ T cells for antibody-dependent cellular cytotoxicity (ADCC). Although the expanded γδ T cells alone induced cell death in MM cell lines, the addition of the anti-SLAMF7 monoclonal antibody elotuzumab (ELO) further enhanced their cytotoxic activity only against SLAMF7-expressing MM cell lines and primary MM cells. Intriguingly, ELO was also able to enhance γδ T cell-induced cell death against OCs cultured alone, and against both MM cells and OCs in their coculture settings. SLAMF7 was found to be highly expressed in OCs differentiated in vitro from monocytes by receptor activator of nuclear factor-κ B ligand and M-CSF, although monocytes only marginally expressed SLAMF7. These results demonstrate that SLAMF7 is highly expressed in both MM cells and OCs, and that the ex vivo-expanded γδ T cells can exert ELO-mediated ADCC against SLAMF7-expressing MM cells and OCs besides their direct cytotoxic activity. Further study is warranted for the innovative utilization of γδ T cells.

Keywords: Elotuzumab; SLAMF7; Vγ9Vδ2 T cell; multiple myeloma; osteoclast.

FIGURE 1

Ex vivo‐expanded Th1‐like γδ T cells induce elotuzumab (ELO)‐mediated antibody‐dependent cellular cytotoxicity (ADCC). Th1‐like γδ T cells were expanded with peripheral blood mononuclear cells (PBMCs) from normal donors (#1–4) by zoledronic acid (ZA) in combination with IL‐2. (A) CD16 expression in the γδ T cells was analyzed by flow cytometry. (B–D) After expansion of γδ T cells, the PBMC fractions were cocultured with PKH26‐labeled multiple myeloma (MM) cell lines as indicated (B, C) and primary CD138⁺ cells isolated from patients with MM (#1, #2) (D) in the presence or absence of ELO at the indicated concentrations. The effector‐to‐target ratio (E:T) was 5:1. After culturing for 4 h, % distribution of 7‐AAD‐positive dead cells within PKH26‐labeled MM cell lines and primary MM cells were counted. Data represent mean ± SD (n = 3) by Tukey's multiple comparisons test. **p < 0.01; ***p < 0.001; ns, not significant.

FIGURE 2

Elotuzumab (ELO) augments the cytotoxic effect of γδ T cells against osteoclasts (OCs) as well as multiple myeloma (MM) cells. (A–D) OCs were differentiated peripheral blood mononuclear cells (PBMCs) obtained from three healthy donors (#1–3). (A, B) OCs were cocultured with γδ T cells in the presence or absence of 1 μg/mL ELO for 24 h. After washing the plates, the viable and dead OCs in the plates were distinguished by staining with calcein‐AM and ethidium homodimer‐1 (EthD‐1). Alive OCs were detected as green, and dead OCs were as red. (A) Representative images are shown. Scale bars are 100 μm. (B) The percent distribution of dead OCs was evaluated. Data are shown as mean ± SD from three technical replicates, assessed by Tukey's multiple comparisons test. (C) During OC differentiation, cell lysates were extracted at the indicated time points and subjected to immunoblotting. β‐Actin served as a loading control. (D) PKH26‐labeled MM.1S cells were cultured alone or cocultured with the differentiated OCs. The expanded Th1‐like γδ T cells were then added to the mixture in the presence or absence of 1 μg/mL ELO. After 24 h of incubation, the cultured cells were washed with PBS. % distribution of 7‐AAD‐positive cells within PKH26‐labeled MM cells were counted (left). Viable and dead OCs were distinguished by staining with calcein‐AM and EthD‐1, respectively. Alive OC numbers were counted under a fluorescence microscope (right). Data are shown as mean ± SD from three technical replicates, assessed by Tukey's multiple comparisons test. *p < 0.05; **p < 0.01; ***p < 0.001; ns, not significant.