OAB-017: Attenuation of T cell cytotoxicity mediated by CD200 expression on multiple myeloma cells

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<h3>Background</h3> The CD200/CD200 receptor (CD200R) axis is known to entail an immunoregulatory function in myeloid derived cells. The ligand, CD200, is implicated in solid and hematological malignancies to be associated with poor prognosis. Multiple myeloma (MM), a plasma cell malignancy, shows strong expression of CD200 in the majority of patient derived primary cells. However, the downstream mechanism upon CD200 ligand binding to the CD200R in T cells is not well understood. In this study, we evaluate the role of CD200 as a potential immune checkpoint in MM and seek to unravel the mechanism of immune escape mediated by CD200. <h3>Methods</h3> CD200 expression on patient-derived primary MM cells and MM cell lines was tested using flow cytometry. A Sleeping Beauty Transposon vector system was used to overexpress CD200 on MM cell lines. CD3/CD28-activated healthy donor T cells were co-cultured with CD200+/- MM cell lines L363, U266 and MM.1s. Using flow cytometry or luciferase assay, the co-culture was analyzed to assess cytotoxicity. To study downstream signaling effects of CD200R activation, CD3/CD28-activated T cells were treated with recombinant human CD200 (rhCD200) and/or anti-CD200 blocking antibody. Western blotting was performed to analyze potential downstream effector signaling. <h3>Results</h3> Approximately three-quarters of patient-derived primary MM cells (n=120) expressed CD200. In MM cell lines (n=9), no surface or cytoplasmic expression of CD200 was observed. Hence, we stably expressed CD200 on MM cell lines using a Sleeping Beauty transposon vector system. In both, flow cytometric analysis and luciferase assay, we observed up to 50% decrease in CD3+ T cell-mediated cytotoxicity in the presence of CD200-expressing MM cells. Possible differences in proliferation rates of MM cell lines due to CD200 overexpression were ruled out by performing an Alamar blue assay. In myeloid derived cells, docking protein-2 (DOK2) is known to directly interact with CD200R. DOK2 was phosphorylated upon CD200 binding in activated T cells when treated with rhCD200 in a time and concentration-dependent manner. This effect could be reversed with an anti-CD200 blocking antibody, providing a mechanistic explanation for the observed attenuation of T cell function. <h3>Conclusions</h3> CD200 expression on MM cell lines leads to attenuated cytotoxicity from primary healthy donor CD3+ T cells. Here, we provide evidence that this inhibitory mechanism in CD3+ T cells is mediated via DOK2.