Multiplex immunohistochemistry elucidates increased distance between cytotoxic T cells and plasma cells in relapsed myeloma, and identifies Lag-3 as the most common checkpoint receptor on cytotoxic T cells of myeloma patients.

A dysfunctional immune tumor microenvironment facilitates disease progression in multiple myeloma (MM). Using multiplex immunohistochemistry (mIHC), we describe the quantitative and qualitative changes in CD3+CD8+ cytotoxic T cells and assess their proximity to malignant plasma cells (PC) in patients with monoclonal gammopathy of undetermined significance (MGUS), and newly diagnosed (ND) and relapsed and/or refractory (RR) MM. Formalin-fixed, paraffin-embedded trephine sections from patients with MGUS (N=32), NDMM (N=65), and RRMM (N=59) were sequentially stained for CD138, CD3, CD8, and checkpoint receptors (CPR) Tim-3, Lag-3, and PD-1. The Halo® image analysis platform was used for cell segmentation and phenotyping, facilitating enumeration of cytotoxic T cells and analysis of proximity to PC. The percentage of CD8+ cytotoxic T cells in proximity to PC is greater in patients with NDMM than patients with RRMM (at 50 μm distance, 90.8% vs. 81.5%; P=0.038). There is a trend for more CD3+ T cells in MGUS (P=0.08) but no difference was observed in the prevalence of CD8+ cytotoxic T cells (P=0.48). Lag-3 is the most common CPR expressed on cytotoxic T cells in myeloma (P<0.0001), while PD-1 is the most common CPR on CD8- T cells of patients with MGUS and RRMM. Our study is the first to report on the spatial relationship between T cells and PC using mIHC on FFPE bone marrow trephine sections from patients with PC dyscrasia. The proximity of T cells to PC during early stages of MM, and overexpression of Lag-3, validate the move of immune therapeutic strategies, including T-cell engagers and checkpoint inhibitors, to upfront treatment or in early-line treatment of MM.