Single cell analysis of the CD8<sup>+</sup> T-cell compartment in multiple myeloma reveals disease specific changes are chiefly restricted to a CD69<sup>-</sup> subset suggesting potent cytotoxic effectors exist within the tumor bed.

Multiple Myeloma (MM) is an incurable disease of the bone marrow (BM) characterized by the uncontrolled proliferation of neoplastic plasma cells. While CD8+ T-cells have an established role in disease control, few studies have focused on these cells within the MM tumor microenvironment (TME). We analyzed CD8+ T-cells in the BM and peripheral blood (PB) of untreated patients with MM and non-myeloma controls using flow cytometry, mass cytometry and single-cell RNA sequencing, using several novel bioinformatics workflows. Inter-tissue differences were most evident in the differential expression of granzymes B and K, which were strongly associated with two distinct subsets of CD8+ T-cells delineated by the expression of CD69, accounting for roughly 50% of BM-CD8+ T-cells of all assessed cohorts. While few differences were observable between health and disease in the BM-restricted CD8CD69+ T-cell subset, the CD8+CD69- T-cell subset in the BM of untreated MM patients demonstrated increased representation of highly differentiated effector cells and evident compositional parallels between the PB, absent in age-matched controls, where a marked reduction of effector cells was observed. We demonstrate the transcriptional signature of BM-CD8+ T-cells from patients with MM more closely resembles TCR-activated CD8+ T-cells from age-matched controls than their resting counterparts.