Activated and Bone-marrow Resident Treg Alterations Underlie Malignant Transformation from MGUS to Multiple Myeloma

Multiple Myeloma (MM) is preceded by the pre-malignant, clonal plasma cell disorder monoclonal gammopathy of undetermined significance (MGUS). Altered immune surveillance during malignant transformation from MGUS to myeloma may involve changes in the regulatory T cell (Treg) compartment which are permissive to myeloma immune escape. To address this hypothesis, we used mass cytometry and unsupervised clustering algorithm Flow Self-organizing Map (FlowSOM) to interrogate at high resolution the heterogeneity within the Treg (CD25+CD27low/neg cells) compartment, in matched bone marrow (BM) and peripheral blood (PB) of MGUS and newly diagnosed NDMM patients. Both mass cytometry and flow cytometry confirmed a trend toward prevalence of CD39-Treg within the Treg compartment in BM and PB of NDMM patients compared to CD39-Treg in MGUS patients. FlowSOM clustering which displayed Treg in 25 metaclusters suggested Treg heterogeneity in both MGUS and NDMM patients, and discovered two subsets which emerged within CD39-Treg of NDMM patients but were negligible or absent in CD39-Treg of MGUS patients. One subset resembled activated Treg based on CD45RO, CD49d and CD62L expression and was found in both BM and PB; another subset resembled BM-resident Treg based on its tissue-resident CD69+CD62L-CD49d- phenotype and restricted location within the BM. Both subsets co-expressed PD-1 and TIGIT, but PD-1 was expressed at higher levels on BM-resident Treg then on activated Treg. Within BM, both subsets had limited Perforin and Granzyme B production, whilst activated Treg in PB acquired high Perforin and Granzyme B production. In conclusion, the use of mass cytometry revealed two discrete subsets of CD39-Treg which are discordant in MGUS and NDMM patients. These subsets may be permissive of plasma cell growth and thus play a role in malignant transformation from MGUS to myeloma, which warrants further study. Understanding the regulatory properties of these Treg subsets may have diagnostic and prognostic significance in MGUS and MM, including the definition of risk in smoldering MM, as well as therapeutic implications.