P800: characterization of regulatory t cells in patients with multiple myeloma

Topic: 13. Myeloma and other monoclonal gammopathies - Biology & Translational Research Background: Despite recent therapeutic advances with the development of proteasome inhibitors, immune modulators, monoclonal antibodies, chimeric antigen receptor T cells, and bispecific T cell engagers, multiple myeloma (MM) is incurable and most patients suffer from relapse by measurable residual disease (MRD) and impaired anti-tumor immune response. However, the roles of CD4 T cells in bone marrow (BM) of MM patients remain largely unknown. Aims: We aimed to characterize the CD4 T cells in BM of MM patients to better understand the mechanism governing immunologic escape. Methods: We performed single-cell RNA sequencing (scRNA-seq) of sorted CD4+ T cells from BM mononuclear cells (BMMCs) of healthy donor (HD), patients with monoclonal gammopathy of undetermined significance (MGUS), smoldering MM (SMM), and MM at diagnosis, and patients with or without minimal residual disease (MRD) after treatment with bortezomib/thalidomide/dexamethasone (VTD). In addition, we examined the abundance and immunophenotypic feature of T regulatory (Treg) cells in peripheral blood (PB) and BM of MM patients by multicolor flow cytometry (MFC). Results: We identified 17 clusters of BM CD4 T cells by scRNA-seq,, which were subdivide into classical CD4 T subsets; naïve (CCR7+SELL+ HNRNPLL-), central memory (CCR7+SELL+HNRNPLL+), tissue-resident memory T (TRM, CD69highKLF3-S1PR1-), Th1 (TBX21+CXCR3+IFNG+), Th2 (GATA3+CCR4high), Th17(RORC+CCR6high), cytotoxic T (GZMB+PRF1+GNLY+NKG7+) and regulatory T cells (Tregs, FOXP3+CTLA4+). Notably, the frequency of cytotoxic T was increased in MM patients at diagnosis compared with HD, while the frequency of cytotoxic T was significantly reduced after VTD in both patients with or without MRD. Interestingly, frequency of activated Tregs (HLA-DRhighCTLA4high) among total Tregs was increased in MM at diagnosis compared with HD. Although activated Tregs significantly decreased in patients without MDR after VTD, this decrease was not observed in patients with MRD. We therefore examined Tregs in MM patients by MFC and found that the frequency of CD45RA−FoxP3high effector Tregs (eTregs) among Tregs was significantly increased in BM of MM patients compared with HD, while the frequency of CD45RA+ naïve Tregs (nTregs) was significantly decreased in PB and BM of MM patients compared with HD. We also observed the significant upregulation of TIGIT, CD38, Ki-67, CD226, GITR, CTLA-4, and PD-1 expressions on Tregs in BM of MM patients compared with HD. Among the TIGIT+ Tregs, the proportion of eTregs was increased in BM of MM patients compared with the control. Also, the higher frequency of TIGIT+ cells among Tregs was prominent within eTregs compared with CD45RA−FoxP3low cytokine-secreting Tregs (non-Tregs). Summary/Conclusion Our results show that decrease in activated Treg is associated with achieving MRD negativity and that TIGIT blockade may support clearing MRD by selectively depleting eTergs in MM patients. Keywords: Regulatory T cell, Bone marrow microenvironment, Multiple myeloma, CD4+ T cells