P813: malignant plasma cells promote expansion of regulatory t cells to create a protective “immune shield”

Topic: 13. Myeloma and other monoclonal gammopathies - Biology & Translational Research Background: Multiple myeloma (MM) remains an incurable disease due to expanding clonal plasma cells (PCs). CD4+CD25+FoxP3+ regulatory T cells (Treg) appear to support bone marrow (BM) PC maintenance (Glatman Z.A., et al. - Cell Reports 2017). Furthermore, Treg depletion in mice bearing MM evokes a potent cell-mediated response by CD8+ T cells and natural killer (NK) cells resulting in complete remission of the neoplastic disease (Dahlhoff J. et al. – Leukemia 2021). Aims: To analyze the immune interactions between Tregs, neoplastic PCs and BM tumor microenvironment. Our goal is to understand whether Tregs promote the formation of an immunological niche for malignant PCs that can further support MM maintenance and progression. Methods: CD4+CD25+FoxP3+ Tregs were analyzed by FACS and histology in BM samples from 29 MM patients and 8 healthy controls. Purified murine CD4+ T cells were co-cultured with wt PCs and irradiated 5TGM1 MM mouse cell line (30 Gy). We used FTR mouse strains (C57BL/6-FoxP3DTR), which express the diphtheria toxin (DT) receptor conjugated to the GFP fluorescent protein under the control of the FoxP3 promoter to in vivo track and deplete Tregs. PCs in BM and secondary lymphoid organs were analyzed by FACS. Results: FACS analysis performed in the BM of MM patients showed Tregs (FoxP3+/CD4+ cells) significantly increased compared with healthy controls (p<.001). Tregs expressing CD150 have been reported to be the subset that build an immune niche for BM hematopoietic stem cells (Hirata Y, et al – Cell Stem Cell 2018). Indeed, CD150+ Tregs were the prevalent subpopulation in BM of MM patients (p<.01). In IHC analysis, CD138/FoxP3 double staining on BM biopsies revealed Tregs preferentially accumulate in close proximity of malignant PC clusters (mean distance 200 μm), while in healthy BM normal Tregs localize at a greater distance from PC islets (mean distance 600 μm). This distance is proportionally inverse to the BM neoplastic infiltration (p<.0001). We aimed to confirm the key role of BM Tregs in murine normal and neoplastic PC development. Prolonged (10 days) Treg-depletion of FTR mice resulted in mature (B220-negative) PCs reduction in BM (p<.01) and their accumulation in the spleen (p<.02). In Treg-depleted transplanted mice mature (B220-negative) PCs infused at the time of transplantation accumulated mainly in secondary lymphoid organs (p<.0001) and showed reduced ability to migrate to the BM (p<.01). Such results suggest Tregs are required for normal BM PC development and maturation. Analysis of in vitro activated spleen-derived CD4+ T cells, when co-cultered with irradiated murine 5TGM1 MM cell line in comparison to wt PCs, showed MM cells support CD4+ T cells activation and proliferation and induced Treg polarization (increased CD25 and FoxP3 expression) and proliferation (p<.001). Indeed, our results taken together with previous mouse studies (Dahlhoff J. et al. – Leukemia 2021), indicate Tregs support MM cell growth. Summary/Conclusion: Our studies support the hypothesis that neoplastic PCs are able to influence BM microenvironment by stimulating CD4+ T activation and proliferation and their Tregs polarization. Indeed, MM cells induce Treg polarization to possibly provide an “immune shield” that facilitate their growth by protecting them from immune-mediated attack (CD8+ T cells and NK cells). Studies are ongoing to demonstrate the mechanism by which neoplastic cells induce Tregs from CD4+ lymphocytes and to confirm our findings on murine MM models (5TMM). Finally, these preclinical experiments suggest Tregs would be an attractive target for MM treatment. Keywords: Immunity, Multiple myeloma, Regulatory T cell, Plasma cells