PF565 MM–BMSCS INDUCE IMMUNOSENESCENT NAÏVE CD4+ T‐CELLS THROUGH THE NOVEL INTERCELLULAR COMMUNICATION – TUNNELING NANOTUBES

Abstract

Background:Multiple myeloma‐bone marrow mesenchymal stromal cells (MM–BMSCs) can help escape immune surveillance. Several studies found that the accumulation of senescent T‐cells in certain types of cancers might be used by tumor cells to escape immune surveillance. However, the modulatory function of MM–BMSCs on senescent T‐cells has been little explored.Aims:This study aimed to investigate the modulatory effect of MM‐BMSCs on immunosenescent naïve CD4+ T‐cell and explore the role of tunneling nanotubes (TNTs) in the immunosuppressive effect of MM–BMSCs.Methods:Naïve CD4+ T‐cells were co‐cultured with BMSCs (derived from healthy donors and patients with MM). Senescence‐associated beta‐galactosidase (SA‐β‐Gal) staining was used to identify senescent CD4+ T‐cells. As a telomerase catalytic subunit, the expression of human telomerase reverse transcriptase (hTERT) was analyzed by reverse transcriptase–polymerase chain reaction. The senescent CD4+ T‐cells were characterized by a loss of expression of CD28; therefore, the expression of CD28 was examined using flow cytometry. Then, MM–BMSCs and CD4+ T‐cells were separately stained with DiO‐green and DiI‐yellow to observe the TNTs between MM–BMSCs and CD4+ T‐cells using confocal laser scanning microscopy. This study detected the fluorescence intensity using flow cytometry at different times to evaluate the cytoplasmic component exchange. Further, to evaluate the contribution of TNTs signaling to the senescent CD4+ T‐cells induced by MM–BMSCs, the TNTs were inhibited using three independent co‐culture systems: (1) Transwell co‐culture system, (2) contact co‐culture system with F‐actin inhibitor (cytochalasin D), and (3) shaken co‐culturesystem.Results:The MM–BMSCs upregulated the expression of SA‐β‐Gal in co‐cultured CD4+ T‐cells more significantly compared with healthy donors (HD)–BMSCs (P = 0.015). The level of CD28 dramatically lowered in CD4+ T‐cells co‐cultured with MM–BMSCs compared with HD–BMSCs (P = 0.004). The expression of hTERT was distinctly downregulated by MM–BMSCs compared with HD–BMSCs (P = 0.011). As shown in Fig 1, nanotubes were formed between MM–BMSCs and CD4+ T‐cells. CD4+ T‐cells accumulated small amounts of DiO‐green despite the long‐term, close cell‐cell contacts (Fig. 2, P> 0.05). However, a large amount of DiI‐yellow was observed in MM–BMSCs in a time‐dependent manner (Fig. 2; P = 0.009, 0.036, and 0.006, at 8 h, 12 h, and 24 h, respectively).The percentage of senescent CD4+ T‐cells co‐cultured with MM–BMSCs decreased nearly 40% in the Transwell system (P < 0.01; Fig. 3A). The disruption of TNTs structures by gentle shaking reduced the number of senescent CD4+ T‐cells induced by MM–BMSCs obviously (P < 0.05; Fig. 3B). Cytochalasin D caused a reduction in the percentage of senescent CD4+ T‐cells after 6 h of co‐culture (P < 0.05; Fig. 3C).Summary/Conclusion:MM–BMSCs could induce immunosenescent naïve CD4+ T‐cells. This study demonstrated the existence of TNTs and cytoplasmic component transmission from MM–BMSCs to CD4+ T‐cells. Inhibition of TNTs rescued the immunosuppression effect of MM–BMSCs. A better understanding of TNTs between MM–BMSCs and T‐cells might help in proposing a novel mechanism involved the immunomodulatory effect of MM–BMSCs.image