Objective: Chimeric antigen receptor T cells (CART) emerged as a robust therapeutic approach for refractory or relapsed B acute lymphoblastic leukaemia (B-ALL) in recent years. However, 30-50% of patients experienced leukaemia relapse within 1 year after CART cells therapy. Bone marrow derived mesenchymal stromal cells (MSCs) have been demonstrated to have immunosuppressive properties on T cell-mediated immune responses, but the impacts of MSCs on CART cells are not clear. Here we address the role of galectin-9 secreted from MSCs in immunosuppression of CART cells.
Methods: MSCs and T cells were isolated from bone marrow and peripheral blood of healthy donors, respectively, and CD19 targeted CART cells containing 4-1BB costimulatory construction were prepared as previously reported. The proliferation of CART cells was evaluated by cell counting and Ki-67 expression. The expression of PD1, TIM3, LAG3 and FasL was detected by flow cytometry. The cytotoxicity of CART cells was determined by luciferase-based assays. Transwell was used to assess the contribution of soluble factors. The mRNA expression of COX-2, IL-6, IL-10, TGFβ and galectin-9 was detected by real time PCR. We used the lentivirus-based shRNA interference to assess the role of Galetin-9 on immunosuppression of CART cells.
Results: In this work, we discovered that CAR-T cells co-cultured with MSCs for 72 hours in vitro showed a decreased proliferation rate and Ki-67 expression. And the inhibition effect became more significant with the increase in the proportion of MSCs (Fig.a,b,c,d). Notably, the expression of inhibitory receptor TIM3 upregulated remarkably while no change of PD1, LAG3 and FasL were observed(Fig.e,f). We routinely use luciferase-based cytotoxic assay for functional testing. By this measure, we found that with the increase in the proportion of MSC, CAR-T cells behave even weaker cytotoxic effect, achieving lower cytolysis rate of Nalm6 cells(Fig.g).To further determine the possible factors that contribute to the changes mentioned above, CART cells were separated from MSCs by transwell. As expected, the phenomenon of inhibited CART cells proliferation and cytotoxicity, increased expression of TIM3 was observed as well, although without direct contact of CART cells with MSCs. The contribution of soluble factors was in consideration and further experiments of real time PCR revealed that COX-2 and galectin-9 were strongly induced by CART cells and pro-inflammatory cytokines(IFNγ, TNFα). In light of that galectin-9 is the ligand of inhibitory receptor TIM3, which increased obviously in the presence of MSCs, we speculated that galectin-9 might be responsible for immunosuppression of CART cells. A knockdown approach with shRNA demonstrated the immunosuppressive activity of galectin-9 deficient MSCs on CART cells decreased significantly. We provided experimental evidence that galectin-9 may contribute to MSC mediated immunosuppression on CART cells by binding to its receptor TIM-3.
Conclusion: Our findings demonstrated for the first time that galectin-9 is involved in MSC mediated immunosuppression on CART cells, and represented a potential therapeutic target for enhancing the efficacy of CART cells and reducing the incidence of leukaemia relapse after CART cells therapy.
Key words: Chimeric antigen receptor T cells; mesenchymal stromal cells; immunosuppression; galectin-9.
Fig. a. Co-culture of CART cells and MSCs. b,c,d. Cell proliferation and Ki-67 expression of CART cells co-cultured with MSCs at different ratios. e,f. Expression of TIM3, PD1, LAG3, FasL of CART cells co-cultured with MSCs at different ratios. g. Cytotoxicity of CART cells co-cultured with MSCs at different ratios (CART without MSC, CART:MSC=1:5, CART:MSC=1:10, CART:MSC=1:20).
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Disclosures
No relevant conflicts of interest to declare.
Phase I study of F16IL2 antibody–cytokine fusion with very low‐dose araC in acute myeloid leukaemia relapse after allogeneic stem cell transplantation