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Chimeric antigen receptor T cells (CAR-T) therapy has achieved remarkable therapeutic success in treating a variety of hematopoietic malignancies. However, the high relapse rate and poor in vivo persistence, partially caused by CAR-T cell exhaustion, are still important barriers against CAR-T therapy. It remains largely elusive on the mechanisms of CAR-T exhaustion and how to attenuate exhaustion to achieve better therapeutic efficacy. In this study, we initially observed that CAR-T cells showed rapid differentiation and increased exhaustion after co-culture with tumor cells in vitro, and then performed single-cell ATAC-seq to depict the comprehensive and dynamic landscape of chromatin accessibility of CAR-T cells during tumor cell stimulation. Analyses of differential chromatin accessible regions and motif accessibility revealed that TFs were distinct in each cell type and reconstituted a coordinated regulatory network to drive CAR-T exhaustion. Furthermore, we performed scATAC-seq in patient-derived CAR-T cells and identified BATF and IRF4 as pivotal regulators in CAR-T cell exhaustion. Finally, knockdown of BATF or IRF4 enhanced the killing ability, inhibited exhaustion, and prolonged the persistence of CAR-T cells in vivo. Together, our study unraveled the epigenetic regulatory mechanisms of CAR-T exhaustion and provided new insights into CAR-T engineering to achieve better clinical treatment benefits.

Relapses of CD19-expressing leukemia in patients who achieved initial remission after CART cell treatment have been reported to correlate with poor CART cells persistence. Sustained tonic signaling or strong activation drives CART cell differentiation and exhaustion, which limit the therapeutic efficacy and persistence of CART cells. Here, we identified dasatinib as the optimal candidate to prevent or reverse both CD28/CART and 4-1BB/CART cell differentiation and exhaustion during ex vivo expansion, which profoundly enhanced the therapeutic efficacy and in vivo persistence. Moreover, strong activation-induced CART cells differentiation, exhaustion and apoptosis driven by CD3/CD28 stimulation or antigen exposure were dramatically prevented or reversed by dasatinib treatment. Mechanistically, dasatinib markedly reduced the phosphorylation of Src and Lck, and downregulated the expression of genes involved in CAR signaling pathways, which resulted in the optimization of cell differentiation, exhaustion and apoptosis-related gene expression. Our study proposes a promising pharmacological approach for optimizing CART cells manufacture, and provides an experimental basis for reinvigorating CART cells in clinical application. Supplementary Information The online version contains supplementary material available at 10.1186/s13045-021-01117-y.

Successful chimeric Ag receptor modified T cell therapy for isolated testicular relapse after hematopoietic cell transplantation in an acute lymphoblastic leukemia patient

Chronic myeloid leukemia (CML) is a clonal disorder of multipotent hematopoietic progenitor cells and is characterized by t (9; 22) (q34; q11) that produce the BCR-ABL fusion protein with constitut...

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). Figure Disclosures No relevant conflicts of interest to declare.

Introduction: Chimeric antigen receptor (CAR) T-cell therapy has displayed potent anti-leukemia activity in refractory/relapsed acute lymphocytic leukemia (ALL). However, the influence of CAR-T therapy on host systemic and local immunity has not been well examined. We therefore applied high-throughput T cell receptor β chain sequencing to track dynamic change of T-cell repertoire in vivo induced by CAR-T therapy in B-cell acute lymphocytic leukemia patients. Patients and Methods: Six patients with 45 samples were under observation. The samples obtained to be tested were the peripheral blood mononuclear cell (PBMC) samples and bone marrow mononuclear cell (BMMC) samples before and after CAR-T administration, as well as the CAR-transduced autologous T cell samples on the day when they were to be infused to patients. The information of samples and patients was summarized in Table 1. The TCR full length mRNAs of these samples were deeply sequenced using the ImmunHubTM TCR profiling system (ImmunQuad Biotech). Briefly, a 5'RACE unbiased amplification protocol was used. An algorithm was applied to raw sequencing data for PCR and sequencing errors correction and V, D, J, C gene segments mapping with IMGT®. The inverse Simpson index and the clonality index was calculated to estimate TCRβ clonotype diversity and the state of clonal proliferation of T cells. The donut chart and clone tracking heat map were generated by R. Result: Compared to preinfusion TCR diversity, we observed inverse Simpson's index of 4 of 6 patients' PBMC samples (Fig. 1A) and of 3 of 4 patients' BMMC samples (Fig. 1B) had been increasing to day 7 after CAR-T treatment. As time went by, a decline of TCR diversity from day 7 to day 28 was detected in both PBMC and BMMC samples (Fig. 1). Of note, a relative rising tide of TCR diversity was observed after the decline in PBMC samples (Fig. 1A). The decreased TCR diversity leaded us to test the change of TCR clonality. As shown by Fig. 2A and 2B, in comparison with the pre-treatment and the CAR-T samples, we detected highly clonal proliferation of T cells in both blood and bone marrow. Next, we applied the clonality index to quantitatively define T cell clonal expansion (Fig. 2C and 2D). The clonality index was raised from 0.16±0.07 and 0.14±0.05 before treatment to 0.27±0.09 and 0.3±0.15 at last time point post infusion in PBMCs and BMMCs, respectively (mean ± SD, student's t test, P=0.03 and P=0.17, respectively).CAR-T-induced T-cell clonal expansion triggered us to trace back the original clonal source. For tracking clonal evolution, we selected the 100 most prevalent T cell clonotypes found at the last time point in both PBMCs and BMMCs and tracked their frequencies at earlier time points. As was displayed by the heat map, the top 100 T cell clones dominating in PBMCs and BMMCs at the last time point were barely found in CAR-T cell pool and were the low-frequency clonotypes in preinfusion samples (Fig. 3A and 3B). In order to quantitatively describe this phenomenon, we compared the total frequency of top 10 T cell clones in PBMC and BMMC samples at the last time point to their corresponding total frequency in preinfusion samples and the infused CAR-T pool (Fig. 3C, 3D, 3E and 3F). The total frequency of the top 10 T cell clones was 23%±11% and 27%±14% at last time point, 4%±6% and 8%±7% before treatment and 0.05%±0.09% and 0.01%±0.01% in the infused CAR-T samples in PBMCs and BMMCs, respectively (mean ± SD, Fig. 3C, 3D, 3E and 3F; student's t test, P=0.01, P=0.01, P=0.08 and P=0.03, respectively). Only one patient's (patient 6's) top 10 T cell clones in PBMCs at last time point were not found in both CAR-T pool and preinfusion sample, which may be caused by the limitation of sequencing detection (Fig. 3C). Conclusion: For the first time, we demonstrated CAR-T therapy could stimulate the clonal proliferation of endogenous non-CAR T cells in patients. Along with other groups' animal results (Barber A et al. J Immunol. 2009) indicating that CAR-T therapy could facilitate the infiltrating of tumor antigen-specific T cells, these expanded T cell clones of patients in our trial are most likely tumor antigen specific, which could provide synergistic anti-tumor effect following CAR-T adoptive transfer. The trial was registered in Chinese Clinical Trial Registry and the registration number is ChiCTR-OCC-15007008. Disclosures No relevant conflicts of interest to declare.