- Here, blinatumomab-induced transcriptional changes reflected the functional immune activity of the blinatumomab-activated T cells, including the upregulation of pathways such as the immune system, glycolysis, IFNA signaling, gap junctions, and IFNG signaling. - The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. - If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. - Full list of author information is available at the end of the article. - In addition, full hematologic recovery occurred significantly more frequently in the blinatumomab group than in the chemotherapy group [9].. - Despite these encouraging results, not all patients with B-ALL respond to blinatumomab therapy based on eval- uations of the response rate to blinatumomab in mul- tiple studies . - Notably, T cells cannot be activated by blinatumomab nor other BiTE antibodies in the absence of target cells [20]. - However, there are still patients showing a poor response to blinatumomab therapy even in the presence of the immune checkpoints inhibitors PD-1 and CTLA4 [29]. - Therefore, it has been hypothesized that T EM cells account for most of the blinatumomab-mediated cytotoxicity . - In addition, the priming and activation of nạve T cells strongly rely on signaling through CD28 and other co-stimulatory molecules [32], leading to the conclusion that nạve T cells will not be activated by blinatumomab in the absence of any costimulatory factors. - Recently, single-cell RNA-seq (scRNA-seq) has been widely used in the analysis of T cell subpopulations [35 – 37]. - The dose-dependent specific lysis induced by blinatumomab in RS4;11 and SUP-B15 cells are shown in Additional file 1, Fig. - By applying unsupervised clustering in the principal component space of this dataset, we identi- fied five cell clusters (Fig. - Cluster C1 was composed of T cells exhibiting a highly specific expression of the T cell markers CD3D and CD3E.. - S4D) in the. - 2c, Additional file 1, Fig. - S5), were found to be higher in the RS4;11 group than in the SUP-B15 group after blinatumomab treatment for 16 and 48 h. - Our re- sults show that the T cell changes in individual cell lines and at different time points are in agreement with the observations of the combined one. - The blinatumomab responsive T cell clusters (TC3, TC10 and TC13) con- sistently expanded after blinatumomab treatment in both cell lines, while the magnitude of the expansions in the RS4;11 group were larger than those in SUP-B15 group at both 16 and 48 h. - Similarly, the TC10-CD4+ Activated T cluster was chronologically ordered at the terminal end of the CD4+ activation trajectory (Additional file 1, Fig. - Genes associated with activation were mapped onto the activation trajectory, confirming an increased expression of the genes IL2RA, CD69, TNFRSF18 and TNFRSF4 at the end of the trajectories (Additional file 1, Fig. - c The proportion of each cluster in the untreated (RU-16 h, RU-48 h, SU-16 h, and SU-48 h) and blinatumomab-treated (RT-16 h, RT-48 h, ST-16 h, and ST-48 h) groups. - The clusters were placed in descending order based on the proportion of each cluster in the blinatumomab-treated group. - 1.5) were common across all four blinatumomab-activated T cell clusters, although several unique DEGs were found in each of the activated T cell clusters (Fig. - The majority of the common. - d Pathway enrichment analysis revealed 36 differentially expressed genes listed in the reactome database (https://reactome.. - The size of the circles is proportional to the Gene Count in the corresponding category. - Cytokines induced by interferons were also upregulated, including CXCL10, ISG15, IFIT3, IFI35, and IFI6, confirming the activation of IFNA and IFNG regulation signaling path- ways in the blinatumomab-activated clusters.. - In order to validate the T cell responses in the cell line model in a more heterogeneous system, we analyzed a total of 2271 T cells from 13,240 sequenced single B- ALL PBMCs and BMMCs from two different donors (Additional file 1, Table S1, Fig. - We calculated the numbers of shared signature genes of clusters from both cell line model and patient samples in order to compare the similarities of the relevant clus- ters from these two models (Additional file 1, Fig. - In addition, the activated T cell clusters PTC2 and PTC6 also exhibit a high expression of the 36 common DEGs identified in the cell line model (Fig. - 4e, Additional file 1, Fig. - Additionally, the fold change of TNFRSF4 (Δ = 0.9) in the PTC6 cluster was significantly higher than that of TNFRSF18 ( Δ = 0.6) or LAG3 (Δ = 0.1) (Fig. - This evident change in the amount of TNFRSF4 underlines its func- tional roles in the modulation of blinatumomab-induced T cell activation.. - In order to cor- roborate the observed expression of TNFSF4 on B-ALL tumor cells, we examined the expression of the gene in both B-ALL cell lines. - The changes in the TNFS F4 mRNA levels were also analyzed in the SUP-B15 and RS4;11 groups after blinatumomab treatment. - We de- tected a decrease in the amount of TNFSF4 expression in RS4;11. - a A t-SNE projection of all T cells in the patient derived model with 9 subclusters. - d Heatmap of the 36 common genes identified in cell line models, differentially expressed between PTC2-CD8+ Activated and PTC0-CD8+ TEM, PTC6-Activated T and PTC3-Nạve T single cells. - e Bar plot of the expression of TNFRSF4 , TNFRSF18 and LAG3 in clusters PTC3-Nạve T and PTC6-Activated T. - The y-axis shows the log2 value of the expression value. - Δ represents the log2 value of the fold change. - In order to determine the impact of upregulation of TNRFSF4/TNFSF4 signaling on the cytolytic activity of blinatumomab, we used a recombinant human TNFSF4 protein in the cytotoxicity model. - At first, PBMCs were co-cultured with target cells for 16 or 48 h at a ratio of 10:1 in the presence of the recombinant human TNFSF4 protein. - No significant additive effects were found in the sensitive cell line RS4;11 by adding TNFSF4, as its. - b Relative expression of TNFSF4 in RS4;11 and SUP-B15 cells as measured by qPCR. - c The combined density distribution curve of the relative expression of TNFSF4 (probe set id: 207426_s_at) in 576 patients with B-ALL from Microarray Innovations in LEukemia study (GEO accession: GSE13204) using of 2-component Gaussian mixture model. - Dashed curves represents the 2-component distribution in the mixture model (blue: low expression, red: high expression). - The boxplot shows the expression distribution of TNFSF4 in the two groups. - Increased blinatumomab-directed lysis of the less sensitive SUP-B15 cells was observed after 48 but not 16 h of co-culturing, suggesting that the effect of TNFSF4 to the cytolytic activity is better detected in the long-time co-culturing condition (Additional file 1, Fig. - With an optimized assay condition of 1:2 effector-to-target cell ratio and 5 days of co-culturing, the recombinant human TNFSF4 protein significantly increased blinatumomab-directed lysis of the less sensitive SUP-B15 cells in a dose-dependent manner (Fig. - The functions of different T cell populations in blinatumomab-mediated cytotoxicity are a topic of active discussion in the field. - Single cell transcriptomics allowed a comprehensive and precise analysis of the impact of different T cell populations on blinatumomab-mediated cytotoxicity. - The ligands in the tumor cells bind to the co-signaling receptors on T cells and modulate T cell responses after activation. - Previous studies have shown the importance of the co-inhibitory receptors PD-1 and CTLA4 for the cytolytic activity of blinatumomab . - Cur- rently, a phase I trial combining blinatumomab with in- hibitors of the co-inhibitory receptors PD-1 and CTLA4 is ongoing. - samples from only one healthy donor and two patients were used in single-cell transcriptome analysis, which may not be fully representative of the patient population.. - Thus, a larger cohort may need to be used in the future in order to generalize the findings.. - In agreement with these observa- tions, few T cells were fully activated in the SUP-B15 group, which resulted in poor blinatumomab-directed lysis of SUP-B15 cells (Fig. - Moreover, the ligands of the co-stimulatory signaling pathways are rarely present in tumor cells [62]. - In this study, we found that a higher expression of TNFSF4, the ligand of the co-stimulatory receptor TNFRSF4, was present among the sensitive tar- get cells RS4;11. - TNFRSF4 belongs to the next generation of immune therapeutic targets in the field of oncology. - More- over, co-stimulatory signaling transduction by TNFRSF4 has been designed in the third-generation CARs.. - Further studies are needed in the future to. - Thus, the PBMCs that were used in the different assays came from different donors. - C34564) and then co-cultured with PBMCs for 16 or 48 h at an effector-to-target cell ratio of 10:1 and in the presence of blinatumomab. - In order to observe a significant dose-response effect of TNFSF4, the PBMCs were co-cultured with target cells for 5 days at a ratio of 1:2 in the presence of recombin- ant human TNFSF4 protein (R&D system, #1054-OX- 010). - PBMCs were co-cultured with target cells for 16 or 48 h at a ratio of 10:1 in the presence of either 0 or 0.1 ng/. - B-ALL patient PBMCs and bone marrow mononuclear cells (BMMCs) (N = 3, Proteo- genex, #ALL205BM, #ALL207BM, #ALL207L) were cul- tured for 16 h in the presence of either 0 or 10 ng/mL blinatumomab. - Preprocessing of the single-cell RNA-Seq data. - Batch effects were corrected by a mutual nearest neighbor-based method available in the scater package [75].. - Based on the Euclidean distance between cells in the first 20 principal components, a k-nearest neighbor graph was built and the Louvain algorithm was used to calculate modularity, as available in the Seurat R package [76]. - Here, the resolution parameter, which sets the granularity of the subsequent cluster extraction, was chosen to optimally segregate all cells and T cells in the two datasets in our analysis, respectively. - 207426_s_at) in the bone marrow samples of 576 pa- tients with acute B-lymphoblastic leukemia were re- trieved from Microarray Innovations in the LEukemia (MILE) study (GEO accession: GSE13204). - Removal of the cluster composed of doublets.. - (c) Heatmap showing the expression of the top 20 signature genes in each Treg subcluster of sin- gle cells. - (d) Matrix showing the number of the top 20 signature genes shared by each cluster in cell line model samples and each cluster in patient derived model. - (e) Bar plot of the expression of TNFRSF4 , TNFRSF18 and LAG3 in clusters PTC0-CD8+ T EM. - The y-axis showed the log2 value of the ex- pression value. - Δ represented the log2 value of the fold change. - Expression levels of TNFSF4 in the RS4;11 and SUP-B15 before and after Blinatumomab treatment for 48 h. - The CD19 expression in RS4;11 and SUP-B15. - Additional file 3. - Detailed description of cell clustering and definition in the two models.. - h: Blinatumomab-treated SUP-B15 cells at 48 h. - This work was funded by Amgen Inc., the National Key Research and Development Program (2016YFC0902800 to R.R. - The single cell RNA-seq datasets generated during the current study are available in the Sequence Read Archive (SRA) repository (Accession Number:. - The expression profiles of BMMC samples from 576 B-ALL pa- tients in Microarray Innovations in LEukemia study are available in the Gene Expression Omnibus (GEO) repository (Accession Number: GSE13204). - Phase II trial of the anti-CD19 bispecific T cell-engager blinatumomab shows hematologic and molecular remissions in patients with relapsed or refractory B-precursor acute lymphoblastic leukemia. - Strictly target cell-dependent activation of T cells by bispecific single-chain antibody constructs of the BiTE class. - Blinatumomab: enlisting serial killer T-cells in the war against hematologic malignancies. - Frequency of regulatory T cells determines the outcome of the T-cell-engaging antibody blinatumomab in patients with B-precursor ALL. - Lu DR, Wu H, Driver I, Ingersoll S, Sohn S, Wang S, Li C-M, Phee H: Dynamic changes in the regulatory T-cell heterogeneity and function by murine IL-2 mutein. - unique features of different members of the human guanylate-binding protein family. - Microparticles released from Mycobacterium tuberculosis-infected human macrophages contain increased levels of the type I interferon inducible proteins including ISG15. - A diverse range of gene products are effectors of the type I interferon antiviral response. - Chemokines in the cancer. - CD4+CD25+ T regulatory cells dependent on ICOS promote regulation of effector cells in the Prediabetic lesion. - Adoptive immunotherapy with redirected T cells produces CCR7 − cells that are trapped in the periphery and benefit from combined CD28-OX40 Costimulation. - Lun ATL, Riesenfeld S, Andrews T, Dao TP, Gomes T, participants in the 1st Human Cell Atlas J, Marioni JC
Xem thử không khả dụng, vui lòng xem tại trang nguồn hoặc xem
Tóm tắt