Sequences of 10 cell barcode (BC) of interest are indicated in Supplementary Table?1. Then, using single-cell RNA sequencing (scRNAseq) approach, we demonstrate that CD19neg leukemic cells were present before CAR-T cell therapy and thus that the relapse results from the selection of these rare CD19neg B-ALL clones. In conclusion, our study shows that scRNAseq profiling can reveal pre-existing CD19neg subclones, raising the possibility to assess the risk of targeted therapy failure. mRNA (through de novo frameshift/missense mutations, alternatively spliced CD19 mRNA species and hemizygous deletions spanning the locus)16C19. However, no study has clearly demonstrated whether CAR-T cell therapy is directly responsible of CD19 transcription dysregulation or simply allows the emergence of minor CD19neg clones escaping CD19-targeted therapy. In the second hypothesis, detection of those CD19neg clones may represent a valuable predictor of CD19-negative relapse permitting to assess TX1-85-1 beforehand the risk for CAR-T treatment failure and to adapt accordingly the therapeutic strategy. Here, we report the single-cell RNA-sequencing (scRNAseq) profiling of leukemic samples from a B-ALL patient, at two-time points, before and after CAR-T therapy. Our work reveals the presence of preexisting CD19neg B-ALL clones before the CAR-T treatment. Results Analysis of B-ALL samples prior and after CAR-T cell treatment We used scRNAseq approach to investigate leukemic cells of a B-ALL patient at two-time points; before and after anti-CD19 CAR-T cell therapy. The B-ALL patient underwent a relapse after chemotherapy and CD19pos leukemic cells from bone marrow (BM) were harvested (T1 cells?=?before CAR-T cell therapy; Supplementary Fig. 1a). Then, the patient was treated with anti-CD19 CAR-T. After an initial complete remission, the patient experienced a frank relapse, as noted by circulating blasts and an infiltration ( 90%) of CD10posCD19neg leukemic cells in the BM (Supplementary Fig. 1b) from which cells were harvested (T2 cells?=?after CAR-T cell therapy). Therefore, we asked whether these CD19neg B-ALL clones were present before CAR-T cell therapy. To address this issue, T1 and T2 samples were sorted according to forward scatter and side scatter, a cell viability marker, CD3 and CD19 surface expression (Fig. ?(Fig.1a).1a). We obtained four tubes of sorted cells: T1-CD19pos, T1-CD19neg, T2-CD19pos, and T2-CD19neg. Each of TX1-85-1 those samples were marked with a distinct anti-CD45-hashtag oligonucleotide (HTO) antibody20 (Supplementary Table?1), then mixed and analyzed by scRNAseq using the 5 10 Genomics technology21 (Fig.?1a). After libraries sequencing and data preprocessing, we applied the Seurat graph-based clustering algorithm and identified six main clusters. Then, we used the UMAP nonlinear dimensionality reduction method, to visualize cell transcriptome heterogeneity22. According to various gene markers, we assigned cell type to clusters (Fig.?1b, c). Non-B cells, i.e., myeloid and NK cells are located in clusters 2 and 5, respectively. Physiological B cells are in clusters 3 and 4, while B-ALL cells are in clusters 0 and 1. Sample demultiplexing assigned 1189, 524, 764, and 442 cells to T1-CD19pos, T1-CD19neg, T2-CD19pos, and T2-CD19neg samples, respectively, and allowed us to visualize sample of origin for each cell on the UMAP plot. Notably, T1-CD19pos and T2-CD19neg tumoral cells were detected mainly to clusters TX1-85-1 0 and 1, respectively (Fig.?1d). CaSpER tool23 and B-allele frequency (BAF) analysis indicate that cells from these two clusters displayed common copy number variants (CNV), such as chromosome 9q deletion, indicating that CD19pos and CD19neg B-ALL are related (Supplementary Fig.?2). The most differentially expressed transcription factor between clusters 0 and 1 was gene, a well-known tumor suppressor gene24,25 (Supplementary Fig.?3). Strikingly, CD19 mRNA transcripts were detected in T2-CD19neg tumoral cells albeit at lower level than in T1-CD19pos tumoral cells (Supplementary Fig.?4a, b). The analysis of bulk cDNA from T1-CD19pos and T2-CD19neg sorted cells shows that T2-CD19neg cells do not express full-length transcripts (Fig.?1e). We observed that both samples express TX1-85-1 a previously described18 nonfunctional CD19 transcript retaining intron 2 (Supplementary Fig.?4c). However, in T2-CD19neg cells only this nonfunctional isoform is expressed, explaining the absence of CD19 protein despite the presence of CD19 mRNA. Open in a separate window Fig. 1 CD19neg B-ALL relapse following CAR-T therapy.a Cell sorting strategy. Cells before (T1) and after (T2) CAR-T treatment were gated according to FSC/SSC profile (left FACS plots). Then, Selp live cells (middle FACS plots) were analyzed according to CD3 and CD19 expression (right FACS plots; see also Supplementary Fig.?9). Gates.