Fig. 1

Generation of NA cells derived from activated T lymphocytes. A. CRISPR/Cas9-based strategy performed to obtain ALK+ ALCL model cells from activated T lymphocytes. PBMCs were collected from healthy donor blood and activated with anti-CD3/CD28 for 7 days. Activated cells were transfected with RNP complex (Cas9 + gRNA^NPM1/gRNA^ALK). DNA was extracted at day 0 and then every three days to estimate translocation frequency overtime. B. Translocation frequency assessed by PCR amplification of derivative chromosome 5, Der5, from 2 (D2) to 15 days (D15) post-transfection (n = 4 independent donors). PCR were performed in duplicate on DNA dilutions (dilutions: 25 ng to 0.1 ng). The translocation frequency (F) was calculated as described in [21] presuming that a human diploid cell contains approximately 6 pg of DNA. C. Proliferation curve of control T lymphocytes (control cell groups (CTL): unsorted, CD4 + CD8-, CD8 + CD4-) and CRISPR/Cas9-transfected lymphocytes (NA cell groups: unsorted, CD4 + CD8-, CD8 + CD4-). Median with standard deviation for n = 3. D. Representative image of metaphases (FISH analysis) obtained from NA cells, 1 month post-transfection. Break-apart probe (green + red): ALK gene; blue probe: NPM1 gene. E. Western blot analysis of NPM-ALK, STAT3, phosphorylated NPM-ALK (P-NPM-ALK) and phosphorylated STAT3 (P-STAT3) in NA cells from Donors 1 to 4, at 1 month post-transfection. Activated T lymphocytes were used as negative controls, and an ALK+ ALCL cell line was used as a positive control (CTL+). Vinculin was used as the loading control. F. Evolution of sequencing read numbers for each type of translocation breakpoint junction sequence obtained by targeted sequencing (for der2 and der5) at day 5 and day 15 after transfection. Each curve represents the number of normalized reads of one single sequence. For Der5, one sequence is overrepresented (*) (corresponding to a ΔAG deletion). G. Violin plots of indel size (in bp for deletions and insertions) for Der2 and Der5 breakpoint junction sequences, at day 5 and day 15 for the four donors. H. Schematic diagram of NA cell selection showing the number of breakpoint junctions at day 5 and 15 post-transfection (normalized mean ± SD of number of different junction sequences observed per transfection and calculated from the sequencing of at least 900 reads). F = translocation frequency estimated by PCR of DNA dilutions (see Fig. 1B). I. Analysis of TCRγ clonality via multiplex PCR of activated T lymphocytes (day of transfection: D0) and NA cells 15 days (D15) post-transfection (corresponding to Donor 4)