Buffy coats from healthy individuals were purchased from the Department of Transfusion and Tissues of University Hospital Brno (UHB). RosetteSep Human T Cell Enrichment Cocktail (StemCell) was used to isolate primary T cells according to manufacturer’s protocol. Thirty-two primary CLL samples were selected from the biobank of the Department of Internal Medicine, Hematology and Oncology, UHB. All patients fulfilled the "International Workshop on Chronic Lymphocytic Leukemia"/National Cancer Institute diagnostic criteria for CLL.12 Collected peripheral blood samples were subjected to gradient centrifugation to obtain high-purity B lymphocytes (>98%) using Ficoll-Paque PLUS (GE Healthcare) combined with RosetteSep Kits (StemCell) to deplete non-B cells and subsequently vitally frozen. The selection criteria were based on availability of samples’ genetic information in order to compile genetically defined groups. All selected samples had been screened by Sanger and/or next-generation sequencing for somatic hypermutation in IGHV 13 14 and mutations in TP53,15 16 ATM,17 NOTCH1 18 and SF3B1.19 Only cases with highly abundant TP53 and ATM mutations were included. Conversely, wild-type (WT) cases had no mutation detected above the threshold of a respective method used. All primary cells (T and CLL cells) were cultivated in serum-free AIM-V medium (Thermo Fisher Scientific). T cells were stimulated by interleukin (IL)-2 (50 U/mL, Miltenyi Biotech) and Dynabeads Human T-activator CD3/CD28 (ratio 1:3, bead:cell; Thermo Fisher Scientific). For ‘2S’ stimulation of CLL cells, resiquimod (1 µg/mL, Sigma) and IL-2 (500 U/mL) were supplemented to the cells 3 days prior to starting any experiments.

HG3 (a generous gift from Dr Rosenquist, Sweden), MEC1 (DSZM) and Lenti-X 293T (TakaraBio Inc.) cell lines were used herein. HG3 is WT in TP53 gene20, while MEC1 has a truncated TP53 allele resulting from c.949_950insC mutation and the other allele is deleted21. Both cell lines have unmutated IGHV. HG3 was maintained in RPMI-1640 medium (Biosera), MEC1 in Iscove's Modified Dulbecco's Medium (Biosera) and Lenti-X in Dulbecco's Modified Eagle's Medium (Biosera). All media were supplemented with 10% (v/v) heat‑inactivated fetal bovine serum (FBS; Biosera) and 1% (v/v) penicillin/streptomycin (MP Biomedicals).

Sequence encoding anti-CD19 CAR was previously described22 and synthetized in BlueHeron Biotech Company (USA). This construct was subsequently cloned into BamHI and SalI sites of the lentiviral vector pRRL-CMV-GFP-sin18 (originally generated by D Trono, purchased from Addgene), in which the CMV promotor had been exchanged for EF1α promotor by molecular cloning. The sequence of each genetic fragment was confirmed by Sanger sequencing. Additionally, helper plasmids pCMV-VSV-G and pCMV-dR8.91 were from Addgene (originally generated by D Trono).

To generate lentiviral particles, we first treated Lenti-X cells with 25 µM Chloroquine Diphosphate (Sigma) 5 hours prior to transfection. Subsequently, we used polyethylenimine (PEI, 1 mg/mL; Polysciences) in a ratio of 3:1 (PEI:DNA) for transfection, where we combined the plasmids in equimolar ratio and used a total of 3 µg of DNA per 8×10⁵ Lenti-X cells. The medium was replaced 3 hours later. Conditioned lentiviral medium was harvested 48 and 72 hours later, frozen and stored at −80°C. Lenti-X Concentrator (TakaraBio) was used to concentrate the conditioned medium according to the manufacturer’s protocol. After centrifugation, the pellets were resuspended in AIM-V medium (1/100th of the original volume) and used immediately to transduce target T cells. A 1.5 mL Eppendorf tube was coated with 0.2 mL of Retronectin (TakaraBio) diluted to 100 µg/mL for 2 hours at room temperature and then incubated with 2% bovine serum albumin (Sigma) for 30 min. T cells (8×10⁵), which were prestimulated with IL-2 and CD3/CD28 24 hours prior to transduction, were resuspended in the retronectin-coated tube in 0.5 mL of concentrated virus supplemented with Polybrene (10 µg/mL, Merck) and centrifuged at 1000 g at 32°C for 2 hours. Immediately afterwards, viral medium was replaced. T cells were then left to expand until further use (the total in vitro cultivation time never exceeded 10 days).

MEC1 bears an inherent TP53 impairment. To be able to use it to generate various genetically distinct single-cell clones with either TP53 or ATM knocked out, we first had to reintroduce the TP53 WT allele under an inducible promotor. pCMV-Neo-Bam p53 WT plasmid (#16434, Addgene) was digested by BamHI, and the digestion product (TP53 WT gene) was cloned into a pLenti CMV/TO Hygro empty plasmid (#17484, Addgene) under a doxycyclin-inducible promotor. Lentiviral particles were produced as described previously; MEC1 cells were transduced and selected by 500 μg/mL hygromycin (InvivoGen) for 14 days. Afterwards, the lentiGuide-Puro two-vector system, composed of single guide RNA (sgRNA) containing lentiGuide-Puro vector (#52963, Addgene) and Cas9 expressing LentiCas9-Blast vector (#52962, Addgene), was used to generate CRISPR knockouts of ATM and TP53 genes. The following sgRNAs were cloned into lentiGuide-Puro:

5′-GGATGCTGTTCTCAGACTGA-3′ targeting exon 6 of ATM

5′-TCGACGCTAGGATCTGACTG-3′ targeting exon 2 of TP53

5′-TATCTGAGCAGCGCTCATGG-3′ targeting exon 5 of TP53

Transfection and virus harvesting were performed as described previously. Cas9-transduced cells (HG3 and MEC1 p53 wt) were selected by 10 µg/mL blasticidin (InvivoGen) for 7 days. Immediately afterwards, cells were transduced by lentiviruses containing the sgRNA of interest and selected by 10 µg/mL puromycin (InvivoGen, #ant-pr-1) for 4 days. In order to obtain monoclonal cell populations, the selected cells were plated into 96-well plates as 1 cell per well using the limiting dilution method.

Sanger sequencing and CRISP-ID tool (http://crispid.gbiomed.kuleuven.be/) were used to detect editing events. In the cell clones harboring indels, the loss of the respective protein was verified by western blotting. Only clones with no band visible after prolonged exposure were selected for further study. For ATM protein detection, no prior stimulation was needed. In case of p53, a 24-hour treatment with 2 µM doxorubicin was performed to stabilize the p53 protein. Prior to the treatment with doxorubicin, MEC1 p53 wt cells were also incubated with 2 µg/mL doxycycline for 24 hours in order to induce the WT p53 expression. In order to assess the accumulation of p21, the knockout cells were incubated with 2 µM doxorubicin for 24 hours, lysed and subjected to western blotting. Additionally, the phosphorylation of KAP1 was determined by western blotting in ATM-knockout cells incubated with 1 µM doxorubicin for 4 hours. The antibodies used in the study are listed in online supplementary table 1.

The antileukemic in vitro activity of CAR T cells was assessed in 4-day cultures using 1:5 ratio of target:effector cells. For this assay, the collection of knockout clones, as well as the set of primary CLL cells, were used as targets. In the case of vitally frozen primary CLL cells, these were thawed, and their viability was assessed by flow cytometry. A part of CLL cells was subsequently 2S stimulated and used in cytotoxicity assay only 72 hours later, while the other part was directly mixed with T cells. In detail, 2×10⁴ live B cells were combined with 1×10⁵ T cells (control (CTRL) or CAR) in 96-well U-bottom plates in 200 µL of AIM-V medium without any supplements. The target B-cell survival was monitored at 24 and 96 hours by flow cytometry. The amount of retrieved CD19+ cells from the mixture of B cells with CTRL T cells was set as 100% and compared with the amount of CD19+ cells cultured with CAR T cells. Additionally, supernatants from 4-day coculture of knockout cell clones and CTRL/CAR T cells were collected, and production of interferon (IFN)-γ was measured by ELISA using Human IFN-γ Standard TMB ELISA Development Kit (PeproTech) following manufacturer’s protocol.

Mouse experiments were carried out with equal numbers of male and female NOD-scid IL2Rg^null (NSG) mice (Charles River Laboratories, France) 8–12 weeks old per experimental group.

To assess the penetrance of individual knockout clones, 2.5×10⁶ tumor cells from each individual clone or WT HG3 and MEC1 cells were injected via tail vein in a total volume of 0.1 mL phosphate-buffered saline. For further studies, we preferentially selected clones derived from the HG3 cell line, which had the most similar disease-onset time. Two experimental settings to assess CAR T-cell in vivo performance were used: low-tumor and high-tumor burden settings. These scenarios differed in the number of tumor cells (5×10⁵ cells and 2×10⁶ cells, respectively). Moreover, CTRL or CAR T cells were injected intravenously 5 or 7 days later (low or high scenarios, respectively). Mice were closely monitored for any manifestation of the disease and were sacrificed when symptoms of severe disease (lethargy, excessive weight loss, change in fur quality, apathy and hind leg paralysis) occurred. Spleen was harvested, mechanically homogenized and passed through a 30 µm nylon filter, and erythrocytes were lysed using Tris-buffered ammonium chloride. Samples were then analyzed by staining with fluorescently conjugated monoclonal antibodies. Blood samples from the surviving CAR-treated animals were obtained from the tail vein approximately a week after the mice regained their starting weight. DNA from blood and spleen tissues was extracted using NucleoSpin Tissue DNA extraction kit (Macherey-Nagel) following the manufacturer’s instructions.

Quantitative real-time PCR was performed on 150 ng of DNA with three replicates for each sample using the 2X Taqman Gene Expression Master Mix (Applied Biosystems). Specific primers and probe were designed to amplify the junction region between the light and heavy chain of the single-chain variable fragment of anti-CD19 antibody (clone FMC63) that is contained within the CAR construct:

Forward primer: 5′-ACAGGGTAATACGCTTCCGT-3′

Reverse primer: 5′-CCCTGAGACAGTGCATGTGA-3′

MGB probe: 5′-FAM CCACCTGTGATCTCCAGCTTGGTC-3′

PCR with real-time fluorescence detection was performed on a 384-well Thermo Scientific QuantStudio 12 k (Thermo Fisher Scientific). The number of copies for each vector, expressed as copies/ng DNA, was determined by comparison of the measured cycle threshold for each well to the cycle threshold of a standard curve prepared by dilution of a linearized receptor-encoding plasmid spiked into 150 ng of CTRL mouse genomic DNA (obtained from a healthy NSG animal) per well. The R² value of the standard curve was 0.9945, and the slope was −3.4134 (corresponding to efficiency of reaction of 96%). Known spike controls ranged from 1×10¹ to 1×10⁷ copies per well.

All flow-cytometric analyses were performed using FACS Verse flow cytometer (BD Biosciences). Antibodies used in the study are listed in online supplementary table 1. To confirm the CAR expression, double staining was used: biotinylated goat antimouse polyclonal IgG antibody (Thermo Fisher Scientific) was visualized with AlexaFluor647-conjugated streptavidin (Invitrogen). In all flow-cytometric analyses, cell viability was monitored with 7-aminoactinomycin D (Invitrogen). Data were analyzed in FlowJo V.10 software.

All statistical analyses were performed using software incorporated in GraphPad Prism V.5. Specific statistical tests used for different study variables are described in the figure legends. All tests were two-sided. P values of <0.05 were considered statistically significant.

For the present study, we selected the anti-CD19 CAR incorporating 4-1BB costimulation molecule in the design.5 22 Our optimized protocol led to the production of a high yield of CAR-expressing T cells (>85%, online supplementary figure 1A). The functionality of CAR T cells was validated in various in vitro tests (online supplementary figure 1B). In order to comprehensively assess the prognostic value of CLL genetic background on the response to CAR T-cell therapy, we compiled a set of genetically defined 32 primary CLL samples. Of these, 26 were IGHV unmutated (9 ATM-mutated, 8 TP53-mutated, and 9 without mutations in ATM, TP53, NOTCH1 or SF3B1) and 6 bore hypermutation in the IGHV gene but no mutations in the other studied genes. After thawing, no difference in cell viability was observed among the genetic classes (online supplementary figure 2). B cells were then either directly mixed with T cells or stimulated through IL-2 and nuclear factor kappa B-activating signals23 prior to exposure to T cells in order to mimic a leukemic microenvironment and thus increase the in vitro fitness. After 96 hours of cocultivation, while there still were viable unstimulated CLL cells surviving in culture with CTRL T cells, no viable unstimulated B cells were detected in the mixture with CAR T cells (figure 1A). 2S stimulation proved to have a positive effect on CLL in vitro fitness, as some CLL cells survived even in coculture with CAR T cells (figure 1B). However, after 96 hours of coculture, no significant differences in the survival of the different genetic classes were observed (mean percentage of surviving unmutated IGHV WT cells, 2%; TP53-mutated, 1.5%; ATM-mutated, 3.3%; and IGHV-mutated WT cells, 3.2%; figure 1B). Unsurprisingly, exposure to CAR T cells led to a gradual decrease in the number of target CLL cells throughout the cocultivation period in both studied set-ups (figure 1C).

Primary CLL cells strive in vitro and, so far, no successful strategy for their reliable xenotransplantation has been described. Therefore, we used the CRISPR/Cas9 system to generate a collection of cell lines with a specific genetic background from commercially available CLL-derived cell lines (HG3 and MEC1). We aimed to introduce TP53 or ATM mutations that would result in disruption of the targeted gene expression and thus to generate a set of genetically distinct knockout single-cell clones. However, MEC1 cells already express a truncated p53. Since constitutive WT p53 introduction into p53-defficient tumors could induce senescence24 25, we introduced WT TP53 under an inducible promotor. Short-term expression of WT p53 protein could be induced by treating the cells with doxycycline, and it was further stabilized upon DNA damage triggered by doxorubicin (online supplementary figure 3A). These cells were denoted as ‘MEC1 p53 WT’ and used subsequently to generate individual MEC1 ATM -knockout and TP53-knockout clones. In total, we were able to create 7 TP53 single-cell knockout clones (three and four, respectively) and four ATM single-cell knockout clones (three and one, respectively) from both HG3 and MEC1 cells. As shown in figure 2A, no expression of p53 was detectable upon induction of DNA damage in the TP53-knockout cells, and there was a complete loss of ATM protein in the generated knockouts. Even the downstream effector genes were not activated in the generated cell lines upon adequate stimulus (p21 in case of TP53 knockouts, p21 and pKAP1 in ATM knockouts; online supplementary figure 3B), demonstrating true functional impairment of the targeted genes and their downstream signaling pathways in our generated cell lines. In vitro, no other obvious phenotypical changes were associated with acquiring the desired gene mutations; no major differences in cell proliferation were noted (online supplementary figure 3C); and no differences in expression of serine protease inhibitor PI-9, an inhibitor of granzyme B-mediated T-cell cytotoxicity, were found (online supplementary figure 3D).

When mixed with available knockout cell lines, CAR T cells, but not CTRL T cells, became specifically activated in their presence as evidenced by the production of IFN-γ (p=0.0020, figure 2B). In line with previous observations from primary CLL cells, after 96 hours of in vitro coculture with CAR T cells, a similar proportion of CD19+ target cells were surviving in the two genetically defined groups (mean percentage of surviving tumor cells was 12.9% and 14% in TP53 and ATM knockouts, respectively; figure 2C). However, when we examined the target elimination in time, we observed that TP53-knockout clones more often exhibited the ability to maintain proliferation in coculture with CAR T cells than their ATM-knockout counterparts (4/7 TP53 clones vs 1/4 ATM clone, figure 2D).

Since primary CLL cells engraft inefficiently in immunocompromised mice, we chose to evaluate the in vivo efficacy of anti-CD19 CAR T-cell therapy in NSG mice injected with CRISPR/Cas9 generated knockout cell lines. First, we evaluated the kinetics of the disease of all generated cell lines as well as the original HG3 and MEC1 cells in vivo (figure 3A, HG3 cell line and knockout clones derived from it are depicted with off-color shapes). In this model system, intravenous injection of tumor cell lines led to development of progressive leukemia with significant involvement of the spleen, liver and kidneys. Two HG3-derived TP53-knockout clones and an additional ATM clone did not engraft (clones 2, 3 and 6, respectively) even though these mice were followed for 60 days. In the remaining cases, the kinetics of TP53-knockout-driven disease clearly showed a more severe course than the ATM-knockout one (median days of succumbing to the disease were 19 and 27, respectively; p=0.0262), similar to the original cell lines (median day 20).

We selected HG3-derived clones 1 (TP53 knockout) and 4 (ATM knockout) for further in vivo studies. First, we inoculated NSG mice with 2×10⁶ tumor cells (this setting was termed ‘high-tumor burden’; figure 3B). Seven days later, CAR or CTRL T cells were injected via tail vein. On day 14, mice injected with the TP53-knockout clone started showing signs of the disease, which was quickly progressing in the following days (figure 3C). In line with the kinetics experiment, ATM-knockout disease onset was delayed (figure 3C, p<0.0001), and its progression was slower. At the moment of succumbing to the disease (median days 16 and 23 in TP53 and ATM CTRL mice, respectively), there was no significant difference in the disease morbidity among the animals injected with CTRL T cells from the two genetic classes (assessed here by weight loss, figure 3D). However, mice injected with the TP53-knockout clone had a significantly higher tumor burden compared with the ATM knockout (p=0.0002, figure 3D). Due to these disease-related differences, the positive treatment effect was solely apparent in mice with ATM-knockout disease (p<0.0001, figure 3E). Quantification of the integrated CAR-expressing vector in spleens of the treated mice additionally uncovered inefficient engraftment of CAR T cells in the animals with TP53-knockout tumors, plausibly caused by shorter time for their proliferation in these mice (p=0.0012, figure 3F). Eighty-five percent of mice bearing ATM-knockout disease treated with CAR T cells regained their original weight, were considered cured and afterwards had high levels of CAR T cells detectable in their circulation (figure 3F).

Finally, in an attempt to alleviate the disease burden caused by the TP53 knockout, we inoculated NSG mice with 5×10⁵ tumor cells (setting termed ‘low-tumor burden’) and started the treatment 5 days later (figure 4A). In this setting, mice injected with TP53-knockout cells started showing signs of the disease only 1 day later than in the above-mentioned high-tumor burden scenario (day 15, figure 4B). Similarly, severe disease course was observed among mice injected with HG3 WT cells. On the other hand, the low-tumor cell dose was suboptimal to induce leukemia with ATM-knockout cells, since only 4/7 of animals injected with CTRL T cells developed the disease. Nevertheless, in this setting, the treatment partially prolonged survival of animals with TP53-knockout disease (p=0.0204), ATM-knockout disease (p=0.0217), as well as those injected with HG3 WT tumor cells (p=0.0485, figure 4C). One of five (20%) mice injected with HG3 WT cells and treated with CAR T cells was considered cured of disease due to regaining of the original weight and high levels of CAR T cells in blood (assessed by flow cytometry, online supplementary figure 4).