Abstract
Chimeric antigen receptor (CAR) therapy has had a transformative effect on the treatment of haematologic malignancies1,2,3,4,5,6, but it has shown limited efficacy against solid tumours. Solid tumours may have cell-intrinsic resistance mechanisms to CAR T cell cytotoxicity. Here, to systematically identify potential resistance pathways in an unbiased manner, we conducted a genome-wide CRISPR knockout screen in glioblastoma, a disease in which CAR T cells have had limited efficacy7,8. We found that the loss of genes in the interferon-γ receptor (IFNγR) signalling pathway (IFNGR1, JAK1 or JAK2) rendered glioblastoma and other solid tumours more resistant to killing by CAR T cells both in vitro and in vivo. However, loss of this pathway did not render leukaemia or lymphoma cell lines insensitive to CAR T cells. Using transcriptional profiling, we determined that glioblastoma cells lacking IFNγR1 had lower upregulation of cell-adhesion pathways after exposure to CAR T cells. We found that loss of IFNγR1 in glioblastoma cells reduced overall CAR T cell binding duration and avidity. The critical role of IFNγR signalling in susceptibility of solid tumours to CAR T cells is surprising, given that CAR T cells do not require traditional antigen-presentation pathways. Instead, in glioblastoma tumours, IFNγR signalling was required for sufficient adhesion of CAR T cells to mediate productive cytotoxicity. Our work demonstrates that liquid and solid tumours differ in their interactions with CAR T cells and suggests that enhancing binding interactions between T cells and tumour cells may yield improved responses in solid tumours.
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Data availability
The screening data gathered in this study are available at the Gene Expression Omnibus under accession code GSE179147.
Code availability
The custom Macro used for analysis of time-lapse spinning disk confocal microscopy is available on GitHub (https://github.com/prajuvikas/Rebecca_Immune_Synapse_analysis_2022Jan).
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Acknowledgements
R.C.L. was supported by T32 GM007306, T32 AI007529, and the Richard N. Cross Fund. M.B.L. was supported by T32 2T32CA071345-21A1. S.R.B. was supported by T32CA009216-38. N.J.H. was supported by the Landry Cancer Biology Fellowship. J.J. is supported by a NIH F31 fellowship (1F31-MH117886). A.S. received support from a John Hansen Research Grant from DKMS (no. DKMS-SLS-JHRG-2020-04). G.G. was partially funded by the Paul C. Zamecnik Chair in Oncology at the Massachusetts General Hospital Cancer Center and NIH R01CA 252940. M.V.M. and this work are supported by the Damon Runyon Cancer Research Foundation, Stand Up to Cancer, NIH R01CA 252940, R01CA238268 and R01CA249062. We acknowledge the advice of J. Doench and work from the Genetic Perturbation Platform at the Broad Institute on the CRISPR screen; and the Massachusetts General Hospital Flow Cytometry Core at the Charlestown Navy Yard. We thank the Lumicks team for assistance with cell binding avidity experiments; D.E. Grinshpun from the laboratory of B. Ebert for the ICAM-1 overexpression vector; and B. Choi and K. Gallagher for technical advice in experimental design.
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Contributions
R.C.L., T.L., T.O., A.R. and M.V.M. conceived the study. R.C.L., J.J., T.L., T.O., A.R., G.G. and M.V.M. developed the initial study design. R.C.L., T.R.B. and M.V.M. wrote the manuscript. R.C.L., M.C.K., A.A.B., M.B.L. and I.S. performed the experiments. R.C.L., M.C.K., N.J.H., P.M.L., K.S., P.V.A. and T.L. analysed the experiments. All authors contributed intellectually to the experiments as well as editing and approval of the final version of the manuscript.
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Competing interests
N.J.H. is a consultant for Constellation Pharmaceuticals. I.S. is now an employee of Arsenal Bio. T.O. is an employee of Flagship Labs 69, Inc. A.R. is a founder and equity holder of Celsius Therapeutics, an equity holder in Immunitas Therapeutics and until 31 August 2020 was a member of the scientific advisory boards of Syros Pharmaceuticals, Neogene Therapeutics, Asimov and ThermoFisher Scientific. From 1 August 2020, A.R. has been an employee of Genentech, a member of the Roche Group. G.G. receives research funds from IBM and Pharmacyclics, and is an inventor on patent applications related to MuTect, ABSOLUTE, MutSig, MSMuTect, MSMutSig, MSIDetect, POLYSOLVER and TensorQT. G.G. is a founder, consultant and holds privately held equity in Scorpion Therapeutics. M.V.M. receives sponsored research support from KitePharma and Novartis. M.V.M. is an inventor on patents related to adoptive cell therapies held by Massachusetts General Hospital and University of Pennsylvania (some licensed to Novartis). M.V.M. holds equity in 2Seventy Bio, TCR2, Century Therapeutics, Genocea, Oncternal and Neximmune, and has served as a consultant for multiple companies involved in cell therapies. M.V.M. serves on the Board of Directors of 2Seventy Bio. The other authors declare no competing interests.
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Extended data figures and tables
Extended Data Fig. 1 Glioblastoma screening conditions were determined to have a cytotoxicity efficiency of IC50.
CAR T cells were generated and utilized in a cytotoxicity assay against U87 glioblastoma cells to evaluate screening conditions. a, Timeline of EGFR-CAR T cell growth of 6 different normal donors used in the screen and experiments throughout. Arrows indicate media and IL-2 addition. b, Flow-based cytotoxicity assay of EGFR-CAR or untransduced T cells (UTD) from the same donors to determine IC50 conditions for screen (n = 3ND; mean +/− s.e.m. shown; two-way ANOVA, Sidak’s multiple comparisons test). The 1:10 effector to target cell ratio was chosen. c, Schematic of genome wide CRISPR screen in U87 glioblastoma cells using the Brunello library. d, Distribution of coverage across guides. A histogram is shown for the number of reads detected for each guide in our dataset. Binning was performed and data was plotted on log scale. All tests were two-sided. p = *<0.05, **<0.01, ***<0.001, ****<0.0001.
Extended Data Fig. 2 U87 cell line knockouts are confirmed and cell growth independent of T cells is characterized in vitro.
U87 cells were evaluated for antigen expression and functional knockout in vitro and monitored over time for resistance to CAR T cell killing. Flow cytometry of U87 knockout lines stained for EGFR (a), IFNγR1 (b), and phospho-STAT1 (c) after IFNγ stimulation. d, Proliferation of U87-KO cell lines independent of CAR coculture as determined by GFP area normalized to time 0 on IncuCyte (n = 2). e, Representative images from IncuCyte cytotoxicity assays (representative of 16 images taken total: 4 in technical replicate, 4 biological replicates). f, Quantified IncuCyte cytotoxicity assays shown in e. over 72 h of EGFR-CAR with IFNγR1-KO, JAK1-KO, or JAK2-KO cell lines compared to WT U87 cells (n = 4ND, paired t-test at 72 h, maximum p-value from KO1 and 2 shown). g, Western blots of single cell sorts of U87 JAK1 and JAK2-KO cell lines (n = 1, full scans available in Supplementary Information 3). h, Luciferase-based cytotoxicity assays of U87 single cell (sc) JAK1 and JAK2-KO from g (n = 4ND in 6 technical replicates, two-way ANOVA; maximum p-value from KO1 and 2 shown; Dunnett’s multiple comparisons test. Calculated as a percentage of luminescence of tumor only wells). i, Cytokine measurements from supernatants of EGFR-CAR and U87 tumor coculture of the indicated KO for 24 h (n = 3ND in technical duplicate, mean shown in pg/mL; white = below limit of detection). Mean +/− s.e.m. shown. All tests were two-sided.
Extended Data Fig. 3 Glioblastoma cell lines IFNγR1-KO, JAK1-KO, and JAK2-KO are resistant to EGFR-CAR treatment.
U87 KO cell lines were tested in vivo for response to CAR T cell treatment and a second glioblastoma cell line was tested for resistance to EGFR-CAR cytotoxicity. a, Schematic of in vivo subcutaneous model of glioblastoma using the U87 cell line. WT, EGFR-KO, IFNγR1-KO1, JAK1-KO1, and JAK2-KO1 U87 tumor growth based on luciferase imaging from animals treated with EGFR-CAR or UTD from the same donor (4 animals per group, unpaired t-test at Day 28). b, Flow cytometry of U251 knockout lines stained for EGFR, IFNγR1 and pSTAT1. Note for pSTAT1 isotype control is secondary alone. c, IncuCyte cytotoxicity assays of U251 KO tumor cell lines cocultured with EGFR-CAR for 72 h at a 1:1 ratio with 2 different guides for each KO (n = 4ND, paired t-test at 72 h). d, Proliferation of KO cell lines independent of CAR coculture as determined by GFP area normalized to time 0 on IncuCyte (n = 2). e, Weekly imaging of one representative cage from each group from Fig. 1f–h. f, Survival curves from Fig. 1f–h (Log-rank Mantel-Cox test). Mean +/− s.e.m. shown.
Extended Data Fig. 4 Loss of IFNγR1 signaling is resistant to CAR T cell cytotoxicity in GBM irrespective of the CAR target or costimulation domain and pharmacologic inhibition of JAK1 and JAK2 leads to decreased CAR T cell killing of GBM.
Flow cytometry of U251 knockout lines stained for IL13Rα2 (a) and U87-CD19-KO lines stained for CD19 (b). Luciferase-based cytotoxicity assays of the IL13Rα2-CAR on U251 tumor cell lines (c), CD19-CAR on U87-CD19 positive KO cells (d), or EGFR-28ζ CAR on U87 KO cells (e) at the indicated CAR:Target cell ratios (n = 4ND in 6 technical replicates, two-way ANOVA; maximum p-value from KO1 and 2 shown; Dunnett’s multiple comparisons test. Calculated as a percentage of luminescence of tumor only wells). Impedance-based cytotoxicity assays of U87 (f) or U251 (g) cells with EGFR-CAR after treatment with ruxolitinib or DMSO (n = 4ND in technical duplicate). h, U87 impedance cytotoxicity assay of U87 cells treated with ruxolitinib-treated T cells (n=4ND in technical dupilcate; paired t-test at 72 h). phospho-STAT1 staining of U87 (i) or U251 (j) cells after treatment with ruxolitinib or DMSO and IFNγ stimulation. c Mean +/− s.e.m. shown. All tests were two-sided.
Extended Data Fig. 5 Disruption of IFNγR signaling pathway confers resistance to CAR T cell cytotoxicity in several solid tumor models.
Glioblastoma, pancreatic, ovarian, and lung cells were assessed for effects of loss of IFNγR signaling on CAR T cell efficacy. a, Schematic of TRAC-KO EGFR-CAR and IFNγ-KO TRAC-KO EGFR-CAR vector design. b, IFNγ expression measured by flow of stimulated edited EGFR-CARs (n = 3ND, biological triplicate, paired t-test). c, Luciferase-based killing assays of U87 KOs cocultured with the indicated CAR (n = 3ND, 4 technical replicates; two-way ANOVA; maximum p-value from KO1 and 2 shown; Dunnett’s multiple comparisons test). d, Luciferase-based cytotoxicity assay of AsPC-1 treated with Meso-CAR for 18 h at the indicated E:T ratios (n = 4ND, 6 technical replicates, two-way ANOVA, Dunnett’s multiple comparisons test, maximum p-value shown from KO1 and KO2). e, Images from Fig. 2a–c. IncuCyte cytotoxicity assays of pancreatic AsPC-1 (f), pancreatic BxPC-3 (g,h), and lung A549 (i) cell lines targeted with the indicated CARs at a 1:1 ratio for 72 h (n = 4ND, paired t-test at 72 h with the maximum p-value shown for KO1 and KO2). Representative images at 72 h shown (representative of 16 images taken total: 4 in technical replicate, 4 biological replicates). Mean +/− s.e.m. shown. All tests were two-sided.
Extended Data Fig. 6 Loss of IFNγR signaling does not affect CAR T cell efficacy across hematological tumors.
a, Flow cytometry of Nalm6 KO lines stained for CD19 and IFNγR1. b, Luciferase-based cytotoxicity assay of Nalm6 cells treated with CD19-CAR T cells for 18 h at the indicated E:T ratios (n = 4ND in 6 technical replicates, two-way ANOVA, Sidak’s multiple comparison’s test). c, Cytokine measurements from supernatants of CD19-CAR cocultured with the indicated Nalm6 tumor cell line for 24 h (n = 4ND, mean shown in pg/ml). d, phospho-STAT1 flow cytometry intracellular staining of ruxolitinib/DMSO treated Nalm6 cells stimulated with IFNγ. e, Luciferase-based cytotoxicity assay of Nalm6 cells treated with ruxolitinib or DMSO prior to CAR T cell exposure for 18 h at the indicated E:T ratios (n = 3ND in technical duplicate, two-way ANOVA, Sidak’s multiple comparison’s test). f, Imaging from the CAR treated groups from Fig. 2e–g. g, Schematic of JeKo-1 lymphoma orthotopic model. h, Survival of the CAR treated groups from g (Log-rank Mantel-Cox test). i, Imaging from the CAR treated groups from g at the indicated days. j, BLI quantification from g for each tumor group treated with either CD19-CAR or UTD from the same donor (5 animals per group, unpaired t-test at Day 21). k, Luciferase-based cytotoxicity assay of MM1S cells treated with BCMA-CAR T cells for 18 h at the indicated E:T ratios (n = 4ND in 6 technical replicates, two-way ANOVA, Sidak’s multiple comparison’s test). l, Schematic of MM1S multiple myeloma orthotopic model. m, Imaging from the CAR treated groups from l at the indicated days. n, BLI quantification from l for each tumor group treated with either BCMA-CAR or UTD (5 animals per group, unpaired t-test at Day 14). Mean +/− s.e.m. shown. All tests were two-sided.
Extended Data Fig. 7 Loss of IFNγR signaling in GBM tumor does not impact growth, exhaustion, or phenotype of CAR T cells; however, U87 and Nalm6 IFNγR1-KOs show different expression profiles when exposed to CAR T cells.
a, Schematic of experimental set up prior to transcriptional analysis. CAR T cells were cocultured with tumor cells at a 1:10 effector:target ratio, sorted on live CAR or tumor cells, then lysed for RNA analysis (n = 3ND). b, Heatmap of signature pathways of CAR T cells incubated with U87 WT, IFNγR1-KO, or EGFR-KO. c, Schematic of long-term stimulation of CAR T cells with a 1:1 E:T ratio of U87 WT, IFNγR1-KO, or EGFR irradiated tumor cells. d, CAR T cell growth over time (paired t-test at each time point, max p-value shown for KO1 and KO2). e, Flow cytometry of CAR T cells for Lag3, Tim3, and PD-1 (paired t-test at each time point, max p-value shown for KO1 and KO2). f, Memory phenotype of CAR T cells at Day 28 (n = 4ND for d-f). Transcriptional analysis of EGFR, IFNγR1, and PD-L1 in U87 (g) or Nalm6 (h) WT, IFNγR1-KO, or EGFR/CD19-KO cells cocultured with EGFR or CD19-CAR T cells (respectively) for 12 h (n = 3ND, line at median with bars for min and max). PD-L1 protein expression on U87 (i) or Nalm6 (j) WT, IFNγR1-KO, or EGFR/CD19-KO cells cocultured with EGFR or CD19-CAR T cells (respectively) (n = 4ND, one-way ANOVA, Dunnett’s multiple comparison’s test). k, Cytotoxicity assays of U87 and Nalm6 cells exposed to increasing concentrations of IFNγ calculated as a percentage of luminescence from untreated tumor wells (n = 12). l, PD-L1 surface expression on WT U87 or Nalm6 tumor measured by flow cytometry after treatment with 20ng/mL IFNγ. Mean +/− s.e.m. shown unless otherwise stated. All tests were two-sided.
Extended Data Fig. 8 Solid and liquid tumors have differential responses to death receptor and IFNγR1 KO.
Western blots of U87 (a) and Nalm6 (b) BID and FADD-KO (full scans available in Supplementary Information 4). c, Competitive survival assay measured by flow cytometry using violet stained WT U87 cocultured at a 3:1 ratio with BID (ns all timepoints), FADD, or EGFR-KO cells in the presence of a 1:5 ratio with EGFR-CAR or UTD from the same donor and monitored every 24 h (n = 4ND, two-way ANOVA, KO1 and KO2 combined, Sidak’s multiple comparisons test). d, Competitive survival assay with Nalm6 BID, FADD, CD19-KO in the presence of CD19-CAR with same setup and analysis as in c. Competitive survival assays of WT AsPC-1 pancreatic targeted with Mesothelin or EGFR-CAR (e), JeKo-1 lymphoma targeted with CD19-CAR (f), or RPMI-8226 multiple myeloma targeted with BCMA-CAR (g) cocultured at a 3:1 ratio with IFNγR1-KO or antigen-KO cells with same setup and analysis as in c. EGFR-CAR T cell interactions with U87 WT, IFNγR1-KO or EGFR-KO tumors were imaged every 2.5 min over a 30-min period using live imaging spinning disk confocal microscopy. Synapses were defined with mCherry expressing CAR T cells and GFP expressing tumor cells interaction and monitored over time. h, Representative image of tracks of CAR T cell synapses with each tumor type over time defined in seconds (855 µm x 855 µm). Shorter synapses are blue (150 s) and longer synapses are red (1650 s). Videos of timelapses are available as Supplementary Videos 1-3 . i, Quantified duration of each synapse across tumors (>150 synapses observed in each, one-way ANOVA, Dunnett’s multiple comparisons test). Mean +/− s.e.m. shown. All tests were two-sided.
Extended Data Fig. 9 ICAM-1 expression has differential upregulation post-CAR T cell exposure across tumors.
a, Volcano plot of differential expression of IFNγR1-KO2 versus WT for U87 and Nalm6 cocultures with CAR T cells as described in Ext. Data Fig. 7a. Line drawn at adjusted p-value of 0.01. b, Transcriptional analysis of ICAM-1 expression by NanoString of U87 or Nalm6 WT, IFNγR1-KO, or EGFR/CD19-KO cells cocultured with EGFR or CD19-CAR T cells (respectively) for 12 h (n = 3ND, line at median with bars for min and max, one-way ANOVA, Dunnett’s multiple comparisons test). c, Soluble ICAM-1 from supernatant of cocultures of U87 cells with EGFR-CAR T cells (n = 3ND in technical duplicate) and Nalm6 cells with CD19-CAR (n = 4ND) after 24 h (one-way ANOVA, Dunnett’s multiple comparisons test). Gating strategy for expression of ICAM-1 shown in Fig. 4e on U87 (d) and Nalm6 (e) with and without exposure to CAR T cells. f, ICAM-1 surface expression on WT U87 or Nalm6 tumor measured by flow cytometry after 18hr treatment with 20ng/mL IFNγ. g, Flow cytometry of ICAM-1 expression on various cell lines post-CAR T cell exposure. Shown as a ratio of CAR-exposed/tumor only (U87 n = 4ND in technical triplicate, BxPC3 and RPMI n = 4ND in technical duplicate, Nalm6, AsPC-1, JeKo-1 and MM1S n = 4ND). h, Quantification of CAR T cell target antigen across tumor types. Mean +/− s.e.m. shown. All tests were two-sided.
Extended Data Fig. 10 Blockade of adhesion axes in the presence of CAR T cells has differential effects on U87 and Nalm6 cells.
Tumor cells were targeted with CAR T cells in the presence of blocking antibodies against various adhesion molecules. Cytotoxicity assay of U87 or Nalm6 cells treated with LFA-1 (CD11a/CD18) (a) blocking antibody prior to exposure to EGFR-or CD19-CAR respectively (n = 4ND; paired t-test at 72 h). Cytokines from 24 h cocultures of EGFR-CAR with U87 cells (b) or CD19-CAR and Nalm6 cells (c) in the presence of the indicated blocking antibodies or isotype control. d, Competitive survival assay measured by flow cytometry using violet stained WT U87 targeted with EGFR-CAR cocultured at a 3:1 ratio with ICAM-1 KO cells in the presence of a 1:5 CAR to target cell ratio with CAR or untransduced T cells from the same donor and monitored every 24 h (n = 4ND, two-way ANOVA, ICAM-1 KO1 and KO2 combined, Sidak’s multiple comparisons test). e, Cytotoxicity of EGFR-CAR treated U87 WT or EGFR-KO engineered to overexpress ICAM-1 compared to non-overexpressing WT and EGFR-KO U87 tumors (n = 4ND; paired t-test at 72 h). U87 glioblastoma cells were assessed for CD58 expression and its impact on CAR T cell cytotoxicity. Flow cytometry of CD58 expression on U87 cells before and after CAR T cell exposure displayed as a percentage of total cells (f) and MFI (g) (n = 4ND in technical duplicate). h, IncuCyte cytotoxicity assay of U87 cells cocultured at a 1:1 ratio of CAR T cell:tumor cell for 72 h in the presence of blocking antibodies to either CD58 or CD2 (n = 4ND, paired t-test at 72 h). Mean +/− s.e.m. shown. All tests were two-sided.
Supplementary information
Supplementary Information 3–5
Full scans of western blots from Extended Data Fig. 2. (Supplementary Information 3.) JAK1 (a), β-actin (top, b), JAK2 (c), and β-actin (bottom, d) western blot full scans of U87 single cell (sc) JAK1 and JAK2 knockouts from Extended Data Fig. 2g. Full scans of western blots from Extended Data Fig. 8. (Supplementary Information 4.) β-actin + BID (top, a), β-actin + FADD (bottom, b) western blot full scans of U87 BID and FADD-KO cells from Extended Data Fig. 8a. β-actin + BID (top, c), β-actin + FADD (bottom, d) western blot full scans of Nalm6 BID and FADD-KO cells from Extended Data Fig. 8b. Image analysis workflow for confocal tracking analysis. (Supplementary Information 5.)
Supplementary Table 1
Fig. 1 in vivo raw data.
Supplementary Table 2
Fig. 2 in vivo raw data.
Supplementary Table 3
Extended Data Fig. 3 in vivo raw data.
Supplementary Table 4
Extended Data Fig. 6 in vivo raw data.
Supplementary Video 1
CAR T cells have shorter synapse duration with IFNγR1-KO U87 compared to WT. Time-lapse video of EGFR-CAR T cells incubated with WT U87 tumours from Extended Data Fig. 8. Left panel shows tumour cells in green and CAR T cells in magenta. The right panel exhibits the masking analysis of synapse duration with defined tumour cells in red, CAR T cells that do not interact with tumour cells in blue and interaction between the two cell types in yellow-green. Movies are displayed as 10 fps. Time interval 2.5 min. Scale bar is 200 μm.
Supplementary Video 2
CAR T cells have shorter synapse duration with IFNγR1-KO U87 compared to WT. Time-lapse video of IFNγR1-KO U87 tumours from Extended Data Fig. 8. Left panel shows tumour cells in green and CAR T cells in magenta. The right panel exhibits the masking analysis of synapse duration with defined tumour cells in red, CAR T cells that do not interact with tumour cells in blue and interaction between the two cell types in yellow-green. Movies are displayed as 10 fps. Time interval 2.5 min. Scale bar is 200 μm.
Supplementary Video 3
CAR T cells have shorter synapse duration with IFNγR1-KO U87 compared to WT. Time-lapse video of EGFR-KO U87 tumours from Extended Data Fig. 8. Left panel shows tumour cells in green and CAR T cells in magenta. The right panel exhibits the masking analysis of synapse duration with defined tumour cells in red, CAR T cells that do not interact with tumour cells in blue and interaction between the two cell types in yellow-green. Movies are displayed as 10 fps. Time interval 2.5 min. Scale bar is 200 μm.
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Larson, R.C., Kann, M.C., Bailey, S.R. et al. CAR T cell killing requires the IFNγR pathway in solid but not liquid tumours. Nature 604, 563–570 (2022). https://doi.org/10.1038/s41586-022-04585-5
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DOI: https://doi.org/10.1038/s41586-022-04585-5
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