Mapping variant outcomes on anti-tumor hallmarks of main human T cells with depraved-making improvements to screens

Mapping variant outcomes on anti-tumor hallmarks of main human T cells with depraved-making improvements to screens

   May 29, 2024  Posted in Health 55 Views
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Files availability

All processed screening files are supplied as Supplementary Tables. Supply files are supplied with this paper.

Code availability

Code for producing in silico predicted structures is deposited right here: https://github.com/gnikolenyi/izar_vis (ref. 73).

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Acknowledgements

N.Okay. and S.B.S. are equally contributing 2d authors. B.I. is supported by Nationwide Institute of Health grants (R37CA258829, R01CA280414, R01CA266446, U54CA274506); and moreover by the Pershing Square Sohn Cancer Evaluate Alliance Award; the Burroughs Wellcome Fund Occupation Award for Medical Scientists; a Tara Miller Melanoma Evaluate Alliance Young Investigator Award; the Louis V. Gerstner, Jr. Students Program; and the V Foundation Students Award. This work used to be supported by a Herbert Irving Comprehensive Cancer Heart (HICCC) Velocity Grant (to B.I.), the HICCC Human Tissue Immunology and Immunotherapy Initiative and NIH Grant P30CA013696. Medical illustrations had been ready by U. Mackensen. The illustration in Prolonged Files Fig. 9a used to be created with https://www.biorender.com.

Writer files

Writer notes

  1. These authors contributed equally: Zachary H. Walsh, Parin Shah.

  2. These authors jointly supervised this work: Johannes C. Melms, Benjamin Izar.

Authors and Affiliations

  1. Columbia College Vagelos Faculty of Physicians and Surgeons, Unusual York, NY, USA

    Zachary H. Walsh, Parin Shah, Neeharika Kothapalli, Shivem B. Shah, D. Zack Brodtman, Meri Rogava, Michael Mu, Patricia Ho, Sinan Abuzaid, Johannes C. Melms & Benjamin Izar

  2. Division of Medication, Division of Hematology and Oncology, Columbia College Irving Medical Heart, Unusual York, NY, USA

    Zachary H. Walsh, Parin Shah, D. Zack Brodtman, Meri Rogava, Michael Mu, Patricia Ho, Sinan Abuzaid, Neil Vasan, Johannes C. Melms & Benjamin Izar

  3. Columbia Heart for Translational Immunology, Unusual York, NY, USA

    Zachary H. Walsh, Parin Shah, D. Zack Brodtman, Meri Rogava, Michael Mu, Patricia Ho, Sinan Abuzaid, Johannes C. Melms & Benjamin Izar

  4. Herbert Irving Comprehensive Cancer Heart, Columbia College Irving Medical Heart, Unusual York, NY, USA

    Zachary H. Walsh, Parin Shah, D. Zack Brodtman, Giuseppe Leuzzi, Meri Rogava, Michael Mu, Patricia Ho, Sinan Abuzaid, Neil Vasan, Alberto Ciccia, Johannes C. Melms & Benjamin Izar

  5. Division of Systems Biology, Columbia College Irving Medical Heart, Unusual York, NY, USA

    Gergo Nikolenyi, Mohammed AlQuraishi & Benjamin Izar

  6. Division of Genetics and Constructing, Columbia College Medical Heart, Unusual York, NY, USA

    Giuseppe Leuzzi & Alberto Ciccia

  7. Division of Pediatrics, Columbia College Irving Medical Heart, Unusual York, NY, USA

    Joshua D. Milner

Contributions

B.I. and Z.H.W. conceived the peruse. B.I. supplied overall supervision with toughen from J.C.M. Z.H.W., P.S. and J.C.M. deliberate, designed and executed all key experiments. S.B.S., M.M., P.H., M.R. and S.A. performed experiments. N.Okay. performed computational analyses of screens with toughen from Z.H.W. and D.Z.B. G.N. performed structural modeling and visualizations. N.V., M.A., J.D.M., A.C. and G.L. supplied extra guidance for the accomplish, execution and interpretation of screens. Z.H.W., P.S., J.C.M. and B.I. wrote the manuscript with input and approval from all authors.

Corresponding creator

Correspondence to
Benjamin Izar.

Ethics declarations

Competing interests

B.I. is a specialist for or received honoraria from Volastra Therapeutics, Johnson & Johnson (Janssen), Novartis, Eisai, AstraZeneca and Merck and has received assessment funding to Columbia College from Agenus, Alkermes, Arcus Biosciences, Checkmate Prescribed tablets, Compugen, Immunocore, Regeneron and Synthekine. Z.H.W. and B.I. filed a patent utility consistent with this work. The diversified authors assemble no longer win competing interests.

Peep review

Peep review files

Nature Biotechnology thanks Dimitrios Wagner and the diversified, nameless, reviewer(s) for their contribution to the glance review of this work.

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Prolonged files

Prolonged Files Fig. 1 Optimization of workflows for depraved making improvements to in main human T cells.

a, Overview of approach for centered depraved making improvements to in main human T cells. b-d, Target web sites of sgRNAs towards CD2, B2M, and TRBC1/2 web sites predicted to generate gene knockout thru several mechanisms (SPLd = splice donor space mutation, SPLa = splice acceptor space mutation, SM = originate codon mutation, ES = conversion to early terminate codon). e, Representative dart cytometry histograms from one human donor showing ABE-mediated knockout of CD2 and B2M the utilization of sgRNAs indicated in (b-c), and f, CBE-mediated knockout of CD2, TRBC1/2, and B2M the utilization of sgRNAs indicated in (b-d). g, Quantification of depraved making improvements to effectivity in (e) (n = 3 self sustaining human donors). h, Quantification of depraved making improvements to effectivity in (f), (n = self sustaining human 4 donors for B2M_ES and TRBC1/2_ES; n = 2 self sustaining human donors for B2M_SPLd and CD2_SM). i, Representative dart cytometry dotplots and histograms demonstrating CBE-mediated knockout of TCRab. For histograms, crimson signifies gated mTurquoise-detrimental cells, and blue signifies gated mTurquoise-sure cells. j, Quantification of ABE-mediated knockout of B2M with lentiviral integration of B2M_SM_1 sgRNA and electroporation of ABE mRNA in CD4 and CD8 T cell subsets (n = 2 self sustaining human donors). okay, Enhancing effectivity (measured by % B2M loss on dart cytometry) and viability of T cells transduced with B2M_SM_1 sgRNA and electroporated with varying doses of ABE. Vertical dotted line represents ABE dose selected (per 1e6 T cells) for screens. Error bars symbolize point out +/− SD (panels g, h, j).

Supply files

Prolonged Files Fig. 2 Tiling conceal targets, library transduction, and pooled depraved making improvements to of T cells.

a, Classification of sgRNAs in the ClinVar library consistent with mutation subtype. b, Schematic of gene targets for the 12-Gene tiling conceal and their feature in T cells. c, Classification of sgRNAs in the 12-Gene tiling library consistent with mutation subtype. d, Schematic for abilities of library depraved-edited T cells. e, Transduction effectivity of ClinVar depraved editor library in n = 2 self sustaining human donors.

Prolonged Files Fig. 3 Metrics for rigor and reproducibility of major-scale depraved making improvements to screens.

a, Density plots showing LFC values of diversified categories of guides from the ClinVar library at Day 35 put up-electroporation of the lengthy-term growth conceal arm. Dashed line represents the underside 5% of the distribution of blended empty window and quiet mutation controls. Indicated are the percentages of guides in every category falling below this threshold. sgRNAs producing variants in CD3D, CD3E, CD3G, or CD3Z had been binned into the ‘CD3 advanced’ category. The 2d donor from the conceal is shown (in partner to Fig. 2a). b, Scatter scheme showing LFC values of detrimental help watch over sgRNAs (alongside with both empty window and quiet mutations) in both donors from the ClinVar Library at Day 28 put up-electroporation in the lengthy-term growth conceal arm. c-d, Distribution of grand depraved aggregation (RRA) ratings for gene-smart dropout prognosis in the c, CD25 hi vs lo (activation) kind and d, CFSE lo vs hi (fast-term proliferation) kind hands of the ClinVar library across both donors. The terminate 5 negatively selected genes in CD25 hi vs lo and in CFSE lo vs hi are listed. e, Shared sure help watch over sgRNA (n = 600) had been identified between the ClinVar and 12-gene tiling screens and sgRNA LFCs from matched lengthy-term proliferation arm timepoints (Day 28 of ClinVar Show conceal, Day 26 of 12-gene Tiling Show conceal) are plotted. For every conceal, the everyday LFC of every sgRNA across both donors is plotted. Simple linear regression with two-sided Pearson take a look at (panel e).

Prolonged Files Fig. 4 Prognosis of ClinVar conceal across readouts.

a, Scatterplot showing LFC of selected sgRNAs producing mutations in LCK, SOS1, and PTPRC. Timepoint shown is Day 28 put up-electroporation in the ClinVar lengthy-term growth conceal arm. b, Volcano scheme showing enriched and depleted guides in the CFSE lo vs hi proliferation kind. For visualization capabilities, one mutation for every labeled sgRNA is shown. One book donor is shown. Unfounded discovery price (FDR) cutoff <0.05. c, Volcano scheme showing enriched and depleted guides in the CD25 hi vs lo proliferation kind. For visualization capabilities, one mutation for every labeled sgRNA is shown. FDR cutoff <0.05. One book donor is shown.

Prolonged Files Fig. 5 Characterization of variant outcomes by ClinVar classification.

a, (Top) distribution of detrimental help watch over sgRNAs in the ClinVar library at day 28 of the lengthy-term proliferation conceal arm. (Bottom) sgRNA LFC distributions for selected genes centered in the ClinVar library. Crimson traces present sgRNAs producing amino acid mutations which are linked to ClinVar-annotated pathogenic variants. b-j, Scatterplots of sgRNAs focusing on selected genes in the ClinVar library at day 28 of the lengthy-term proliferation conceal arm. Dotted traces symbolize top and bottom 5% cutoffs of detrimental help watch over sgRNA distribution. sgRNAs are binned into four particular categories: predicted to generate an linked mutation to a ClinVar ‘VUS’ (‘Identical VUS’; darkish blue), predicted to generate a particular mutation at an amino acid with a ClinVar ‘VUS’ (‘Diff VUS’; gentle blue), predicted to generate an linked mutation to a ClinVar ‘pathogenic’, ‘pathogenic/seemingly pathogenic’, or ‘seemingly pathogenic’ variant (‘Identical P’; crimson), and sgRNAs predicted to generate a particular mutation at an amino acid with a ClinVar ‘pathogenic’, ‘pathogenic/seemingly pathogenic’, or ‘seemingly pathogenic’ variant (‘Diff P’; yellow). Chosen sgRNAs are annotated. Simple linear regression (panels b-j).

Prolonged Files Fig. 6 Self reliant prognosis of 12-gene tiling conceal and integration of outcomes with ClinVar conceal.

a, sgRNA LFCs across both donors at Day 26 of the lengthy-term proliferation arm of the 12-gene tiling conceal are plotted. Dotted traces symbolize top and bottom 10% cutoffs of the distribution of detrimental help watch over sgRNAs (empty window and quiet simplest sgRNAs) for every donor. Chosen sgRNAs, with predicted arrangement gene and mutation, are shown. b, sgRNA LFCs as in a, with blue overlay filtered by arrangement gene. c, Shared sgRNAs (n = 325) had been identified between the ClinVar and 12-gene tiling screens and sgRNA LFCs from matched lengthy-term proliferation arm timepoints (Day 28 of ClinVar Show conceal, Day 26 of 12-gene Tiling Show conceal) are plotted. For every conceal, the everyday LFC of every sgRNA across both donors is plotted. Chosen sgRNAs with shared enrichment/depletion patterns across donors and screens are annotated. Simple linear regression (panel c).

Prolonged Files Fig. 7 Enrichment and building-feature relationship of variants promoting T cell proliferation.

a, Lollipop scheme showing LFC of sgRNAs focusing on PIK3CD at Day 15 put up-electroporation in the lengthy-term proliferation arm of the ClinVar conceal. sgRNAs are mapped to the centered space of the canonical isoform of PIK3CD (p110δ) and purposeful domains of the protein are annotated. Chosen variants and their predicted mutational consequences are annotated. b,c, Timecourse line graphs of LFC of sgRNAs focusing on PIK3CD in both donors in the lengthy-term proliferation arm of the ClinVar conceal. d, Lollipop scheme for sgRNAs focusing on AKT1 at Day 35 put up-electroporation in the lengthy-term proliferation arm of the ClinVar conceal, mapped to the canonical AKT1 isoform. e, Timecourse line graphs of LFC of sgRNAs focusing on AKT1 in the lengthy-term growth arm of the ClinVar conceal. f, Structure and situation of mutations in AKT1. (ethical) General predicted building of AKT1 (blue) and mutated residues (crimson). (left) Wild-kind (WT) and mutated (mut) residues (crimson). D323G is expected to localize subsequent to L14 (darkish blue). g, Lollipop plots for sgRNAs focusing on LCK at day 26 put up-electroporation in the lengthy-term proliferation arm of the 12-gene tiling conceal, mapped to the canonical LCK isoform. h, Structure and situation of mutations in LCK. (top) General predicted building of LCK (blue) and mutated residues (crimson). (bottom) Wild-kind (WT) and mutated (mut) residues (crimson).

Prolonged Files Fig. 8 Signaling and impression of subtle variations in making improvements to effectivity and phenotypic readouts.

a, Quantification of S6 phosphorylation (pS235/S236) and b, AKT phosphorylation (pS473) measured by dart cytometry in T cells with indicated genotypes (x axis) after 10 minutes of stimulation with anti-CD3/CD28 antibodies. c, For all validated sgRNAs focusing on PIK3CD (that’s, Cys416Arg, Tyr524Cys, Glu525Gly_His526Arg, and Glu527Gly_Lys528Glu), sgRNA making improvements to effectivity and enact dimension on AKT phosphorylation (pS473), d, TNFα MFI, and e, IL2 expression are plotted for every of the three donors extinct in preliminary validation experiments in Fig. 3. In cases where sgRNAs generated extra than one edits contained in the making improvements to window (as an instance, PIK3CD Glu525Gly_His526Arg), the everyday making improvements to effectivity across all centered bases in the making improvements to window used to be extinct. Files in (a-b) used to be generated from n = 3 self sustaining human donors. Inner every donor this files used to be normalized to the quiet help watch over situation. One-scheme ANOVA with Dunnett’s take a look at for extra than one comparisons (panels a,b). Simple linear regression (panels c-e). Error bars symbolize point out +/− SD (panels a, b).

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Prolonged Files Fig. 9 Experimental accomplish, purposeful assays, and melanoma co-culture experiments with NY-ESO-1 TCR T cells engineered with variants identified in depraved making improvements to screens.

a, Schematic for engineering and rising NY-ESO-1 particular T cells. b, Representative dart cytometry dotplot of NY-ESO-1 particular T cells sooner than sorting. c, Viable A375-dsRed cells relative to time t0 after culture with NY-ESO-1 particular T cells for forty eight hours at varying effector to arrangement ratios. aMHCI = MHC class I-blocking antibody (n = 3 self sustaining organic replicates). d, Representative dart cytometry histograms of ABE-mediated knockout of B2M or CD2 in NY-ESO-1 particular T cells. e, Representative contour plots of single or multiplexed depraved making improvements to of B2M and CD2. f, AKT phosphorylation (pS473), in NY-ESO-1 particular T cells after both 15 minutes of co-culture with A375 cells (+) or media alone (-) (n = 3 self sustaining organic replicates). g, MFI of TNFα and h, GrzB in NY-ESO-1 particular T cells with indicated depraved edits after 8-hour co-culture with A375-dsRed cells at a 1:1 effector to arrangement ratio (n = 3 self sustaining organic replicates). i, Frequency of NY-ESO-1 particular T cells with indicated genotypes co-expressing TNFα, IL2, and GrzB after 8 hours of co-culture with A375 cells at a 1:1 effector to arrangement ratio (n = 3 self sustaining organic replicates.). NT = non-focusing on help watch over sgRNA j, Viable wild-kind (WT) or CD58-KO A375 cells relative to time t0 after forty eight hours of co-culture with NY-ESO-1 particular T cells at a 1:1 effector to arrangement ratio (n = 3 self sustaining organic replicates.) okay, Viable B2M-KO A375 cells relative to time t0 after forty eight hours of co-culture with NY-ESO-1 particular T cells with indicated genotypes. Dotted traces in (f-i) symbolize point out of the help watch over. Dotted traces in (j-okay) symbolize relative viable cell count at time t0. One-scheme ANOVA with Tukey’s take a look at for extra than one comparisons (panel c). One-scheme ANOVA with Dunnett’s take a look at for extra than one comparisons (panels f-i, okay). Pupil’s t take a look at (panel j). Error bars symbolize point out +/− SD (panels c, f-okay).

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Prolonged Files Fig. 10 Regain and outcomes of leukemia co-culture with CD19 CAR-T cells geared up with variants identified in depraved making improvements to screens.

a, Representative histograms of GFP expression, indicating transduction effectivity of first- and 2d-abilities CD19-CAR constructs (CD19-CD3z or CD19-BBz, respectively) in main human T cells. Blue histograms symbolize untransduced help watch over T cells. b, Expression of CTLA4 on CD19-CAR T cells, edited with a help watch over non-focusing on sgRNA (NT) or CTLA4-KO sgRNA, following forty eight-hour co-culture with Nalm6 leukemia cells at an 0.5:1 effector to arrangement ratio. c, Relative cell numbers of Nalm6 cells forty eight hours after co-culture with CD19-BBz CAR T variants at several E:T ratios, when when in contrast with time t0. d, Relative sequence of CD19-KO Nalm6 cells forty eight hours after co-culture with CD19-BBz CAR T cells at an 0.25:1 effector to arrangement ratio, when when in contrast with time t0. e, Quantification of CD19-CD3z CAR T cell AKT phosphorylation (pS473) by dart cytometry after 15 minutes of culture with both Nalm6 leukemia (+) or media alone (−) with book dart histograms. f, Quantification of CD19-CD3z CAR T cell intracellular expression of TNFα and g, IL2 after 8-hour culture with Nalm6 leukemia (+) or in media simplest (-). h, Relative cell numbers of untamed-kind and i, CD19-KO Nalm6 cells forty eight hours after co-culture with CD19-CD3z CAR T cells at an 0.5:1 effector to arrangement ratio, when when in contrast with time t0. Dotted traces in (e-g) symbolize point out of the help watch over population. Dotted traces in (panel c, d, h, i) symbolize relative viable cell count at time t0. One-scheme ANOVA with Tukey’s take a look at for extra than one comparisons (panel b), one-scheme ANOVA with Dunnett’s take a look at for extra than one comparisons (panels d-i). Error bars symbolize point out +/- SD (panels b-i).

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Walsh, Z.H., Shah, P., Kothapalli, N. et al. Mapping variant outcomes on anti-tumor hallmarks of main human T cells with depraved-making improvements to screens.
Nat Biotechnol (2024). https://doi.org/10.1038/s41587-024-02235-x

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