Journal: The Journal of experimental medicine

Article Title: Induction of immortal-like and functional CAR T cells by defined factors.

doi: 10.1084/jem.20232368

Figure Lengend Snippet: Figure 1. Ablation of ZC3H12A and BCOR induces immortal-like and functional CAR19TIF cells. (A) The composition of the anti-mCD19 CAR. (B) Vector design of pMSCV-sgRNA-CAR19. Thy1.1 is coexpressed with CAR at the protein level via P2A, serving as a marker of CAR expression. (C) Experimental design. CD8+Cas9+ T cells were activated by anti-CD3/anti-CD28 (1 μg/ml), infected with retrovirus expressing CAR19 with sgRNA targeting indicated genes. 1 million CD8+Thy1.1+ CAR19T cells were transferred into B6 mice. CAR19T cells and B cells from peripheral blood were monitored by flow cytometry. (D–F) Rep- resentative plots (D) and statistical analysis (E and F) of Thy1.1+ CAR19T cells and CD19+ B cells among single live cells from peripheral blood 7 and 28 days after transfer are shown (n = 4 mice for each group). (G) Experimental design for genome-wide dual sgRNA screening for genes whose loss of function confers persistence to ZC3H12A-deficient CAR19T cells. The indicated sgRNA library was used to infect activated CD8+ T cells, and 150 million CAR19T cells were transferred into 50 B6 mice (3 million per mouse). 7 days and 3 mo later, Thy1.1+ CAR19T cells were sorted from the spleen for sgRNA analysis. (H) Screening result. (I and J) Representative plots (I) and statistical analysis (J) of Thy1.1+ CAR19T cells and CD19+ B cells among single live cells from peripheral blood of mice transferred with CAR19TIF cells expressing indicated sgRNAs are shown (n = 3 mice in PBS group, n = 6 mice in sgNT group, n = 5 mice in sgZc3h12a group, n = 5 mice in sgBcor/Zc3h12a group). (K) Percentages of CD19+ B cells from the spleen of mice 6 mo after transferring CAR19TIF cells expressing indicated sgRNAs (n = 4 mice in sgNT group, n = 5 mice in sgBcor/Zc3h12a group). (L and M) Representative plots (L) and statistical analysis (M) of Thy1.1+ CAR19T cells ex- pressing indicated sgRNA and CD19+ B cells among single live cells from spleen 7 days after transfer are shown (n = 4 mice for each group). (E, F, J, K, and M) Data represent mean ± SEM from one of three independent experiments. *P < 0.05, ****P < 0.0001, ns, not significant, one-way ANOVA multiple-comparisons test in E and F, two-way ANOVA multiple-comparisons test in J, two-tailed unpaired Student’s t test in K and M.

Article Snippet: Subsequently, the sgRNA portion of the Brie genome-wide sgRNA library (Cat# 73632; Addgene) for mice was PCR-amplified and ligated into this vector using Gibson assembly (Cat# E2621S; NEB).

Techniques: Functional Assay, Plasmid Preparation, Marker, Expressing, Infection, Flow Cytometry, Genome Wide, Transferring, Two Tailed Test

Journal: Cell

Article Title: A membrane-associated MHC-I inhibitory axis for cancer immune evasion

doi: 10.1016/j.cell.2023.07.016

Figure Lengend Snippet: (A-D) Representative histograms (left) and bar plots (right) showing the surface levels of HLA-A2:AFP (A) or HLA-A2 (B) in human THP-1-Cas9-AFP-BFP cells, and the surface levels of H-2Kb:OVA (C) or H-2Kb (D) in mouse RN2-Cas9-OVA-BFP cells transduced with the indicated sgRNAs. (n=3) (E and F) Schematic of the T cell activation assay (E) and bar plot showing the IL-2 secreted by the B3Z T cell hybridoma incubated with sgRNA-transduced RN2-Cas9-OVA-BFP cells (F). (n=3) (G and H) Schematic of the mouse T cell killing assay (G) and bar plot showing the percentages of sgRNA-transduced RN2-Cas9-OVA-BFP cells killed by the OT-I T cells (H). (n=3) (I and J) Schematic of the human T cell killing assay (I) and bar plot showing the percentages of sgRNA-transduced NY-ESO-1-expressing THP-1-Cas9 cells killed by the NY-ESO-1 TCR-T cells (J). (n=3) (K) Schematic of the in vivo validations of SUSD6 functions in a mouse syngeneic AML model. (L-P) Quantification of the tumor volumes (L and O) and Kaplan-Meier survival curves (M and P) of immunocompetent (L and M) or CD8+ T cell-depleted (O and P) mice transplanted with sgRNA-transduced C1498-Cas9-GFP cells as described in (K). (for L and M: n=4 for sgNT and n=6 for sgSusd6; for O and P: n=5 for sgNT and n=6 for sgSusd6) (R) Violin plot of SUSD6 mRNA levels in normal HSPCs, MDS cells, and AML cells with different karyotypes from patient samples. HSPCs, hematopoietic stem and progenitor cells; HSC, hematopoietic stem cell; MPP, multipotent progenitor; CMP, common myeloid progenitor; GMP, granulocyte-monocyte progenitor; MEP, megakaryocyte-erythrocyte progenitor; MDS, myelodysplastic syndromes. Data were obtained from BloodSpot.51 (S) Survival of AML patients with high or low expression of SUSD6 in the TCGA-LAML cohort. (T) Pearson correlation of SUSD6 expression levels in AML cells and T cell activation signature in CD8+ T cells from the bone marrow immuno-microenvironments from AML patients. Data were generated by single-cell RNA-seq.53 Data are presented as the mean ± SEM. ns, not significant; *, p< 0.05; **, p< 0.01; and ***, p< 0.001 by two-tailed unpaired Student’s t-test (A-D, F, H, and J), two-way ANOVA for the last time point (L and O), or Log-rank Mantel-Cox test (M and P). Rosa26-targeting sgRNA (sgRosa, for human) and non-targeting sgRNA (sgNT, for mouse) were used as controls.

Article Snippet: THP-1-Cas9-AFP-BFP and RN2-Cas9-cOVA-BFP cells were transduced with the Brunello sgRNA library (Addgene #73179) or Brie sgRNA library (Addgene #73632) virus, 41 respectively, at a low MOI (~0.3).

Techniques: Transduction, Activation Assay, Incubation, Expressing, In Vivo, Generated, RNA Sequencing, Two Tailed Test

Journal: Cell

Article Title: A membrane-associated MHC-I inhibitory axis for cancer immune evasion

doi: 10.1016/j.cell.2023.07.016

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: THP-1-Cas9-AFP-BFP and RN2-Cas9-cOVA-BFP cells were transduced with the Brunello sgRNA library (Addgene #73179) or Brie sgRNA library (Addgene #73632) virus, 41 respectively, at a low MOI (~0.3).

Techniques: Control, Purification, Blocking Assay, Virus, Recombinant, Protease Inhibitor, Transfection, Immunoprecipitation, Activation Assay, Magnetic Beads, Reverse Transcription, SYBR Green Assay, Enzyme-linked Immunosorbent Assay, Double Knockout, shRNA, Real-time Polymerase Chain Reaction, Genome Wide, Plasmid Preparation, Software, Flow Cytometry

Journal: Cell

Article Title: A membrane-associated MHC-I inhibitory axis for cancer immune evasion

doi: 10.1016/j.cell.2023.07.016

Figure Lengend Snippet: (A-D) Representative histograms (left) and bar plots (right) showing the surface levels of HLA-A2:AFP (A) or HLA-A2 (B) in human THP-1-Cas9-AFP-BFP cells, and the surface levels of H-2Kb:OVA (C) or H-2Kb (D) in mouse RN2-Cas9-OVA-BFP cells transduced with the indicated sgRNAs. (n=3) (E and F) Schematic of the T cell activation assay (E) and bar plot showing the IL-2 secreted by the B3Z T cell hybridoma incubated with sgRNA-transduced RN2-Cas9-OVA-BFP cells (F). (n=3) (G and H) Schematic of the mouse T cell killing assay (G) and bar plot showing the percentages of sgRNA-transduced RN2-Cas9-OVA-BFP cells killed by the OT-I T cells (H). (n=3) (I and J) Schematic of the human T cell killing assay (I) and bar plot showing the percentages of sgRNA-transduced NY-ESO-1-expressing THP-1-Cas9 cells killed by the NY-ESO-1 TCR-T cells (J). (n=3) (K) Schematic of the in vivo validations of SUSD6 functions in a mouse syngeneic AML model. (L-P) Quantification of the tumor volumes (L and O) and Kaplan-Meier survival curves (M and P) of immunocompetent (L and M) or CD8+ T cell-depleted (O and P) mice transplanted with sgRNA-transduced C1498-Cas9-GFP cells as described in (K). (for L and M: n=4 for sgNT and n=6 for sgSusd6; for O and P: n=5 for sgNT and n=6 for sgSusd6) (R) Violin plot of SUSD6 mRNA levels in normal HSPCs, MDS cells, and AML cells with different karyotypes from patient samples. HSPCs, hematopoietic stem and progenitor cells; HSC, hematopoietic stem cell; MPP, multipotent progenitor; CMP, common myeloid progenitor; GMP, granulocyte-monocyte progenitor; MEP, megakaryocyte-erythrocyte progenitor; MDS, myelodysplastic syndromes. Data were obtained from BloodSpot.51 (S) Survival of AML patients with high or low expression of SUSD6 in the TCGA-LAML cohort. (T) Pearson correlation of SUSD6 expression levels in AML cells and T cell activation signature in CD8+ T cells from the bone marrow immuno-microenvironments from AML patients. Data were generated by single-cell RNA-seq.53 Data are presented as the mean ± SEM. ns, not significant; *, p< 0.05; **, p< 0.01; and ***, p< 0.001 by two-tailed unpaired Student’s t-test (A-D, F, H, and J), two-way ANOVA for the last time point (L and O), or Log-rank Mantel-Cox test (M and P). Rosa26-targeting sgRNA (sgRosa, for human) and non-targeting sgRNA (sgNT, for mouse) were used as controls.

Article Snippet: Mouse pooled genome-wide sgRNA library (Brie) sequences , Addgene , Addgene: 73632.

Techniques: Transduction, Activation Assay, Incubation, Expressing, In Vivo, Generated, RNA Sequencing, Two Tailed Test

Journal: Cell

Article Title: A membrane-associated MHC-I inhibitory axis for cancer immune evasion

doi: 10.1016/j.cell.2023.07.016

Figure Lengend Snippet: (A) Representative western blots (left) and normalized band intensities (right) of HLA-A and B2m in sgRNA-transduced THP-1 cells. (n=5). (B) Schematic of the surface HLA-A2 internalization assay in sgRNA-transduced THP-1 cells. (C) Quantifications of the surface-remaining HLA-A2. (n=3) (D-F) Time course studies of the surface HLA-A2 expression on shRNA-transduced THP-1 cells treated with Cycloheximide (CHX, D), Bafilomycin A1 (BafA1, E), or Epoxomicin (Epox, F) (n=4). (G-J) Representative confocal images (top) and quantifications (bottom) of the surface-derived MHC-I colocalized with the plasma membrane markers (G and I) or the lysosomal marker (H and J) in THP-1 cells (G and H) or MutuDC cells (I and J) transduced with indicated shRNAs. At least 100 cells were quantified in each group. All scale bars: 5 μm. (K and L) Flow cytometric analyses of intracellular MHC-I storage (K) and recycle (L) in shRNA-transduced THP-1 cells as described in Figures S5H and S5I, respectively. (for K: n=6; for L: n=4) Data are presented as the mean ± SEM (A-F, K, and L) or box and whiskers with all data points (G-J). ns, not significant; ***, p<0.001 by two-tailed unpaired Student’s t-test (A), two-way ANOVA for the last time point (C-F, K, and L), or Mann-Whitney test (G-J). sgRosa and shRen were used as controls.

Article Snippet: Mouse pooled genome-wide sgRNA library (Brie) sequences , Addgene , Addgene: 73632.

Techniques: Western Blot, Expressing, shRNA, Derivative Assay, Clinical Proteomics, Membrane, Marker, Transduction, Two Tailed Test, MANN-WHITNEY

Journal: Cell

Article Title: A membrane-associated MHC-I inhibitory axis for cancer immune evasion

doi: 10.1016/j.cell.2023.07.016

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Mouse pooled genome-wide sgRNA library (Brie) sequences , Addgene , Addgene: 73632.

Techniques: Control, Purification, Blocking Assay, Virus, Recombinant, Protease Inhibitor, Transfection, Immunoprecipitation, Activation Assay, Magnetic Beads, Reverse Transcription, SYBR Green Assay, Enzyme-linked Immunosorbent Assay, Double Knockout, shRNA, Real-time Polymerase Chain Reaction, Genome Wide, Plasmid Preparation, Software, Flow Cytometry