Journal: The Journal of Clinical Investigation

Article Title: IL-2–inducible T cell kinase deficiency sustains chimeric antigen receptor T cell therapy against tumor cells

doi: 10.1172/JCI178558

Figure Lengend Snippet: “nt-KO” indicates the control group of CAR-T cells electroporated with RNP complex containing nontargeting sgRNA, while “ITK-KO” indicates the group that received ITK -targeting sgRNA. ( A ) Schematic representation of the anti–human CD19–CAR molecule. CMV, cytomegalovirus promoter; CD8αSP, signal peptide of human CD8α; anti-CD19-scFv, single-chain fragment variable of anti–human CD19 antibody (clone: FMC63); CD8αTM, transmembrane domain of human CD8α; CD28, 4-1BB, and CD3ζ, signal transduction domains of human CD28, 4-1BB, and CD3ζ, respectively. ( B ) Generation of ITK-deficient CAR-T cells. Briefly, T cells were enriched from PBMCs and activated with anti-CD3/CD28 beads for 24 hours. Then, T cells were transduced with CAR-encoding lentivirus. Forty-eight hours after transduction, CAR-T cells were electroporated with RNP complex. ( C ) Gene editing efficiency of ITK locus by sgRNA1 targeting ITK (ITK-sgRNA1). CAR-T cells were collected for analysis 3 days after electroporation of RNP complex. ( D ) Validation of ITK deficiency at protein level by Western blotting. CAR-T cells were collected for Western blotting 5 days after electroporation. ( E – G ) In vitro killing assay against the indicated target tumor cells using control and ITK-KO CAR-T cells ( n = 4). Luciferase-expressing MEC1, HG3, and Raji cells were mixed at the indicated ratios with CAR-T cells and analyzed 48 hours after coculture. ( H ) Representative flow cytometric plots of IFN-γ, TNF-α, and granzyme B expression in CAR-T cells stimulated as indicated. E, effector (CAR-T cells); T, target (MEC1 cells). ( I ) Summary of percentages of CAR-T cells expressing different cytokines in H ( n = 4). Compiled data from 1 independent experiment for E – G and I . Technical replicates are shown in E – G and I . Data represent results of at least 2 independent experiments in C – I .

Article Snippet: Briefly, 3 days after CAR transduction, anti-CD3/CD28 beads were removed, and 5 × 10 6 CAR-T cells were resuspended in 100 μL electroporation buffer (82 μL P3 Primary Cell Solution mixed with 18 μL Supplement; catalog V4XP-3024, Lonza).

Techniques: Control, Transduction, Electroporation, Biomarker Discovery, Western Blot, In Vitro, Luciferase, Expressing

Journal: The Journal of Clinical Investigation

Article Title: IL-2–inducible T cell kinase deficiency sustains chimeric antigen receptor T cell therapy against tumor cells

doi: 10.1172/JCI178558

Figure Lengend Snippet: ( A ) Experimental design of CAR-T cell therapy against intraperitoneally injected MEC1 cells in NPG mice. CAR-T cells were expanded for 11 days after electroporation before intravenous injection into mice. PB, peripheral blood. ( B ) Representative flow cytometric plots of CAR-GFP and CD3 in PB samples collected from nt-KO or ITK-KO CD19-CAR-T cell recipients at the indicated time points. ( C ) Summary of percentages of CAR-T cells (CD3 + GFP + ) shown in B ( n = 3 for day 77 ITK-KO group and n = 4 for the rest). ( D ) Representative flow cytometric plots of expression of the indicated molecules by CAR-T cells in PB samples collected from nt-KO or ITK-KO-CAR T cell recipients 28 days after CAR-T cell infusion. ( E ) Summary of percentages of CAR-T cells that are LAG-3 + , PD-1 + , TIM-3 + , TIGIT + , or CTLA4 + , as shown in D ( n = 4). ( F and G ) Summary of percentages of CAR-T cells that are Ki-67 + ( F ) and annexin V + ( G ) as shown in , C and D ( n = 5). ( H ) Statistical analysis of different populations of cells shown in ( n = 4). ( I ) Experimental design of CAR-T cell therapy against subcutaneously injected MEC1 cells in NPG mice. CAR-T cells were expanded for 11 days after electroporation before intravenous injection into mice. ( J ) Representative flow cytometric plots of CAR-GFP and CD3 in PB samples collected from nt-KO or ITK-KO CD19-CAR recipients at the indicated time points. ( K ) Summary of percentages of CAR-T cells (CD3 + GFP + ) shown in J ( n = 4, mean ± SEM). Compiled data from 1 independent experiment for C , E – H , and K . Statistical differences were determined by 2-tailed unpaired Student’s t test. Data represent results of at least 2 independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: Briefly, 3 days after CAR transduction, anti-CD3/CD28 beads were removed, and 5 × 10 6 CAR-T cells were resuspended in 100 μL electroporation buffer (82 μL P3 Primary Cell Solution mixed with 18 μL Supplement; catalog V4XP-3024, Lonza).

Techniques: Injection, Electroporation, Expressing

Journal: The Journal of Clinical Investigation

Article Title: IL-2–inducible T cell kinase deficiency sustains chimeric antigen receptor T cell therapy against tumor cells

doi: 10.1172/JCI178558

Figure Lengend Snippet: ( A ) Experimental design of CAR-T cell therapy against intraperitoneally injected Raji cells in NPG mice. CAR-T cells were expanded for 11 days after electroporation. ( B ) Flow cytometric plots of CAR-GFP and CD3 in PB samples collected from indicated recipients at the indicated time points. ( C ) Summary of CAR-T cell percentages (CD3 + GFP + ) in B ( n = 1 for day 95 nt-KO group, n = 3 for day 95 ITK-KO group, n = 4 for the rest). ( D ) Representative flow cytometric plots of annexin V and 7-AAD expression by CAR-T cells from PB samples 28 days after CAR-T cell injection. ( E ) Summary of annexin V + CAR-T cells percentages in D ( n = 4). ( F ) Flow cytometric plots of LAG-3 and TIGIT expression by CAR-T cells from PB samples 24 days after CAR-T cell infusion. ( G ) Summary of LAG-3 + or TIGIT + CAR-T cell pecentages in F ( n = 4). ( H ) Bioluminescence images of NPG mice xenografted with Raji cells as in A . Representative figures from 1 independent experiment. ( I ) Kaplan-Meier survival of Raji-bearing NPG mice ( n = 9) (log-rank Mantel-Cox test with Bonferroni’s correction for multiple comparisons). Compiled data from 2 independent experiments. ( J ) Statistical analysis of CD45RO and/or CCR7 expression by CAR-T cells in ( n = 4). ( K , L ) Statistical analysis of Raji to CAR-T cells ratios ( K ) and CAR-T cell number fold changes ( L ) as shown in (n = 4). ( M ) Statistical analysis of IFN-γ, TNF-α, and granzyme B expression in CAR-T cells shown in ( n = 3). Compiled data from 1 independent experiment for C , E , G , and J – M . Two-tailed unpaired Student’s t test was performed in C , E , G , and J – M . Data represent at least 2 independent experiments. * P < 0.05, ** P < 0.01, **** P < 0.0001.

Article Snippet: Briefly, 3 days after CAR transduction, anti-CD3/CD28 beads were removed, and 5 × 10 6 CAR-T cells were resuspended in 100 μL electroporation buffer (82 μL P3 Primary Cell Solution mixed with 18 μL Supplement; catalog V4XP-3024, Lonza).

Techniques: Injection, Electroporation, Expressing, Two Tailed Test

Journal: The Journal of Clinical Investigation

Article Title: IL-2–inducible T cell kinase deficiency sustains chimeric antigen receptor T cell therapy against tumor cells

doi: 10.1172/JCI178558

Figure Lengend Snippet: ( A ) Experimental design of CAR-T cell therapy against intravenously injected MEC1 cells expressing luciferase in NPG mice via the lateral tail vein. CAR-T cells were expanded for 12 days following electroporation before intravenous injection into mice. ( B ) Representative flow cytometric plots of CAR-GFP and CD3 in PBMCs collected from CLL-CAR-T cell recipients at the indicated time points. ( C ) Summary of percentages of CAR-T cells (CD3 + GFP + ) as shown in B (mean ± SEM; n = 5 for the nt-KO groups on days 26, 33, and 40, as well as the ITK-KO groups on days 40 and 52; n = 4 for nt-KO day 52 group; n = 6 for the rest). ( D ) Representative flow cytometric plots of TIM-3 expression on CAR-T cells in PBMCs collected from nt-KO or ITK-KO CLL-CAR-T recipients 26 days after infusion. ( E ) Summary of percentages of CAR-T cells that are TIM-3 + as shown in D ( n = 5). ( F ) Representative flow cytometric plots of CD62L and CD45RA expression in the indicated CAR-T cells collected. PBMCs were collected on day 33 after CAR-T cell injection. ( G ) Statistical analysis of different cell populations as shown in F ( n = 5). ( H ) Representative bioluminescence images of NPG mice xenografted with MEC1 cells as designed in A . ( I ) Survival of MEC1-bearing NPG mice treated with PBS or nt-KO CLL-CAR-T or ITK-KO CLL-CAR-T cells ( n = 6) (log-rank Mantel-Cox test with Bonferroni’s correction for multiple comparisons). Compiled data from 2 independent experiments in C , E , G , and I . Statistical differences were determined by 2-tailed unpaired Student’s t test in C , E , and G . Data represent results of 2 independent experiments. * P < 0.05, ** P < 0.01, **** P < 0.0001.

Article Snippet: Briefly, 3 days after CAR transduction, anti-CD3/CD28 beads were removed, and 5 × 10 6 CAR-T cells were resuspended in 100 μL electroporation buffer (82 μL P3 Primary Cell Solution mixed with 18 μL Supplement; catalog V4XP-3024, Lonza).

Techniques: Injection, Expressing, Luciferase, Electroporation