Journal: Nature immunology

Article Title: BATF and IRF4 cooperate to counter exhaustion in tumor-infiltrating CAR T cells

doi: 10.1038/s41590-021-00964-8

Figure Lengend Snippet: a , MA plots of basic region-leucine zipper (bZIP) transcription factor gene expression in TOX-depleted ( TOX DKO, left ) or NR4A-depleted ( Nr4a TKO, right ) CAR TILs , — which mount increased anti-tumor responses— relative to control CAR TILs. Differentially expressed genes (adjusted p- value < 0.1, log 2 fold-change ≥ 0.5 or ≤ −0.5) are highlighted; selected genes are labeled. b , Basis of the experiment to identify bZIP transcription factors that activate an NFAT:AP-1 reporter through a positive feedback loop, either directly by binding adjacent to NFAT on the NFAT:AP-1 composite site or indirectly by increasing the expression or activity of NFAT or AP-1. Mouse CD8 + T cells were transduced with retroviral expression vectors encoding a Thy1.1 reporter, separated by a P2A sequence from a co-expressed bZIP transcription factor, and under the control of six tandem NFAT:AP1 sites upstream of a minimal IL-2 promoter. Transcription factors for testing were chosen based on the data analysis in a . c , Gating strategy for the experiments. d, g , Histograms of Thy1.1 expression after CD8 + T cell transduction. No Thy1.1 , transduced with empty retrovirus without Thy1.1 or bZIP transcription factor; No bZIP , transduced with retroviral vector encoding Thy1.1 but no bZIP transcription factor, a condition that assesses the background induction of Thy1.1 by endogenous NFAT and AP-1; Jun , Maff , Batf , and Batf3 ( d ) and JunD, Fosl2 , and Nfil3 ( g ), transduced with vector encoding the indicated bZIP transcription factor. e , h , Percentage of Thy1.1 + cells in three replicate experiments. f , i , MFI of Thy1.1 expression in these experiments. j , k , Results for each sample, normalized to those of the No bZIP control from the same experiment. Each circle in e, f, h, I, j and k represents one mouse. Data are representative of ( c, d, g ) or obtained from ( e, f, h-k ) three biological replicate experiments. Data in e and h were analyzed by one-way ANOVA. *p≤0.05; **P≤0.01; ***P≤0.001; ****P≤0.0001.

Article Snippet: The Platinum-E Retroviral Packaging Ecotropic (PlatE) cell line was purchased from Cell Bio Labs. All tumor cell lines were tested frequently to be sure they were negative for mycoplasma contamination and were used at passage 4 after thawing from stock.

Techniques: Gene Expression, Control, Labeling, Binding Assay, Expressing, Activity Assay, Transduction, Retroviral, Sequencing, Plasmid Preparation

Journal: Nature immunology

Article Title: BATF and IRF4 cooperate to counter exhaustion in tumor-infiltrating CAR T cells

doi: 10.1038/s41590-021-00964-8

Figure Lengend Snippet: a , Retroviral transduction efficiencies for CAR and MSCV-IRES-eGFP retroviral expression plasmids, assessed as expression of Thy1.1 and GFP respectively. FMO, fluorescence-minus-one control. b , Histograms showing BATF ( left ), JUN ( middle ), and MAFF ( right ) expression after retroviral transduction of CD8 + T cells with the corresponding retroviral expression plasmids or pMIG empty-vector control, assessed by flow cytometry with antibodies to the endogenous proteins. c, Histograms showing CAR expression (assessed by staining for the Myc tag) in pMIG- ( grey ), BATF- ( red ), JUN- ( sky blue ) and MAFF- ( orange ) transduced CAR T cells. d-e, Replicate tumor growth experiments using B16F0-hCD19 ( d ) and MC38-hCD19 ( e ) tumor cells. 1×10 5 tumor cells were injected subcutaneously into the left flank of C57BL/6 mice at day 0 (D0) in 100 μl phosphate-buffered saline (PBS); 3×10 6 control pMIG-, JUN-, MAFF-, or BATF-transduced CAR T cells were adoptively transferred by retro-orbital injection at day 7. Tumor sizes were measured by caliper. f , Histograms showing expression of the indicated markers by each group of CAR TILs, assessed by flow cytometry. g , Left panels , Histograms showing expression of CD44, CD62L, CD127 and KLRG1. Middle panels , Overlaid contour plots of CD44 and CD62L ( top ) and CD127 and KLRG1 ( bottom ) in pMIG- ( grey ) and BATF- ( red ) transduced CAR TILs. Right panel , expression of the markers quantitated as MFI. h , Left panels , Histograms showing expression of TNF, IFNγ, granzyme B, and CD107a after resting in T cell media or after stimulation with PMA and ionomycin for 4 h. Right panel , expression of the markers quantitated as MFI. i , pMIG (n=6) and BATF (n=6). Quantitation of TCF1 + and TCF1 – CAR TILs. Each circle in g, h, and i represents one mouse, and the bar graphs represent the mean ± standard error of mean (s.e.m.). Data in d-i were obtained from two independent biological experiments. Data in g, h , and i were analyzed by two-tailed unpaired Student’s t -test.

Article Snippet: The Platinum-E Retroviral Packaging Ecotropic (PlatE) cell line was purchased from Cell Bio Labs. All tumor cell lines were tested frequently to be sure they were negative for mycoplasma contamination and were used at passage 4 after thawing from stock.

Techniques: Retroviral, Transduction, Expressing, Fluorescence, Control, Plasmid Preparation, Flow Cytometry, Staining, Injection, Saline, Quantitation Assay, Two Tailed Test

Journal: Nature immunology

Article Title: BATF and IRF4 cooperate to counter exhaustion in tumor-infiltrating CAR T cells

doi: 10.1038/s41590-021-00964-8

Figure Lengend Snippet: a , Tumor growth curves for the individual mice from – (PBS (n=12), pMIG (n=16), BATF (n=25) and HKE (n=12)). b - f , 1×10 5 B16F0-hCD19 tumor cells were subcutaneously injected into the left flank of C57BL/6 mice at day 0 (D0). 100 μl of PBS, without cells or containing 1.5×10 6 CAR T cells transduced with retroviral expression plasmids encoding either pMIG control, BATF, or BATF HKE-mutant, were adoptively transferred into C57BL/6 recipient mice by retro-orbital injection on day 12. TILs were isolated on days 13, 16, 19, and 22. c , Expression of CAR T cell marker Thy1.1 on CD8 + TILs on the indicated days. d - f , Frequencies and MFIs of the indicated PD-1- and TIM3-expressing populations from , . g - m , 1×10 5 B16F0-hCD19 tumor cells were injected subcutaneously into the left flank of C57BL/6 mice at day 0 (D0). 1.5×10 6 wild-type (WT, n=4) or BATF-deficient (BATF KO, n=4) CAR T cells were adoptively transferred at day 12. Tumor-infiltrating lymphocytes were isolated at day 20. h , Tumor growth curves for individual mice ( dashed lines ) and average ( bold lines ) of all tumor growth curves in a group. i-k , Contour plots of Thy1.1 expression in CD8 + TILs ( i ), percentages of Thy1.1 + CAR TILs ( j ) and numbers of Thy1.1 + CAR TILs normalized to tumor size ( k ) in tumor-bearing BATF WT or BATF KO mice. l , Contour plots of PD-1 and TIM3 expression ( left ) and percentage of PD-1 hi TIM3 + cells ( right ) in WT or BATF KO CAR TILs. m , Contour plots of PD-1 and TOX expression ( top left ), TIM3 and TCF1 expression ( bottom left ), and percentages of the indicated populations ( right ) in WT or BATF KO CAR TILs. Data in a were obtained from three independent experiments. Each circle in d - f and j - m represents one mouse, and the bar graphs represent the mean ± standard error of mean (s.e.m.). Data in d-m are representative of two independent experiments. Data in j - m were analyzed by two-tailed unpaired Student’s t -test.

Article Snippet: The Platinum-E Retroviral Packaging Ecotropic (PlatE) cell line was purchased from Cell Bio Labs. All tumor cell lines were tested frequently to be sure they were negative for mycoplasma contamination and were used at passage 4 after thawing from stock.

Techniques: Injection, Transduction, Retroviral, Expressing, Control, Mutagenesis, Isolation, Marker, Two Tailed Test

Journal: Nature immunology

Article Title: BATF and IRF4 cooperate to counter exhaustion in tumor-infiltrating CAR T cells

doi: 10.1038/s41590-021-00964-8

Figure Lengend Snippet: a - e , 2.5×10 5 B16F10-OVA tumor cells were injected subcutaneously into the left flank of C57BL/6 mice at day 0 (D0). 100 μl of PBS (n=10), without cells or containing 3×10 6 OT-I T cells transduced with retroviral expression plasmids encoding pMIG control (n=10), BATF(n=10), IRF4(n=10), or BATF+IRF4(n=10), were adoptively transferred by retro-orbital injection at day 7. b , Averaged tumor growth curves for all mice in the indicated groups. c , Tumor sizes measured in individual mice at day 18. d , Mouse survival curves. e , Tumor growth curves in individual mice. f - m , 2.5×10 5 B16F10-OVA tumor cells were injected subcutaneously into the left flank of C57BL/6 mice at day 0. 1×10 6 pMIG control (n=4)-, BATF(n=5)-, IRF4(n=5)-, or BATF+IRF4(n=5)-transduced OT-I cells were adoptively transferred at day 12. TILs were isolated at day 20. g , Gating strategy for flow cytometric analysis of OT-I TILs. h , Averaged tumor growth curves for all mice in the indicated groups. i , Left , Contour plots of CD8α and CD45.1 expression in OT-I TILs. Middle , Percentage of OT-I TILs in CD8 + TILs. Right , Number of OT-I TILs normalized to tumor size. j , Left, Contour plots of PD-1 and TIM3 expression in each group of OT-I TILs. Right, Percentages of the indicated PD-1- and TIM3-expressing cell populations. k , Left, Contour plots of PD-1 and TOX expression in the indicated OT-I TILs. Right, Percentage of PD-1 + TOX + cells in OT-I TILs. l , m , Left, Contour plots of expression of granzyme B ( l ) and the indicated cytokines ( m ) under resting conditions or after PMA/ionomycin stimulation for 4 h. Right, Percentages of OT-I TILs expressing granzyme B ( l ) or the indicated cytokines ( m ) under resting conditions or after PMA/ionomycin stimulation for 4 h. Data obtained from two biological experiments n , Histograms showing JUN and BATF expression in the indicated groups of transduced OT-I T cells. o , Tumor growth curves for individual mice given pMIG control-, BATF-, JUN-, or BATF+JUN-transduced OT-1 cells ( top ) and averaged tumor growth curves for all mice in each group ( bottom ). Experimental scheme as in a . Each circle in i , j , k , l , and m represents one mouse, and the bar graphs represent the mean ± standard error of mean (s.e.m.). Data were obtained from two independent biological experiments. Data in h , j , l , and m were analyzed by two-way ANOVA test; data from i and k , by one-way ANOVA test. *p≤0.05; **p≤0.01; ***p≤0.001; ****p≤0.0001.

Article Snippet: The Platinum-E Retroviral Packaging Ecotropic (PlatE) cell line was purchased from Cell Bio Labs. All tumor cell lines were tested frequently to be sure they were negative for mycoplasma contamination and were used at passage 4 after thawing from stock.

Techniques: Injection, Transduction, Retroviral, Expressing, Control, Isolation

Journal: BioTechniques

Article Title: Spin infection for efficient gene delivery in muscle stem cells for intramuscular cell transplantation

doi: 10.2144/000114576

Figure Lengend Snippet: Transfection reagents Lipofectamine 2000, Lipofectamine LTX, TransIT-293, TransIT-2020, TransIT-LT1, PolyJet, or LipoJet, were used for pMX-GFP retroviral vector transfection. A. GFP expression in Plat-E cells 2 days after transfection by each transfection reagent, and following infection to primary myoblasts with indicated viral supernatants for 2 days. These phase contrast images show primary myoblasts after infection. C. The number of GFP positive primary myoblasts out of total cells. Data are presented as the mean ± SEM. (n=3)

Article Snippet: Retroviral supernatants were produced by transfection of pMX-GFP (Cell Biolabs, San Diego, CA, USA) or a pMX-mCherry retroviral vector into a 293T Platinum-E Retroviral Packaging Cell Line (Plat-E) (Cell Biolabs).

Techniques: Transfection, Plasmid Preparation, Expressing, Infection