Journal: Nature biomedical engineering

Article Title: Enhanced intratumoural activity of CAR T cells engineered to produce immunomodulators under photothermal control

doi: 10.1038/s41551-021-00781-2

Figure Lengend Snippet: a , Schematic representation of transduced primary human T cells and thermal delivery profiles. b , Gluc activity of the 7H-YB thermal switch in primary human T cells after continuous (light grey) and pulsed (dark grey) heat treatments with temperatures, total durations and heating profiles as indicated. * P = 0.033; ** P = 0.0021; two-tailed t -test; mean ± s.e.m. is depicted; n = 3 biologically independent wells. c , PI and AnV flow staining of CD3 + T cells. Bars represent viable populations (PI − AnV − ) normalized to unheated samples. * P = 0.0455; ** P = 0.0034; **** P < 0.0001; two-way ANOVA and Šidák post-test and correction; mean ± s.e.m. is depicted; n = 3 biologically independent wells; FSC-A, forward scatter area; SSC-A, side scatter area. Two independent experiments were performed with similar results. d , CellTrace Violet (CTV) flow histograms of T cells after heat treatments and incubation with CD3/28 beads at a 3:1 bead to T cell ratio. Values in histogram boxes represent percentage in indicated gate. Two independent experiments were performed with similar results. e , Number of cells in lower well of a transwell plate containing CXCL12. T cells were heated and loaded into the top well before sampling at indicated timepoints. Two-way ANOVA and Tukey post-test and correction; mean ± s.e.m. is depicted; n = 3 biologically independent wells. Two independent experiments were performed with similar results. f , Percent cytotoxicity observed in CD19− or CD19+ luciferized K562 cells after incubation with T cells constitutively expressing CARs after heating, with effector to target ratios as indicated. * P = 0.0166 at a 1:1 CAR T cell to target cell ratio; * P = 0.0251 at a 5:1 CAR T cell to target cell ratio; two-way ANOVA and Šidák post-test and correction; mean ± s.e.m. is depicted; n = 3 biologically independent wells. Two independent experiments were performed with similar results.

Article Snippet: To detect CD19 CAR expression, biotinylated CD19 (10 μg ml −1 ; Acro Biosystems CD9-H8259) and streptavidin-APC (ThermoFisher S868) were used according to the manufacturer’s instructions.

Techniques: Activity Assay, Two Tailed Test, Staining, Incubation, Sampling, Expressing

Journal: Nature biomedical engineering

Article Title: Enhanced intratumoural activity of CAR T cells engineered to produce immunomodulators under photothermal control

doi: 10.1038/s41551-021-00781-2

Figure Lengend Snippet: a , Thermal and luminescence images of wells containing TS-Fluc T cells after irradiation with NIR laser light. Thermal images (left) were acquired using a FLIR thermal camera while luminescence images (right) were acquired using an IVIS Spectrum CT system 6 h after heating. b , Schematic representation of TS-Fluc αCD19 CAR construct transduced into primary human T cells before transfer into NSG mice with two flank (K562 or Raji) tumours followed by photothermal heating of single tumour. c , Thermal images of mouse during laser irradiation of tumour site at 0 and 3 min. d , Kinetic traces (coloured lines) showing average skin temperature of a 3 × 3 pixel region of interest (ROI) centred on laser site. Shaded regions show s.d. of three heating runs. e , Left: luminescence images of heated mice bearing either K562 (CD19−) or Raji (CD19+) tumours. Signal indicates luciferase activity by transferred TS-Fluc T cells. Dotted circles represent ROIs. Right: luminescence of each tumour site relative to the luminescence from the unheated tumour in the same animal. A separate experiment with repeated heating of Raji tumours was conducted to confirm reproducibility of experimental results ( Supplementary Fig. 8 ). f , Mice bearing two Raji (CD19+) tumours with one site heated. Left: luminescence images of excised tumours (heated and unheated) and spleen. Right: quantification of luminescence following heat treatments (0, 6, 12, 18, 24 h). **** P < 0.0001; two-way ANOVA and Tukey post-test and correction; mean ± s.e.m. is depicted; n = 4 to 5 biologically independent mice.

Article Snippet: To detect CD19 CAR expression, biotinylated CD19 (10 μg ml −1 ; Acro Biosystems CD9-H8259) and streptavidin-APC (ThermoFisher S868) were used according to the manufacturer’s instructions.

Techniques: Activation Assay, In Vivo, Irradiation, Construct, Luciferase, Activity Assay

Journal: Nature biomedical engineering

Article Title: Enhanced intratumoural activity of CAR T cells engineered to produce immunomodulators under photothermal control

doi: 10.1038/s41551-021-00781-2

Figure Lengend Snippet: a , Schematic of co-culture assay of heated TS-IL-15 αCD19 CAR T cells and CFSE-labelled wild-type cells. CD3/28 beads were added at 1:10 bead to T cell ratio. b , Representative flow histograms (left), quantified proliferation (middle) and division indices (right) of the CFSE-labelled wild-type T cell population, as calculated by FlowJo proliferation tool, after a 30 min thermal treatment at indicated temperatures. *** P = 0.0009; * P = 0.0352; two-tailed t -test; mean ± s.e.m. is depicted; n = 3 biologically independent wells. c , IL-15 superagonist concentrations in supernatant of TS-IL-15 αCD19 CAR T cells following heat treatments. Temperature and duration of treatments are as indicated. ** P = 0.0032, **** P < 0.0001; two-way ANOVA and Tukey post-test and correction; mean ± s.e.m. is depicted; n = 3 biologically independent wells, comparisons displayed between sample and unheated control. Two independent experiments were performed with similar results. d , Schematic of TS-IL-15 αCD19 CAR vector used to transduce primary human T cells before transfer into tumour-bearing NSG mice. e , Tumour growth curves of CD19+ K562s following transfer of TS-IL-15 αCD19 CAR T cells on day 0 and heat treatments on days 2, 6, 9, 13 and 16. *** P = 0.0004 between cells only and untreated cohort on day 23; * P = 0.0157 between cohorts receiving cells and heat versus cells only on day 20; ** P = 0.0047 between cohorts receiving cells and heat versus cells only on day 23; ** P = 0.0012 between cohorts receiving cells and heat versus cells only on day 27; two-way ANOVA and Tukey post-test and correction; mean ± s.e.m. is depicted; n = 7 biologically independent mice. Three independent experiments were performed with similar results. f , Survival curves of tumour-bearing mice in d and e following transfer of TS-IL-15 αCD19 CAR T cells and heat treatments. ** P = 0.000022, log-rank (Mantel–Cox) test with correction for six multiple comparisons, n = 7 biologically independent mice. g , Schematic of TS-IL-15 vector transduced into primary murine Pmel-1 T cells transferred into tumour-bearing C57BL/6 J mice. h , Tumour growth curves following inoculation of B16-F10 after transfer of TS-IL-15 Pmel-1 T cells on day 0 and heat treatments on days 1 and 3. * P = 0.0438 between untreated and heat-only cohorts on day 10; * P = 0.0166 between cohorts receiving cells and heat versus cells only on day 12; ** P = 0.0058 between cohorts receiving cells and heat versus cells only on day 15; two-way ANOVA and Tukey post-test and correction; mean ± s.e.m. is depicted; n = 6 to 7 biologically independent mice. i , Survival curves of tumour-bearing mice in g and h following transfer of TS-IL-15 Pmel-1 T cells and heat treatments. ** P = 0.000043, log-rank (Mantel–Cox) test with correction for six multiple comparisons, n = 6 to 7 biologically independent mice.

Article Snippet: To detect CD19 CAR expression, biotinylated CD19 (10 μg ml −1 ; Acro Biosystems CD9-H8259) and streptavidin-APC (ThermoFisher S868) were used according to the manufacturer’s instructions.

Techniques: Control, Co-culture Assay, Two Tailed Test, Plasmid Preparation, Transduction

Journal: Nature biomedical engineering

Article Title: Enhanced intratumoural activity of CAR T cells engineered to produce immunomodulators under photothermal control

doi: 10.1038/s41551-021-00781-2

Figure Lengend Snippet: a , Schematic of TS-BiTE and TS-Fluc thermal switches containing heat-triggered BiTE or Fluc reporters. Both constructs contained constitutive αCD19 CARs. b , Histograms for HisTag flow staining in TS-BiTE and TS-Fluc primary T cells following heating. c , NKG2DL flow staining on primary human T cells and K562s using an NKG2D-Fc chimera and an αFc-A488 secondary antibody. Stain, full staining; 2° Ctrl, secondary antibody only. d , Schematic depicting BiTE-mediated targeting of K562 target cells lacking the CAR target antigen via BiTE binding to NKG2DL and CD3. e , Flow-gating strategy for defining bystander cells on the basis of CD19 CAR expression using phycoerythrin (PE) in Jurkat co-culture assays with K562s. UTD controls were gated on the lower (CAR−) population for graphing in g . f , g Flow staining of CD69 on Jurkat T cells following heating and incubation with K562s. TS-BiTE CAR+ histograms ( f ) and summary data of indicated populations ( g ) are graphed. Statistics show comparison to UTD; ** P = 0.0025 between bystander and UTD populations after 30 min heating; ** P = 0.0044 between bystander and UTD populations after 45 min heating; **** P < 0.0001; two-way ANOVA and Tukey post-test and correction; mean + s.e.m. is depicted; n = 3 biologically independent wells. Two independent experiments were performed with similar results. MFI, median fluorescent intensity. h , Cytokine concentrations in supernatant of primary human T cells after heat treatments and incubation with K562 cells. T cells were either UTD or transduced with the indicated thermal switches. **** P < 0.0001; two-way ANOVA and Tukey post-test and correction; mean + s.e.m. is depicted; n = 3 biologically independent wells. i , Cytotoxicity against K562s as quantified by luciferase assay after incubation with primary human T cells. T cells were either UTD or transduced with the indicated thermal switches. ** P = 0.0043 between UTD and TS-BiTE cells; **** P < 0.0001; two-way ANOVA and Tukey post-test and correction; comparisons are to UTD control; mean + s.e.m. is depicted; n = 3 biologically independent wells.

Article Snippet: To detect CD19 CAR expression, biotinylated CD19 (10 μg ml −1 ; Acro Biosystems CD9-H8259) and streptavidin-APC (ThermoFisher S868) were used according to the manufacturer’s instructions.

Techniques: Construct, Staining, Binding Assay, Expressing, Co-Culture Assay, Incubation, Comparison, Transduction, Luciferase, Control