supt1 cells Search Results


supt1 cells  (European Collection of Authenticated Cell Cultures)
90
European Collection of Authenticated Cell Cultures supt1 cells
A split CAR associates through the interaction of TetRB and TIP and dissociates upon minocycline addition. ( a ) Overview of the split CAR approach (TetCAR), incorporating the tetracycline repressor protein B (TetRB) and the peptide TIP. Addition of the small molecule antibiotic minocycline reversibly disrupts TetRB-TIP binding, displaces the endodomain and inhibits CAR activation. ( b ) Schematic of the CAR constructs with eGFP endodomains. CARs contain an anti-human CD19 scFv from FMC63, CD8 stalk regions, CD28 transmembrane domains and eGFP endodomain. TetCARs have a TetRB endodomain with eGFP as a separate protein with or without TIP. ( c ) Representative widefield fluorescent images of HEK293T cells transduced with eGFP-tagged CAR structures, ± 100 nM minocycline. ( d ) Schematic of the CAR constructs with 41BB-CD3ζ endodomains. CARs contain an anti-human CD19 scFv from FMC63, with a CD8 stalk and transmembrane domain and 41BB-CD3ζ endodomain. TetCARs have a TetRB endodomain with 41BB-CD3ζ as a separate protein, with or without TIP. E) Killing of <t>SupT1</t> cells engineered to express CD19 and GFP (SupT1-CD19-GFP) after 24 h co-culture with CAR-T cells at a 1:1 effector:target ratio. 100 nM of minocycline was added to relevant wells. Data shows mean percentage (± SD) of live cells compared to non-transduced (NT) T-cell control, n = 4 donors from 1 experiment. Statistical analysis was through a two-way ANOVA with Tukey’s multiple comparisons between each group at 0 nM, or with Šidák’s multiple comparisons within each group ± minocycline. P values = FMC63-Tet-BBz 0 nM versus 100 nM (****, < 0.0001). ( f ) IFN-γ or ( g ) IL-2 release after 24 h of co-culture with SupT1-CD19-GFP at 1:1 E:T ratio. Data shows mean ± SD, n = 4 donors from 1 experiment. Statistical analysis was through a two-way ANOVA with Tukey’s multiple comparisons between FMC63-Tet-BBz and TIP-less-Tet-BBz. P values = FMC63-Tet-BBz 0 nM versus 100 nM (**, 0.0013) and FMC63-Tet-BBz 0 nM versus TIP-less-Tet-BBz 0 nM (***, 0.0001).
Supt1 Cells, supplied by European Collection of Authenticated Cell Cultures, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/supt1+cells/supt1+cells/pmc08578617-225-0-6
Average 90 stars, based on 1 article reviews
supt1 cells - by Bioz Stars, 2026-09
90/100 stars
  Buy from Supplier
supt1 cells  (KOKEN CO)
90
KOKEN CO supt1 cells
A split CAR associates through the interaction of TetRB and TIP and dissociates upon minocycline addition. ( a ) Overview of the split CAR approach (TetCAR), incorporating the tetracycline repressor protein B (TetRB) and the peptide TIP. Addition of the small molecule antibiotic minocycline reversibly disrupts TetRB-TIP binding, displaces the endodomain and inhibits CAR activation. ( b ) Schematic of the CAR constructs with eGFP endodomains. CARs contain an anti-human CD19 scFv from FMC63, CD8 stalk regions, CD28 transmembrane domains and eGFP endodomain. TetCARs have a TetRB endodomain with eGFP as a separate protein with or without TIP. ( c ) Representative widefield fluorescent images of HEK293T cells transduced with eGFP-tagged CAR structures, ± 100 nM minocycline. ( d ) Schematic of the CAR constructs with 41BB-CD3ζ endodomains. CARs contain an anti-human CD19 scFv from FMC63, with a CD8 stalk and transmembrane domain and 41BB-CD3ζ endodomain. TetCARs have a TetRB endodomain with 41BB-CD3ζ as a separate protein, with or without TIP. E) Killing of <t>SupT1</t> cells engineered to express CD19 and GFP (SupT1-CD19-GFP) after 24 h co-culture with CAR-T cells at a 1:1 effector:target ratio. 100 nM of minocycline was added to relevant wells. Data shows mean percentage (± SD) of live cells compared to non-transduced (NT) T-cell control, n = 4 donors from 1 experiment. Statistical analysis was through a two-way ANOVA with Tukey’s multiple comparisons between each group at 0 nM, or with Šidák’s multiple comparisons within each group ± minocycline. P values = FMC63-Tet-BBz 0 nM versus 100 nM (****, < 0.0001). ( f ) IFN-γ or ( g ) IL-2 release after 24 h of co-culture with SupT1-CD19-GFP at 1:1 E:T ratio. Data shows mean ± SD, n = 4 donors from 1 experiment. Statistical analysis was through a two-way ANOVA with Tukey’s multiple comparisons between FMC63-Tet-BBz and TIP-less-Tet-BBz. P values = FMC63-Tet-BBz 0 nM versus 100 nM (**, 0.0013) and FMC63-Tet-BBz 0 nM versus TIP-less-Tet-BBz 0 nM (***, 0.0001).
Supt1 Cells, supplied by KOKEN CO, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/supt1+cells/supt1+cells/pm10544082-89-58-60
Average 90 stars, based on 1 article reviews
supt1 cells - by Bioz Stars, 2026-09
90/100 stars
  Buy from Supplier
cd8+ supt1 cells transduced with the wild-type tcr (wt)  (BioMimetic Therapeutics)
90
BioMimetic Therapeutics cd8+ supt1 cells transduced with the wild-type tcr (wt)
A split CAR associates through the interaction of TetRB and TIP and dissociates upon minocycline addition. ( a ) Overview of the split CAR approach (TetCAR), incorporating the tetracycline repressor protein B (TetRB) and the peptide TIP. Addition of the small molecule antibiotic minocycline reversibly disrupts TetRB-TIP binding, displaces the endodomain and inhibits CAR activation. ( b ) Schematic of the CAR constructs with eGFP endodomains. CARs contain an anti-human CD19 scFv from FMC63, CD8 stalk regions, CD28 transmembrane domains and eGFP endodomain. TetCARs have a TetRB endodomain with eGFP as a separate protein with or without TIP. ( c ) Representative widefield fluorescent images of HEK293T cells transduced with eGFP-tagged CAR structures, ± 100 nM minocycline. ( d ) Schematic of the CAR constructs with 41BB-CD3ζ endodomains. CARs contain an anti-human CD19 scFv from FMC63, with a CD8 stalk and transmembrane domain and 41BB-CD3ζ endodomain. TetCARs have a TetRB endodomain with 41BB-CD3ζ as a separate protein, with or without TIP. E) Killing of <t>SupT1</t> cells engineered to express CD19 and GFP (SupT1-CD19-GFP) after 24 h co-culture with CAR-T cells at a 1:1 effector:target ratio. 100 nM of minocycline was added to relevant wells. Data shows mean percentage (± SD) of live cells compared to non-transduced (NT) T-cell control, n = 4 donors from 1 experiment. Statistical analysis was through a two-way ANOVA with Tukey’s multiple comparisons between each group at 0 nM, or with Šidák’s multiple comparisons within each group ± minocycline. P values = FMC63-Tet-BBz 0 nM versus 100 nM (****, < 0.0001). ( f ) IFN-γ or ( g ) IL-2 release after 24 h of co-culture with SupT1-CD19-GFP at 1:1 E:T ratio. Data shows mean ± SD, n = 4 donors from 1 experiment. Statistical analysis was through a two-way ANOVA with Tukey’s multiple comparisons between FMC63-Tet-BBz and TIP-less-Tet-BBz. P values = FMC63-Tet-BBz 0 nM versus 100 nM (**, 0.0013) and FMC63-Tet-BBz 0 nM versus TIP-less-Tet-BBz 0 nM (***, 0.0001).
Cd8+ Supt1 Cells Transduced With The Wild Type Tcr (Wt), supplied by BioMimetic Therapeutics, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/supt1+cells/cd8++supt1+cells+transduced+with+the+wild+type+tcr++wt+/pm30339020-151-10-50
Average 90 stars, based on 1 article reviews
cd8+ supt1 cells transduced with the wild-type tcr (wt) - by Bioz Stars, 2026-09
90/100 stars
  Buy from Supplier

Image Search Results


A split CAR associates through the interaction of TetRB and TIP and dissociates upon minocycline addition. ( a ) Overview of the split CAR approach (TetCAR), incorporating the tetracycline repressor protein B (TetRB) and the peptide TIP. Addition of the small molecule antibiotic minocycline reversibly disrupts TetRB-TIP binding, displaces the endodomain and inhibits CAR activation. ( b ) Schematic of the CAR constructs with eGFP endodomains. CARs contain an anti-human CD19 scFv from FMC63, CD8 stalk regions, CD28 transmembrane domains and eGFP endodomain. TetCARs have a TetRB endodomain with eGFP as a separate protein with or without TIP. ( c ) Representative widefield fluorescent images of HEK293T cells transduced with eGFP-tagged CAR structures, ± 100 nM minocycline. ( d ) Schematic of the CAR constructs with 41BB-CD3ζ endodomains. CARs contain an anti-human CD19 scFv from FMC63, with a CD8 stalk and transmembrane domain and 41BB-CD3ζ endodomain. TetCARs have a TetRB endodomain with 41BB-CD3ζ as a separate protein, with or without TIP. E) Killing of SupT1 cells engineered to express CD19 and GFP (SupT1-CD19-GFP) after 24 h co-culture with CAR-T cells at a 1:1 effector:target ratio. 100 nM of minocycline was added to relevant wells. Data shows mean percentage (± SD) of live cells compared to non-transduced (NT) T-cell control, n = 4 donors from 1 experiment. Statistical analysis was through a two-way ANOVA with Tukey’s multiple comparisons between each group at 0 nM, or with Šidák’s multiple comparisons within each group ± minocycline. P values = FMC63-Tet-BBz 0 nM versus 100 nM (****, < 0.0001). ( f ) IFN-γ or ( g ) IL-2 release after 24 h of co-culture with SupT1-CD19-GFP at 1:1 E:T ratio. Data shows mean ± SD, n = 4 donors from 1 experiment. Statistical analysis was through a two-way ANOVA with Tukey’s multiple comparisons between FMC63-Tet-BBz and TIP-less-Tet-BBz. P values = FMC63-Tet-BBz 0 nM versus 100 nM (**, 0.0013) and FMC63-Tet-BBz 0 nM versus TIP-less-Tet-BBz 0 nM (***, 0.0001).

Journal: Scientific Reports

Article Title: Tunable control of CAR T cell activity through tetracycline mediated disruption of protein–protein interaction

doi: 10.1038/s41598-021-01418-9

Figure Lengend Snippet: A split CAR associates through the interaction of TetRB and TIP and dissociates upon minocycline addition. ( a ) Overview of the split CAR approach (TetCAR), incorporating the tetracycline repressor protein B (TetRB) and the peptide TIP. Addition of the small molecule antibiotic minocycline reversibly disrupts TetRB-TIP binding, displaces the endodomain and inhibits CAR activation. ( b ) Schematic of the CAR constructs with eGFP endodomains. CARs contain an anti-human CD19 scFv from FMC63, CD8 stalk regions, CD28 transmembrane domains and eGFP endodomain. TetCARs have a TetRB endodomain with eGFP as a separate protein with or without TIP. ( c ) Representative widefield fluorescent images of HEK293T cells transduced with eGFP-tagged CAR structures, ± 100 nM minocycline. ( d ) Schematic of the CAR constructs with 41BB-CD3ζ endodomains. CARs contain an anti-human CD19 scFv from FMC63, with a CD8 stalk and transmembrane domain and 41BB-CD3ζ endodomain. TetCARs have a TetRB endodomain with 41BB-CD3ζ as a separate protein, with or without TIP. E) Killing of SupT1 cells engineered to express CD19 and GFP (SupT1-CD19-GFP) after 24 h co-culture with CAR-T cells at a 1:1 effector:target ratio. 100 nM of minocycline was added to relevant wells. Data shows mean percentage (± SD) of live cells compared to non-transduced (NT) T-cell control, n = 4 donors from 1 experiment. Statistical analysis was through a two-way ANOVA with Tukey’s multiple comparisons between each group at 0 nM, or with Šidák’s multiple comparisons within each group ± minocycline. P values = FMC63-Tet-BBz 0 nM versus 100 nM (****, < 0.0001). ( f ) IFN-γ or ( g ) IL-2 release after 24 h of co-culture with SupT1-CD19-GFP at 1:1 E:T ratio. Data shows mean ± SD, n = 4 donors from 1 experiment. Statistical analysis was through a two-way ANOVA with Tukey’s multiple comparisons between FMC63-Tet-BBz and TIP-less-Tet-BBz. P values = FMC63-Tet-BBz 0 nM versus 100 nM (**, 0.0013) and FMC63-Tet-BBz 0 nM versus TIP-less-Tet-BBz 0 nM (***, 0.0001).

Article Snippet: SupT1 cells were purchased from the European Collection of Authenticated Cell Cultures.

Techniques: Binding Assay, Activation Assay, Construct, Transduction, Co-Culture Assay, Control

Optimization of TetCAR surface expression and signaling. ( a ) Schematic overview of TetCAR constructs containing 41BB-ζ or CD28-ζ endodomains. Antigen recognition is provided by the FMC63 scFv or Fab fragment. ( b ) Transduction efficiency as measured by CD34 staining of the RQR8 marker gene. Data shows mean ± SD, n = 5 donors from 2 independent experiments. ( c ) Median fluorescent intensity (left) and representative histograms (right) of CAR expression on surface of RQR8 + cells as measured by staining with soluble, Fc-tagged CD19 protein. Data shows mean ± SD, n = 3 donors from 1 experiment. Unpaired T tests were used for statistical analysis. P values = FMC63-BBz versus Fab-Tet-BBz (***, 0.0003) or Fab-Tet-28z (***, 0.0002), FMC63-Tet-BBz versus Fab-Tet-BBz (ns, 0.089), FMC63-Tet-28z versus Fab-Tet-28z (*, 0.045). ( d ) Killing of SupT1-CD19-GFP after 24 h co-culture with CAR-T cells at a 1:1 effector:target ratio. 100 nM of minocycline was added to relevant wells. Data shows mean percentage (± SD) of live cells compared to non-transduced (NT) control, n = 5 donors from 2 independent experiments. Statistical analysis was through a two-way ANOVA with Šidák’s multiple comparisons within each group ± minocycline. P values for each construct + /- minocycline were: FMC63-Tet-BBz (**, 0.0023), FMC63-Tet-28z (***, 0.0003) and Fab-Tet-28z (*, 0.0279). ( e ) IFN-γ and ( f ) IL-2 release after 24 h of co-culture with SupT1-CD19-GFP at 1:1 E:T ratio. Data shows mean ± SD, n = 5 donors from 2 independent experiments. Statistical analysis was through two-way ANOVAs between the TetCAR groups at 0 nM minocycline (with Tukey’s multiple comparisons) or within these groups ± minocycline (with Šidák’s multiple comparisons). P values were; between FMC63-Tet-BBz and Fab-Tet-BBz (**, 0.0097, IFN-γ) and between FMC63-Tet-28z and Fab-Tet-28z (****, < 0.0001, IFN-γ and **, 0.0017, IL-2). P values between the TetCAR constructs ± minocycline were: FMC63-Tet-BBz (***, 0.0007, IFN-γ), FMC63-Tet-28z (*, 0.0409, IFN-γ), Fab-Tet-BBz (****, < 0.0001, IFN-γ and *, 0.0152, IL-2) and Fab-Tet-28z (****, < 0.0001 both IFN-γ and IL-2).

Journal: Scientific Reports

Article Title: Tunable control of CAR T cell activity through tetracycline mediated disruption of protein–protein interaction

doi: 10.1038/s41598-021-01418-9

Figure Lengend Snippet: Optimization of TetCAR surface expression and signaling. ( a ) Schematic overview of TetCAR constructs containing 41BB-ζ or CD28-ζ endodomains. Antigen recognition is provided by the FMC63 scFv or Fab fragment. ( b ) Transduction efficiency as measured by CD34 staining of the RQR8 marker gene. Data shows mean ± SD, n = 5 donors from 2 independent experiments. ( c ) Median fluorescent intensity (left) and representative histograms (right) of CAR expression on surface of RQR8 + cells as measured by staining with soluble, Fc-tagged CD19 protein. Data shows mean ± SD, n = 3 donors from 1 experiment. Unpaired T tests were used for statistical analysis. P values = FMC63-BBz versus Fab-Tet-BBz (***, 0.0003) or Fab-Tet-28z (***, 0.0002), FMC63-Tet-BBz versus Fab-Tet-BBz (ns, 0.089), FMC63-Tet-28z versus Fab-Tet-28z (*, 0.045). ( d ) Killing of SupT1-CD19-GFP after 24 h co-culture with CAR-T cells at a 1:1 effector:target ratio. 100 nM of minocycline was added to relevant wells. Data shows mean percentage (± SD) of live cells compared to non-transduced (NT) control, n = 5 donors from 2 independent experiments. Statistical analysis was through a two-way ANOVA with Šidák’s multiple comparisons within each group ± minocycline. P values for each construct + /- minocycline were: FMC63-Tet-BBz (**, 0.0023), FMC63-Tet-28z (***, 0.0003) and Fab-Tet-28z (*, 0.0279). ( e ) IFN-γ and ( f ) IL-2 release after 24 h of co-culture with SupT1-CD19-GFP at 1:1 E:T ratio. Data shows mean ± SD, n = 5 donors from 2 independent experiments. Statistical analysis was through two-way ANOVAs between the TetCAR groups at 0 nM minocycline (with Tukey’s multiple comparisons) or within these groups ± minocycline (with Šidák’s multiple comparisons). P values were; between FMC63-Tet-BBz and Fab-Tet-BBz (**, 0.0097, IFN-γ) and between FMC63-Tet-28z and Fab-Tet-28z (****, < 0.0001, IFN-γ and **, 0.0017, IL-2). P values between the TetCAR constructs ± minocycline were: FMC63-Tet-BBz (***, 0.0007, IFN-γ), FMC63-Tet-28z (*, 0.0409, IFN-γ), Fab-Tet-BBz (****, < 0.0001, IFN-γ and *, 0.0152, IL-2) and Fab-Tet-28z (****, < 0.0001 both IFN-γ and IL-2).

Article Snippet: SupT1 cells were purchased from the European Collection of Authenticated Cell Cultures.

Techniques: Expressing, Construct, Transduction, Staining, Marker, Co-Culture Assay, Control

Reconfiguration of endodomain positions enhances TetCAR function. ( a ) Schematic overview of Fab-TetCAR constructs containing membrane-proximal 41BB or CD28 endodomains, with a TIP-CD3ζ or TIP-41BB-CD3ζ domains. ( b ) Transduction efficiency as measured by CD34 staining of the RQR8 marker gene. Data shows mean ± SD, n = 5 donors from 2 independent experiments. ( c ) Median fluorescent intensity of CAR expression on surface of RQR8 + cells as measured by staining with soluble, Fc-tagged CD19 protein. Data shows mean ± SD, n = 5 donors from 2 experiments. Statistical analysis was through one-way ANOVA between the groups, p values were; between FMC63-BBz and each Fab-TetCAR (****, < 0.0001). Differences between Fab-TetCARs alone were analyzed by one-way ANOVA but were not significant. ( d ) Killing of SupT1-CD19-GFP after 24 h co-culture with CAR-T cells at 1:1 E:T ratio. 100 nM of minocycline was added to relevant wells. Data shows mean ± SD, n = 5 donors from 2 independent experiments. Statistical analysis was through a two-way ANOVA comparing each group ± minocycline (with Šidák’s multiple comparisons). P values were; FMC-Tet-BBz (*, 0.0119). ( e ) IFN-γ and ( f ) IL-2 release after 24 h of co-culture with SupT1-CD19 at 1:1 E:T ratio (± 100 nM minocycline). Data shows mean ± SD, n = 5 donors from 2 independent experiments. Statistical analysis was through a 2-way ANOVA comparing each group ± minocycline (with Šidák’s multiple comparisons). P values for IFN-γ = 28-Tet-z (*, 0.0484), 28BB-Fab-Tet-z and 28BB-Fab-Tet-BBz (****, < 0.0001). P values for IL-2 = 28-Tet-z (*, 0.0150), 28BB-Fab-Tet-z (****, < 0.0001), 28-Fab-Tet-BBz (**, 0.0029) and 28BB-Fab-Tet-BBz (***, 0.0007). ( g ) Killing of NALM6 after 48 h co-culture with CAR-T cells at 1:1 E:T ratio. 100 nM of minocycline was added to relevant wells. Data shows mean ± SD, n = 5 donors from 2 independent experiments. Statistical analysis was through a two-way ANOVA comparing each group ± minocycline (with Šidák’s multiple comparisons). P values were; BB-Fab-Tet-z (**, 0.0083), BB-Fab-Tet-BBz (*, 0.0189) and 28-Fab-Tet-z, 28BB-Fab-Tet-z, 28-Fab-Tet-BBz and 28BB-Fab-Tet-BBz (****, < 0.0001). ( h ) IFN-γ and I) IL-2 release after 48 h of co-culture with NALM6 at 1:1 E:T ratio (± 100 nM minocycline). Data shows mean ± SD, n = 5 donors from 2 independent experiments. Statistical analysis was through a 2-way ANOVA comparing each group ± minocycline (with Šidák’s multiple comparisons). P values for IFN-γ = BB-Fab-Tet-z (*, 0.0433), 28-Fab-Tet-z (*, 0.0387), 28BB-Fab-Tet-z (****, < 0.0001) and 28BB-Fab-Tet-BBz (**, 0.0020). P values for IL-2 = 28BB-Fab-Tet-z (*, 0.0450).

Journal: Scientific Reports

Article Title: Tunable control of CAR T cell activity through tetracycline mediated disruption of protein–protein interaction

doi: 10.1038/s41598-021-01418-9

Figure Lengend Snippet: Reconfiguration of endodomain positions enhances TetCAR function. ( a ) Schematic overview of Fab-TetCAR constructs containing membrane-proximal 41BB or CD28 endodomains, with a TIP-CD3ζ or TIP-41BB-CD3ζ domains. ( b ) Transduction efficiency as measured by CD34 staining of the RQR8 marker gene. Data shows mean ± SD, n = 5 donors from 2 independent experiments. ( c ) Median fluorescent intensity of CAR expression on surface of RQR8 + cells as measured by staining with soluble, Fc-tagged CD19 protein. Data shows mean ± SD, n = 5 donors from 2 experiments. Statistical analysis was through one-way ANOVA between the groups, p values were; between FMC63-BBz and each Fab-TetCAR (****, < 0.0001). Differences between Fab-TetCARs alone were analyzed by one-way ANOVA but were not significant. ( d ) Killing of SupT1-CD19-GFP after 24 h co-culture with CAR-T cells at 1:1 E:T ratio. 100 nM of minocycline was added to relevant wells. Data shows mean ± SD, n = 5 donors from 2 independent experiments. Statistical analysis was through a two-way ANOVA comparing each group ± minocycline (with Šidák’s multiple comparisons). P values were; FMC-Tet-BBz (*, 0.0119). ( e ) IFN-γ and ( f ) IL-2 release after 24 h of co-culture with SupT1-CD19 at 1:1 E:T ratio (± 100 nM minocycline). Data shows mean ± SD, n = 5 donors from 2 independent experiments. Statistical analysis was through a 2-way ANOVA comparing each group ± minocycline (with Šidák’s multiple comparisons). P values for IFN-γ = 28-Tet-z (*, 0.0484), 28BB-Fab-Tet-z and 28BB-Fab-Tet-BBz (****, < 0.0001). P values for IL-2 = 28-Tet-z (*, 0.0150), 28BB-Fab-Tet-z (****, < 0.0001), 28-Fab-Tet-BBz (**, 0.0029) and 28BB-Fab-Tet-BBz (***, 0.0007). ( g ) Killing of NALM6 after 48 h co-culture with CAR-T cells at 1:1 E:T ratio. 100 nM of minocycline was added to relevant wells. Data shows mean ± SD, n = 5 donors from 2 independent experiments. Statistical analysis was through a two-way ANOVA comparing each group ± minocycline (with Šidák’s multiple comparisons). P values were; BB-Fab-Tet-z (**, 0.0083), BB-Fab-Tet-BBz (*, 0.0189) and 28-Fab-Tet-z, 28BB-Fab-Tet-z, 28-Fab-Tet-BBz and 28BB-Fab-Tet-BBz (****, < 0.0001). ( h ) IFN-γ and I) IL-2 release after 48 h of co-culture with NALM6 at 1:1 E:T ratio (± 100 nM minocycline). Data shows mean ± SD, n = 5 donors from 2 independent experiments. Statistical analysis was through a 2-way ANOVA comparing each group ± minocycline (with Šidák’s multiple comparisons). P values for IFN-γ = BB-Fab-Tet-z (*, 0.0433), 28-Fab-Tet-z (*, 0.0387), 28BB-Fab-Tet-z (****, < 0.0001) and 28BB-Fab-Tet-BBz (**, 0.0020). P values for IL-2 = 28BB-Fab-Tet-z (*, 0.0450).

Article Snippet: SupT1 cells were purchased from the European Collection of Authenticated Cell Cultures.

Techniques: Construct, Membrane, Transduction, Staining, Marker, Expressing, Co-Culture Assay

TetCAR activity can be fine-tuned in vitro with a dose-dependent response to minocycline. ( a ) Killing of SupT1-CD19-GFP after 24 h of co-culture with CAR-T cells at 1:4 E:T ratio. A range of minocycline doses from 0.02-1600 nM were added to relevant wells. Data shows mean % of live targets relative to an inert TetCAR control, ± SD. n = 4 donors from 1 experiment. ( b ) IFN-γ and ( c ) IL-2 release after 24 h of co-culture with SupT1-CD19-GFP at various minocycline doses. Data shows mean ± SD, n = 4 donors from 1 experiment. ( d ) IL-2 secretion from FMC63-BBz or 28BB-Fab-Tet-z CARs 1–5 h after co-culture with SupT1-CD19-GFP at a 2:1 E:T ratio. 100 nM minocycline was added to separate wells every hour. Data shows the mean (± SD) secretion of IL-2 at each time-point in groups that received minocycline at the beginning of the experiment, or every hour afterwards. Color coded bars indicate the number of hours that the co-cultures were exposed to minocycline for. n = 4 donors from 2 independent experiments. ( e ) Cytotoxicity or ( f ) IL-2 secretion by 28BB-Fab-Tet-z CARs after coculture with SupT1-CD19 at a 1:1 E:T ratio. Inhibition by minocycline was removed by washing cells with complete media at 48, 24 and 2 h before addition of SupT1-CD19 targets. Wash steps are indicated by “[W]”. Data shows mean (± SD) % of live targets relative to NT T cells ( e ) or mean (± SD) IL-2 secretion ( f ) after 24 h. n = 3 donors from 1 experiment. Statistical analysis was through a one-way ANOVA with multiple comparisons between the 28BB-Fab-Tet-z CAR under different conditions. P values for cytotoxicity ( e ) were: 48 h wash versus no wash (*, 0.0118), 24 h wash versus no wash (**, 0.0050) and no drug versus no wash (**, 0.0044). P values for IL-2 secretion ( f ) were: 48 h wash versus no wash (**, 0.0015), no drug versus no wash (**, 0.0044), no drug versus 2 h wash (*, 0.0103) and 48 h wash versus 2 h wash (**, 0.0033).

Journal: Scientific Reports

Article Title: Tunable control of CAR T cell activity through tetracycline mediated disruption of protein–protein interaction

doi: 10.1038/s41598-021-01418-9

Figure Lengend Snippet: TetCAR activity can be fine-tuned in vitro with a dose-dependent response to minocycline. ( a ) Killing of SupT1-CD19-GFP after 24 h of co-culture with CAR-T cells at 1:4 E:T ratio. A range of minocycline doses from 0.02-1600 nM were added to relevant wells. Data shows mean % of live targets relative to an inert TetCAR control, ± SD. n = 4 donors from 1 experiment. ( b ) IFN-γ and ( c ) IL-2 release after 24 h of co-culture with SupT1-CD19-GFP at various minocycline doses. Data shows mean ± SD, n = 4 donors from 1 experiment. ( d ) IL-2 secretion from FMC63-BBz or 28BB-Fab-Tet-z CARs 1–5 h after co-culture with SupT1-CD19-GFP at a 2:1 E:T ratio. 100 nM minocycline was added to separate wells every hour. Data shows the mean (± SD) secretion of IL-2 at each time-point in groups that received minocycline at the beginning of the experiment, or every hour afterwards. Color coded bars indicate the number of hours that the co-cultures were exposed to minocycline for. n = 4 donors from 2 independent experiments. ( e ) Cytotoxicity or ( f ) IL-2 secretion by 28BB-Fab-Tet-z CARs after coculture with SupT1-CD19 at a 1:1 E:T ratio. Inhibition by minocycline was removed by washing cells with complete media at 48, 24 and 2 h before addition of SupT1-CD19 targets. Wash steps are indicated by “[W]”. Data shows mean (± SD) % of live targets relative to NT T cells ( e ) or mean (± SD) IL-2 secretion ( f ) after 24 h. n = 3 donors from 1 experiment. Statistical analysis was through a one-way ANOVA with multiple comparisons between the 28BB-Fab-Tet-z CAR under different conditions. P values for cytotoxicity ( e ) were: 48 h wash versus no wash (*, 0.0118), 24 h wash versus no wash (**, 0.0050) and no drug versus no wash (**, 0.0044). P values for IL-2 secretion ( f ) were: 48 h wash versus no wash (**, 0.0015), no drug versus no wash (**, 0.0044), no drug versus 2 h wash (*, 0.0103) and 48 h wash versus 2 h wash (**, 0.0033).

Article Snippet: SupT1 cells were purchased from the European Collection of Authenticated Cell Cultures.

Techniques: Activity Assay, In Vitro, Co-Culture Assay, Control, Inhibition

Effector function of CD28-containing TetCAR matches 41BBζ control CAR. ( a ) Killing of SupT1-CD19-GFP after 24 h or NALM6 after 48 h of co-culture with CAR-T cells at 1:1–1:32 E:T ratio. Data shows mean ± SD, n = 4 donors from 2 independent experiments. ( b ) SupT1-CD19, NALM6, Raji or Raji-CD19KO targets were incubated with mitomycin C, then co-cultured with CAR-T cells at 1:2 E:T ratio for 7 days. To relevant wells, 400 nM of minocycline was added on day 0. Graphs show mean (± SD) number of RQR8 + T cells (filled bars) or total CD3 + T cells (white bars) for each target. n = 4 donors (SupT1-CD19, Raji and Raji-CD19KO) or n = 3 (NALM6) from 2 independent experiments. Statistical analysis was through a two-way ANOVA comparing mean RQR8 number in each group ± minocycline (with Šidák’s multiple comparisons). P values for SupT1-CD19 were 28-Fab-Tet-z (**, 0.0028) and 28BB-Fab-Tet-z (**, 0.0050). P values for Raji were 28-Fab-Tet-z (*, 0.0320) and 28BB-Fab-Tet-z (*, 0.0304). ( c ) Mean fluorescent intensity of Tim3 and Lag3 after 7 days coculture with SupT1-CD19 targets, ± 400 nM minocycline. Data shows geometric mean (± SD) in CD3 + T cells. n = 4 donors, from 2 independent experiments. Statistical analysis was through a two-way ANOVA comparing each group ± minocycline (with Šidák’s multiple comparisons). P values for Lag3 expression were 28-Fab-Tet-z (**, 0.0078) and 28BB-Fab-Tet-z (*, 0.0207). ( d ) Percentage of naïve (CD62L + , CD45RA + ), Tcm (central memory; CD62L + , CD45RA - ), Tem (effector memory; CD62L - , CD45RA - ) or Temra (terminally differentiated effector memory; CD62L - , CD45RA + ) memory T cell populations after 7 days coculture with SupT1-CD19 targets, ± 400 nM minocycline. Data shows mean (± SD) in CD3 + T cells. n = 4 donors, from 2 independent experiments.

Journal: Scientific Reports

Article Title: Tunable control of CAR T cell activity through tetracycline mediated disruption of protein–protein interaction

doi: 10.1038/s41598-021-01418-9

Figure Lengend Snippet: Effector function of CD28-containing TetCAR matches 41BBζ control CAR. ( a ) Killing of SupT1-CD19-GFP after 24 h or NALM6 after 48 h of co-culture with CAR-T cells at 1:1–1:32 E:T ratio. Data shows mean ± SD, n = 4 donors from 2 independent experiments. ( b ) SupT1-CD19, NALM6, Raji or Raji-CD19KO targets were incubated with mitomycin C, then co-cultured with CAR-T cells at 1:2 E:T ratio for 7 days. To relevant wells, 400 nM of minocycline was added on day 0. Graphs show mean (± SD) number of RQR8 + T cells (filled bars) or total CD3 + T cells (white bars) for each target. n = 4 donors (SupT1-CD19, Raji and Raji-CD19KO) or n = 3 (NALM6) from 2 independent experiments. Statistical analysis was through a two-way ANOVA comparing mean RQR8 number in each group ± minocycline (with Šidák’s multiple comparisons). P values for SupT1-CD19 were 28-Fab-Tet-z (**, 0.0028) and 28BB-Fab-Tet-z (**, 0.0050). P values for Raji were 28-Fab-Tet-z (*, 0.0320) and 28BB-Fab-Tet-z (*, 0.0304). ( c ) Mean fluorescent intensity of Tim3 and Lag3 after 7 days coculture with SupT1-CD19 targets, ± 400 nM minocycline. Data shows geometric mean (± SD) in CD3 + T cells. n = 4 donors, from 2 independent experiments. Statistical analysis was through a two-way ANOVA comparing each group ± minocycline (with Šidák’s multiple comparisons). P values for Lag3 expression were 28-Fab-Tet-z (**, 0.0078) and 28BB-Fab-Tet-z (*, 0.0207). ( d ) Percentage of naïve (CD62L + , CD45RA + ), Tcm (central memory; CD62L + , CD45RA - ), Tem (effector memory; CD62L - , CD45RA - ) or Temra (terminally differentiated effector memory; CD62L - , CD45RA + ) memory T cell populations after 7 days coculture with SupT1-CD19 targets, ± 400 nM minocycline. Data shows mean (± SD) in CD3 + T cells. n = 4 donors, from 2 independent experiments.

Article Snippet: SupT1 cells were purchased from the European Collection of Authenticated Cell Cultures.

Techniques: Control, Co-Culture Assay, Incubation, Cell Culture, Expressing