Structured Review

Jackson Immuno mouse t cells
IHC staining for FAP in various human tumors, and design and in vitro activity of FAP-reactive CARs. Representative IHC staining for FAP in human melanoma (A), colorectal (B), pancreatic (C), and breast (D) adenocarcinomas. Isotype stains were negative (not depicted). Bars: 400 µm (A); 200 µm (B–D). Schematic of the FAP-reactive CAR constructs FAP5-CAR (E) and Sibro-CAR (F). LS, GM-CSFR leader sequence; V H and V L , variable heavy and light chains; L, 218 linker; CD8, transmembrane domain; CD28, 4-1BB, and CD3-ζ, intracellular signaling domains; m, murine; h, human. Both constructs were cloned into the MSGV1 retroviral vector. Retrovirus containing FAP5-CAR or Sibro-CAR constructs were generated and used to transduce mouse and human T cells, respectively, and flow cytometry was used to assess transduction efficiency at day 2 after transduction for FAP5-CAR (G) and day 8–10 after transduction for Sibro-CAR (H). Solid line is isotype control and filled histogram is FAP5 or Sibrotuzumab stained. Day 5-stimulated untransduced (UnTd) and FAP5-CAR–transduced (Td) <t>mouse</t> T cells were assessed for reactivity against plate-bound BSA, α-CD3 mAb, and recombinant human FAP (r-huFAP), and against HEK293 cell lines expressing or not expressing FAP. After an overnight stimulation, supernatants were assessed for IFN-γ with an IFN-γ ELISA (I), and cells were further assessed for cell surface CD107a expression, and production of IFN-γ and TNF by ICS (J). For ICS, cells are gated on FAP5-CAR Td cells. Day ∼14-stimulated UnTd or Sibro-CAR Td human T cells were assessed for in vitro reactivity as described for mouse. IFN-γ ELISA (K), and ICS results gated on Sibro-CAR Td T cells (L) are shown. Mean ± SD. All results are representative of at least three independent experiments.
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1) Product Images from "Immune targeting of fibroblast activation protein triggers recognition of multipotent bone marrow stromal cells and cachexia"

Article Title: Immune targeting of fibroblast activation protein triggers recognition of multipotent bone marrow stromal cells and cachexia

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20130110

IHC staining for FAP in various human tumors, and design and in vitro activity of FAP-reactive CARs. Representative IHC staining for FAP in human melanoma (A), colorectal (B), pancreatic (C), and breast (D) adenocarcinomas. Isotype stains were negative (not depicted). Bars: 400 µm (A); 200 µm (B–D). Schematic of the FAP-reactive CAR constructs FAP5-CAR (E) and Sibro-CAR (F). LS, GM-CSFR leader sequence; V H and V L , variable heavy and light chains; L, 218 linker; CD8, transmembrane domain; CD28, 4-1BB, and CD3-ζ, intracellular signaling domains; m, murine; h, human. Both constructs were cloned into the MSGV1 retroviral vector. Retrovirus containing FAP5-CAR or Sibro-CAR constructs were generated and used to transduce mouse and human T cells, respectively, and flow cytometry was used to assess transduction efficiency at day 2 after transduction for FAP5-CAR (G) and day 8–10 after transduction for Sibro-CAR (H). Solid line is isotype control and filled histogram is FAP5 or Sibrotuzumab stained. Day 5-stimulated untransduced (UnTd) and FAP5-CAR–transduced (Td) mouse T cells were assessed for reactivity against plate-bound BSA, α-CD3 mAb, and recombinant human FAP (r-huFAP), and against HEK293 cell lines expressing or not expressing FAP. After an overnight stimulation, supernatants were assessed for IFN-γ with an IFN-γ ELISA (I), and cells were further assessed for cell surface CD107a expression, and production of IFN-γ and TNF by ICS (J). For ICS, cells are gated on FAP5-CAR Td cells. Day ∼14-stimulated UnTd or Sibro-CAR Td human T cells were assessed for in vitro reactivity as described for mouse. IFN-γ ELISA (K), and ICS results gated on Sibro-CAR Td T cells (L) are shown. Mean ± SD. All results are representative of at least three independent experiments.
Figure Legend Snippet: IHC staining for FAP in various human tumors, and design and in vitro activity of FAP-reactive CARs. Representative IHC staining for FAP in human melanoma (A), colorectal (B), pancreatic (C), and breast (D) adenocarcinomas. Isotype stains were negative (not depicted). Bars: 400 µm (A); 200 µm (B–D). Schematic of the FAP-reactive CAR constructs FAP5-CAR (E) and Sibro-CAR (F). LS, GM-CSFR leader sequence; V H and V L , variable heavy and light chains; L, 218 linker; CD8, transmembrane domain; CD28, 4-1BB, and CD3-ζ, intracellular signaling domains; m, murine; h, human. Both constructs were cloned into the MSGV1 retroviral vector. Retrovirus containing FAP5-CAR or Sibro-CAR constructs were generated and used to transduce mouse and human T cells, respectively, and flow cytometry was used to assess transduction efficiency at day 2 after transduction for FAP5-CAR (G) and day 8–10 after transduction for Sibro-CAR (H). Solid line is isotype control and filled histogram is FAP5 or Sibrotuzumab stained. Day 5-stimulated untransduced (UnTd) and FAP5-CAR–transduced (Td) mouse T cells were assessed for reactivity against plate-bound BSA, α-CD3 mAb, and recombinant human FAP (r-huFAP), and against HEK293 cell lines expressing or not expressing FAP. After an overnight stimulation, supernatants were assessed for IFN-γ with an IFN-γ ELISA (I), and cells were further assessed for cell surface CD107a expression, and production of IFN-γ and TNF by ICS (J). For ICS, cells are gated on FAP5-CAR Td cells. Day ∼14-stimulated UnTd or Sibro-CAR Td human T cells were assessed for in vitro reactivity as described for mouse. IFN-γ ELISA (K), and ICS results gated on Sibro-CAR Td T cells (L) are shown. Mean ± SD. All results are representative of at least three independent experiments.

Techniques Used: Immunohistochemistry, Staining, In Vitro, Activity Assay, Construct, Sequencing, Clone Assay, Plasmid Preparation, Generated, Transduction, Flow Cytometry, Cytometry, Recombinant, Expressing, Enzyme-linked Immunosorbent Assay

2) Product Images from "The Bacterial Enzyme IdeS Cleaves the IgG-Type of B Cell Receptor (BCR), Abolishes BCR-Mediated Cell Signaling, and Inhibits Memory B Cell Activation"

Article Title: The Bacterial Enzyme IdeS Cleaves the IgG-Type of B Cell Receptor (BCR), Abolishes BCR-Mediated Cell Signaling, and Inhibits Memory B Cell Activation

Journal: The Journal of Immunology Author Choice

doi: 10.4049/jimmunol.1501929

IdeS cleaves IgG-type, but not IgM-type, of BCR on B cells. ( A ) Flow cytometry analysis of cells stained with biotinylated anti-Fab Ab, followed by streptavidin-allophycocyanin, after treatment of Nu-DUL-1 cells (IgG-type) and Daudi cells (IgM-type) with
Figure Legend Snippet: IdeS cleaves IgG-type, but not IgM-type, of BCR on B cells. ( A ) Flow cytometry analysis of cells stained with biotinylated anti-Fab Ab, followed by streptavidin-allophycocyanin, after treatment of Nu-DUL-1 cells (IgG-type) and Daudi cells (IgM-type) with

Techniques Used: Flow Cytometry, Cytometry, Staining

3) Product Images from "Preclinical Assessment of CAR T-Cell Therapy Targeting the Tumor Antigen 5T4 in Ovarian Cancer"

Article Title: Preclinical Assessment of CAR T-Cell Therapy Targeting the Tumor Antigen 5T4 in Ovarian Cancer

Journal: Journal of Immunotherapy (Hagerstown, Md. : 1997)

doi: 10.1097/CJI.0000000000000203

IFNγ production by 5T4 CAR T cells in response to immortalized ovarian cell lines expressing 5T4 and autologous tumor cells. Peripheral T cells were successfully transduced from 11 patients. T cells were transduced with the H8-CAR or 2E4-CAR or no CAR (Mock) vector. T cells (1×10 5 ) were co-cultured for 24 hours with 1×10 5 SKOV-3, OVCAR-3 (A) and primary autologous tumor cells (B). After 24 hours, supernatant was collected and IFNγ quantitified by enzyme-linked immunosorbent assay. Error bars represent the mean and SD of triplicate results. Two-way analysis of variance with Sidak’s correction; * P
Figure Legend Snippet: IFNγ production by 5T4 CAR T cells in response to immortalized ovarian cell lines expressing 5T4 and autologous tumor cells. Peripheral T cells were successfully transduced from 11 patients. T cells were transduced with the H8-CAR or 2E4-CAR or no CAR (Mock) vector. T cells (1×10 5 ) were co-cultured for 24 hours with 1×10 5 SKOV-3, OVCAR-3 (A) and primary autologous tumor cells (B). After 24 hours, supernatant was collected and IFNγ quantitified by enzyme-linked immunosorbent assay. Error bars represent the mean and SD of triplicate results. Two-way analysis of variance with Sidak’s correction; * P

Techniques Used: Expressing, Transduction, Plasmid Preparation, Cell Culture, Enzyme-linked Immunosorbent Assay

Comparison of higher versus lower affinity CAR constructs. NSG mice were inoculated with ovarian cancer cell lines on day 0. Seven days later mice were treated with either the higher affinity H8-CAR or the lower affinity 2E4-CAR T cells. Kaplan-Meier survival curves of NSG mice bearing SKOV-3 tumors and treated with 1×10 7 CAR T 5T4 cells. Log-rank (Mantel-Cox) test; * P
Figure Legend Snippet: Comparison of higher versus lower affinity CAR constructs. NSG mice were inoculated with ovarian cancer cell lines on day 0. Seven days later mice were treated with either the higher affinity H8-CAR or the lower affinity 2E4-CAR T cells. Kaplan-Meier survival curves of NSG mice bearing SKOV-3 tumors and treated with 1×10 7 CAR T 5T4 cells. Log-rank (Mantel-Cox) test; * P

Techniques Used: Construct, Mouse Assay

Dose escalation of 5T4 CAR T cells in NSG ovarian cancer model. NSG mice were challenged with 2.5×10 6 SKOV-3 tumor cells on day 0 and 7 days later were treated with either ascending doses of H8-CAR T cells or saline. A, In-life bioluminescence images of NSG mice treated with ascending doses of H8-CAR T cells are shown over time alongside control animals. B, Kaplan-Meier survival curves of NSG mice receiving ascending doses of H8-CAR T cells. CAR indicates chimeric antigen receptor.
Figure Legend Snippet: Dose escalation of 5T4 CAR T cells in NSG ovarian cancer model. NSG mice were challenged with 2.5×10 6 SKOV-3 tumor cells on day 0 and 7 days later were treated with either ascending doses of H8-CAR T cells or saline. A, In-life bioluminescence images of NSG mice treated with ascending doses of H8-CAR T cells are shown over time alongside control animals. B, Kaplan-Meier survival curves of NSG mice receiving ascending doses of H8-CAR T cells. CAR indicates chimeric antigen receptor.

Techniques Used: Mouse Assay

4) Product Images from "Preclinical Assessment of CAR T-Cell Therapy Targeting the Tumor Antigen 5T4 in Ovarian Cancer"

Article Title: Preclinical Assessment of CAR T-Cell Therapy Targeting the Tumor Antigen 5T4 in Ovarian Cancer

Journal: Journal of Immunotherapy (Hagerstown, Md. : 1997)

doi: 10.1097/CJI.0000000000000203

IFNγ production by 5T4 CAR T cells in response to immortalized ovarian cell lines expressing 5T4 and autologous tumor cells. Peripheral T cells were successfully transduced from 11 patients. T cells were transduced with the H8-CAR or 2E4-CAR or no CAR (Mock) vector. T cells (1×10 5 ) were co-cultured for 24 hours with 1×10 5 SKOV-3, OVCAR-3 (A) and primary autologous tumor cells (B). After 24 hours, supernatant was collected and IFNγ quantitified by enzyme-linked immunosorbent assay. Error bars represent the mean and SD of triplicate results. Two-way analysis of variance with Sidak’s correction; * P
Figure Legend Snippet: IFNγ production by 5T4 CAR T cells in response to immortalized ovarian cell lines expressing 5T4 and autologous tumor cells. Peripheral T cells were successfully transduced from 11 patients. T cells were transduced with the H8-CAR or 2E4-CAR or no CAR (Mock) vector. T cells (1×10 5 ) were co-cultured for 24 hours with 1×10 5 SKOV-3, OVCAR-3 (A) and primary autologous tumor cells (B). After 24 hours, supernatant was collected and IFNγ quantitified by enzyme-linked immunosorbent assay. Error bars represent the mean and SD of triplicate results. Two-way analysis of variance with Sidak’s correction; * P

Techniques Used: Expressing, Transduction, Plasmid Preparation, Cell Culture, Enzyme-linked Immunosorbent Assay

Comparison of higher versus lower affinity CAR constructs. NSG mice were inoculated with ovarian cancer cell lines on day 0. Seven days later mice were treated with either the higher affinity H8-CAR or the lower affinity 2E4-CAR T cells. Kaplan-Meier survival curves of NSG mice bearing SKOV-3 tumors and treated with 1×10 7 CAR T 5T4 cells. Log-rank (Mantel-Cox) test; * P
Figure Legend Snippet: Comparison of higher versus lower affinity CAR constructs. NSG mice were inoculated with ovarian cancer cell lines on day 0. Seven days later mice were treated with either the higher affinity H8-CAR or the lower affinity 2E4-CAR T cells. Kaplan-Meier survival curves of NSG mice bearing SKOV-3 tumors and treated with 1×10 7 CAR T 5T4 cells. Log-rank (Mantel-Cox) test; * P

Techniques Used: Construct, Mouse Assay

5T4 CAR construct and transduction efficiency. A, Anti-5T4 CAR construct shown in the integrated form. B, Percentage of CD3 T cells from healthy donors and patients transduced with H8-CAR and 2E4-CAR. C, Percentage of patient-derived and healthy donor-derived CD4 and CD8 T cells transduced with H8-CAR and 2E4-CAR. The Student t test, * P
Figure Legend Snippet: 5T4 CAR construct and transduction efficiency. A, Anti-5T4 CAR construct shown in the integrated form. B, Percentage of CD3 T cells from healthy donors and patients transduced with H8-CAR and 2E4-CAR. C, Percentage of patient-derived and healthy donor-derived CD4 and CD8 T cells transduced with H8-CAR and 2E4-CAR. The Student t test, * P

Techniques Used: Construct, Transduction, Derivative Assay

Dose escalation of 5T4 CAR T cells in NSG ovarian cancer model. NSG mice were challenged with 2.5×10 6 SKOV-3 tumor cells on day 0 and 7 days later were treated with either ascending doses of H8-CAR T cells or saline. A, In-life bioluminescence images of NSG mice treated with ascending doses of H8-CAR T cells are shown over time alongside control animals. B, Kaplan-Meier survival curves of NSG mice receiving ascending doses of H8-CAR T cells. CAR indicates chimeric antigen receptor.
Figure Legend Snippet: Dose escalation of 5T4 CAR T cells in NSG ovarian cancer model. NSG mice were challenged with 2.5×10 6 SKOV-3 tumor cells on day 0 and 7 days later were treated with either ascending doses of H8-CAR T cells or saline. A, In-life bioluminescence images of NSG mice treated with ascending doses of H8-CAR T cells are shown over time alongside control animals. B, Kaplan-Meier survival curves of NSG mice receiving ascending doses of H8-CAR T cells. CAR indicates chimeric antigen receptor.

Techniques Used: Mouse Assay

5) Product Images from "The Syk-binding Ubiquitin Ligase c-Cbl Mediates Signaling-dependent B Cell Receptor Ubiquitination and B Cell Receptor-mediated Antigen Processing and Presentation *"

Article Title: The Syk-binding Ubiquitin Ligase c-Cbl Mediates Signaling-dependent B Cell Receptor Ubiquitination and B Cell Receptor-mediated Antigen Processing and Presentation *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M112.357640

c-Cbl mediates Ag·BCR ubiquitination and processing. Upper panel , B cells were pulsed with anti-human IgM-btn for the indicated time (minutes) at 37 °C. The cells were lysed and ubiquitinated ligand-BCR complexes isolated by ubiquitin
Figure Legend Snippet: c-Cbl mediates Ag·BCR ubiquitination and processing. Upper panel , B cells were pulsed with anti-human IgM-btn for the indicated time (minutes) at 37 °C. The cells were lysed and ubiquitinated ligand-BCR complexes isolated by ubiquitin

Techniques Used: Isolation

6) Product Images from "Immune targeting of fibroblast activation protein triggers recognition of multipotent bone marrow stromal cells and cachexia"

Article Title: Immune targeting of fibroblast activation protein triggers recognition of multipotent bone marrow stromal cells and cachexia

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20130110

Murine and human multipotent BMSCs express FAP and are recognized by T cells expressing FAP-reactive CARs. Passage-5 in vitro–expanded murine BMSCs were stained with antibodies specific for Sca-1, PDGFR-α, and FAP, and assessed by flow cytometry (A). “Q” represents quadrant. Solid lines are isotype controls and filled histograms are FAP stained. UnTd or FAP5-CAR Td T cells were cultured overnight with murine BMSCs and supernatants were assessed for IFN-γ by ELISA (B), and cells were further analyzed for expression of CD107a and production of IFN-γ and TNF by ICS (C). Mean ± SD. Data are representative of two independent experiments. Flow cytometric phenotype of in vitro–expanded human BMSCs derived from three different donors (D). BMSCs from D were stained with the FAP-specific monoclonal antibodies Sibrotuzumab (E) and FAP5 (F) and assessed by flow cytometry. Solid lines are isotype or secondary antibody controls and filled histograms are FAP or Sibrotuzumab stained. UnTd or Sibro-CAR Td T cells were cultured overnight with BMSCs and the supernatants assessed for IFN-γ by ELISA (G), and cells were further analyzed for CD107a expression and IFN-γ and TNF production by ICS (H). Mean ± SD. Similar results were seen with two additional T cell donors.
Figure Legend Snippet: Murine and human multipotent BMSCs express FAP and are recognized by T cells expressing FAP-reactive CARs. Passage-5 in vitro–expanded murine BMSCs were stained with antibodies specific for Sca-1, PDGFR-α, and FAP, and assessed by flow cytometry (A). “Q” represents quadrant. Solid lines are isotype controls and filled histograms are FAP stained. UnTd or FAP5-CAR Td T cells were cultured overnight with murine BMSCs and supernatants were assessed for IFN-γ by ELISA (B), and cells were further analyzed for expression of CD107a and production of IFN-γ and TNF by ICS (C). Mean ± SD. Data are representative of two independent experiments. Flow cytometric phenotype of in vitro–expanded human BMSCs derived from three different donors (D). BMSCs from D were stained with the FAP-specific monoclonal antibodies Sibrotuzumab (E) and FAP5 (F) and assessed by flow cytometry. Solid lines are isotype or secondary antibody controls and filled histograms are FAP or Sibrotuzumab stained. UnTd or Sibro-CAR Td T cells were cultured overnight with BMSCs and the supernatants assessed for IFN-γ by ELISA (G), and cells were further analyzed for CD107a expression and IFN-γ and TNF production by ICS (H). Mean ± SD. Similar results were seen with two additional T cell donors.

Techniques Used: Expressing, In Vitro, Staining, Flow Cytometry, Cytometry, Cell Culture, Enzyme-linked Immunosorbent Assay, Derivative Assay

Expression of FAP on freshly isolated murine BMSCs from OS cells. BM (A) and OS cells (B) were isolated from untreated wild-type C57BL/6 mice and stained with antibodies against CD45, TER119, Sca-1, PDGFR-α, and FAP, followed by flow cytometry analysis. CD45 + /TER119 + cells demarcate hematopoietic and erythroid lineage cells (Lin + ). Expression of FAP in various populations of OS cells stained with antibodies specific for Sca-1 and PDGFR-α (B). Irradiated non–tumor-bearing mice were treated with 2 × 10 7 UnTd or FAP5-CAR Td T cells and, 7 d later, OS cells were isolated and analyzed as in B. Expression of FAP on various OS cell populations isolated from mice treated with UnTd (C) or FAP5-CAR Td (D) T cells is shown. (E) Mean fluorescence intensity (MFI) of FAP in the various Sca-1 and PDGFR-α subsets found in OS cells. All data are gated on live, single cells. Data for C–E are further gated on Lin − (CD45 − /TER119 − ) cells. Solid lines are isotype controls and filled histograms are FAP5 stained. All data are representative of at least two independent experiments.
Figure Legend Snippet: Expression of FAP on freshly isolated murine BMSCs from OS cells. BM (A) and OS cells (B) were isolated from untreated wild-type C57BL/6 mice and stained with antibodies against CD45, TER119, Sca-1, PDGFR-α, and FAP, followed by flow cytometry analysis. CD45 + /TER119 + cells demarcate hematopoietic and erythroid lineage cells (Lin + ). Expression of FAP in various populations of OS cells stained with antibodies specific for Sca-1 and PDGFR-α (B). Irradiated non–tumor-bearing mice were treated with 2 × 10 7 UnTd or FAP5-CAR Td T cells and, 7 d later, OS cells were isolated and analyzed as in B. Expression of FAP on various OS cell populations isolated from mice treated with UnTd (C) or FAP5-CAR Td (D) T cells is shown. (E) Mean fluorescence intensity (MFI) of FAP in the various Sca-1 and PDGFR-α subsets found in OS cells. All data are gated on live, single cells. Data for C–E are further gated on Lin − (CD45 − /TER119 − ) cells. Solid lines are isotype controls and filled histograms are FAP5 stained. All data are representative of at least two independent experiments.

Techniques Used: Expressing, Isolation, Mouse Assay, Staining, Flow Cytometry, Cytometry, Irradiation, Fluorescence

Bone toxicity and cachexia in mice treated with FAP5-CAR T cells. Irradiated mice were treated with 2 × 10 7 UnTd or FAP5-CAR Td T cells, and 7 d later subjected to a comprehensive necropsy. H E-stained cross section of the femurs from mice treated with UnTd (A) or FAP5-CAR Td (B) T cells. Bars, 400 µm. Femurs and tibias of irradiated and nonirradiated mice that did not undergo adoptive cell transfer (No Tx) or that underwent adoptive transfer with UnTd or FAP5-CAR Td T cells were harvested at day 7 (2 mice pooled per group), and BM (C) and OS (D) cells were isolated and live cells quantitated. Mean ± SD. Data are the average number of cells isolated from the femurs and tibiae of one mouse, and are representative of at least two independent experiments. *, P
Figure Legend Snippet: Bone toxicity and cachexia in mice treated with FAP5-CAR T cells. Irradiated mice were treated with 2 × 10 7 UnTd or FAP5-CAR Td T cells, and 7 d later subjected to a comprehensive necropsy. H E-stained cross section of the femurs from mice treated with UnTd (A) or FAP5-CAR Td (B) T cells. Bars, 400 µm. Femurs and tibias of irradiated and nonirradiated mice that did not undergo adoptive cell transfer (No Tx) or that underwent adoptive transfer with UnTd or FAP5-CAR Td T cells were harvested at day 7 (2 mice pooled per group), and BM (C) and OS (D) cells were isolated and live cells quantitated. Mean ± SD. Data are the average number of cells isolated from the femurs and tibiae of one mouse, and are representative of at least two independent experiments. *, P

Techniques Used: Mouse Assay, Irradiation, Staining, Adoptive Transfer Assay, Isolation

IHC staining for FAP in various human tumors, and design and in vitro activity of FAP-reactive CARs. Representative IHC staining for FAP in human melanoma (A), colorectal (B), pancreatic (C), and breast (D) adenocarcinomas. Isotype stains were negative (not depicted). Bars: 400 µm (A); 200 µm (B–D). Schematic of the FAP-reactive CAR constructs FAP5-CAR (E) and Sibro-CAR (F). LS, GM-CSFR leader sequence; V H and V L , variable heavy and light chains; L, 218 linker; CD8, transmembrane domain; CD28, 4-1BB, and CD3-ζ, intracellular signaling domains; m, murine; h, human. Both constructs were cloned into the MSGV1 retroviral vector. Retrovirus containing FAP5-CAR or Sibro-CAR constructs were generated and used to transduce mouse and human T cells, respectively, and flow cytometry was used to assess transduction efficiency at day 2 after transduction for FAP5-CAR (G) and day 8–10 after transduction for Sibro-CAR (H). Solid line is isotype control and filled histogram is FAP5 or Sibrotuzumab stained. Day 5-stimulated untransduced (UnTd) and FAP5-CAR–transduced (Td) mouse T cells were assessed for reactivity against plate-bound BSA, α-CD3 mAb, and recombinant human FAP (r-huFAP), and against HEK293 cell lines expressing or not expressing FAP. After an overnight stimulation, supernatants were assessed for IFN-γ with an IFN-γ ELISA (I), and cells were further assessed for cell surface CD107a expression, and production of IFN-γ and TNF by ICS (J). For ICS, cells are gated on FAP5-CAR Td cells. Day ∼14-stimulated UnTd or Sibro-CAR Td human T cells were assessed for in vitro reactivity as described for mouse. IFN-γ ELISA (K), and ICS results gated on Sibro-CAR Td T cells (L) are shown. Mean ± SD. All results are representative of at least three independent experiments.
Figure Legend Snippet: IHC staining for FAP in various human tumors, and design and in vitro activity of FAP-reactive CARs. Representative IHC staining for FAP in human melanoma (A), colorectal (B), pancreatic (C), and breast (D) adenocarcinomas. Isotype stains were negative (not depicted). Bars: 400 µm (A); 200 µm (B–D). Schematic of the FAP-reactive CAR constructs FAP5-CAR (E) and Sibro-CAR (F). LS, GM-CSFR leader sequence; V H and V L , variable heavy and light chains; L, 218 linker; CD8, transmembrane domain; CD28, 4-1BB, and CD3-ζ, intracellular signaling domains; m, murine; h, human. Both constructs were cloned into the MSGV1 retroviral vector. Retrovirus containing FAP5-CAR or Sibro-CAR constructs were generated and used to transduce mouse and human T cells, respectively, and flow cytometry was used to assess transduction efficiency at day 2 after transduction for FAP5-CAR (G) and day 8–10 after transduction for Sibro-CAR (H). Solid line is isotype control and filled histogram is FAP5 or Sibrotuzumab stained. Day 5-stimulated untransduced (UnTd) and FAP5-CAR–transduced (Td) mouse T cells were assessed for reactivity against plate-bound BSA, α-CD3 mAb, and recombinant human FAP (r-huFAP), and against HEK293 cell lines expressing or not expressing FAP. After an overnight stimulation, supernatants were assessed for IFN-γ with an IFN-γ ELISA (I), and cells were further assessed for cell surface CD107a expression, and production of IFN-γ and TNF by ICS (J). For ICS, cells are gated on FAP5-CAR Td cells. Day ∼14-stimulated UnTd or Sibro-CAR Td human T cells were assessed for in vitro reactivity as described for mouse. IFN-γ ELISA (K), and ICS results gated on Sibro-CAR Td T cells (L) are shown. Mean ± SD. All results are representative of at least three independent experiments.

Techniques Used: Immunohistochemistry, Staining, In Vitro, Activity Assay, Construct, Sequencing, Clone Assay, Plasmid Preparation, Generated, Transduction, Flow Cytometry, Cytometry, Recombinant, Expressing, Enzyme-linked Immunosorbent Assay

FAP expression in mouse tumors, and in vivo activity of FAP5-CAR–transduced T cells against various murine tumors. In vitro cultured B16, MC38, MC17-51, 4T1, CT26, and Renca murine tumors were assessed for FAP expression by flow cytometry with the FAP-specific antibody FAP5 (A). Solid line is isotype control and filled histogram is FAP5 stained. Results are representative of at least two independent experiments. Established (∼11–16 d) subcutaneously implanted B16 (B), MC38 (C), MC17-51 (D), 4T1 (E), CT26 (F), and Renca (G) tumors were harvested from mice (irradiated before harvest) and assessed for FAP expression by IHC using biotinylated-FAP5 antibody. Bars, 400 µm. Representative of at least two independent experiments. C57BL/6 mice bearing established B16 (H), MC38 (I), MC17-51 (J) tumors, and BALB/c mice bearing established 4T1 (K), CT26 (L), or Renca (M) tumors were left untreated (No Tx) or treated with 10 7 UnTd or 10 7 FAP5-CAR Td T cells, and the perpendicular diameters of the tumors were measured over time. Mean ± SEM. Results are representative of at least two independent experiments for H–J and one experiment for K–M with initially five mice per group. *, P
Figure Legend Snippet: FAP expression in mouse tumors, and in vivo activity of FAP5-CAR–transduced T cells against various murine tumors. In vitro cultured B16, MC38, MC17-51, 4T1, CT26, and Renca murine tumors were assessed for FAP expression by flow cytometry with the FAP-specific antibody FAP5 (A). Solid line is isotype control and filled histogram is FAP5 stained. Results are representative of at least two independent experiments. Established (∼11–16 d) subcutaneously implanted B16 (B), MC38 (C), MC17-51 (D), 4T1 (E), CT26 (F), and Renca (G) tumors were harvested from mice (irradiated before harvest) and assessed for FAP expression by IHC using biotinylated-FAP5 antibody. Bars, 400 µm. Representative of at least two independent experiments. C57BL/6 mice bearing established B16 (H), MC38 (I), MC17-51 (J) tumors, and BALB/c mice bearing established 4T1 (K), CT26 (L), or Renca (M) tumors were left untreated (No Tx) or treated with 10 7 UnTd or 10 7 FAP5-CAR Td T cells, and the perpendicular diameters of the tumors were measured over time. Mean ± SEM. Results are representative of at least two independent experiments for H–J and one experiment for K–M with initially five mice per group. *, P

Techniques Used: Expressing, In Vivo, Activity Assay, In Vitro, Cell Culture, Flow Cytometry, Cytometry, Staining, Mouse Assay, Irradiation, Immunohistochemistry

7) Product Images from "The Bacterial Enzyme IdeS Cleaves the IgG-Type of B Cell Receptor (BCR), Abolishes BCR-Mediated Cell Signaling, and Inhibits Memory B Cell Activation"

Article Title: The Bacterial Enzyme IdeS Cleaves the IgG-Type of B Cell Receptor (BCR), Abolishes BCR-Mediated Cell Signaling, and Inhibits Memory B Cell Activation

Journal: The Journal of Immunology Author Choice

doi: 10.4049/jimmunol.1501929

IdeS cleaves IgG-type, but not IgM-type, of BCR on B cells. ( A ) Flow cytometry analysis of cells stained with biotinylated anti-Fab Ab, followed by streptavidin-allophycocyanin, after treatment of Nu-DUL-1 cells (IgG-type) and Daudi cells (IgM-type) with
Figure Legend Snippet: IdeS cleaves IgG-type, but not IgM-type, of BCR on B cells. ( A ) Flow cytometry analysis of cells stained with biotinylated anti-Fab Ab, followed by streptavidin-allophycocyanin, after treatment of Nu-DUL-1 cells (IgG-type) and Daudi cells (IgM-type) with

Techniques Used: Flow Cytometry, Cytometry, Staining

8) Product Images from "The Bacterial Enzyme IdeS Cleaves the IgG-Type of B Cell Receptor (BCR), Abolishes BCR-Mediated Cell Signaling, and Inhibits Memory B Cell Activation"

Article Title: The Bacterial Enzyme IdeS Cleaves the IgG-Type of B Cell Receptor (BCR), Abolishes BCR-Mediated Cell Signaling, and Inhibits Memory B Cell Activation

Journal: The Journal of Immunology Author Choice

doi: 10.4049/jimmunol.1501929

IdeS cleaves IgG-type, but not IgM-type, of BCR on B cells. ( A ) Flow cytometry analysis of cells stained with biotinylated anti-Fab Ab, followed by streptavidin-allophycocyanin, after treatment of Nu-DUL-1 cells (IgG-type) and Daudi cells (IgM-type) with
Figure Legend Snippet: IdeS cleaves IgG-type, but not IgM-type, of BCR on B cells. ( A ) Flow cytometry analysis of cells stained with biotinylated anti-Fab Ab, followed by streptavidin-allophycocyanin, after treatment of Nu-DUL-1 cells (IgG-type) and Daudi cells (IgM-type) with

Techniques Used: Flow Cytometry, Cytometry, Staining

9) Product Images from "The Bacterial Enzyme IdeS Cleaves the IgG-Type of B Cell Receptor (BCR), Abolishes BCR-Mediated Cell Signaling, and Inhibits Memory B Cell Activation"

Article Title: The Bacterial Enzyme IdeS Cleaves the IgG-Type of B Cell Receptor (BCR), Abolishes BCR-Mediated Cell Signaling, and Inhibits Memory B Cell Activation

Journal: The Journal of Immunology Author Choice

doi: 10.4049/jimmunol.1501929

IdeS cleaves IgG-type, but not IgM-type, of BCR on B cells. ( A ) Flow cytometry analysis of cells stained with biotinylated anti-Fab Ab, followed by streptavidin-allophycocyanin, after treatment of Nu-DUL-1 cells (IgG-type) and Daudi cells (IgM-type) with
Figure Legend Snippet: IdeS cleaves IgG-type, but not IgM-type, of BCR on B cells. ( A ) Flow cytometry analysis of cells stained with biotinylated anti-Fab Ab, followed by streptavidin-allophycocyanin, after treatment of Nu-DUL-1 cells (IgG-type) and Daudi cells (IgM-type) with

Techniques Used: Flow Cytometry, Cytometry, Staining

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Staining:

Article Title: Disruption of PD-1 Enhanced the Anti-tumor Activity of Chimeric Antigen Receptor T Cells Against Hepatocellular Carcinoma
Article Snippet: .. The CAR expression was evaluated by the biotinylated goat anti-human Fab antibody (109-066-006, Jackson ImmunoResearch, United States), followed by PE-conjugated streptavidin (12-4317-87, eBioscience, United States) staining, if not specifically indicated. .. For the intracellular Akt and phospho-Akt (Ser473 ) analysis, CAR T cells were first stained by the biotinylated goat anti-human Fab antibody and FITC-conjugated streptavidin (11-4317-87, eBioscience, United States) on ice after the CAR T cells harvested from the 48-h coculture of GPC3-CAR T and PLC/PRF/5 cells at a ratio of 1:1, and then, the cells were fixed, permeabilized, and stained using the antibodies of an anti-Akt mouse mAb (2920S), an anti-phospho-Akt (Ser473 ) rabbit mAb (4060S), a mouse mAb IgG1 isotype control (5415S) and Rabbit mAb IgG isotype control (3900S) (all from Cell Signaling Technology, United States) according to the manufacturer’s protocol, followed by PE-conjugated secondary antibodies of anti-mouse IgG (8887S, Cell Signaling Technology, United States) and anti-rabbit IgG (8885S, Cell Signaling Technology, United States).

Article Title: The Bacterial Enzyme IdeS Cleaves the IgG-Type of B Cell Receptor (BCR), Abolishes BCR-Mediated Cell Signaling, and Inhibits Memory B Cell Activation
Article Snippet: .. Cells were stained with biotinylated Abs specific for the F(ab′)2 portion (Jackson ImmunoResearch, cat. no. 109-066-097; cross-reacts with L chain present in all Ig subclasses) and the Fc portion of IgG (Jackson ImmunoResearch, cat. no. 109-066-098; specific for IgG H chain) or IgM (GM-80A, ICL). .. Streptavidin-allophycocyanin (Jackson ImmunoResearch, cat. no. 016-130-084) was used to monitor cells in FL4 using an Accuri C6 flow cytometer.

Article Title: The Bacterial Enzyme IdeS Cleaves the IgG-Type of B Cell Receptor (BCR), Abolishes BCR-Mediated Cell Signaling, and Inhibits Memory B Cell Activation
Article Snippet: .. Cells were stained with a biotinylated Ab specific for the F(ab′)2 portion (cat. no. 109-066-097), followed by streptavidin-allophycocyanin (cat. no. 016-130-084; both from Jackson ImmunoResearch), and the cells were monitored in FL4 using an Accuri C6 flow cytometer. .. Peripheral blood was collected in heparin tubes (BD Vacutainer; cat. no. 367876) from healthy volunteers and treated with either 30 μg/ml IdeS or PBS for 1 h at 37°C prior to isolating PBMCs using density gradient separation (Ficoll-Paque PLUS).

Flow Cytometry:

Article Title: The Bacterial Enzyme IdeS Cleaves the IgG-Type of B Cell Receptor (BCR), Abolishes BCR-Mediated Cell Signaling, and Inhibits Memory B Cell Activation
Article Snippet: .. Cells were stained with a biotinylated Ab specific for the F(ab′)2 portion (cat. no. 109-066-097), followed by streptavidin-allophycocyanin (cat. no. 016-130-084; both from Jackson ImmunoResearch), and the cells were monitored in FL4 using an Accuri C6 flow cytometer. .. Peripheral blood was collected in heparin tubes (BD Vacutainer; cat. no. 367876) from healthy volunteers and treated with either 30 μg/ml IdeS or PBS for 1 h at 37°C prior to isolating PBMCs using density gradient separation (Ficoll-Paque PLUS).

Cytometry:

Article Title: The Bacterial Enzyme IdeS Cleaves the IgG-Type of B Cell Receptor (BCR), Abolishes BCR-Mediated Cell Signaling, and Inhibits Memory B Cell Activation
Article Snippet: .. Cells were stained with a biotinylated Ab specific for the F(ab′)2 portion (cat. no. 109-066-097), followed by streptavidin-allophycocyanin (cat. no. 016-130-084; both from Jackson ImmunoResearch), and the cells were monitored in FL4 using an Accuri C6 flow cytometer. .. Peripheral blood was collected in heparin tubes (BD Vacutainer; cat. no. 367876) from healthy volunteers and treated with either 30 μg/ml IdeS or PBS for 1 h at 37°C prior to isolating PBMCs using density gradient separation (Ficoll-Paque PLUS).

Expressing:

Article Title: Disruption of PD-1 Enhanced the Anti-tumor Activity of Chimeric Antigen Receptor T Cells Against Hepatocellular Carcinoma
Article Snippet: .. The CAR expression was evaluated by the biotinylated goat anti-human Fab antibody (109-066-006, Jackson ImmunoResearch, United States), followed by PE-conjugated streptavidin (12-4317-87, eBioscience, United States) staining, if not specifically indicated. .. For the intracellular Akt and phospho-Akt (Ser473 ) analysis, CAR T cells were first stained by the biotinylated goat anti-human Fab antibody and FITC-conjugated streptavidin (11-4317-87, eBioscience, United States) on ice after the CAR T cells harvested from the 48-h coculture of GPC3-CAR T and PLC/PRF/5 cells at a ratio of 1:1, and then, the cells were fixed, permeabilized, and stained using the antibodies of an anti-Akt mouse mAb (2920S), an anti-phospho-Akt (Ser473 ) rabbit mAb (4060S), a mouse mAb IgG1 isotype control (5415S) and Rabbit mAb IgG isotype control (3900S) (all from Cell Signaling Technology, United States) according to the manufacturer’s protocol, followed by PE-conjugated secondary antibodies of anti-mouse IgG (8887S, Cell Signaling Technology, United States) and anti-rabbit IgG (8885S, Cell Signaling Technology, United States).

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    IdeS cleaves IgG-type, but not IgM-type, of BCR on B cells. ( A ) Flow cytometry analysis of cells stained with biotinylated anti-Fab Ab, followed by <t>streptavidin-allophycocyanin,</t> after treatment of Nu-DUL-1 cells (IgG-type) and Daudi cells (IgM-type) with
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    IdeS cleaves IgG-type, but not IgM-type, of BCR on B cells. ( A ) Flow cytometry analysis of cells stained with biotinylated anti-Fab Ab, followed by streptavidin-allophycocyanin, after treatment of Nu-DUL-1 cells (IgG-type) and Daudi cells (IgM-type) with

    Journal: The Journal of Immunology Author Choice

    Article Title: The Bacterial Enzyme IdeS Cleaves the IgG-Type of B Cell Receptor (BCR), Abolishes BCR-Mediated Cell Signaling, and Inhibits Memory B Cell Activation

    doi: 10.4049/jimmunol.1501929

    Figure Lengend Snippet: IdeS cleaves IgG-type, but not IgM-type, of BCR on B cells. ( A ) Flow cytometry analysis of cells stained with biotinylated anti-Fab Ab, followed by streptavidin-allophycocyanin, after treatment of Nu-DUL-1 cells (IgG-type) and Daudi cells (IgM-type) with

    Article Snippet: Cells were stained with a biotinylated Ab specific for the F(ab′)2 portion (cat. no. 109-066-097), followed by streptavidin-allophycocyanin (cat. no. 016-130-084; both from Jackson ImmunoResearch), and the cells were monitored in FL4 using an Accuri C6 flow cytometer.

    Techniques: Flow Cytometry, Cytometry, Staining

    IdeS cleaves IgG-type, but not IgM-type, of BCR on B cells. ( A ) Flow cytometry analysis of cells stained with biotinylated anti-Fab Ab, followed by streptavidin-allophycocyanin, after treatment of Nu-DUL-1 cells (IgG-type) and Daudi cells (IgM-type) with

    Journal: The Journal of Immunology Author Choice

    Article Title: The Bacterial Enzyme IdeS Cleaves the IgG-Type of B Cell Receptor (BCR), Abolishes BCR-Mediated Cell Signaling, and Inhibits Memory B Cell Activation

    doi: 10.4049/jimmunol.1501929

    Figure Lengend Snippet: IdeS cleaves IgG-type, but not IgM-type, of BCR on B cells. ( A ) Flow cytometry analysis of cells stained with biotinylated anti-Fab Ab, followed by streptavidin-allophycocyanin, after treatment of Nu-DUL-1 cells (IgG-type) and Daudi cells (IgM-type) with

    Article Snippet: A total of 10 μg/ml biotinylated anti–CH1-IgG (BAC, cat. no. 7103202100) was used for detection of the F(ab′)2 portion of IgG, and 0.5 μg/ml biotinylated goat anti-human Fc-specific F(ab′)2 fragment (Jackson ImmunoResearch, cat. no. 109-066-098) was used for detection of the Fc portion of IgG.

    Techniques: Flow Cytometry, Cytometry, Staining