human ifn γ detection antibody  (Thermo Fisher)

Journal: Journal for immunotherapy of cancer

Article Title: Utilization of primary tumor samples for cancer neoantigen discovery.

doi: 10.1136/jitc-2024-010993

Figure Lengend Snippet: Figure 2 Example of screening TIL for recognition of cancer neoantigens (patient 4371). (A) The number and distribution of all tumor-specific mutations detected in the primary formalin-fixed, paraffin-embedded sample and three lung metastases resected at the National Cancer Institute. (B) TIL cultures from 24 tumor fragments (F1-24) were co-cultured with autologous DCs that were either pulsed with ten 25-mer peptide pools (PPs) or transfected with corresponding TMGs representing 144 mutations detected in metastatic tumors, as well as 13 mutations detected exclusively in primary tumors. Names of PPs and TMGs containing the latter are underlined. A TMG encoding point mutations from another patient (Irrel. TMG) and DMSO were used as negative controls for TMG and PP testing, respectively. Results of IFN-γ ELISpot are depicted. (C) F13 TIL were co-cultured with DCs that were pulsed with individual peptides contained in PP9; names of genes harboring mutations exclusive to the primary tumors are underlined. Numerical suffixes represent different transcripts of the same genes or, in the case of indels, different overlapping peptides. PMA/ionomycin (PMA) was used as a non-specific positive control. Results of IFN-γ ELISpot and flow cytometric analysis of 4-1BB expression on the cell surface are depicted together. (D) 4-1BB upregulation was analyzed using flow cytometry following an overnight co-culture of F13 TIL with autologous DCs that were pulsed either with PP9 (red dots) or DMSO (black dots). Data was gated on live CD8+ T cells. Cells in the gated area were subjected to single-cell sorting and TCR sequencing. (E) T cells from an unrelated healthy donor were transduced with the PP9-reactive TCR and co-cultured with the patient’s DCs cells that were pulsed with decreasing concentrations of BIRC6P1505T (black squares) and corresponding wildtype peptide (empty squares). The 9-mer BIRC6P1505T peptide was identified from the 25-mer sequence as the minimal epitope in a separate experiment (not shown). IFN-γ production was measured the following day using IFN-γ ELISA. DCs, dendritic cells; DMSO, dimethylsulfoxide; ELISpot, enzyme-linked immunosorbent spot assay; IFN, interferon; PMA, phorbol 12-myristate 13-acetate; TCR, T-cell receptor; TIL, tumor-infiltrating lymphocyte; TMG, tandem minigene.

Article Snippet: The plates were then processed as detailed previously,32 using biotinylated anti- human IFN-γ detection antibody (clone 7- B6- 1) and streptavidin- ALP (both from Mabtech, Cincinnati, Ohio, USA).

Techniques: Immunopeptidomics, Formalin-fixed Paraffin-Embedded, Cell Culture, Transfection, Enzyme-linked Immunospot, Positive Control, Expressing, Flow Cytometry, Co-Culture Assay, FACS, Sequencing, Transduction, Enzyme-linked Immunosorbent Assay, ELISpot Assay

Journal: Journal for immunotherapy of cancer

Article Title: Utilization of primary tumor samples for cancer neoantigen discovery.

doi: 10.1136/jitc-2024-010993

Figure Lengend Snippet: Figure 4 Example of screening PBL-derived memory T cells for recognition of cancer neoantigens (patient 4400). (A) Experimental design. (B) The number and distribution of all tumor-specific mutations detected in the primary FFPE sample and two lung metastases resected at the National Cancer Institute. (C) Memory T cells from PBL were separately stimulated with two PPs (PP-PRIM1 and PP-PRIM2) and a TMG (TMG-PRIM) representing mutations exclusive to primary tumors, along with a control PP and a TMG containing mutations previously recognized by autologous TIL from six tested patients (PP-CTRL and TMG-CTRL). Expanded CD8+ and CD4+ cells were then tested for recognition of B cells that were pulsed or electroporated with corresponding PPs or TMGs. Results of IFN-γ ELISpot are depicted. Sterile water alone (Mock) and DMSO were used as negative controls for TMG and PP testing, respectively. PMA/ionomycin (PMA) was used as a positive control. (D) Reactive cells from (C) were co-cultured with B cells pulsed with individual 25-mer peptides from the three PPs. Results of IFN-γ ELISpot and flow cytometric analysis of surface 4-1BB expression are depicted. (E) 4-1BB upregulation was analyzed using flow cytometry following an overnight co-culture of CD4-PP-PRIM1 cells with autologous B cells that were pulsed either with DDX5R355C or DDX5WT peptide. Peptides used in this and the following assays were high-performance liquid chromatography-purified. Data was gated on live CD4+ T cells. Cells in the shaded areas were subjected to single-cell sorting and TCR sequencing. (F) Results of TCR sequencing performed on the populations from (E). A single TCR, named 4400 TCR1 (CDR3b sequence CASTDPPVNEQYF), was highly enriched in 4-1BB-positive versus negative cells. (G) T cells from an unrelated healthy donor were transduced with TCR1 and were co-cultured with the patient’s DCs pulsed with serial dilutions of either DDX5WT (WT) or DDX5R355C peptide (MUT). IFN-γ concentration was measured in co-culture supernatants by ELISA. Squares represent average reads from duplicate co-culture wells; error bars represent SD. The same procedure was repeated with another healthy donor, yielding consistent results (not shown). Panel (A) was created with www.BioRender.com. DCs, dendritic cells; DMSO, dimethylsulfoxide; ELISpot, enzyme-linked immunosorbent spot assay; FFPE, formalin-fixed, paraffin-embedded; IFN, interferon; PBL, peripheral blood; PMA, phorbol 12-myristate 13-acetate; PP, peptide pool; TCR, T-cell receptor; TIL, tumor infiltrating lymphocyte; TMG, tandem minigene; WT, wild type.

Article Snippet: The plates were then processed as detailed previously,32 using biotinylated anti- human IFN-γ detection antibody (clone 7- B6- 1) and streptavidin- ALP (both from Mabtech, Cincinnati, Ohio, USA).

Techniques: Derivative Assay, Immunopeptidomics, Control, Enzyme-linked Immunospot, Sterility, Positive Control, Cell Culture, Expressing, Flow Cytometry, Co-Culture Assay, High Performance Liquid Chromatography, Purification, FACS, Sequencing, Transduction, Concentration Assay, Enzyme-linked Immunosorbent Assay, ELISpot Assay, Formalin-fixed Paraffin-Embedded

Journal: Journal for Immunotherapy of Cancer

Article Title: Utilization of primary tumor samples for cancer neoantigen discovery

doi: 10.1136/jitc-2024-010993

Figure Lengend Snippet: Example of screening TIL for recognition of cancer neoantigens (patient 4371). ( A ) The number and distribution of all tumor-specific mutations detected in the primary formalin-fixed, paraffin-embedded sample and three lung metastases resected at the National Cancer Institute. ( B ) TIL cultures from 24 tumor fragments (F1-24) were co-cultured with autologous DCs that were either pulsed with ten 25-mer peptide pools (PPs) or transfected with corresponding TMGs representing 144 mutations detected in metastatic tumors, as well as 13 mutations detected exclusively in primary tumors. Names of PPs and TMGs containing the latter are underlined. A TMG encoding point mutations from another patient (Irrel. TMG) and DMSO were used as negative controls for TMG and PP testing, respectively. Results of IFN-γ ELISpot are depicted. ( C ) F13 TIL were co-cultured with DCs that were pulsed with individual peptides contained in PP9; names of genes harboring mutations exclusive to the primary tumors are underlined. Numerical suffixes represent different transcripts of the same genes or, in the case of indels, different overlapping peptides. PMA/ionomycin (PMA) was used as a non-specific positive control. Results of IFN-γ ELISpot and flow cytometric analysis of 4-1BB expression on the cell surface are depicted together. ( D ) 4-1BB upregulation was analyzed using flow cytometry following an overnight co-culture of F13 TIL with autologous DCs that were pulsed either with PP9 (red dots) or DMSO (black dots). Data was gated on live CD8 + T cells. Cells in the gated area were subjected to single-cell sorting and TCR sequencing. ( E ) T cells from an unrelated healthy donor were transduced with the PP9-reactive TCR and co-cultured with the patient’s DCs cells that were pulsed with decreasing concentrations of BIRC6 P1505T (black squares) and corresponding wildtype peptide (empty squares). The 9-mer BIRC6 P1505T peptide was identified from the 25-mer sequence as the minimal epitope in a separate experiment (not shown). IFN-γ production was measured the following day using IFN-γ ELISA. DCs, dendritic cells; DMSO, dimethylsulfoxide; ELISpot, enzyme-linked immunosorbent spot assay; IFN, interferon; PMA, phorbol 12-myristate 13-acetate; TCR, T-cell receptor; TIL, tumor-infiltrating lymphocyte; TMG, tandem minigene.

Article Snippet: The plates were then processed as detailed previously, using biotinylated anti-human IFN-γ detection antibody (clone 7-B6-1) and streptavidin-ALP (both from Mabtech, Cincinnati, Ohio, USA).

Techniques: Immunopeptidomics, Formalin-fixed Paraffin-Embedded, Cell Culture, Transfection, Enzyme-linked Immunospot, Positive Control, Expressing, Flow Cytometry, Co-Culture Assay, FACS, Sequencing, Transduction, Enzyme-linked Immunosorbent Assay, ELISpot Assay

Journal: Journal for Immunotherapy of Cancer

Article Title: Utilization of primary tumor samples for cancer neoantigen discovery

doi: 10.1136/jitc-2024-010993

Figure Lengend Snippet: Example of screening PBL-derived memory T cells for recognition of cancer neoantigens (patient 4400). ( A ) Experimental design. ( B ) The number and distribution of all tumor-specific mutations detected in the primary FFPE sample and two lung metastases resected at the National Cancer Institute. ( C ) Memory T cells from PBL were separately stimulated with two PPs (PP-PRIM1 and PP-PRIM2) and a TMG (TMG-PRIM) representing mutations exclusive to primary tumors, along with a control PP and a TMG containing mutations previously recognized by autologous TIL from six tested patients (PP-CTRL and TMG-CTRL). Expanded CD8 + and CD4 + cells were then tested for recognition of B cells that were pulsed or electroporated with corresponding PPs or TMGs. Results of IFN-γ ELISpot are depicted. Sterile water alone (Mock) and DMSO were used as negative controls for TMG and PP testing, respectively. PMA/ionomycin (PMA) was used as a positive control. ( D ) Reactive cells from ( C ) were co-cultured with B cells pulsed with individual 25-mer peptides from the three PPs. Results of IFN-γ ELISpot and flow cytometric analysis of surface 4-1BB expression are depicted. ( E ) 4-1BB upregulation was analyzed using flow cytometry following an overnight co-culture of CD4-PP-PRIM1 cells with autologous B cells that were pulsed either with DDX5 R355C or DDX5 WT peptide. Peptides used in this and the following assays were high-performance liquid chromatography-purified. Data was gated on live CD4 + T cells. Cells in the shaded areas were subjected to single-cell sorting and TCR sequencing. ( F ) Results of TCR sequencing performed on the populations from ( E ). A single TCR, named 4400 TCR1 (CDR3b sequence CASTDPPVNEQYF), was highly enriched in 4-1BB-positive versus negative cells. ( G ) T cells from an unrelated healthy donor were transduced with TCR1 and were co-cultured with the patient’s DCs pulsed with serial dilutions of either DDX5 WT (WT) or DDX5 R355C peptide (MUT). IFN-γ concentration was measured in co-culture supernatants by ELISA. Squares represent average reads from duplicate co-culture wells; error bars represent SD. The same procedure was repeated with another healthy donor, yielding consistent results (not shown). Panel ( A ) was created with www.BioRender.com . DCs, dendritic cells; DMSO, dimethylsulfoxide; ELISpot, enzyme-linked immunosorbent spot assay; FFPE, formalin-fixed, paraffin-embedded; IFN, interferon; PBL, peripheral blood; PMA, phorbol 12-myristate 13-acetate; PP, peptide pool; TCR, T-cell receptor; TIL, tumor infiltrating lymphocyte; TMG, tandem minigene; WT, wild type.

Article Snippet: The plates were then processed as detailed previously, using biotinylated anti-human IFN-γ detection antibody (clone 7-B6-1) and streptavidin-ALP (both from Mabtech, Cincinnati, Ohio, USA).

Techniques: Derivative Assay, Immunopeptidomics, Control, Enzyme-linked Immunospot, Sterility, Positive Control, Cell Culture, Expressing, Flow Cytometry, Co-Culture Assay, High Performance Liquid Chromatography, Purification, FACS, Sequencing, Transduction, Concentration Assay, Enzyme-linked Immunosorbent Assay, ELISpot Assay, Formalin-fixed Paraffin-Embedded

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Identification of an HLA-A*11:01-restricted neoepitope of mutant PIK3CA and its specific T cell receptors for cancer immunotherapy targeting hotspot driver mutations

doi: 10.1007/s00262-024-03729-y

Figure Lengend Snippet: Identification of HLA-A*11:01-restricted T cell epitopes of mutant PI3Kα. a Stabilization analysis of HLA-A on TAP1-deficient K562 cells expressing HLA-A*11:01, which b median fluorescent intensity (MFI) from three independent experiments. c Naïve CD8 + T cells were isolated from HLA-A*11:01 positive healthy donors (HDs) and stimulated with pE542K and pH1047L peptides. After 2 rounds of stimulation, the response of T cells was determined by IFN-γ ELISpot after pulsed with wild type and mutant peptides. d Tetramer staining showing the percentage of gated tetramer + CD8 + T cells. e Experimental procedure for the isolation of pH1047L-specific T cells from HDs. CD8 + T cells were repetitively stimulated with pH1047L-pulsed DCs. pH1047L-specific T cells were sorted by flow cytometric analysis, and selected TCR clones were sequenced. TCR-T cells were engineered and characterized by cytokine production and cytotoxicity. IFN-γ, interferon gamma; ELISpot, enzyme-linked immunospot. N-P64: SARS-CoV-2 N protein 361-369 (KTFPPTEPK). Differences were tested by student’s t -test ***: p < 0.001; ****: p < 0.0001; ns: denotes not significant

Article Snippet: Subsequently, the plates were incubated with anti-human biotinylated IFN-γ detection antibody (Clone: 7-B6-1, 1 mg/ ml, Mabtech), followed by incubation with Streptavidin-AP (1:1000, Mabtech) and BCIP/NBT-plus substrate (Mabtech).

Techniques: Mutagenesis, Expressing, Isolation, Enzyme-linked Immunospot, Staining, Clone Assay

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Identification of an HLA-A*11:01-restricted neoepitope of mutant PIK3CA and its specific T cell receptors for cancer immunotherapy targeting hotspot driver mutations

doi: 10.1007/s00262-024-03729-y

Figure Lengend Snippet: Validation of pH1047L-specific TCR functionality in primary T cells. Primary CD8 + T cells were transfected on day 2 post-stimulation with TRAC/TRBC knockout (dKO) or not (no KO). Direct comparison of a mTCRβ expression and b tetramer binding rate between primary CD8 + T cells dKO with no KO. Flow cytometric analysis of c the expression and d the percentage of CD137 + pH1047L-specific TCR-T cells after co-cultured with HLA-A*11:01 + K562 cells pulsed with wild type or mutant peptide. DMSO was used as control. e Flow cytometric analysis of dose-dependency of CD137 + cell proportions, pulsed with titrated pH1047L peptide. ELISA of f IFN-γ and g IL-2 release from pH1047L-specific TCR-T cells. Differences were tested by student’s t-test. *: p < 0.05; **: p < 0.01; ****: p < 0.0001

Article Snippet: Subsequently, the plates were incubated with anti-human biotinylated IFN-γ detection antibody (Clone: 7-B6-1, 1 mg/ ml, Mabtech), followed by incubation with Streptavidin-AP (1:1000, Mabtech) and BCIP/NBT-plus substrate (Mabtech).

Techniques: Biomarker Discovery, Transfection, Knock-Out, Comparison, Expressing, Binding Assay, Cell Culture, Mutagenesis, Control, Peptide ELISA

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Identification of an HLA-A*11:01-restricted neoepitope of mutant PIK3CA and its specific T cell receptors for cancer immunotherapy targeting hotspot driver mutations

doi: 10.1007/s00262-024-03729-y

Figure Lengend Snippet: pH1047L-specific TCR-T cells recognize endogenously present PIK3CA H1047L . ( a , b ). Flow cytometric analysis of the percentage of CD137 + dKO-CD8 + T cells following overnight co-culture with K562, MDA-MB-231 and SKOV3 cells expressing PIK3CA H1047L at an effector-to-target (E:T) ratio of 1:1. ELISA of c IFN-γ and d IL-2 release from activated pH1047L-specific TCR-T cells. e The pH1047L-specific TCR1-T cells were co-cultured with HCC827 cancer cells endogenously expressing HLA-A*11:01 overnight. The percentage of CD137 + TCR-T cells was analyzed via flow cytometry. An MHC blocker (clone W6/32) was added to block the TCR–pMHC interaction. Differences were tested by student’s t -test. *: p < 0.05; **: p < 0.01; ***: p < 0.001; ****: p < 0.0001

Article Snippet: Subsequently, the plates were incubated with anti-human biotinylated IFN-γ detection antibody (Clone: 7-B6-1, 1 mg/ ml, Mabtech), followed by incubation with Streptavidin-AP (1:1000, Mabtech) and BCIP/NBT-plus substrate (Mabtech).

Techniques: Co-Culture Assay, Expressing, Enzyme-linked Immunosorbent Assay, Cell Culture, Flow Cytometry, Blocking Assay