p815 mastocytoma cells  (ATCC)

Journal: Computational and Structural Biotechnology Journal

Article Title: The Abundance of α-Chain-Centric TCRs in the Mouse Repertoire of Primarily Activated Effectors and Reactivated Memory T Cells

doi: 10.34133/csbj.0026

Figure Lengend Snippet: The experimental design. A schematic presentation of the workflow for the generation of primarily activated effectors (EF) (A) and reactivated memory T cells (EM) (B). (A) The spleen was recovered from naïve C57BL/6 mice (the MHC haplotype H-2 b ), and spleen cells were cultured in vitro with P815 mastocytoma cells (the MHC haplotype H-2 d ). T cells that recognized allogeneic MHC class I/peptide complexes expanded and generated a pool of primarily activated effectors. (B) Naïve C57BL/6 mice were immunized with P815 mastocytoma. Due to a mismatch in MHC class I molecules between recipient mice (K b D b ) and tumor cells (K d D d ), P815 was rejected in mice, and a pool of long-lived CD44 + CD62L − CD8 + memory T cells formed. Two months after in vivo immunization, the spleen was recovered from the P815-immunized mice, and memory T cells were rechallenged with the same P815 tumor. This resulted in the clonal expansion of reactivated memory T cells specific to tumor alloantigens.

Article Snippet: Murine mastocytoma P815 (K d D d ) [TIB-64, American Type Culture Collection (ATCC)], P815-GFP, lymphoma EL4 (K b D b ) (TIB-39, ATCC), and EL4-GFP were obtained from the collection of N.N.

Techniques: Cell Culture, In Vitro, Generated, In Vivo

Journal: Computational and Structural Biotechnology Journal

Article Title: The Abundance of α-Chain-Centric TCRs in the Mouse Repertoire of Primarily Activated Effectors and Reactivated Memory T Cells

doi: 10.34133/csbj.0026

Figure Lengend Snippet: T cells transduced with the individual α-chain TCR from the repertoires of primarily activated effectors and reactivated memory cells responded to P815 alloantigens in vitro. Twenty α-chain TCRs (TCRα) selected from the repertoire of primarily activated effectors (EF) or reactivated memory T cells (EM) were transduced into T cells from intact C57BL/6 mice. TCRα-modified T cells were then cultured with mitomycin C (MitC)-treated cognate allogeneic mastocytoma P815. Transduced T cells were similarly cultured alone or with MitC-treated syngeneic lymphoma EL4 to evaluate the background proliferation. Nontransduced (NTR) T cells and GFP-modified T cells, similarly cultured in MLTC, were used as negative controls. The proliferative response (OD) in MLTC of T cells transduced with TCRα from the EF repertoire (A) or the EM repertoire (B) was evaluated after 72 h. The data of one of 2 representative experiments were shown as mean ± SD for 3 technical repeats. One-way ANOVA corrected with Tukey’s multiple comparison.

Article Snippet: Murine mastocytoma P815 (K d D d ) [TIB-64, American Type Culture Collection (ATCC)], P815-GFP, lymphoma EL4 (K b D b ) (TIB-39, ATCC), and EL4-GFP were obtained from the collection of N.N.

Techniques: Transduction, In Vitro, Modification, Cell Culture, Comparison

Journal: Computational and Structural Biotechnology Journal

Article Title: The Abundance of α-Chain-Centric TCRs in the Mouse Repertoire of Primarily Activated Effectors and Reactivated Memory T Cells

doi: 10.34133/csbj.0026

Figure Lengend Snippet: T cells transduced with the dominant-active α-chain TCRs killed cognate tumor cells in vitro. T cells of intact C57BL/6 mice (H-2 b ) were modified with identified dominant-active TCRα from the repertoire of primarily activated effectors (EF) or reactivated memory T cells (EM) (Figs. and ). Transduced T cells were mixed with the allogeneic mastocytoma P815-GFP (K d D d ) at a ratio of 2:1. Nontransduced (NTR) T cells were used as the control. Cytotoxic activity of transduced T cells was analyzed by flow cytometry after 24 h of culturing by evaluating the percentage of dead P815 tumor cells as GFP + PI + cells. Data from 2 to 4 independent experiments are shown as mean ± SD. * P < 0.05, ** P < 0.01 versus the NTR control, unpaired Student’s t test.

Article Snippet: Murine mastocytoma P815 (K d D d ) [TIB-64, American Type Culture Collection (ATCC)], P815-GFP, lymphoma EL4 (K b D b ) (TIB-39, ATCC), and EL4-GFP were obtained from the collection of N.N.

Techniques: Transduction, In Vitro, Modification, Control, Activity Assay, Flow Cytometry

Journal: Computational and Structural Biotechnology Journal

Article Title: The Abundance of α-Chain-Centric TCRs in the Mouse Repertoire of Primarily Activated Effectors and Reactivated Memory T Cells

doi: 10.34133/csbj.0026

Figure Lengend Snippet: T cells transduced with the dominant-active α-chain TCRs killed cognate tumor cells in vitro. T cells of intact C57BL/6 mice (H-2 b ) were modified with identified dominant-active TCRα from the repertoire of primarily activated effectors (EF) or reactivated memory T cells (EM) (Figs. and ). Transduced T cells were mixed with the allogeneic mastocytoma P815-GFP (K d D d ) at a ratio of 2:1. Nontransduced (NTR) T cells were used as the control. Cytotoxic activity of transduced T cells was analyzed by flow cytometry after 24 h of culturing by evaluating the percentage of dead P815 tumor cells as GFP + PI + cells. Data from 2 to 4 independent experiments are shown as mean ± SD. * P < 0.05, ** P < 0.01 versus the NTR control, unpaired Student’s t test.

Article Snippet: Murine mastocytoma P815 (K d D d ) [TIB-64, American Type Culture Collection (ATCC)], P815-GFP, lymphoma EL4 (K b D b ) (TIB-39, ATCC), and EL4-GFP were obtained from the collection of N.N.

Techniques: Transduction, In Vitro, Modification, Control, Activity Assay, Flow Cytometry

Journal: Science Advances

Article Title: Unconventional CD8 + T cell surveillance of cytomegalovirus via Qa-1/HLA-E–restricted epitope recognition

doi: 10.1126/sciadv.aea8707

Figure Lengend Snippet: ( A ) Schematic of the bioinformatics workflow used to generate the MCMV peptide library listed in table S1. ( B ) P815 cells were pulsed with peptides (non–Qa-1 binder M45 and known binders Qdm, FL9, and M-SL9), and Qa-1 staining was performed. Histograms are representative of eight independent experiments (gating strategy available in fig. S1). ( C ) Normalized Qa-1 antibody binding on the surface of P815 cells following incubation with each indicated peptide or dimethyl sulfoxide (DMSO) control. Peptides derived from the same MCMV protein are denoted by the first letter(s) of the peptide sequence in parentheses following the viral protein name. Data are pooled from eight independent experiments, n = 3 to 8 per group. Data are presented as the means ± standard error of the mean (SEM). Statistical significance was determined using a one-way analysis of variance (ANOVA), and significant differences are * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001. ( D ) Peptide sequence schematic highlighting anchor positions between known Qa-1 binders and MCMV peptides. Solid lines indicate the same or extremely similar amino acid (AA) usage, while dotted lines indicate somewhat similar AA usage, based on biochemical properties. The strongest m59 peptide, m59(RL), is indicated as the two m59 peptides differed by one AA.

Article Snippet: P815 [American Type Culture Collection (ATCC) no. TIB-64, RRID:CVCL_2154] and K562 (ATCC no. CCL-243, RRID:CVCL_0004) cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM)/high glucose with l -glutamine (4 mM), glucose (4500 mg/liter), and sodium pyruvate (Thermo Fisher Scientific no. SH30243.01).

Techniques: Staining, Binding Assay, Incubation, Control, Derivative Assay, Sequencing

Journal: Science Advances

Article Title: Unconventional CD8 + T cell surveillance of cytomegalovirus via Qa-1/HLA-E–restricted epitope recognition

doi: 10.1126/sciadv.aea8707

Figure Lengend Snippet: ( A ) Normalized Qa-1 antibody binding on the surface of P815 cells or ( B ) HLA-E expression on the surface of K562-HLA-E cells following incubation with each indicated peptide or DMSO control. Data are pooled from (A) 11 or (B) 5 independent experiments, n = 3 to 11 per condition. Data are presented as the means ± SEM. Statistical significance was determined using a one-way ANOVA, and significant differences are denoted by ** P < 0.01 or **** P < 0.0001. ( C ) AlphaFold3 was used to model Qa-1-peptide complexes with HLA-E binding peptides that bound to Qa-1 in (A). ( D ) AlphaFold3 was used to model HLA-E-peptide complexes with Qa-1 binding peptides that bound to HLA-E in (B). ( E ) AlphaFold3 was used to model m139 in the binding groove of HLA-E:01*03. pLDDT score calculations are presented for each peptide, and the peptide residues are colored blue when the score was >90 (very high confidence), cyan when the score was 70 to 90 (confident prediction), yellow when the score was 50 to 70 (low confidence, indicating possible disorder or flexibility), or orange when the score was <50 (very low confidence).

Article Snippet: P815 [American Type Culture Collection (ATCC) no. TIB-64, RRID:CVCL_2154] and K562 (ATCC no. CCL-243, RRID:CVCL_0004) cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM)/high glucose with l -glutamine (4 mM), glucose (4500 mg/liter), and sodium pyruvate (Thermo Fisher Scientific no. SH30243.01).

Techniques: Binding Assay, Expressing, Incubation, Control