Journal: bioRxiv

Article Title: Inducing an oxidized redox-balance improves anti-tumor CD8 + T cell function

doi: 10.1101/2023.03.27.533229

Figure Lengend Snippet: ( A ) CD8 + T cells isolated from punch biopsies of adjacent skin, tumor-margin or -center were assessed for mitochondrial content using MitoTracker Green n = 15 biological independent samples. ( B ) analysis of p38 phosphorylation in CD8 + T cells isolated from matched adjacent and tumor margin skin, ( C ) CD3 + tumor infiltrating T cells were isolated from KDSC tumors, expanded by stimulation with αCD2/3/28 tetramers, followed by restimulation with αCD2/3/28 tetramers in the absence or presence of DMNQ (1 μM) for 24 hours. ( D ) Representative flowcytometry contour plot showing p-p38 T180/Y182 and IFN-γ in restimulated TILs treated with or without DMNQ. ( E ) Quantification of p-p38 T180/Y182 and ( F ) IFN-γ expression in restimulated CD8 + TILs pre-treated with or without DMNQ. Data in panels A-F are presented as mean ± SEM and statistical significance is determined by paired t -test. See also Figure S5.

Article Snippet: Next, cells were restimulated with αCD2/3/28 tetramers (STEMCELL Technologies) in RPMI containing 10% FCS and 200U/ml rhIL-2 for 5 hours.

Techniques: Isolation, Expressing

Journal: STAR Protocols

Article Title: Screening self-peptides for recognition by mouse alloreactive CD8 + T cells using direct ex vivo multimer staining

doi: 10.1016/j.xpro.2022.101943

Figure Lengend Snippet:

Article Snippet: ∗QuickSwitchTM Quant H-2 Kb Tetramer Kit-PE , MBL International , Cat#TB-7400-K1.

Techniques: Purification, Virus, Plasmid Preparation, Recombinant, Saline, Quantitation Assay, Enzyme-linked Immunosorbent Assay, Software, Adhesive, Sterility, Cytometry

Journal: PLoS ONE

Article Title: Co-Stimulation through 4-1BB/CD137 Improves the Expansion and Function of CD8 + Melanoma Tumor-Infiltrating Lymphocytes for Adoptive T-Cell Therapy

doi: 10.1371/journal.pone.0060031

Figure Lengend Snippet: Melanoma TIL from HLA-A0201 + patients with a significant population of CD8 + MART-1 tetramer + cells were rapidly expanded with or without anti-4-1BB as before. The post-REP TIL were sorted by FACS for CD8 + T cells and assayed for tumor antigen-specific CTL activity using a flow-cytometry-based assay that measures caspase-3 cleavage in target cells. The results of three different patient TIL lines are shown. The top panels ( A ) show the CTL activity of TIL #2292 using the melanoma cell line 624 (HLA-A0201 + ) and the control HLA-A-unmatched line 938 as targets (left side), or MART-1 peptide-pulsed T2 target cells as targets (right side). The bottom panels ( B ) show the CTL activity of two other HLA-A0201 + TIL lines (#2276 and #2122) against 624 or 938 cells with similar results. In all cases ( A and B ) the levels of non-specific killing were markedly lower.

Article Snippet: After 7 days, the cells were stained for CD8 and MART-1 tetramer and analyzed for cell division using a FACSCanto II (BD Biosciences).

Techniques: Activity Assay, Flow Cytometry

Journal: PLoS ONE

Article Title: Co-Stimulation through 4-1BB/CD137 Improves the Expansion and Function of CD8 + Melanoma Tumor-Infiltrating Lymphocytes for Adoptive T-Cell Therapy

doi: 10.1371/journal.pone.0060031

Figure Lengend Snippet: TIL from HLA-A0201 + patients were rapidly expanded with or without added anti-4-1BB antibody. The cells were labeled with eFluor670 dye and re-stimulated with HLA-A0201 + MART-1 peptide-pulsed DC, as previously described . The post-REP TIL were labeled with the cell division dye eFluor670 (Invitrogen) and re-stimulated with HLA-A0201-matched mature DC pulsed with MART-1 peptide for 7 days. The cells were harvested and stained for CD8 and MART-1 tetramer and analyzed by flow cytometry for the frequency of divided (eFluor670 low ) in the gated CD8 + MART-1 tetramer + subset. One representative experiment out of two is shown. As shown in A , CD8 + TIL isolated from 4-1BB costimulated REP cultures (#2183) had an enhanced response to MART-1 peptide re-stimulation, as shown by the increased number of cell divisions measured by eFluor670 dilution in the CD8 + MART-1 tetramer + gated cells. TIL isolated from REP cultures that received anti-4-1BB also exhibited an increase in the fold expansion of CD8 + MART-1 tetramer + cells following re-stimulation of the post-REP TIL with MART-1 peptide pulsed DC as shown in experiments with two separate TIL lines (#2183 and #2559) ( B ).

Article Snippet: After 7 days, the cells were stained for CD8 and MART-1 tetramer and analyzed for cell division using a FACSCanto II (BD Biosciences).

Techniques: Labeling, Staining, Flow Cytometry, Isolation