Peptide-based CD8+ T cell assay

RNEU_420–429 (PDSLRDLSVF) and the negative control peptide LCMV NP_118–126 (RPQASGVYM) were produced in the Johns Hopkins Biosynthesis and Sequence Facility at a purity >95%. Antibodies used for flow cytometry studies were: anti-CD8-FITC (BD Biosciences), anti-CD8-PE (BD Biosciences), anti-CD8-PErCP (BD Biosciences), anti-CD8-APC (BD Biosciences), anti-galectin-3-AF647 (Biolegend), anti-galectin-3-PE (R&D), anti-PD1-PE (eBioscience), anti-LAG-3-PE (eBioscience), anti-Thy1.2-PerCP (Biolegend), anti-CD44-Pacific Blue (eBioscience), anti-CD11b-PE (BD Biosciences), anti-CD11c-FITC (BD Biosciences), anti-B220-APC (BD Biosciences), anti-Ly6C-PerCP-Cy5.5 (eBioscience), anti-IFNγ–PE (BD Biosciences), anti-IFNγ-Pacific Blue (eBioscience), and anti-Granzyme B-APC (BD Biosciences). Cellular division was assessed by labeling of high-avidity neu-specific CD8⁺ T cells with 1.5 μM CellTrace CFSE cell proliferation kit (Invitrogen) prior to adoptive transfer (8 (link)). Permeability was assessed with LIVE/DEAD Fixable Aqua Dead Cell Stain (Invitrogen). Antibody staining was conducted at 4°C for 20 minutes in FACS buffer (PBS, 5%FBS, 0.02% NaAzide).

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Kouo T., Huang L., Pucsek A.B., Cao M., Solt S., Armstrong T, & Jaffee E. (2015). Galectin-3 shapes antitumor immune responses by suppressing CD8+ T cells via LAG-3 and inhibiting expansion of plasmacytoid dendritic cells. Cancer immunology research, 3(4), 412-423.

Publication 2015

anti-CD8-FITC anti-CD8-PE anti-CD8-PerCP anti-CD8-APC anti-PD1-PE anti-LAG-3-PE anti-Thy1.2-PerCP anti-CD11b-PE anti-CD11c-FITC anti-B220-APC anti-Ly6C-PerCP-Cy5.5 anti-IFNγ–PE anti-IFNγ-Pacific Blue anti-Granzyme B-APC CellTrace CFSE cell proliferation kit LIVE/DEAD Fixable Aqua Dead Cell Stain

Adoptive transfer Af647 Anti thy1 Antibodies Antibody Biosynthesis Buffer Cd11b Cd44 Cd8 t cells Cell Cell proliferation Cellular division Cfse Cy5 5 Fitc Flow cytometry Galectin 3 Granzyme b Ifnγ Lcmv np118 126 Peptide Permeability Stain

Corresponding Organization :

Other organizations : Johns Hopkins University, Johns Hopkins Medicine

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Variable analysis

independent variables

RNEU_420–429 (PDSLRDLSVF) peptide
LCMV NP_118–126 (RPQASGVYM) peptide (negative control)

dependent variables

Cellular division of high-avidity neu-specific CD8+ T cells
Permeability assessed with LIVE/DEAD Fixable Aqua Dead Cell Stain

control variables

Antibody staining conducted at 4°C for 20 minutes in FACS buffer (PBS, 5%FBS, 0.02% NaAzide)
High-avidity neu-specific CD8+ T cells labeled with 1.5 μM CellTrace CFSE cell proliferation kit prior to adoptive transfer

controls

Positive control: RNEU_420–429 (PDSLRDLSVF) peptide
Negative control: LCMV NP_118–126 (RPQASGVYM) peptide

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