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6 protocols using fitc anti cd107a

1

Intracellular Cytokine Profiling of Splenocytes

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For intracellular cytokine staining, 2 × 106 splenocytes were stimulated in the presence of protein transport inhibitor, GolgiStopTM GolgiPlugTM (BD Bioscience) with the same peptide pools as the ELISpots. Media alone and phorbol 12-myristate 13-acetate (PMA) and ionomycin stimulations (BD Bioscience) were used as negative and positive controls respectively. To test for degranulation of cells, FITC anti-CD107a (Biolegend) antibody was also added during stimulation. After 6 h, cells were washed and stained with LIVE/DEAD violet. Surface staining was then added containing BV510 anti-CD4, APC-Cy7 anti-CD8, BV711 anti-CD62L, and AF700 anti-CD44 (Biolegend). After 30 min incubation, cells were spun, washed, and fixed using the CytoPerm CytoWash kit following the manufacturer’s protocol (BD Bioscience). Intracellular staining was then prepared using APC anti-IFNγ, BV650 anti-TNFα, PE-Cy7 anti-IL-2, and PE-Cy5 anti-CD3 (Biolegend). All data were collected on a modified LSRII flow cytometer (BD Bioscience) with FACS DIVA 6.0 GUI followed by analysis with FlowJo software 9.0 (BD Bioscience).
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2

PBMC Activation and Cytokine Analysis

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Peripheral blood mononuclear cells (PBMC) were isolated over a density
gradient using Ficoll-Paque Plus (GE Healthcare). Isolated PBMC were washed in
phosphate buffered saline and incubated for 5 hours with FITC-anti-CD107a
(Biolegend) and Protein Transport Inhibitor (eBioscience) in RPMI1640 as above
with or without 50ng/mL phorbol 12-myristate 13-acetate (PMA), and 1μ
g/mL ionomycin (Sigma-Aldrich). After incubation the cells were washed and
stained with APC-Cy7-CD8 (clone HIT8a), PerCP-Cy5.5-CD19 (clone HIB19),
PerCP-Cy5.5-HLA-DR (clone L243), and Zombie Red (Biolegend). The cells were then
washed, fixed and permeabilized using the BD Biosience Cytoperm/Cytofix kit per
the manufacture’s protocol, and stained with BV421-IFNγ (clone
4S.B3, Biolegend). Flow cytometric analysis was analyzed as above. A total of 4
patients from the Ipilimumab only group and 3 from Ipilimumab plus ATRA were
included in this analysis. There was insufficient sample from one of the
Ipilimumab plus ATRA treated patients and the T cell response assay was not
performed.
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3

Intracellular Cytokine Staining of Splenocytes

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For intracellular cytokine staining, 2x10 6 splenocytes were stimulated in the presence of protein transport inhibitor, GolgiStop TM GolgiPlug TM (BD Bioscience) with the same peptide pools as the ELISpots.
Media alone and phorbol 12-myristate 13-acetate (PMA) and ionomycin stimulations (BD Bioscience) were used as negative and positive controls respectively. To test for degranulation of cells, FITC anti-CD107a (Biolegend) antibody was also added during stimulation. After 6hrs, cells were washed and stained with LIVE/DEAD violet. Surface staining was then added containing BV510 anti-CD4, APC-Cy7 anti-CD8, BV711 anti-CD62L and AF700 anti-CD44 (Biolegend). After 30 minutes incubation, cells were spun, washed, and fixed using the CytoPerm CytoWash kit following manufacturer's protocol (BD Bioscience).
Intracellular staining was then prepared using APC anti-IFNγ, BV650 anti-TNFα, PE-Cy7 anti-IL2, and PE-Cy5 anti-CD3 (Biolegend). All data was collected on a modified LSRII flow cytometer (BD Bioscience) followed by analysis with FlowJo software (BD Bioscience).
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4

Quantifying CAR-T Cell Cytotoxicity

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An E:T ratio of 1:1 was used when co‐culturing CD44v6 CAR‐T (or NT) cells with target cells in complete RPMI 1640 media containing anti‐CD107a‐FITC (Biolegend, USA) and protein transport inhibitor (BD Bioscience, USA) for 4 h. CD3‐APC/Cy7(Biolegend, USA), CD4‐APC (Biolegend, USA) and CD8‐BV605 (BD Bioscience, USA) were used to stain the harvested cells.
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5

Activated T cell Immunophenotyping in Mice

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Adequate single cells were isolated from the spleen, peripheral blood, colon adjacent tissues, and adenoma tissues of mice. These cells were incubated with the appropriate surface antibodies: anti-CD45-PE/Cy7 (Biolegend, Cat# 103113), anti-CD3-PE/Cy7 (Biolegend, Cat# 100219), anti-CD4-FITC (Invitrogen, 11-0041-85), anti-CD8a-APC (Invitrogen, 47-0081-82), anti-CD11b-FITC (Biolegend, Cat# 101206), anti-Gr-1-PE (Biolegend, Cat# 108408), anti-CD107a-FITC (Biolegend, Cat# 121605), and anti-CD137-APC (Biolegend, Cat# 106109). For intracellular staining, cells were treated with the Fixation/Permeabilization solution (Invitrogen, 00-5223-56) overnight at 4 °C before incubating with intracellular antibodies: anti-IFNγ-PE (Biolegend, Cat# 163503). CD4+ T cells were defined as CD45+CD4+CD8a; CD8+ T cells were defined as CD45+CD4CD8a+; activated CD4+ T cells were defined as CD3+CD4+CD137+IFNγ+; and activated CD8+ T cells were defined as CD3+CD8a+CD107a+IFNγ+ [23 (link),24 (link)]. Data acquisition was performed on an Accuri C6 Plus and analyzed using FlowJo (RRID:SCR_008520).
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6

Comprehensive PBMC Cytokine Analysis

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About 150,000 peripheral blood mononuclear cells (PBMCs) per well were stimulated in a 96-well plate with peptides (2.5 ng/ml), 1 μg/ml anti-CD28, anti-CD49d, and anti-CD107a FITC (BioLegend) for 6 hr. After 30 min, 5 μg/ml Brefeldin A and 1× Monensin (BioLegend) were added. The positive control contained 2.5 ng/ml PMA and 25 ng/ml ionomycin, and the negative cells and CD28/CD49d. Cells were washed and stained with LIVE/DEAD Fixable Violet stain (Invitrogen), fixed, and permeabilized with BD Fix/Perm solution (BD) and stained with a mix of fluorochrome-conjugated antibodies: CD3 EDC, CD8 PerCP (BD), interleukin (IL)-2 PE, IFN-γ, Pe-Cy7, and tumor necrosis factor (TNF)-α APC (BioLegend). Unstained and singly stained cells were used as controls to calculate compensation. The cell fluorescence intensities were acquired on a Cyan ADP Cytometer (Beckman Coulter) and analyzed with Summit software (Dako).
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