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Paloma Riquelme

James A. Hutchinson

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Figure 2

Standard protocols for detection of human CMV-specific T cells

This protocol describes detection CMV-reactive T cells in human PBMC. Briefly, PBMC are stimulated with CMV lysate for 18 hours. Afterwards, CMV-reactive CD4+ and CD8+ T cells are detected by intracellular staining of cytokines, including IFN-γ. Our group has applied this method to frozen PBMC samples from patients with advanced melanoma.

ReagentsRPMI1640 without phenol red (BE12918F, Lonza-Biozym)FCS (10270, Gibco)DMSO (D2650, Sigma)Glutamax100X (35050038, Gibco-life)Pen-Strep 100X (15140-122, Gibco-life)Human AB Serum, heat-inactivated pooled male-only (ZKT Tübingen)CMV Lysate (AR03041PU-N, Origene)αCD28 (340975, BD)αCD49d (340976, BD)αPD-1 (MAB10864, R&amp;D)αCTLA-4 (MAB3254, R&amp;D)Mouse IgG1 Isotype Control (MAB002, R&amp;D)Protein Transport Inhibitor Cocktail 500X (00-4980-93, eBioscience)Cell Stimulation Cocktail 500X (00-4970-93, eBioscience)SEB (S4881-1MG, Sigma-Aldrich)DPBS without Ca2+ or Mg2+ (D8537, Sigma)Cell Staining Buffer (420201, BioLegend)FcR Blocking Reagent (120-003-855, Miltenyi Biotec)IC Fixation Buffer (00-8222, eBioscience)Permeabilization Buffer (00-8333, eBioscience)Ecotainer Aqua dest. (0082423E, Braun)ViaKrome 808 Fixable Viability Dye (C36628, Beckman Coulter)Human FcR blocking reagent (130-059-901, Miltenyi)Brilliant Stain Buffer Plus (566385, BD)Antibodies: see Figure 1 &nbsp;&nbsp;MaterialsCryotubes (72.380, Sarstedt)Freezing Container, “Mr. Frosty” (5100-0001, Nalgene)96-well plate round bottom (3399, Corning)96-well plate polypropylene, V-bottom (3357, Corning)

Cytoflex LX cytometer: U3-V5-B3-Y5-R3-I2 (Beckman Coulter)CytExpert version 2.4 Acquisition and Analysis Software (Beckman Coulter)Kaluza analysis software v.2.1 (Beckman Coulter)

1. REAGENT PREPARATION1.1 Freezing Medium 11.&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Add 20 ml FCS to 30 ml RPMI 16402.&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Freezing medium 1 can be stored up to 1 month at 4°C&nbsp;1.2 Freezing Medium 21.&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Add 10 ml DMSO to 40 ml FCS2.&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Freezing medium 2 can be stored up to 1 month at 4°C&nbsp;1.3 Culture medium1.&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Add 9.3 ml of RPMI 1640 into a falcon tube.2.&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Add 0.5 ml of human AB serum (HABS)3.&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Add 0.1 ml of GlutaMax4.&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Add 0.1 ml of Pen-Strep&nbsp;2. CELL FREEZING1.&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Pre-cool cryotubes, freezing container and all reagents to 4°C,2.&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Work on ice using cold reagents in the laminar flow hood3.&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Add 1 ml Freezing Medium 1 to up to 20 x 106 pelleted PBMC4.&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Incubate 20 min on ice5.&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Slowly add 1 ml cold Freezing Medium 26.&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Mix by gently pipetting7.&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Transfer 1 ml of cell suspension (10 x 106 cells/ml) per cryotube8.&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Place cryotubes in a freezing container and store at -80°C9.&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;After 1 day, remove cryotubes from freezing container and store them at -80°C for up to 6 months10.&nbsp;&nbsp;Alternatively, samples can be stored in liquid nitrogen for longer than 6 months 3. CELL THAWING1.&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Pre-warm water bath to 37°C2.&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Thaw cells quickly by swirling the cryotube in the water bath (&lt;60 seconds)3.&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Before last ice crystal melts, place cryotube in a cold rack4.&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Transfer the content of the cryotube into a falcon tube5.&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Add 10 ml of cold PBS containing 10% human AB serum dropwise6.&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Count cells7.&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Centrifuge at 200 g for 10 min8.&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Aspirate supernatant using vacuum pump9.&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Resuspend cell pellet in culture medium at up to 10 x 106/ml10.&nbsp;&nbsp;&nbsp;Add 150 µl of cell suspension per well in a 96-well plate11.&nbsp;&nbsp;&nbsp;Incubate for 6 hours at 37°C 5% CO2 before proceeding with stimulation 4. IN VITRO RESTIMULATION WITH CMV LYSATE1.&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Add 5 µg/ml CMV lysate to each well. 2.&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Add 1µg/ml αCD28 and αCD49d to each well.3.&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;As a negative control, leave one unstimulated well4.&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Optionally, add 5µg/ml αPD1, αCTLA-4 or isotype control to the corresponding wells5.&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Incubate for 18 hours at 37°C 5% CO26.&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;In the last 4 hours of incubation, add 2 µl/ml Protein Transport Inhibitor Cocktail to all the wells (0.3 µl 500X stock per well containing 150 µl medium)7.&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;As a positive control, add 10 µg/ml SEB for 18 hours or alternatively 2 µl/ml Cell Stimulation Cocktail to one well for the last 4 hours. &nbsp;5. INTRACELLULAR CYTOKINE STAINING1.&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Transfer cells from culture wells into a polypropylene V bottom 96 well plate2.&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Centrifuge at 1400rpm for 3 min 3.&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Resuspend pellet in 20 µl PBS containing ViaKrome Fixable Viability Dye 808 (32,5µl dye/ml)4.&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Incubate for 20 min at RT in the dark5.&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Wash samples with 180 µl Cell Staining Buffer (CSB) by centrifugation at 1400 rpm for 3 min 6.&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Aspirate supernatant using vacuum pump7.&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Add 10 µl 10% FcR block in CSB and shake at 800 rpm for 60'' in a rotary shake8.&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Incubate at 4 °C for 15 min in the dark9.&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Add 20 µl surface antibodies mastermix as indicated in Figure 110.&nbsp; Shake at 800 rpm for 5 min and incubate for further 25 min at 4°C in the dark11. &nbsp;Wash samples with 170 µl CSB by centrifugation at 1400 rpm for 3 min 12. &nbsp;Aspirate supernatant using vacuum pump 13. &nbsp;Add 40 µl CSB to each well and resuspend cell pellets14.&nbsp; Add 50 µl IC Fixation Buffer 15.&nbsp; Incubate for 30 min at RT in the dark16.&nbsp; &nbsp;Wash samples with 110 µl Perm Buffer by centrifugation at 1400 rpm for 3 min 17.&nbsp; Aspirate supernatant using vacuum pump 18.&nbsp; Add 40 µl intracellular antibody mastermix as indicated in Figure 1. 19.&nbsp; Shake at 800 rpm for 5 min and incubate for further 25 min at RT in the dark20. &nbsp;Wash samples with 160 µl Perm Buffer by centrifugation at 1400 rpm for 3 min21.&nbsp; Aspirate supernatant using vacuum pump 22. &nbsp;Wash samples with 200 µl Perm Buffer by centrifugation at 1400 rpm for 3 min23.&nbsp; Aspirate supernatant using vacuum pump 24.&nbsp; Resuspend in 150 µl CSB for analysis by flow cytometry &nbsp;6. ANALYSIS OF DATA Our gating strategy for analysis of IFN-γ-producing T cells is shown in Figure 2. Samples were acquired using a Cytoflex LX cytometer (Beckman Coulter) and analysed with Kaluza analysis software v.2.1

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This document describes protocols used for in vitro restimulation of cryopreserved PBMC with CMV lysates and subsequent detection of CMV-specific CD4+ and CD8+ T cells by intracellular staining of cytokines.

Standard protocols for detection of human CMV-specific T cells

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Method Article

Paloma Riquelme, James A. Hutchinson

Paloma Riquelme

Department of Surgery, University Hospital Regensburg, Germany

Corresponding Author

ORCiD: https://orcid.org/0000-0001-8962-3156

James A. Hutchinson

Department of Surgery, University Hospital Regensburg, Germany

ORCiD: https://orcid.org/0000-0002-1199-4210

DOI:

10.21203/rs.3.pex-758/v1

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Flow Cytometry, Cytomegalovirus, CMV, intracellular cytokine staining, IFN-γ, TNF-α, IL-17, IL-4, CD69

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Method Article

Paloma Riquelme, James A. Hutchinson

Paloma Riquelme

Department of Surgery, University Hospital Regensburg, Germany

Corresponding Author

ORCiD: https://orcid.org/0000-0001-8962-3156

James A. Hutchinson

Department of Surgery, University Hospital Regensburg, Germany

ORCiD: https://orcid.org/0000-0002-1199-4210

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10.21203/rs.3.pex-758/v1

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