CyTOF Measurement of Immunocompetence Across Major Immune Cell Types

Priyanka B. Subrahmanyam1, Holden T. Maecker2

1 Post‐doctoral Research Fellow, Immunity, Transplantation and Infection, Stanford University School of Medicine, Stanford, 2 Professor, Department of Microbiology & Immunology, Stanford University School of Medicine, Stanford
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 9.54
DOI:  10.1002/cpcy.27
Online Posting Date:  October, 2017
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Abstract

The central role of the immune system is becoming appreciated in a wide variety of diseases. Cancer immunotherapy is one area that has yielded much recent success, although not all patients benefit equally. At the same time, recent studies have highlighted the heterogeneity of the human immune system. Despite this heterogeneity, we do not routinely measure immune competence in clinical practice, and there are no consensus assays of healthy immune function. Using mass cytometry (CyTOF), we can simultaneously detect ∼40 markers to identify various cell subsets and determine their function by the expression of cytokines, cytotoxicity, and activation markers. This can help assess ‘immunocompetence’ and facilitate better implementation of immunotherapies, both in specific disease settings and perhaps eventually as a prognostic tool in healthy subjects. Here we introduce the concepts behind this assay and provide a protocol that we have successfully implemented to identify possible predictive biomarkers of immunotherapy outcome. © 2017 by John Wiley & Sons, Inc.

Keywords: CyTOF; immunotherapy; immune profiling; biomarkers

     
 
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Table of Contents

  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1:

  Materials
  • Complete medium (see recipe)
  • Pierce Universal Nuclease, 25 kU (ThermoFisher Scientific, at. no. 88701)
  • Human PBMC sample
  • Stimulation reagents and secretion inhibitors (Table 9.54.2)
  • EDTA, 0.5 M (Gibco, cat. no. 15575)
  • CyFACS (see recipe)
  • Maleimide‐DOTA loaded with 115In, 5 mg/ml (Macrocyclics, cat. no. B‐272)
  • CyPBS (see recipe)
  • Paraformaldehyde, 16% (w/v) (Alfa Aesar, cat. no. 43368)
  • 10× permeabilization buffer (eBioscience, cat. no. 00‐8333‐56)
  • Brefeldin A (Sigma‐Aldrich, cat. no. B7651)
  • Monensin, 1000× (BioLegend, cat. no. 420701)
  • Phorbol 12‐myristate 13‐acetate (PMA; Sigma‐Aldrich, cat. no. P8139)
  • Ionomycin (Sigma‐Aldrich, cat. no. I0634)
  • Cell ID Ir‐Intercalator (193Ir/195Ir), 1000× (Fluidigm, cat. no. 201192A)
  • EQ Four Element calibration beads (Fluidigm, cat. no. 201078)
  • 1.5‐ml, 15‐ml, and 50‐ml conical tubes
  • Centrifuge with adaptors to fit 15‐ml conical tubes and 1.5‐ml microcentrifuge tubes
  • 96‐well U‐bottom plates (Corning Falcon, cat. no. 353077)
  • Centrifugal filter units, 0.1 μm (Millipore, cat. no. UFC30VV00)
  • 5‐ml polystyrene tubes with cell‐strainer caps (Corning Falcon, cat. no. 352235)
  • Additional reagents and equipment for counting cells ( appendix 3A; Phelan & Lawler, )
Table 9.4.2   MaterialsStimulation of Cells

Reagent Intermediate stock dilution Final concentration (unstimulated) Final concentration (PMA + ionomycin)
Brefeldin A (5 mg/ml in DMSO) 1:10 in CyPBS 5 μg/ml 1:100 of intermediate stock 5 μg/ml 1:100 of intermediate stock
Monensin (1000×) 1:10 in CyPBS 1× 1:100 of intermediate stock 1× 1:100 of intermediate stock
PMA (1 mg/ml in DMSO) 1:1000 in CyPBS 10 ng/ml 1:100 of intermediate stock
Ionomycin (1 mg/ml in DMSO) 1:10 in CyPBS 1 μg/ml 1:100 of intermediate stock

NOTE: All solutions should be stored in metal‐free containers. Typically, sterile plasticware, tubes, filters, and new, never‐washed glassware are metal‐free. Glassware used should never have been washed, since soap can cause barium contamination.
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Figures

Videos

Literature Cited

Literature Cited
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