Application protocol

Rapid antigen-specific T cell enrichment (Rapid ARTE)

Direct ex vivo characterization of human antigen-specific CD154+CD4+ T cells  

 CD154 is transiently up-regulated on activated CD4+ T cells and plays an important role as a co-stimulatory molecule in T cell/ag-presenting cell interactions through ligation of CD40. Due to its transient expression within hours after activation, CD154 can be used as a marker for activated ag-specific CD4+ T cells. Adding a CD40-blocking antibody during the stimulation of cell suspensions prevents down-regulation of CD154 expression induced by the interaction with CD40 expressed on ag-presenting cells. Combining magnetic cell enrichment and multiparameter flow cytometry analysis of CD154+CD4+ T cells allows the direct ex vivo detection and characterization of rare ag-specific T cells.

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Materials

The following is a listing of reagents, instruments, and consumables needed for each step of this protocol. These products are for research use only.

Generation of PBMCs and in vitro stimulation for induction of CD154 expression

PBMC cultivation

  • RPMI 1640 medium
  • 100× L-glutamine stock solution (200 mM)
  • Human AB serum
  • 24-well plate (e.g., Gas-permeable Culture Plate # 150-000-362)
    Note: With the Gas-permeable Culture Plate # 150-000-362, up to 2.5×107 PBMCs/well/mL can be stimulated as opposed to 1×107 PBMCs/well/mL in standard 24-well plates.

PBMC stimulation

  •  CD40 pure – functional grade, human (# 130-094-133)
  • Reagents for  ag-specific T cell stimulation (e.g., PepTivator BKV VP1 – research grade, human, # 130-097-272 and PepTivator BKV LT – research grade, human, # 130-096-504)
  • Brefeldin A
  • FcR Blocking Reagent, human (# 130-059-901)

Magnetic and immunofluorescent surface labeling

Buffer (standard wash and dilution buffer)

  • autoMACS® Rinsing Solution (# 130-091- 222)
  • Bovine serum albumin (MACS® BSA Stock Solution; # 130-091-376)
  • CD154 MicroBead Kit, human (# 130-092-658)
  • Inside Stain Kit (# 130-090-477)
  • Orbital shaker
  • CD3-VioBlue®, human (#130-094-363)
  • CD4-APC, human (# 130-092-374)
  • CD8-PerCP, human (# 130-094-972)
  • CD14-PerCP, human (# 130-094-969)
  •  CD20-PerCP, human (# 130-094-976)

Magnetic enrichment and intracellular staining

  • MS Columns (# 130-042-201)
  • MACS Separator for MS columns (e.g. MiniMACS™ Separator # 130-042-102)
  • MACS Multistand  (#130-042-303)
  • CD154-FITC (# 130-096-233)
  • (Optional) Anti-cytokine antibodies for intracellular staining (e.g., Anti-IFN-g-PE # 130-091-653 or Anti-IL-17A-PE # 130-094-521)
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Protocol


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This protocol includes detailed instructions for preparing necessary reagents and materials and then carrying out each step.