Detection of CMV pp65-specific T cells by intracellular staining with anti-INF-γ-PE

Title

Detection of CMV pp65-specific CD8⁺ T cells by intracellular IFN-γ staining following peptide stimulation

Overview

This protocol describes the intracellular detection of interferon-gamma (IFN-γ) in CMV pp65-specific CD8⁺ T cells from peripheral blood mononuclear cells (PBMCs), using short-term peptide stimulation and flow cytometry. The protocol was used in the publication “Human thymopoiesis produces polyspecific CD8⁺ α/β T cells responding to multiple viral antigens” (eLife, 2017).

Materials

• PBMCs from human peripheral blood (fresh or thawed from cryopreservation)
• RPMI 1640 + 10% fetal bovine serum (FBS)
• Peptivator® CMV pp65 peptide pool (Miltenyi Biotec)
• Brefeldin A (10 μg/mL final; e.g., Sigma or BD GolgiPlug)
• Anti-IFN-γ-PE antibody (clone 4S.B3, BD Biosciences)
• Surface antibodies: CD3, CD8 (fluorochromes as desired)
• Fixation/Permeabilization Kit: BD Cytofix/Cytoperm
• Flow cytometer

Protocol

1. Cell Preparation
   • Resuspend PBMCs in complete RPMI medium.
   • Sort the CD8⁺ memory T cell subset (e.g., CD3⁺ CD8⁺ CD45RO⁺) by flow cytometry (FACS) under sterile conditions.
   • Plate 1–2×10⁶ cells per well (e.g., in 96-well round-bottom plates).
2. Stimulation
   • Add CMV pp65 peptide pool to a final concentration of 1 μg/mL.
   • Incubate at 37 °C, 5% CO₂.
   • After 2 hours, add Brefeldin A to reach 10 μg/mL final concentration.
   • Continue incubation for an additional 4 hours (total 6 h).
3. Surface staining
   • Wash cells with PBS.
   • Stain for CD3 and CD8 (and viability dye if needed) for 30 min at 4 °C.
4. Fixation and Permeabilization
   • Fix and permeabilize using the Cytofix/Cytoperm kit, following the manufacturer’s instructions.
5. Intracellular staining
   • Stain for IFN-γ (clone 4S.B3, PE-conjugated) for 30 min at 4 °C in the dark.
   • Wash and resuspend in PBS + 2% FBS before acquisition.
6. Flow cytometry
   • Acquire on a cytometer and analyze IFN-γ⁺ CD3⁺ CD8⁺ T cells.

Controls

• Negative control: PBMCs unstimulated.
• Positive control: PBMCs unstimulated PMA/Ionomycin can be used if needed.