Home › Immunology › HLA-Ig Based Artificial Antigen Presenting Cells for Efficient ex vivo Expansion of Human CTL
Immunology JoVE (Open Access) Citable · DOI

HLA-Ig Based Artificial Antigen Presenting Cells for Efficient ex vivo Expansion of Human CTL

DOI: 10.3791/2801-v
What you'll learn
  • ✓ Construct HLA-A2-Ig-based artificial antigen presenting cells (aAPC) from mammalian cell lines
  • ✓ Load peptide antigens onto aAPC and validate quality control metrics
  • ✓ Isolate human CTL and establish DC-independent ex vivo expansion cultures
  • ✓ Assess expanded CTL polyfunctionality and proliferation capacity without exhaustion
Protocol

Biopharma Insights A new DC independent method for induction and expansion of antigen-specific T cells is described. HLA A2-Ig based artificial Antigen Presenting Cells (aAPC) are loaded with HLA-A2 restricted peptides to efficiently expand CTL of diverse antigen specificity. This technology holds great potential for CTL-based adoptive immunotherapy.

Difficulty
advanced
Total time
~4–6 days (aAPC generation ~2–3 days; CTL isolation and expansion culture ~3–5 days depending on endpoint analysis)
Model organism
HEK293 or similar mammalian cell line (human donor PBMCs for CTL isolation)
Biosafety
BSL-2

Steps

1
Generate HLA-A2-Ig-based artificial antigen presenting cells

Transfect or use stable mammalian cell lines expressing HLA-A2-Ig fusion protein construct. Confirm expression and harvest aAPC for downstream use.

▶ 01:39
2
Perform quality control and peptide loading on aAPC

Validate aAPC surface expression via flow cytometry or equivalent method. Load HLA-A2-restricted peptides and confirm stable peptide–MHC complex formation; store appropriately.

▶ 02:47
3
Isolate human cytotoxic T lymphocytes from PBMCs

Obtain human peripheral blood mononuclear cells and enrich for CTL population using magnetic bead selection or flow cytometry sorting. Assess purity and viability before culture.

▶ 03:43
4
Establish in vitro aAPC-based CTL expansion culture

Co-culture isolated CTL with peptide-loaded aAPC in defined medium with appropriate cytokine supplementation. Maintain culture conditions and monitor cell proliferation over time.

▶ 05:22
5
Assess expanded CTL polyfunctionality and activation state

Analyze expanded CTL populations by intracellular cytokine staining, phenotyping, or functional assays to confirm polyfunctionality and absence of exhaustion markers after prolonged expansion.

▶ 06:25
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