Isolation and expansion of human cytomegalovirus- specific cytotoxic T lymphocytes using interferon-γ secretion assay

Abstract 

Objective

The aim of this study was to isolate and expand donor-derived human cytomegalovirus (HCMV)-specific cytotoxic T lymphocytes (CTLs) for adoptive transfer of 10⁷ cells per m² of body surface area to restore protective immunity after stem cell transplantation.

Materials and Methods

A new strategy to generate HCMV-specific CTLs using the interferon-γ (IFN-γ) secretion assay, followed by expansion to numbers sufficient for clinical application with interleukin-2 and feeder cell stimulation, is described.

Results

From 1 to 5 × 10⁴ HCMV peptide-specific T lymphocytes (greater than 90% CD3⁺CD8⁺) were isolated from 1 to 2 × 10⁸ peripheral blood mononuclear cells comparable to 50 to 100 mL of blood from HLA-A*0201 HCMV seropositive blood donors and expanded ex vivo after a median of 16 days (range 8–28 days; ) to greater than 10⁷/m² HCMV peptide-specific CTLs using autologous or allogeneic feeder cell stimulation. In three experiments, expansion to 6 weeks was performed, achieving a median of 1.6 × 10⁹ cells (range 6.1 × 10⁸–3.3 × 10⁹). Characterization of these HCMV-specific CTL lines revealed an average purity of 89.2% (range 66.2–99.3%) using HCMV pp65 peptide HLA-A*0201 tetramer staining and 89.4% (range 64.4–99.5%) by peptide-specific IFN-γ secretion . A median of 82.6% (range 76.0–88.0%) showed perforin secretion and 57.5% (range 22.2–80.7%) specific lysis of peptide-pulsed T2 cells . A median of 52.2% (range 35.2–7.3%) revealed specific killing of HCMV-infected autologous, but not allogeneic, fibroblasts .

Conclusion

IFN-γ secretion assay allows development of a simple and rapid protocol with short expansion times for generation of greater than 10⁷/m² HCMV-specific CTLs for adoptive immunotherapy.