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Updated: Aug 11, 2026

Measurement of T Cell Alloreactivity Using Imaging Flow Cytometry
Published on: April 19, 2017
[Detection, enrichment and expansion of T lymphocytes mediating alloresponse based on cytokine]
Xiao-jian Meng1, Mao-fang Lin, Zhen Cai
1Department of Hematology, The First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou 310003, China.
Objective:
To detect, enrich and expand the cytokine secreting T lymphocytes after allogeneic PBMNCs stimulation.
Methods:
The novel cytokine secretion assay (CKSA) was applied to detect T lymphocytes secreting IFN-gamma at single cell level in human mixed lymphocytes reaction. IFN-gamma secreting T cells were enriched by means of magnetic sorting system and expanded with OKT(3), anti-CD(3)mAb and IL-2 combination. Antigen specificity of the expanded cells was confirmed using enzyme linked immunospot assay.
Results:
A sizable proportion of IFN-gamma secreting T lymphocytes could be detected [(1.12 +/-0.13)% compared with (0.23 +/-0.07)%] and be further enriched to (67.3 +/-10.5)%, or (93.8 +/-22.1) fold. T lymphocytes could be expanded up to 600-fold within 21-28 days and the specific IFN-gamma response of expanded cells was confirmed with stimulation of the relevant allogeneic PBMNC, which was significantly higher than the irrelevant PBMNC control.
Conclusion:
It is feasible to detect significantly increased IFN-gamma secreting T lymphocytes after allogeneic PBMNCs stimulation based on the CKSA technique at single cell level and these cells can be efficiently enriched and expanded for further research.
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