[Detection, enrichment and expansion of T lymphocytes mediating alloresponse based on cytokine].
To detect, enrich and expand the cytokine secreting T lymphocytes after allogeneic PBMNCs stimulation.



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.



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.



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.
Department of Hematology, The First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou 310003, China.
Xiao-jian
XJ
Meng
Mao-fang
MF
Lin
Zhen
Z
Cai
D000911:Antibodies, Monoclonal / Q000494:pharmacology; D018106:CD28 Antigens / Q000276:immunology; D049109:Cell Proliferation; D002478:Cells, Cultured; D016207:Cytokines / Q000378:metabolism*; D006086:Graft vs Host Disease / Q000276:immunology*; D006801:Humans; D007371:Interferon-gamma / Q000378:metabolism*; D007376:Interleukin-2 / Q000378:metabolism; D007963:Leukocytes, Mononuclear / Q000166:cytology* / Q000276:immunology; D007959:Lymphocyte Culture Test, Mixed; D016853:Muromonab-CD3 / Q000494:pharmacology; D013601:T-Lymphocytes / Q000166:cytology* / Q000276:immunology
D000911:Antibodies, Monoclonal; D018106:CD28 Antigens; D016207:Cytokines; D007376:Interleukin-2; D016853:Muromonab-CD3; D007371:Interferon-gamma