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Flow cytometric analysis of T cell proliferation in a mixed lymphocyte reaction with dendritic cells.
- Source :
-
Journal of immunological methods [J Immunol Methods] 2003 Apr 01; Vol. 275 (1-2), pp. 57-68. - Publication Year :
- 2003
-
Abstract
- Background: Dendritic cells (DCs) are the most potent antigen-presenting cells. They can be generated in vitro from CD14+ cells, and also from CD34+ progenitor cells. Although T cell proliferation using [3H] thymidine incorporation assay has been used widely to check DC function, this technique only provides limited information about the T cell proliferation. Here, we describe a novel method for quantitative analysis of T cell proliferation using flow cytometry.<br />Materials and Methods: DCs were generated from CD14+ cells from six healthy blood donors. Monocytes were isolated using positive selection with magnetic cell sorting (MACS) and then cultured with IL-4, GM-CSF, IL-1beta, IL-6, TNF-alpha and PGE(2) to yield fully mature DCs. Allogeneic naive T lymphocytes with known mismatches in HLA classes I and II were cocultured with DCs. Naive T cells without DC stimulation served as negative controls. T cells were harvested on days 0, 3, 5, 7, 9, 11 and analysed by flow cytometry. CD3-ECD and CD4-fluorescein isothiocyanate (FITC) or CD8-FITC antibodies were used to distinguish T cell subsets, whereas T cell activation was measured by assessment of HLA-DR, CD45RO, CD25 and CD71 expression. For T cell quantification, fluorescent microparticles were used. Dead cells were excluded with 7-AAD. The bromdeoxyridine (BrdU)-incorporation ELISA procedure was also performed in order to compare with the T cell proliferation assay with regard to absolute cell counts and CD71 expression.<br />Results: The initial T cell concentration on day 1 was 203.9+/-39.7 (173-265) CD3+/CD4+ cells/micro l and 184.5+/-41.6 (148-260) CD3+/CD8+ cells/micro l. The maximal T cell proliferation was recorded on day 7 with a five- to tenfold T cell expansion which resulted in 1994.9+/-383 (1446-2404) CD3+/CD4+ cells/micro l and 944+/-303.7 (560-1483) CD3+/CD8+ cells/micro l. Furthermore, activation markers of both cell lineages were upregulated and reached maxima on days 7 (CD71) and 9 (CD25, HLA-DR). T cell count/micro l as well as CD71 expression both correlated significantly with BrdU incorporation.<br />Conclusion: Flow cytometric analysis permits simple, precise and rapid quantification of T cell proliferation in a mixed lymphocyte reaction with DCs. Activation, proliferation and cell viability can be simultaneously determined. CD71 is particularly well suited as an activation marker for the simultaneous measurement of T cell proliferation. Thus, specific T cell subsets involved in antigen-specific proliferation can be evaluated in detail.
- Subjects :
- Bromodeoxyuridine metabolism
CD3 Complex metabolism
CD4 Antigens metabolism
CD8 Antigens metabolism
Enzyme-Linked Immunosorbent Assay methods
Humans
In Vitro Techniques
Lipopolysaccharide Receptors metabolism
T-Lymphocytes cytology
T-Lymphocytes metabolism
T-Lymphocytes, Cytotoxic immunology
T-Lymphocytes, Helper-Inducer immunology
Dendritic Cells immunology
Flow Cytometry methods
Lymphocyte Activation
Lymphocyte Culture Test, Mixed methods
T-Lymphocytes immunology
Subjects
Details
- Language :
- English
- ISSN :
- 0022-1759
- Volume :
- 275
- Issue :
- 1-2
- Database :
- MEDLINE
- Journal :
- Journal of immunological methods
- Publication Type :
- Academic Journal
- Accession number :
- 12667670
- Full Text :
- https://doi.org/10.1016/s0022-1759(03)00002-4