Enrichment of nucleofected primary human CD4+ T cells: a novel and efficient method for studying gene function and role in human primary T helper cell differentiation.
Identification of key factors mediating the differentiation of naïve CD4(+) T helper cells into Th1 and Th2 subsets is important for understanding the molecular mechanisms of the development of autoimmune diseases as well as asthma and allergy. Functional importance of a given gene in the initiation of human T helper cell differentiation has been hard to study due to the difficulty in transfecting primary resting human T lymphocytes. In this study we have successfully transfected human primary CD4(+) T helper cells using Amaxa's Nucleofection technology. To overcome the background caused by untransfected cells, we have developed a system for enriching nucleofected unstimulated human primary T helper cells that express the gene of interest. This is achieved by introducing a plasmid construct containing a bicistronic unit coding for a truncated mouse MHC class l H-2K(k) cell surface marker followed by selection of H-2K(k) positive cells using antibody coated beads. We demonstrate that the nucleofected and enriched H-2K(k) positive T helper cells differentiate into Th1 and Th2 cells as well as the non-transfected control cells. We also show that by using this novel method, introduction of an shRNA targeting Stat6, a key molecule driving the Th2 cell development, results in impaired Th2 cell differentiation, as expected. The method described here, enables fast and feasible preparation of highly pure transfected primary CD4(+) T cell cultures ideal for studying the influence of overexpression or knockdown of a given gene on T helper cell differentiation and other primary human T cell functions.
Tahvanainen
Johanna
J
Turku Centre for Biotechnology, Turku University and Abo Akademi University, Finland. johanna.tahvanainen@btk.fi
Pykäläinen
Maritta
M
Kallonen
Teemu
T
Lähteenmäki
Hanna
H
Rasool
Omid
O
Lahesmaa
Riitta
R
D015153:Blotting, Western; D015496:CD4-Positive T-Lymphocytes / Q000166:cytology / Q000276:immunology*; D002454:Cell Differentiation / Q000235:genetics / Q000276:immunology; D018274:Electroporation / Q000379:methods; D005434:Flow Cytometry; D006183:H-2 Antigens / Q000235:genetics / Q000276:immunology; D006801:Humans; D018189:Immunomagnetic Separation; D019169:Jurkat Cells; D010957:Plasmids / Q000235:genetics / Q000276:immunology; D034622:RNA Interference; D020133:Reverse Transcriptase Polymerase Chain Reaction; D050800:STAT6 Transcription Factor / Q000235:genetics / Q000276:immunology; D018417:Th1 Cells / Q000166:cytology / Q000276:immunology*; D018418:Th2 Cells / Q000166:cytology / Q000276:immunology*; D014162:Transfection / Q000379:methods*
D006183:H-2 Antigens; D050800:STAT6 Transcription Factor; C494101:STAT6 protein, human