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

Journal of Immunological Methods
Johanna TahvanainenRiitta Lahesmaa

Abstract

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 d...Continue Reading

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Citations

Dec 5, 2012·The Journal of Biological Chemistry·Johanna TahvanainenRiitta Lahesmaa
Sep 18, 2013·The Journal of Experimental Medicine·Zhen YangCornelia M Weyand
Jun 3, 2009·Human Gene Therapy·T MaggM H Albert
Jan 26, 2011·Human Gene Therapy·Cecilia N Barese, Cynthia E Dunbar
Jun 14, 2008·Blood·Eduardo HuarteJose R Conejo-Garcia
Jun 10, 2014·Journal of Immunological Methods·Mingce ZhangRandy Q Cron
Aug 31, 2013·Journal of Immunological Methods·Michael Freeley, Aideen Long
Feb 27, 2008·International Reviews of Immunology·Fatih M Uckun
May 20, 2008·Annals of Medicine·Kirsi J RautajokiRiitta Lahesmaa
Apr 15, 2008·The Journal of Allergy and Clinical Immunology·Emily ClarkeGraham J Jones
Nov 20, 2013·Seminars in Immunology·Soile Tuomela, Riitta Lahesmaa
Sep 9, 2011·Bioconjugate Chemistry·Jessica G BorgerRosario M Sanchez-Martin
Mar 23, 2010·The Journal of Immunology : Official Journal of the American Association of Immunologists·Sanna FilénRiitta Lahesmaa
Mar 7, 2007·The Journal of Immunology : Official Journal of the American Association of Immunologists·Riikka J LundRiitta Lahesmaa

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