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.

Association analysis of common variants of STAT6, GATA3, and STAT4 to asthma and high serum IgE phenotypes.

BACKGROUND: Immune responses characterized by T H 2 type cells and IgE are important for the development of asthma and atopy. The transcription factors STAT6, GATA3, and STAT4 mediate the cytokine-induced development of naive CD4 + T cells into either T H 1 or T H 2 type. OBJECTIVE: We studied genetic variation of the STAT6, GATA3, and STAT4 genes and examined whether single nucleotide polymorphisms (SNPs) in these loci were associated with asthma or serum high IgE levels in the Finnish asthmatic families. METHODS: With denaturing high-performance liquid chromatography we screened all exons and exon-intron boundaries of the genes in 14 to 22 patients. All identified SNPs were genotyped in 120 nuclear families, and the haplotypes were analyzed by Haplotype Pattern Mining based statistical analysis. When potential association was observed, the analysis was replicated among 245 asthmatic patients and 405 population-based control subjects. RESULTS: A total of 23 SNPs were identified, of which 8 were not previously listed in the SNP database. Interestingly, a haplotype analysis of GATA3 showed 3 related haplotypes that associated with different asthma and atopy related phenotypes among both the family and case-control data sets. For STAT6 and STAT4, no significant association to asthma or serum total IgE levels was observed. CONCLUSIONS: We identified a panel of novel SNPs in genes coding for proteins important in the T H 1/T H 2 cell differentiation. SNPs of the GATA3 gene showed an initial association to asthma-related phenotypes. Elucidation of the importance of the identified panel of SNPs in other T H 1/T H 2 mediated diseases will be of great interest.