Activation requirements and responses to TLR ligands in human CD4 super(+) T cells: Comparison of two T cell isolation techniques

• Published in: Journal of immunological methods Vol. 344; no. 1; pp. 15 - 25
• Main Authors: Lancioni, CL, Thomas, J J, Rojas, R E
• Format: Journal Article
• Language: English
• Published: 15.05.2009
• ISSN: 0022-1759
• DOI: 10.1016/j.jim.2009.02.005

Direct regulation of T cell function by microbial ligands through Toll-like receptors (TLR) is an emerging area of T cell biology. Currently either immunomagnetic cell sorting (IMACS) or fluorescence-activated cell sorting (FACS), are utilized to isolate T-cell subsets for such studies. However, it is unknown to what extent differences in T cell purity between these isolation techniques influence T cell functional assays. We compared the purity, response to mitogen, activation requirements, and response to TLR ligands between human CD4 super(+) T cells isolated either by IMACS (IMACS-CD4 super(+)) or by IMACS followed by FACS (IMACS /FACS-CD4 super(+)). As expected, IMACS-CD4 super(+) were less pure than IMACS /FACS-CD4 super(+) (92.5%+/-1.4% versus 99.7%+/-0.2%, respectively). Consequently, IMACS-CD4 super(+) proliferated and produced cytokines in response to mitogen alone and had lower activation requirements compared to IMACS/FACS-CD4 super(+). In addition IMACS-CD4 super(+) but not IMACS/FACS-CD4 super(+) responses were upregulated by the TLR-4 ligand lipopolysaccharide (LPS). On the other hand, TLR-2 and TLR-5 engagement induced costimulation in both IMACS-CD4 super(+) and highly purified IMACS-/FACS-CD4 super(+). Altogether these results indicate that small differences in cell purity can significantly alter T cell responses to TLR ligands. This study stresses the importance of a stringent purification method when investigating the role of microbial ligands in T cell function.