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

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⁺ T cells isolated either by IMACS (IMACS-CD4⁺) or by IMACS followed by FACS (IMACS/FACS-CD4⁺). As expected, IMACS-CD4⁺ were less pure than IMACS/FACS-CD4⁺ (92.5% ± 1.4% versus 99.7% ± 0.2%, respectively). Consequently, IMACS-CD4⁺ proliferated and produced cytokines in response to mitogen alone and had lower activation requirements compared to IMACS/FACS-CD4⁺. In addition IMACS-CD4⁺ but not IMACS/FACS-CD4⁺ 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⁺ and highly purified IMACS-/FACS-CD4⁺. 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.