title: Activation requirements and responses to TLR ligands in human CD4+ T cells: comparison of two T cell isolation techniques.
abstract: 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.
authors.affiliation: Department of Pediatrics, Division of Pediatric Infectious Diseases, Case Western Reserve University & University Hospitals, Cleveland, Ohio 44106, USA. Christina.Lancioni@uhhospitals.org
authors.forename: Christina L
authors.initials: CL
authors.lastname: Lancioni
authors.forename: Jeremy J
authors.initials: JJ
authors.lastname: Thomas
authors.forename: Roxana E
authors.initials: RE
authors.lastname: Rojas
mesh_terms: D015496:CD4-Positive T-Lymphocytes / Q000187:drug effects / Q000276:immunology*; D049109:Cell Proliferation / Q000187:drug effects; D002469:Cell Separation / Q000379:methods*; D003545:Cysteine / Q000031:analogs & derivatives / Q000494:pharmacology; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D007371:Interferon-gamma / Q000096:biosynthesis / Q000276:immunology; D007376:Interleukin-2 / Q000096:biosynthesis / Q000276:immunology; D053645:Interleukin-2 Receptor alpha Subunit / Q000276:immunology / Q000378:metabolism; D008024:Ligands; D008070:Lipopolysaccharides / Q000494:pharmacology; D008934:Mitogens / Q000494:pharmacology; D010835:Phytohemagglutinins / Q000494:pharmacology; D051193:Toll-Like Receptors / Q000819:agonists / Q000276:immunology*
chemical_list: D007376:Interleukin-2; D053645:Interleukin-2 Receptor alpha Subunit; D008024:Ligands; D008070:Lipopolysaccharides; D008934:Mitogens; D010835:Phytohemagglutinins; D051193:Toll-Like Receptors; D007371:Interferon-gamma; D003545:Cysteine