Abstract
Abstract Upon activation naïve T helper (Th) precursor cells are polarized toward various cell subtypes and can be distinguished from each other based on effector function and chemokine expression. Th1 cells regulate cellular immunity via IL-2 plus IFN-γ, activating macrophages, cytotoxic T, NK and B cells in an effort to eliminate intracellular pathogens. Th1 cells preferentially express the chemokine receptors CXCR3 and CCR5 and can be further characterized by intracellular staining for IFN-γ. We have developed a 2-step EasySepTM immunomagnetic column-free method for the enrichment of CD4+CXCR3+ T cells from fresh peripheral blood nucleated cells. First, non-CD4 T cells are targeted for depletion using dextran-coated magnetic particles and a cocktail of antibody complexes. Labeled cells are separated using an EasySepTM magnet, and pre-enriched CD4 T cells are poured off. CXCR3+ cells are then positively selected from the pre-enriched fraction. The procedure can be automated using RoboSepTM. With an initial frequency of 10 ± 3% CD4+CXCR3+ cells, purities of 90 ± 4% (n=12) can be obtained. When stimulated, a high proportion of these cells produce IFN-γ (68 ± 12%) compared with total CD4 T cells and pre-enriched CD4 T cells. Less than 1% of isolated cells produce cytokines associated with other Th cell subtypes (IL-4, IL-17). Enrichment of unstimulated human Th1 cells enables the investigation of chronic inflammation responses and mechanisms of regulation in human models of disease.
Published Version
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