Rapid, column-free two-step procedure for the enrichment of human Th17 Cells from peripheral blood (56.13)

Abstract

Abstract Human Th17 cells play a critical role in the regulation of autoimmune disease and antimicrobial host defenses through production of pro-inflammatory cytokines IL17A and IL-17F. They can be characterized by expression of surface receptors including IL-23R, CCR6, CD161 and lineage-specific transcription factor RORC, but lack of CXCR3 expression. The major disadvantages of current isolation methods are the requirement for in vitro stimulation and the co-secretion of IFN-γ. We have developed a 2-step EasySepTM immunomagnetic column-free method for the enrichment of CD4+CXCR3-CCR6+ cells from fresh peripheral blood nucleated cells (PBNC). First, non-CD4 T cells and CXCR3+ 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. Next, CCR6+ cells are positively selected from the pre-enriched fraction. The procedure can be automated using RoboSepTM. Starting with a frequency of 5 ± 2% CD4+CXCR3-CCR6+ cells, purities of 94 ± 3% (n=10) can be obtained. The resulting cells produce high levels of IL-17 with minimal IFN-γ when measured by ELISA and intracellular staining and increased RORC mRNA expression over total CD4 T cells and CD4+CXCR3+ T cells (Th1 cells). Enrichment of unstimulated human Th17 cells enables the investigation of adaptive immune responses and regulation mechanisms required for the development of future therapies.