EDTA-treated whole blood was incubated using the corresponding monoclonal antibodies: anti-CCR6-PB, anti-PD1-PCy7, anti-CD3-PerCP5.5, anti-CXCR3-PE and anti-ICOS-APCH7 (all from BDBiosciences, New York, NY, USA); anti-CD4-KO (from Beckman Coulter, Miami, FL, USA). Proportions of Th subsets were analyzed by flow cytometry using a Navios Cytometer and Kaluza Software (Beckman Coulter, Miami, FL, USA).
According to the expression of cell surface markers in CD4+ T cells, they could be classified as: Th1 cells (CXCR3+/CCR6-), Th17 cells (CXCR3-/CCR6+) and Th2 cells (CXCR3-/CCR6-). Regarding the activation status, each one could be divided into quiescent cells (ICOS-/PD1-), early activated cells (ICOS+/PD-1-), late activated cells (ICOS+/PD-1+) and exhausted or senescent cells (ICOS-/PD-1+) [32 (link)].
According to the expression of cell surface markers in CD4+ T cells, they could be classified as: Th1 cells (CXCR3+/CCR6-), Th17 cells (CXCR3-/CCR6+) and Th2 cells (CXCR3-/CCR6-). Regarding the activation status, each one could be divided into quiescent cells (ICOS-/PD1-), early activated cells (ICOS+/PD-1-), late activated cells (ICOS+/PD-1+) and exhausted or senescent cells (ICOS-/PD-1+) [32 (link)].
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