Defining parameters for multi-colour flow cytometry and molecular identification of human T cell subsets

Flow cytometry is widely used to define cell populations in immunology. Here, we describe an optimised multi-parameter flow cytometry protocol that enables T cell subset identification in human blood and tissue, using a comprehensive panel of surface and intracellular markers. Antibody selection, cell membrane permeabilisation, T cell stimulation, controls and surrogate markers were considerations taken in establishing the methodology. With the optimised method, CD4 T helper cell subsets, Th1 (T-bet⁺, IFNγ⁺,) Th2 (GATA3⁺, IL-4⁺, IL-13⁺), Th17 (RORγt⁺, IL-17A⁺) and regulatory T cells (FoxP3⁺CD25⁺TNFR2⁺, IL-10⁺,TGFβ⁺), as well as CD8 T cell subsets Tc1 (IFNγ⁺) and Tc2 (IL-4⁺), are detectable at varying frequencies in healthy human blood and in blood and stromal vasculature fractions from lymphoedema patients. Suggested surrogate markers for Th1 (CD183 [CXCR3]), and Th2(CD194 [CCR4]), are evaluated as well. Furthermore, naïve and memory T cells are comprehensively assessed using a combination of antibodies to CD45RA/RO, CD27, CD62L,CCR7 and CD31. CCR7 expression further distinguishes the central and effector memory subsets, while CD31 expression differentiates the recent thymic emigrants from peripherally expanded CD4 naïve T cells. These two naïve T cell subsets can be further investigated for Tcell receptor diversity by way of next-gen sequencing, to confirm if clonality decreases due to homeostatic expansion, especially as thymic output is said to decrease with age. A thorough definition of these T cell subsets will be valuable to a wide range of human immunology studies where the T cells play a role in exacerbating or in alleviating a condition.