T cells present in human peripheral blood mononuclear cells (PBMC) are complex populations of different subtypes with unique functions. CD4+ and CD8+ T cells are most simply classified as naïve, central memory, effector memory, and terminally differentiated effector subtypes, and T cells are currently thought to differentiate along this progression path following antigen experience. In a previous post, I discussed using CD3, CD4, CD8, CD45RA or CD45RO, and CCR7 or CD62L as markers for differentiating T cells into these subtypes by multiparametric flow cytometry.
In addition, CD4+ T cells can be even further characterized as to their specific functions. The cytokine milieu present during T cell activation through the T cell receptor (TCR) guides T cells to differentiate into memory and effector T helper subtypes (TH) including TH1, TH2, and TH17 subsets, all of which are present in PBMC.
Each of these TH subtypes functions by expressing a unique set of cytokines and chemokine receptors in order to elicit specific responses in different tissue settings. It is important to note that memory T cells maintain flexibility in their functionality and can be re-polarized into different subtypes if re-stimulated under an alternate TH inducing condition.
Using multiparametric flow cytometry, each of these TH subsets can be identified by expression of specific cytokines, transcription factors, and surface markers including chemokine receptors, although the various markers used in the literature to classify the same subset do not overlap entirely. Identification of TH subsets by surface markers simply requires surface staining and is the easiest assay to perform. Transcription factor expression can be assessed, which requires intracellular/nuclear permeabilization protocols such as BD Biosciences’ FoxP3 Buffer Set. Finally, intracellular cytokine production can be detected following TCR stimulation with anti-CD3 and anti-CD28 antibodies or the combination of Phorbol 12-Myristate 13-Acetate (PMA) and the Ca2+ ionophore, ionomycin. This generally requires 4-6 hours of stimulation in the presence of protein transport inhibitors: either brefeldin A or monensin, which are optimal for different cytokines.
TH1 cells promote cellular immunity against viruses and intracellular bacteria. TH1 cells have been characterized in many ways. Production of IFNg following TCR stimulation is probably the most common and direct functional representation to assess TH1 in PBMC. Alternatively, the expression of the chemokine receptors CXCR3 and CCR5 have been associated with TH1 but not TH2 or TH17 cells. The transcription factor T-bet (TBX21) is the major factor regulating TH1 differentiation and is also a marker of TH1 cells that can be assessed by flow cytometry, although T-bet expression is promiscuous and can be expressed in TH17 cells. Interestingly, expression of any of these markers may not overlap entirely with IFNg expression. For instance, in central memory T cells, CXCR3+ cells are considered pre-TH1 cells and do not express IFNg, while in the effector memory population, a fraction of CCR5+ and CXCR3+ cells express IFNg. As always, care must be taken when making comparisons between percentiles and functions of populations identified using different markers.
TH2 cells are the CD4 helper subtype that promotes immunity against extracellular pathogens and are involved in allergic inflammation. TH2 cells in PBMC can be classified by production of TH2-specific cytokines including IL-4, IL-5, and IL-13 following TCR stimulation or PMA/ionomycin. However, expression of these cytokines can be difficult to detect and expression of the surface receptors CRTH2, CCR4, or CCR3 are alternatives. Again, expression of CRTH2, CCR4, or CCR3 does not mark all existing IL-4, IL-5, and IL-13 expressing TH2 cells and vice versa, although CRTH2 has been demonstrated to be quite effective.
TH17 cells are known as the subtype associated with inflammatory autoimmune diseases and protection from fungal infections. TH17 cells are classified by expression of IL-17 or surface marker expression, being CCR6+CCR4+. Expression of the TH17-specific transcription factor RORg/gt can also be assessed. A fraction of IL-17 expressing cells can also express IFNg and TH17 associated markers CCR6 and RORg/gt. Some have even suggested that IL-17+IFNg+ cells are a transitional state when more plastic TH17 cells differentiate into effector TH1 cells.
As cytokine expression may be the most discrete method of describing a TH cell regarding its function, I have used cytokine expression as a measure of TH1 and TH17 cells in human PBMC. However in the case of TH2 cells, expression of IL-4 and IL-13 was hard to detect by flow cytometry, even with PMA and ionomycin stimulation and thus I have instead utilized CRTH2. The protocol that I have optimized for identification of TH1, TH2, and TH17 cells involves a 4 hour stimulation of PBMCs with anti-CD3 and anti-CD28 antibodies or with PMA + ionomycin in the presence brefeldin A. Following this, I first stain for surface markers including CRTH2 to identify TH2 cells. Then the cells are fixed and permeabilized using BD Biosciences’ Cytofix Cytoperm buffer set and stained for IFNg (TH1) and IL-17 (TH17). Using this method I have never seen co-expression for any of these factors with the exception of a small population of cells that co-stain with IFNg and IL-17, as commonly described in the literature.
Taken together, selection of markers for assessing these populations must be done carefully, bearing in mind that cytokines characteristic of a TH subtype are not always co-expressed in all cells identified as that TH subtype by the surface markers and transcription factors discussed here. Flow cytometry panels containing markers for all of these TH subtypes can allow measurement of more specific TH populations from PBMC by gating based on specific expression as well as exclusion of the other TH subtype markers.
Heterogeneity of CD4+ memory T cells: functional modules for tailored immunity. Sallusto F, Lanzavecchia A. Eur J Immunol. 2009 Aug;39(8):2076-82.
Chemokine receptor expression identifies Pre-T helper (Th)1, Pre-Th2, and nonpolarized cells among human CD4+ central memory T cells. Rivino L, Messi M, Jarrossay D, Lanzavecchia A, Sallusto F, Geginat J. J Exp Med. 2004 Sep 20;200(6):725-35.
Human T cells that are able to produce IL-17 express the chemokine receptor CCR6. Singh SP, Zhang HH, Foley JF, Hedrick MN, Farber JM. J Immunol. 2008 Jan 1;180(1):214-21.
Selective expression of a novel surface molecule by human Th2 cells in vivo. Nagata K, Tanaka K, Ogawa K, Kemmotsu K, Imai T, Yoshie O, Abe H, Tada K, Nakamura M, Sugamura K, Takano S. J Immunol. 1999 Feb 1;162(3):1278-86.