T Helper 2 Cell Overview

As their name suggests, T helper (Th) cells provide helper functions to other cells of the immune system—especially the antigen-presenting cells (APCs) such as macrophages, dendritic cells, and B cells—and are important for their activation and maturation. There are distinct subsets of CD4⁺ Th cells, including Th1, Th2, Th9, Th17, Th22, Tfh, and Treg cells, each activated by a specific set of cytokines and transcription factors and characterized by the cytokines they secrete and effector functions they perform.

Th2 cells mediate the activation and maintenance of the humoral, or antibody-mediated, immune response against extracellular parasites, bacteria, allergens, and toxins. Th2 cells mediate these functions by producing various cytokines such as IL-4, IL-5, IL-6, IL-9, IL-13, and IL-17E (IL-25). These cytokines are responsible for a strong antibody production, eosinophil activation, and inhibition of several macrophage functions, thus providing phagocyte-independent protective responses. These cytokines also counteract the Th1 responses that allow for the Th2 responsiveness to IL-4. Functionally, Th2 cytokines have effects on many cell types in the body as the cytokine receptors are widely expressed on numerous cell types. Th2 cells stimulate and recruit specialized subsets of immune cells, such as eosinophils and basophils, to the site of infection or in response to allergens or toxins leading to tissue eosinophilia and mast cell hyperplasia. They induce mucus production, goblet cell metaplasia, and airway hyper-responsiveness. Th2 cells also control the regulation of B cell class-switching to IgE. Because of their influence on the production of antibodies and allergic responses, over activation of Th2 cells appears to be responsible for the exacerbation of allergies (Type-1, immediate hypersensitivity reactions), autoimmune reactions such as chronic graft-versus host disease, progressive systemic sclerosis, and systemic lupus erythematosus. Additionally, Th2 cells are also known to be responsible for the development of asthma and other allergic inflammatory diseases. Interestingly, Th2 cells also produce the growth factor amphiregulin and IL-24 which have anti-tumor effects.

Naive CD4⁺ T cells are activated by simultaneous engagement of the T cell receptor (TCR) and co-stimulatory molecule, cluster of differentiation (CD)28, by antigen-presenting cells (APCs) expressing major histocompatibility complex (MHC) class II, such as macrophages, dendritic cells, and B cells [1]. Activation signals are conveyed upon recognition and binding of the TCR to its cognate antigenic peptide displayed on the surface of the MHC class II molecule and through multiple co-stimulatory signals. Once fully activated, naive CD4⁺ T cells in the lymphoid tissue rapidly proliferate, undergoing clonal expansion and differentiation into helper T cells, such as Th1, Th2, or Th17 cells [1]. The surrounding cytokine environment drives polarization of naive T cells into specific cell lineages, where the Th2 cell type is specifically triggered by interleukin (IL)-4 and IL-2 [2].

IL-4 stimulation drives signal transducers and activators of transcription (STAT)6 phosphorylation and upregulation of GATA-binding protein 3 (GATA3), a key transcription factor for Th2 cell development [2]. GATA3 interacts with IL-2-induced STAT5 signaling to upregulate expression of IL-4, the canonical Th2 cytokine. This signaling pathway is followed by an IL-4–driven positive feedback loop whereby the Th2 gene expression profile is amplified by continued IL-4-STAT6-GATA3 and IL-5-STAT5 signaling. The IL-4-dependent STAT6 activation of GATA3 is the canonical Th2 cell differentiation pathway; however, there are a number of nonclassical pathways thought to be important for Th2 polarization that include a variety of transcription factors (such as c-Maf, NF-κB, IRF4, AP1) and cytokines (thymic stromal lymphopoietin (TSLP), IL-5, IL-25, and IL-33) [2,3,4,5].

Differentiated effector Th cells migrate to sites of inflammation in the periphery, where they re-encounter their cognate antigen and secrete effector cytokines, thus driving an antigen-specific immune response. IL-5, IL-9, and IL-13 are secreted by Th2 cells only once they have reached inflamed tissue sites (Figure 1) [3]. Epithelial and innate cells at the site of inflammation also play a role in shaping the Th2 adaptive immune response. Innate lymphoid cells (ILC), macrophages, and dendritic cells secrete proinflammatory cytokines, including IL-1, IL-18, IL-25, IL-33, GM-CSF, M-CSF, and TSLP, which signal through different pathways to promote Th2 differentiation [2,3]. The cytokine milieu in the surrounding inflammatory microenvironment drives different Th2 effector activities. As a result, there are several phenotypically and functionally distinct Th2 memory cell subsets that carry out different effector roles depending on the cytokine influence. For instance, Th2 cells that produce high levels of IL-5 have been shown to contribute to the pathogenesis of allergic asthma, while cells that produce IL-5, IL-17, and IFN-γ, in addition to IL-4 and IL-13, have been identified as noncanonical memory Th2 cells that drive chronic allergic inflammatory diseases and prevent lymphocytic choriomeningitis virus (LCMV) persistence [4]. Memory Th2 cells also express IL17RB, TSLP receptor (TSLPR), chemoattractant receptor-homologous molecule (CRTh2), and CCR8 at similar levels [2,4].

Memory Th2 cells can be divided into at least four distinct subpopulations based on the levels of expressed chemokine (C-X-C motif) receptor 3 (CXCR3) and CD62L [5]. All four subpopulations characteristically produce large amounts of IL-4 and IL-13 in response to antigenic re-stimulation. However, only the CXCR3^low CD62L^low subpopulation produces IL-5 and is considered pathogenic, denoted as memory-type Tpath cells [4]. IL-33 stimulation induces production of IL-5 and IL-13 in memory Th2 cells and type 2 innate lymphoid cells (ILC2), thereby driving a type 2 immunological response [2,4]. Similar to IL-33, the combination of IL-2 and IL-25 also drives IL-5 production in Th2 memory cells and ILC [4,6]. ILC are immune cells derived from common lymphoid progenitors and are grouped based on their cytokine and transcription factor profiles, where ILC2 function in part to regulate Th2 phenotype [6]. Whereas IL-33 can induce a strong Th2 immunity and eosinophilic inflammation by amplifying the Th2 cytokine response in lung and intestine tissue, IL-33 has little effect on the Th2 effector subset and instead mediates inflammation via memory-type Tpath cells [4]. Exposure to an allergen, for example, may initiate a Th2 response in the airway by stimulating epithelial and endothelial cells to produce IL-33, thereby driving IL-5 production by Th2 memory cells and exacerbating the eosinophilic inflammation [4].

Table 1. Subpopulation of Th2 cells and key cytokine profile.

The exact mechanisms regulating maintenance of Th2 memory cells remains unclear. It has been shown that antigen-specific memory Th2 cells driving lung allergic responses reside within the lung tissue and that the Th2 memory response is dependent on IL-7 and IL-33-producing lymphatic endothelial cells (LECs) found within localized structures called inducible bronchus-associated lymphoid tissue (iBALT) [4]. Th2 cells producing IL-4, IL-5, and IL-13 are implicated in a number of inflammatory diseases, including asthma, chronic rhinosinusitis, atopic dermatitis, and eosinophilic gastrointestinal disorders such as ulcerative colitis [3,4]. A major cell type shown to drive murine chronic airway inflammation has been identified as CD44^hi CD62L^low CXCR3^low CCR4^hi CCR8^hi IL7Rα^hi ST2^hi memory-type Tpath2 cells, whereas CCR8^hi Tpath2 cells and CRTH2^hi CD161^hi PGDS^hi Tpath2 cells have been shown to drive chronic atopic dermatitis and eosinophilic gastrointestinal diseases, respectively [4]. Th2 cytokines drive pathogenesis of human asthma and murine allergic airway inflammation models by promoting eosinophilic infiltration, increased mucus production through goblet cell metaplasia, and tissue fibrosis [3,4]. Monoclonal antibodies directed against IL-5 and IL-13 have been used successfully as a therapies against asthma, and antibodies against Th2 cells show potential for future development in therapeutics [4]. Th2 cell-derived IL-4, IL-5, and IL-13 contribute to B cell proliferation and isotype class switching from immunoglobulin (Ig)G1 to IgE, a key antibody involved in parasitic helminth infection and certain allergic diseases associated with Th2 cells such as asthma [3,5]. Th2 cells have also been shown to induce the alternate activation (M2) macrophage phenotype, which mediates resolution of the inflammatory stage and initiates tissue repair in wound healing processes. Effector Th2 cells mediate immune responses to parasitic helminth infections, venoms, certain bacterial infections, and promote wound healing by inducing alternate activation (M2) macrophage phenotype [3].

Cytokine profiling is commonly used to classify the Th cell subtype and also to quantify the amounts of cytokines secreted. Cytokine ELISAs can be used to monitor T cell dependent cytokine secretion in response to activation and lineage-specific differentiation at the population level. Cytokine ELISA kits suitable for the detection and quantitation of hundreds of individual cytokines are commercially available. These kits are typically sold as 96-well plates pre-coated with the capture antibody and contain the detection antibody, as well as standards, buffers, and accessory reagents. Assay sensitivities are commonly in the picogram range.

While ELISAs can be used to measure the secretion of individual cytokines, advances in Luminex multiplexing technology allow for the high-throughput detection of multiple cytokines in a single sample or reaction well. The simultaneous measurement of multiple cytokines is achieved using a bank of antibodies bound to microspheres dyed with fluorophores of differing intensities. Quantitation is accomplished using a sandwich assay approach in combination with a Luminex detection system. A list of those relevant to Th2 biology can be found below in Table 2.

Table 2. Invitrogen ProcartaPlex Luminex Panels for Th2 study.

In addition to ELISA-based methods, another common and powerful tool to study Th2 and other immune populations is flow cytometry. Whereas an ELISA measures the amounts of cytokines secreted, flow cytometry can be used to profile cells based on both surface-expressed or intracellular markers, as well as cytokine expression. Th2 cells are defined by the combined expression of surface and intracellular targets such as: CD45⁺ CD3⁺ CD4⁺ IL-4⁺ CCR4⁺ CRTH2⁺ (CD8^- CD19^- CCR6^- CXCR5^- CXCR3^- CCR10^-) [1,2,3,4]. In addition, flow cytometry can be used to quantify the Th2 population with respect to other populations.

CD4⁺ Th2 cell specific markers

Optimized Multicolor Immunofluroscence Panels (OMIPs) published in the journal Cytometry Part A (Wiley Online Library) describe the use of a combination of specific antibodies and fluorophores for the extensive characterization of cell types by flow cytometry. The articles below provide panels that have been thoroughly tested and use validated set of antibodies and reagents that can be used together for the multicolor characterization of Th2 cell populations.

Table 3. Cytometry Part A OMIPs designed for Th2 cell characterization and analysis.