Functional quartet by CD4⁺ T cells: a concerto of multiple cytokines

The differentiation of thymus-derived naive CD4⁺ T cells into distinct cell lineages upon activation enables them to provide help or to suppress other immune cells, thereby orchestrating varied immune responses against foreign and self antigens. The generation of two distinct subsets of CD4⁺ T helper (Th) cells (Th1 and Th2) was described over 20 years ago on the basis of different sets of cytokines secreted after activation and differentiation of naive CD4⁺ cells; each subset proved to mediate different patterns of immune responses [1]. Th1 cells secrete IFN-γ to protect against intracellular microbe infections, mediate organ-specific autoimmunity and eliminate cancer cells. In contradistinction, Th2 cells secrete IL-3, IL-4, IL-5 and IL-13 to regulate humoral immune responses, defend against parasites and mediate allergy and asthma. It was discovered that dendritic cells (DCs) recognize different pathogens via microbial pattern-recognition receptors (PRRs) and translate these signals into factors polarizing Th1 and Th2 cells [2]. Toll-like receptors (TLRs), which discriminate between different types of pathogens, are the most common PRRs involved in this process. For instance, the binding of TLRs with bacteria ligands, such as unmethylated CpG and lipopolysaccharide, turn immature DCs into IL-12-producing and Th1-promoting DCs (DC1). The PRRs that mediate Th2-promoting DCs (DC2) still need to be determined [2,3].

Cytokines that are secreted from DCs or other cells are critical for the polarization of Th1 and Th2 cells. For Th1 cell differentiation upon T-cell receptor (TCR) stimulation, IFN-γ activates signal transducer and activator of transcription (STAT)1, which in turn induces the transcription factor T-box expressed in T cells (T-bet). T-bet promotes IFN-γ production and expression of IL-12 receptor (IL-12Rβ2), facilitating IL-12-induced IFN-γ production and continuous development of Th1 cells. By contrast, Th2 differentiation is initiated by coordinate signaling through the TCR and IL-4 receptor/STAT6, which induce the expression of the zinc finger transcription factor GATA3; GATA3 is the master regulator that enables expression of Th2 cytokines [4]. Interestingly, Th1 and Th2 differentiation are mutually antagonistic. High levels of IFN-γ, IL-12 and T-bet inhibit GATA3 expression and Th2 differentiation [5], whereas increased levels of IL-4 and GATA3 downregulate T-bet expression and Th1 differentiation [6]. Therefore, IFN-γ and IL-4 serve as autocrines for Th1 and Th2 subsets, respectively, and are inhibitors to the opposite subsets. Owing to genetic imprinting, dividing committed subsets maintain polarization throughout the process of clonal expansion, which facilitates the development of more cells of the same subset for optimal effector function of desired immune responses.

Over the past few years, it has been suggested that the Th1–Th2 paradigm cannot completely elucidate the diversity of CD4⁺ effector T-cell responses. The recently discovered IL-17-producing CD4⁺ Th17 cells are distinct from Th1 and Th2 cells [7,8], revealing additional complexity for T-cell differentiation. Th17 cells secrete IL-17A, IL-17F and IL-22, which are critical in regulating tissue inflammation [8]. Th17 cells have been shown to protect against extracellular bacteria and fungal infection, as well as contributing to certain autoimmune responses [9]. The combination of TGF-β and IL-6 induces the in vitro differentiation of murine Th17 cells [10]. At the same time, TGF-β suppresses Th1 and Th2 cell differentiation. Upon naive T-cell activation, IL-6 induces IL-21 production, which acts in concert with TGF-β to induce the expression of retinoic orphan receptor (ROR)γt via a STAT3-dependent mechanism [11–13]. As the master regulator for Th17 cells, RORγt induces transcription of genes encoding IL-17A and IL-17F. Another cytokine, IL-23, is also essential for Th17, possibly by maintaining its survival [10]. While IL-1 enhances the development of murine Th17 cells [10], it is more important for the development of human Th17 cells [14,15]. It has been only 2 years since Th17 cells were recognized as a distinct T-cell lineage [7,8] and, therefore, further characterization of these cells will continue.

Prior to the finding that TGF-β and IL-6 are involved in the generation of Th17 cells, it has been shown that TGF-β and IL-2 induce forkhead box protein (Foxp)3 in naive CD4⁺ T cells after activation in vitro [16]. Foxp3 expression is required for both the development and function of naturally occurring thymus-borne CD4⁺CD25⁺ T regulatory (natural Treg) cells [17]. TGF-β-induced Foxp3 expression in TCR-challenged naive T cells converts them into adaptive regulatory T cells (adaptive Treg) with potent immunosuppressive function [16]. Indeed, overexpression of the TGF-β transgene under the IL-2 promoter created mice with elevated levels of natural and adaptive Treg cells [18]. When multiple cytokines were tested for conversion of naive T cells into adaptive Treg cells, IL-6 inhibited conversion of CD4⁺Foxp3^-cells into Foxp3⁺ cells but instead induced Th17 cell differentiation, documenting antagonistic interaction between natural and adaptive Treg cells versus Th17 cells [19]. Interplay between Treg and Th17 cells was apparent when IL-6-deficient mice displayed a very high frequency of Treg cells [19]. Depletion of Treg cells in these mice showed reappearance of Th17 cells, suggesting IL-6-independent pathways in the generation of Th17 cells. These results suggest that the differentiation of adaptive Treg cells from naive T cells is a novel differentiation pathway for naive CD4⁺ T cells, and that IL-6-mediated signaling regulates adaptive Treg and Th17 functions.

It seems that ‘naivety’ of the immune system consisting of naive CD4⁺ T cells is maintained by thymus-derived natural Treg cells with higher affinity to self-antigens than naive CD4⁺ T cells. Exposure to foreign or self antigen may launch four different subsets (Th1, Th2, Th17 and adaptive Treg) from naive CD4⁺ T cells dependent upon involvement of specialized DCs (DC1, DC2 and immature DC), activation conditions and cytokine environment. Therefore, the trio of effector cells (Th1, Th2 and Th17) may be shaped into the dominance of one of these subsets executing the most effective response against a particular antigen (e.g., a type of bacteria or virus or organ-specific self antigen) and defensive requirements (e.g., a targeted organ, such as skin or lungs). The fourth subset of adaptive Treg cells always seems to be activated (independently of the effector cell bias) to appropriately downregulate the immune response. Using a musical analogy, the immune concerto of the polyphonic (three different) melodies is played by differently shaped contributions of effector cells but always in the presence of a counterpoint (contrapuntal) melody balancing the overall harmony. This quartet of CD4⁺ T cells generates a myriad of ‘sounds’ produced by multiple cytokines shaping the outcomes of immune responses. Thus, adaptive Treg cells together with natural Treg cells contribute to the maintenance of immunologic self tolerance and negative control of various immune responses, including autoimmune disease, infection, allergy, cancer and allograft rejection [17].

Acknowledgements

The authors thank Natasha Green, MA, ELS, for skillful preparation of the manuscript.

Financial & competing interests disclosure

This work was supported by grants from the National Institutes of Health HL069723. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

No writing assistance was utilized in the production of this manuscript.