A complex issue on CD4⁺ T‐cell subsets

This article introduces a series of reviews covering CD4+ T-cell Subsets appearing in Volume 252 of Immunological Reviews. CD4+ T-cell-mediated immune responses are instrumental to eliminate viral, bacterial, fungal, or parasitic infections or malignant cells through secretion of cytokines and activation and recruitment of macrophages, neutrophils, eosinophils, and basophils. CD4+ T cells also provide help for B cells to make antibodies. CD4+ T-cell-mediated immunity can also involve aberrant recognition of autoantigens, leading to autoimmune diseases. CD4+ regulatory T cells (Tregs) play a central role in the control of autoimmunity, immune homeostasis, and immune responses to pathogens and tumor antigens. We now appreciate that several subsets of CD4+ T cells exist that shape the adaptive immune responses. In the 1980s and 1990s, two seminal discoveries defined the biology of CD4+ T-cell subsets: the concept of the T-helper 1 (Th1)/Th2 paradigm and the identification of CD4+CD25+ Tregs. Since the beginning of new millennium, immunologists have made great strides in our understanding of the immunobiology of CD4+ T cells, and consequently more CD4+ T-cell subsets have been identified, namely Th17, Th9, and Th22 cells. Together with the re-appreciation of follicular T-helper cells (Tfh) which were first observed in the 1970s, we now understand that the CD4+ T-cell subsets play roles in adaptive immunity ranging from the clearance of pathogens and the control of autoimmunity to immune homeostasis and immune responses again tumors (Fig. 1). Herein, we introduce CD4+ T-cell subsets and provide a snapshot of this volume of Immunological Reviews, which updates this rapidly advancing area of immunology that covers diverse aspects of CD4+ T-cell subsets including molecular control of its development and lineage plasticity, function in immunological diseases, and clinical application. In 1986, Mosman and Coffman 1 made the landmark observation that antigen-specific mouse helper T-cell clones could be characterized according to patterns of cytokine production, and two types of helper T cells were distinguished. Type 1 (Th1) cells produced interleukin-2 (IL-2), interferon-γ (IFN-γ), granulocyte-macrophage colony-stimulating factor (GM-CSF), and IL-3 in response to antigen plus antigen-presenting cells (APCs) or to concanavalin A (ConA), whereas type 2 (Th2) cells produced IL-3, IL-4, and a mast cell growth factor (IL-5) 1. Subsequent studies by Mosman and colleagues 2 showed that delayed type hypersensitivity was mediated by Th1 cells but not Th2 cells. Moreover, IL-10 secreted by Th2 cells inhibited cytokine production of IL-2, IL-3, lymphotoxin (LT)/tumor necrosis factor-α (TNF-α), IFN-γ, and GM-CSF by Th1 cells 3. Further study showed that Th2 cells enhanced immunoglobulin E (IgE) and IgG1 synthesis, whereas Th1 cells promoted IgG2a production by B cells 4. This finding was in line with the observation by Paul and Snapper 5, who showed that IL-4 controlled class switching to IgE and IgG1 but markedly inhibited IgM, IgG3, IgG2a, and IgG2b, and IFN-γ controlled class switching to IgG2a, but inhibited the production of IgG3, IgG1, IgG2b, and IgE. In 1990, two teams led by Paul 6 and Swain 7 showed that IL-4 was capable of driving the differentiation of naive CD4+ T cells into IL-4-producing Th2 cells in vitro. In 1993, Murphy and O'Garra and colleagues 8 reported that naive CD4+ T cells could differentiate into IFN-γ-producing Th1 cells in vitro when stimulated with APCs in the presence of IL-12 produced by Listeria monocytogenes-induced macrophages. In 1991, Romagnani and colleagues 9 reported that human CD4+ T-cell clones specific for the intracellular bacterium Mycobacterium tuberculosis were mostly IFN-γ-producing Th1 cells, whereas the CD4+ T-cell clones specific for the extracellular helminth Toxocara canis were mainly IL-4/IL-5-producing Th2 cells. These data established that intracellular pathogens induce Th1 cells and that extracellular pathogens promote the development of Th2 cells 9. Since the establishment of Th1/Th2 paradigm in murine and human systems, the underlying signaling pathways and transcription factors that direct Th1 versus Th2 development were subjected to extensive scrutiny. In 1995, Murphy et al. 10 showed that IL-12 induced Th1 cell differentiation through tyrosine phosphorylation of signal transducer and activator of transcription 4 (Stat4). One year later, three groups reported that IL-4 induced Th2 cell development through Stat6 11-13. Stat6 knockout T cells did not differentiate into Th2 cells in response to either IL-4 or IL-13, and Stat6-deficient B cells failed to produce IgE following in vivo immunization with anti-IgD 11-13. Then, in 1997 and in 2000, Flavell 14 and Glimcher 15 and their colleagues reported that transcription factors GATA-3 and T-bet were required to direct the Th2 and Th1 lineage commitment, respectively. The concept of Th1/Th2 paradigm has provided a concise basis to rationalize many aspects of adaptive immunity against intracellular versus extracellular pathogens and the pathogenesis of immune-mediated tissue damage in infection and allergy (Fig. 1). In 1995, Sakaguchi and colleagues 16 described a distinct population of CD4+ T cells expressing the IL-2 receptor α chain (CD25). These cells represent 5–10% of CD4+ T cells, and they were shown to prevent spontaneous autoimmune diseases. The immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome is multi-organ autoimmune disease that occurs in the first years of life 17. The Scurfy mouse, a murine model of IPEX, was described in 1991 18. In 2001, three laboratories reported that IPEX and Scurfy were the consequence of a deficiency in the gene expressing of the forkhead box protein 3 (Foxp3) transcription factor 19-21. Moreover, Ziegler et al. 22 showed that overexpression of Foxp3 in CD4+ T cells inhibited activation-induced cytokine production and proliferation. In 2003, Sakaguchi 23, Rudensky 24, and Ramsdell 25 independently demonstrated that the generation and function of CD4+CD25+ Tregs were controlled by Foxp3. Scurfy mice were deficient in Tregs, and they developed severe lymphoproliferative autoimmune disease. Ectopic transduction of the Foxp3 gene into conventional T cells induced them to become Tregs 23-25. The expression of Foxp3 could be induced in naive T cells by transforming growth factor-β (TGF-β), and they became Foxp3+ Tregs 26. These findings have established that Foxp3 is the master transcriptional regulator for the development and function of Tregs, and CD4+Foxp3+ Tregs are indeed a separate cell lineage with professional suppressive properties (Fig. 1). Rigorous research on Treg cells has been undertaken 27-32, and in 2008, the biannual China Tregs Conference Series (www.chinatregs.com) was established. As discussed above, Tregs play a central role in the control of autoimmune diseases, and Th1 cells have been associated with several forms of autoimmune diseases including multiple sclerosis, rheumatoid arthritis, type 1 diabetes, and inflammatory bowel disease (IBD) through the production of inflammatory IFN-γ and IL-12. However, mice with selective blockade of IFN-γ and IL-12 function were found to still be sensitive to disease in autoimmune disease models, including experimental autoimmune encephalomyelitis (EAE), collagen-induced arthritis, and type 1 diabetes, arguing for a pathogenic role for Th1 cells in autoimmunity 33-35. In 2003, Dong and colleagues 36 found that mice deficient in inducible T-cell costimulator (ICOS) were resistant to collagen-induced arthritis, which surprisingly was not associated with IFN-γ deficiency but rather impaired expression of IL-17, another proinflammatory cytokine made by T cells. IL-12 is a heterodimeric cytokine, composed of IL-12p40 and IL-12p35 subunits. The IL-12 family-related cytokine IL-23 is composed of IL-23p19 and IL-12p40 subunits 37. In 2003, Cua and colleagues 38 showed that IL-23 p19 knockout mice, in contrast with IL-12 p35-deficient mice, were resistant to the development of EAE, indicating that IL-23 rather than IL-12 is the critical cytokine for the induction of EAE. Cua et al. 39 also demonstrated that IL-23 knockout mice were resistant to collagen-induced arthritis, and this resistance correlated with an absence of IL-17-producing CD4+ T cells. Conversely, IL-12-deficient p35 knockout mice developed more IL-17-producing CD4+ T cells, suggesting that IL-23 may be the differentiation factor for the generation of pathogenic IL-17-producing CD4+ T cells 39. These pursuits for the identification of the cytokines responsible for the pathogenesis of EAE and autoimmune diseases culminated in 2005, 10 years after the discovery of IL-17 40. Two teams led by Weaver 41 and Dong 42 demonstrated that IL-17-producing CD4+ T cells were a distinct cell lineage from Th1 and Th2 cells, and they coined IL-17-producing CD4+ T cells as Th17 cells. Th17 cells, not Th1 cells, were identified to induce EAE, and IL-23 induced Th17 cells to produce IL-17 41-43. One year later, three groups led by Weaver 44, Kuchroo 45, and Stockinger 46 independently reported that Th17 cells could be induced in vitro from naive murine CD4+ T cells by T-cell receptor (TCR) stimulation in the presence of IL-6 and TGF-β. At the same time, Littman and Cua 47 identified that retinoid-acid receptor-related orphan receptor γt (RORγt) was the master regulator gene for the development of Th17 cells. The establishment of Th17 cells as subset of CD4+ T cells has expanded the concept of Th1/Th2 paradigm 48 (Fig. 1). In 2008 and 2009, two more CD4+ T-cell subsets were proposed, namely IL-9-producing Th9 and IL-22-secreting Th22 cells. IL-9 is produced also by Th2 cells, and this cytokine plays an important role in the pathogenesis of asthma, IgE class switching, and clearance of parasitic infections 49. In 2008, the groups led by Stockinger 50 and Kuchroo 51 independently reported that naive CD4+ T cells could differentiate into Th9 cells after TCR stimulation in the presence of TGF-β and IL-4. Interestingly, the combination of TGF-β and IL-4 to induce IL-9 production by CD4+ T cells had been described 14 years earlier 52. Then, in 2010, the ETS family transcription factor PU-1 and IFN regulatory factor 4 (IRF4) were identified as the transcription factors necessary for the development of Th9 cells 53, 54. IL-22 is produced by Th17 cells and a subset of natural killer cells that acts on epithelial cells and keratinocytes, and IL-22 has been linked to skin homeostasis and inflammation 48, 55. In 2009, three groups showed that naive CD4+ T cells became Th22 cells when stimulated with plasmacytoid dendritic cells in the presence of IL-6 and TNF-α 56-58. IL-22-producing human Th22 cells that expressed the chemokine receptors CCR10, CCR6, and CCR4 could be found in human skin during skin disease such as psoriasis 56-58. No transcription factors have been identified for the development of Th22 cells. However, aryl hydrocarbon receptor was reported to play a role in IL-22 production by Th22 cells 56, 57, and genome transcriptome analysis of Th22 cells showed that Foxo4 and BCN2 were upregulated in Th22 cell clones isolated from psoriasis patients 58. Antibody production by B cells requires help from CD4+ T cells 59. These CD4+ T cells are found in the B-cell follicles of secondary lymphoid organs, and they are thus called Tfh cells 60. Tfh cells stimulate antigen-specific naive or memory B-cell activation, which triggers germinal center (GC) formation 60. Tfh cells express distinct surface markers. In 1999, McHeyzer-Williams and Cyster 61 noted that Tfh cells upregulated CXCR5 expression before migrating into follicles after immunization. In 2000, two teams led by Forster 62 and Moser 63 identified CXCR5 as a marker for GC Tfh cells, and the authors provided a full characterization of the Tfh cell phenotype. Tfh cells expressed CD69, HLA-DR, ICOS, and CD40L 62, 63. The SAP (signaling lymphocytic activating molecule-associated protein) gene has been identified as the genetic locus responsible for X-linked lymphoproliferative disease, a fatal immunodeficiency. In 2003, Crotty and Ahmed 64 reported that SAP played a role in Tfh function that was essential for late B-cell help and the development of long-term humoral immunity but was not required for early B-cell help and class switching. SAP knockout mice generated strong acute IgG antibody responses after viral infection but showed a near complete absence of virus-specific long-lived plasma cells and memory B cells, despite the presence of virus-specific memory CD4+ T cells 64. This observation supported the clinical importance of Tfh cells 64. The relevance of Tfh cells to other T-cell subsets was puzzling. In 2008, Dong and coworkers 65 proposed that Tfh cells might be generated via a distinct genetic program than for Th1, Th2 or Th17 cells. In 2009, three groups led by Dong 66, Crotty 67, and Vinuesa 68 independently identified the transcriptional repressor B-cell lymphoma 6 (Bcl-6) as the master regulator for Tfh cell development. The combination of cytokines IL-6 and IL-21 induced naive CD4+ T cells to differentiate into Tfh cells. Bcl-6 expression in Tfh cells was essential for CXCR5 expression, migration into follicles, and GC formation 66-68. Moreover, the transcription factor B-lymphocyte induced maturation protein-1(Blimp-1), an antagonist of Bcl-6, inhibited Tfh cell differentiation and function 66-68. These findings provided convincing evidence that Tfh cells are a separate CD4+ T-cell lineage (Fig. 1). After TCR stimulation, naive CD4+ T cells differentiate into distinct Th cell subsets under different cytokine milieus (Fig. 1). In this volume of Immunological Reviews, there are four reviews that summarize the molecular events that control the differentiation and development of CD4+ T cell subsets. First, Yamane and Paul 69 discuss how TCR signals regulate the development of CD4+ T-cell subsets at the early stages of activation. This review considers TCR signaling strength, TCR proximal signaling intermediates including Src-family tyrosine kinases Lck and Fyn, linker for activation of T cells, Tec-family IL-2-inducible T-cell kinase (Itk), and the Notch pathway. Cytokines activate STAT family transcription factors, master regulators including T-bet, GATA-3, Foxp3, RORγt, Bcl-6, PU-1, as well as other transcription factors including IRF-4. O'Shea and colleagues 70 discuss how this transcription factor network impacts the transcriptome and epigenome of CD4+ T-cell subsets to produce effector cytokines. NF-κB family members control the transcription of DNA in almost all animal cell types. NF-κB regulates a plethora of genes in apoptosis, stress responses, immunity, and inflammation. TCR/CD28 signaling stimulates NF-κB activation. Emerging data indicate that the NF-κB pathway also plays a role in the development and functional divergence of CD4+ T-cell subsets. In this volume, Oh and Ghosh 71 discuss the differential functions of NF-κB during development and activation of CD4+ T-cell subsets. While TCR, cytokine, and NF-κB pathway signaling influence CD4+ T-cell differentiation, environmental factors such as nutrient and oxygen availability, energy, and stress also play a role in the naive CD4+ T-cell fate decision. Pan and colleagues 72 illustrate the influence of metabolic factors on CD4+ T-cell development. They focus on several metabolic sensors: the mammalian target of rapamycin, hypoxia-inducible factor 1α, nuclear hormone receptors peroxisome proliferator-activated receptors, fatty acid-binding proteins, sterol sensing and cholesterol metabolism, and estrogen-related receptor α 72. Taken together, these four reviews summarize the molecular events that control the differentiation and development of CD4+ T-cell subsets in great detail. Th17, Th9, Th22 cells, and Tfh cells all play roles in health and disease. Herein, two reviews by Cua 73 and Weaver 74 and their colleagues illustrate the life cycle of Th17 cells and their biological functions. Th9 and Th22 cells are the most recently identified Th subsets. In this volume, Kaplan 75 addresses how IL-9 is regulated and how Th9 cells fit into the spectrum of CD4+ T-cell subsets in autoimmunity, tumor immunity, and inflammation. IL-22 is produced not only by Th22 cells but also by Th17 cells and innate lymphoid cells (ILCs). Ouyang and colleagues 76 provide an extensive review of the biology of IL-22. Spits and colleagues 77 summarize the current knowledge of Th1 and Th2-like ILCs. These type 1 and type 2 ILCs display a remarkable similarity with Th1 and Th2 cells, with respect to the diversity of cytokine production and the transcription factors governing their development and function 77. The GC is a dynamic site within lymph nodes where B cells fully differentiate into plasma cells and memory B cells. Tfh cells are instrumental in the process by providing help to B cells. Dong and colleagues 78 discuss how transcription factors control Tfh cell differentiation. As in thymus where T cells are generated, GCs give rise to Tfh cells. Ramiscal and Vinuesa 79 summarize the ontogeny, molecular identity, and functional relevance of follicular homing T cells, pre-Tfh cells, Tfh cells, natural killer Tfh cells, and CD4+ and CD8+ follicular Tregs in the GC. Together, these reviews describe the current knowledge of Th9, Th17, Th22 cells, and Tfh cells in health and disease. The discovery of CD4+ T-cell subsets has greatly contributed to our understanding of adaptive immunity in the control of immunological diseases. Three reviews by Kolls 80, Powrie 81, and Jiang 82 and colleagues focus on the translational aspects of CD4+ T-cell subsets. These groups discuss the role of CD4+ T-cell subsets in host defense in the lung, in intestinal inflammation, and in organ transplantation rejection and tolerance, respectively. Through extensive research in the past three decades, we now appreciate that CD4+ T cells consist of several subsets. Th1, Th2, Th9, Th17, Th22, and Tfh cells contribute to immune responses against pathogens and autoimmunity, and CD4+ Tregs control these immune responses. In this volume of Immunological Reviews, the leading experts in the field review the most up to date information on CD4+ T-cell subsets. We very much hope that this volume will accelerate future investigations to translate the knowledge of CD4+ T-cell subsets into the development of effective therapies for immune-mediated diseases.