6.2.2 CD4+ T Cells
The known repertoire of CD4+ helper T cells, characterized by the unique expression of cytokines and master regulator transcription factors, has expanded in recent years.TH1, TH2, TH17,regulatory T (Treg), follicular helper T (TFH), and TH9 are currently known.There has been no report of TFH and TH9 in TIL.
Th1 polarization is involved in antitumor immune responses.TH1 cells differentiate and develop effector functions in response to IL-12 and IFN-c through signal transduction and activation of transcription (STAT)-4 and the transcription factor T-bet.Th1 polarization increases IFN-c production, leading to more robust anti-tumor immunity through improved CTL responses [38].
Effector Th1 cells differentiate from naïve lymphocytes.In general, the effector cell differentiation is driven by the signals received from TCR, costimulatory receptors, and cytokines.In the most simplified model, signals mediated by TCR and costimulatory receptors induce T cell activation and proliferation.Cytokine-derived signals are the main signals determining the differentiation lineage of antigen-activated T cells.Ligation of cytokines with their receptors activates the signal transducer and activator of transcription (STAT) factors.The STATs translocate to the nucleus and bind genes encoding lineage-specifying transcription factors (“master regulators”) and effector cytokines.These events determine the lineage of differentiating T helper cells [39].
The main Th1 inducing cytokines are IL-12 and IFN-γ.IL-12 is produced by antigen-presenting cells.Interaction of IL-12 with the IL-12 receptor, expressed on the surface of T cells, induces STAT4, which in turn induces T-bet, a master regulator of Th1 cells.T-bet binds directly to many Th1-specific genes and positively regulates their expression.T-bet also negatively regulates the expression of Th2- and Th17-specific genes and inhibits the differentiation of Th2 and Th17 cells.In the absence of T-bet, T helper cells differentiate towards Th2-like cells.STAT4 also directly binds to IFN-γ locus and stimulates IFN-γ production.STAT4 and T-bet cooperate to induce maximal IFN-γ production.In the absence of STAT4, T-bet does not induce optimal IFN-γ levels,and the combination of T-bet and STAT4 deficiency abolishes IFN-γ production [40,41].
IFN-γ acts by inducing STAT1.STAT1 synergizes with STAT4 in activating T-bet in T helper cells and also can directly activate Th1-associated genes.IL-4 and IL-10 inhibit Th1 cell differentiation and induce Th2 cells.Thus, generally, Th1 and Th2 are alternate and mutually exclusive lineages of T helper cells.In some conditions, however, Th1 and Th2 cells retain their plasticity, can transdifferentiate, and even co-expressing Th1- and Th2-specific cytokines and transcription factors(Figure 6.3) [42,43].
Figure 6.3 Plasticity of hu man Th1 and Th2 cells.Naive CD4+ T cells are stem-cell-like cells that under the influence of different cytokines can differentiate to various types of effector cells including Th1, Th2, Th9, and TFH cells.Th1 and Th2 central memory cells are arrested at an early stage of differentiation, are highly plastic and some can still switch lineage.Conversely, effector memory cells are more differentiated, less plastic, and rather become polyfunctional.Moreover, Th1 effector cells can acquire IL-10 producing capacities and regulatory functions in chronically inflammed tissues.(Please scan the QR code on the Preface to get original color figures.)
Source: Geginat J, Paroni M, Maglie S, Alfen J S, Kastirr I, Gruarin P, De Simone M, Pagani M, Abrignani S.“Plasticity of Human CD4 T Cell Subsets”.FrontiersinImmunology, 2014, 5:630.
Cytokine milieu is the main factor that determines the lineage of T helper cell differentiation.However, the differentiation is also affected by the dose and the strength of TCR agonistic ligand.It has been shown that stimulation with a high dose of TCR agonistic peptide or a strongly agonistic ligand favors generation of Th1 cells whereas a low dose or a weakly agonistic ligand favors Th2 cells.Therefore, we may speculate that pathogen load, pathogen metabolic activity, and the stage of the disease affect the composition of the responding T helper population [44].
Th2 polarization is thought to promote tumor formation.Th2 polarization is driven by IL-4, STAT-6,and transcription factor Gata-3.Th2 cells secrete IL-4, IL-5, IL-10, and IL-13 and are related to humoral immunity.Th2 differentiation inhibits CD4+ T cells to differentiate toward TH1, resulting in inhibition of antitumor immune responses.In addition, IL-4 may exert direct immunosuppressive effects on CD8+ T cells [45,46].
The development of Th2 cells, which evolve to enhance the clearance of parasites, is coupled to IL-4.Subsequently, the identification of the interleukin 17 (IL-17)-producing T helper (Th17)subset as well as transforming growth factor (TGF-β)-induced regulatory T cells (iTreg cells) has added to the CD4+ effector T cell program (Figure 6.4) [47].
Figure 6.4 CD4+ T cell subsets and Th2 signaling.Naïve T cells can develop into different subsets when facing different cytokine milieus.In Th2 cells, GATA3 remodels IL-4 locus into a more accessible condition.Dec2 caused IL-2Rα up-regulation and can cooperate with GATA3 to enhance Th2 differentiation.GATA3 directly activates ECM1 expression, and ECM1 blocks STAT5 phosphorylation by interaction with IL-2Rβ, by which the negative signal of S1Pr1 re-expression is released, and mature Th2 cells can egress lymph node under the calling of high level of S1P.
Source: Li Z, Zhang Y, Sun B.“Current Understanding of Th2 Cell Differentiation and Function”.ProteinCell, 2011, 2(8):604-11.
By the 1990s, classic studies on helper T cell differentiation had been fully explored.The process by which an uncommitted helper T cell develops into a mature Th1 or Th2 cell is a useful model of developmentally regulated gene expression, T-bet for Th1 cell and GATA3 for Th2 cell.There is good evidence to indicate that this differentiation process is highly plastic.Many factors influence the decision to become a Th1 or Th2 cell.The cytokines IL-12 and IL-4, acting through signal transducer and activator of transcription 4 (STAT4) and STAT6, respectively, are key determinants of the outcome.It also has been reported that antigen dose, co-stimulators, genetic modifiers and other non-cytokine factors have crucial roles in determining the dominance of helper T cell response.However, how each signaling influences the differentiation process of helper T cells is still controversial [47].In the past 20 years, several lines of evidence have indicated that chromatin structure has an integral role in helper T cell differentiation.The induction of competence for effector-cytokine gene expression seems to be involved in de-repression of silent chromatin contexts.For Th2 epigenetic research, Il4 locus is actively repressed in naïve CD4+ T cells by molecules that link methylated DNA to repressive chromatin.Together, methylCpG-binding domain protein 2 (MBD2) and nucleosome remodeling and histone deacetylase (NURD) form the MeCP1 complex.The sites of action of this repression probably overlap with crucial cis-acting elements in the locus, such as the IL-4-IL-13 intergenic region (CNS1), the second intron of IL-4, and the 3′ enhancer (CNS2).Activation of the locus occurs in a stepwise fashion.First, GATA3 mediates displacement of MBD2,leading to chromatin changes such as histone acetylation, an effect that is concurrent with the induction of IL-4 transcription.Later, the acquisition of additional epigenetic changes, such as stable CpG demethylation, occurs along the cytokine gene.A recent study has reported that HS2 is the target of GATA-3 in regulating chromosomal modification of the Il4 locus and is independent of the Il5 and Il13 loci [48].(https://www.daowen.com)
One of the important transcript factors of Th2 development is Dec2, a basic helix-loop-helix(bHLH).We found Dec2 is selectively expressed in Th2 cells, and overexpressing Dec2 by retrovirus-infection or transgenic manipulation could promote Th2 development in Th2 cytokine milieu, while Dec2 deletion leads to less Th2 cytokine expression, such as IL-4, IL-5 and IL-13, in both in vitro and in vivo conditions.In further work, Dec2 is found directly bind to the Junb CNS and the Gata3 promoter to activate their transcription, and can promote CD25 level in Th2, which shows Dec2 may help Th2 development in both differentiation and proliferation aspects [48].
Th17 differentiation is induced during T cell activation in the presence of IL-6, TGF-b, IL-1b, and IL-21, leading to expression of the transcription factor ROR-ct (RORC in human) via activation of STAT-3 and the aryl hydrocarbon receptor (AHR).TH17 cells secrete IL-17A, IL-17F, IL-21, and IL-22 and play roles in driving inflammatory and autoimmune disease and mediating protection against some extracellular pathogens, possibly through neutrophil recruitment via chemokine induction.Tumor-infiltrating TH17 cells have been reported in many human cancers, including those of the ovary, prostate, liver, kidney, and breast.Several studies using mouse models have reported that IL-17 promotes tumor growth mediated by STAT-3 activation.In contrast, a tumor-suppressive effect of IL-17-dependent inflammation has also been reported, although this difference between studies has been attributed to the choice of tumor model [49,50].
The emergence of the distinct Th17 lineage can be attributed to studies of central nervous system autoimmunity.Early studies involving the EAE model revealed that IL-23 knockout (KO) mice were resistant to developing EAE, while IL-12 KO mice remained susceptible.This surprising series of experiments signified that Th1 polarization was not critical to the autoimmune phenotype,as was previously posited.IL-23 was subsequently discovered to drive polarization of a pathogenic CD4+ T cell subset characterized by production of IL-17A and IL-17F, which could induce EAE upon adoptive transfer, while IFN-γ producing Th1 cells could not.These IL-17 producing cells were considered a distinct lineage, when the milieu of cytokines supporting their differentiation was shown to be independent of the environment of cytokines required for Th1 and Th2 development.In fact, the in vitro differentiation of naive CD4+ T cells to Th17 cells is suppressed by Th1/Th2 cytokines IFN-γ and IL-4 and relies on co-stimulation by CD28 and ICOS.The cytokines most important to Th17 differentiation are TGF-β, IL-6, and IL-1β, and the phenotype is maintained long term in the presence of IL-21 and IL-23 [51,52].
Along with IL-6, TGF-β is well known as a critical cytokine for inducing RORγt in naive CD4+ T cells, which in turn drives their differentiation to a Th17 phenotype.However, new findings have provided insight into exactly how TGF-β regulates RORγt.Interestingly, TGF-β was shown to modulate the SKI-SMAD4 complex.The SKI-SMAD4 complex suppresses RORγt, as the SKI protein inhibits acetylation of the Rorc locus.However, in the presence of TGF-β, SKI is degraded,permitting RORγt expression in CD4+ T cells and ultimately driving Th17 differentiation.Low doses of TGF-β1 also inhibit IL-2-mediated activation of STAT5 and reduce T-bet and GATA3 expression, which inhibits Th1/Th2/Treg differentiation while promoting the Th17 lineage.Recent findings have also demonstrated that phosphatase and tensin homolog (PTEN) in Th17 cells suppresses IL-2 signaling, reducing STAT5 and the Treg pathway while upregulating STAT3, a transcription factor that supports the Th17 pathway.It is also important to appreciate that TGF-β and IL-6 induce the IL-23 receptor (IL-23R) in Th17 cells.IL-23 further activates STAT3, RORα and RORγt in Th17 cells to maintain their long term proinflammatory signature.Thus, naive CD4+ T cells cultured with TGF-β and IL-6 but without IL-23 still produce IL-17 but also produce anti-inflammatory cytokine IL-10.These non-pathogenic Th17 cells do not induce EAE and have compromised persistence and phenotypic maintenance in vivo [53-57].
At homeostasis, Th17 cells promote gut barrier defense, granulopoiesis, granulocyte chemotaxis,and immunity against extracellular pathogens.Most Th17 cells reside within the lamina propria of the gut in healthy individuals but are induced at other mucosal sites upon exposure to danger signals, such as infection.To maintain gut defense, IL-17 upregulates claudins for tight junction formation in the intestinal barrier and IL-22 plays a role in epithelial maintenance.IL-17 induces granulopoeisis indirectly through stimulation of epithelial cells, endothelial cells, and fibroblasts to secrete GM-CSF, IL-6, IL-8, and MIP-2.In turn, IL-8, and MIP-2 enhance chemotaxis of neutrophils.Mice deficient in IL-17R have an impaired ability to repopulate these immune cells after irradiation.Th17 cells and IL-17 have been implicated in immunity against extracellular pathogens, such as Klebsiella pneumoniae, Staphylococcus aureus, Salmonella enterica serovar Enteriditus, and Shigella flexneri among other bacterial species.Th17 cells have also been shown to augment Th1 recruitment in Mycobacterium tuberculosis infection, which is critical to granuloma formation and sequestration of bacteria.These collective Th17 functions are critical in preserving the health of the host and when compromised can lead to various disease symptoms, as discussed in Figure 6.5 [58-63].
Figure 6.5 T reg and T h17 cell ba lance.TCR on naïve CD4+ T cells interacts with the antigenic peptide/MHC complex (pMHC) on the surface of APC, which activates CD4+ T cells.With the influence of downstream signalling pathways and cytokines, CD4+ T cells can differentiate into either Treg or Th17 cells.(1) Treg: IL-2 and high concentration of TGF-β activate the STAT5 pathway and CD4+ T cells differentiate into Tregs.At the same time, the levels of IL-10, IL-18 and IL-33 in tissues increase.These cytokines cooperate with Tregs and play a role in the inhibition of immune responses.(2) Th17:IL-6, IL-21, IL-23 and low concentration of TGF-β are involved in the conversion of CD4+ T cells into Th17 cells through the STAT3 pathway.TGF-β at a low concentration induces the expression of IL-23R,which in turn facilitates the differentiation of Th17 cells.Increased numbers of Th17 cells and their secreted cytokines such as IL-17 and IL-22 promote inflammation and the clearance of extracellular pathogens.Abbreviations: APC, antigen-presenting cells; MHC, major histocompatibility complex; TCR,T cell receptor; TGF-β, transforming growth factor-β.(Please scan the QR code on the Preface to get original color figures.)
Source: Wan Z, Zhou Z, Liu Y, Lai Y, Luo Y, Peng X, Zou W.“Regulatory T Cells and T Helper 17 Cells in Viral Infection”.ScandnavianJournalofImmunology, 2020, 91(5): e12873.
Mutations that result in loss of Th17 cell function manifest in disorders such as Job’s syndrome,also named hyper-IgE syndrome, and chronic mucocutaneous candidiasis (CMC) disease.Job’s syndrome is caused by an autosomal dominant STAT3 inactivating mutation and results in increased susceptibility of patients to S.aureus and Candida albicans.Phenotypically, this disease presents as a triad of eosiniphilia, eczema, and recurrent skin and pulmonary infections.CMC disease, manifested by chronic infection of the skin, nails, and mucosa by C.albicans, is related to any of four inheritable gene defects in IL-17RA, IL-17RC, IL-17F, or ACT1.These mutations impair the host Th17 response and increase susceptibility to infection with extracellular pathogens.Thus, Th17 cell function plays a critical role in regulating immune responses in health and in disease within the host.Based on these findings, translational researchers and physician scientists have been actively investigating methods to regulate the Th17 pathway in patients to treat both autoimmunity and infectious disease [64,65].
There are some subsets of Treg; a major one is CD4+, CD25+ Treg expressing the transcription factor FOXP3 that can be “natural Treg” originating in the thymus or “induced Treg” that can be converted in the periphery by exposure to TGF-b.Both types utilize the same mechanisms to mediate immune suppression and may perform overlapping functions.According to previous studies, Treg infiltrates almost all malignant tumors and suppress antitumor immunity, and therefore tumors with marked infiltration of Treg have a poorer clinical outcome [66-68].
Human Treg cells can be classified into the following 3 subfractions: (1) Fraction1, naive/resting Treg cells, defined by FoxP3loCD45RA+CD25lo cells, (2) Fr.2, effector/activated Treg (eTreg)cells, defined by FoxP3hiCD45RA-CD25hicells, and (3) Fr.3, non-Treg cells, defined by FoxP3loCD45RA-CD25lo cells.Naive/resting Treg cells that have just left the thymus have a weak immune suppressive function and differentiate into effector/activated Treg cells following T-cell receptor (TCR) stimulation.eTreg cells are the terminal differentiation state and harbor strong immune suppressive activity.Non-Treg cells do not possess immune suppressive activity, but produce inflammatory cytokines.Typical staining pattern of CD4+ T cells in peripheral blood and lung cancer tissue.In general, the frequency of eTreg cells in humans is 1%-5% in peripheral blood but approximately 10%-50% in the TME.eTreg cells predominantly express various activation cell surface markers including cytotoxic T-lymphocyte antigen-4 (CTLA)-4, programmed cell death(PD)-1, inducible T-cell co-stimulator (ICOS), glucocorticoid-induced tumor necrosis factor receptor-related protein (GITR), OX40, CD15s, CCR4, and CCR8.Naive/resting Treg cells are hardly detected in the tumor microenvironment.Regulatory T cells suppress immune functions through various mechanisms such as CTLA-4-mediated suppression of APC function, consumption of IL-2, production of immunosuppressive cytokines, and production of immune suppressive metabolites [69].
Cytotoxic T-lymphocyte antigen-4 expressed by Treg cells impairs maturation of APCs, such as DCs, by binding to CD80/86.Antigen stimulation by immature DCs or DCs with a low expression of CD80/86 induces T cells with relatively low-affinity TCRs for tumor-antigens derived from self-components to enter an anergic state characterized by hypo-proliferation and hypoproduction of cytokines following antigen re-stimulation.The CTLA-4 binds to CD80/86 on APCs with a higher affinity than CD28, thereby inhibiting costimulatory signals.In addition, CD80/86 bound to CTLA-4 can be physically transferred from APCs to the surface or the cytoplasm of Treg cells by trogocytosis [70,71].
Regulatory T cells highly express CD25 (IL-2 receptor α-chain), dominantly consume IL-2 through high-affinity IL-2 receptors, and hardly produce IL-2, thereby limiting the amount of IL-2 for effector T cell proliferation/activation.Administration of a high dose of IL-2 neutralizes Treg-cell suppressive functions.Treg cells also produce inhibitory cytokines, such as TGF-β, IL-10, and IL-35, to inhibit effector T-cell activation.In addition, cytotoxic substances produced by Treg cells,such as perforin and granzyme, kill effector T cells [72,73].
Well-known immune checkpoint molecules CTLA-4, ICOS, and lymphocyte activation gene-3(LAG-3) are expressed by activated eTreg cells, which inhibits the cytotoxic function and proliferation of effector T cells.Programmed cell death-1 (PD-1) is expressed by activated eTreg cells as well as effector T cells.Yet, the effects of PD-1 inhibition on eTreg cells remain to be determined.Programmed cell death-1 inhibits excessive activation of conventional T cells by suppressing TCR and costimulatory CD28 signaling and renders them dysfunctional or exhausted.Considering the similar expression level of PD-1 by Treg cells in the TME and the similar dependency of TCR and CD28 signaling for their survival and function, PD-1 inhibition could potentiate the activation and immunosuppressive function of Treg cells [74,75].
Indoleamine 2, 3-dioxygenase (IDO), an essential enzyme in the kynurenine pathway of tryptophan metabolism, and tryptophan 2, 3-dioxygenase (TDO) deplete tryptophan in the TME and cause T cell dysfunction.The interaction between CTLA-4 expressed by Treg cells and CD80/86 on APCs promotes IDO secretion.Additionally, Treg cells are more sensitive to oxidative stress than effector T cells because Treg cells have a lower expression of NRF2, which is a key transcription factor for antioxidant responses.Therefore, oxidative stress induces Treg cell apoptosis, and apoptotic Treg cells release a large amount of ATP.Subsequently, ATP is metabolized to adenosine by CD39 and CD73, which are highly expressed by Treg cells, and adenosine binds to the A2A receptor (A2AR),which inhibits effector T cells [76,77].
Tumor-infiltrating lymphocytes (TIL) are one of the representative components of host antitumor immune responses.Both the quality and quantity of TIL determine the effect of the antitumor immune reaction.Previous studies have indicated that patients with cancers showing massive infiltration of CD8+ T cells generally have a better clinical outcome.Conversely, patients with marked infiltration of immunosuppressive cells such as regulatory T cells tend to have a worse prognosis for several types of cancer.The density and distribution of TIL are also strongly affected by the trafficking route.Tumor associated blood vessels in various cancers are structurally and functionally abnormal, and such abnormal vessels reportedly become an obstacle for infiltration of immune effector cells into tumors [77].