Molecular Mechanisms of T Helper Cell Differentiation and

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ABSTRACT

Th cells, which orchestrate immune responses to various pathogens, dierentiate from

naïve CD4 T cells into several subsets that stimulate and regulate immune responses against

various types of pathogens, as well as a variety of immune-related diseases. Decades of

research have revealed that the fate decision processes are controlled by cytokines, cytokine

receptor signaling, and master transcription factors that drive the dierentiation programs.

Since the Th1 and Th2 paradigm was proposed, many subsets have been added to the list.

In this review, I will summarize these events, including the fate decision processes, subset

functions, transcriptional regulation, metabolic regulation, and plasticity and heterogeneity.

I will also introduce current topics of interest.

Keywords: Helper T cell; T-lymphocyte subset; Cell dierentiation; Adaptive immune response;

Transcription factors; Cytokines

INTRODUCTION

Pathogens can infect many dierent tissues and cellular compartments, and the life cycles

of these pathogens can vary considerably. To overcome this, the host immune system

has developed pathogen-specic strategies. For example, Th cells (eector CD4 T cells)

orchestrate immune responses against many dierent pathogens (1-4). Naïve CD4 T cells

recirculate via the blood and lymphatic systems, gathering information about pathogens by

binding to antigens presented by dendritic cells (DCs) in lymphoid tissues. DCs also secrete

cytokines when they present these pathogen-specic antigens to naïve CD4 T cells, thereby

triggering dierentiation pathways (1-4). Activated CD4 T cells proliferate and dierentiate

into dierent immune cell subsets, which determine the type of immune response (1-4).

Research in the eld of Th cell dierentiation and function began about 30 years ago, and it

is still progressing. In this review, I will summarize the basic principles that govern Th cell

dierentiation (Fig. 1). Other topics in this eld, including the microbiota and plasticity/

heterogeneity, will be covered by other articles in this issue.

Immune Netw. 2023 Feb;23(1):e4

https://doi.org/10.4110/in.2023.23.e4

pISSN 1598-2629·eISSN 2092-6685

Review Article

Molecular Mechanisms of T Helper

Cell Diﬀerentiation and Functional

Specialization

Received: Nov 30, 2022

Revised: Jan 17, 2023

Accepted: Jan 29, 2023

Published online: Feb 17, 2023

*Correspondence to

Gap Ryol Lee

Department of Life Science, Sogang University,

35 Baekbeom-ro, Mapo-gu, Seoul 04107,

Korea.

Email: [email protected]

Immunologists

This is an Open Access article distributed

under the terms of the Creative Commons

Attribution Non-Commercial License (https://

creativecommons.org/licenses/by-nc/4.0/)

which permits unrestricted non-commercial

use, distribution, and reproduction in any

medium, provided the original work is properly

cited.

ORCID iDs

Gap Ryol Lee

https://orcid.org/0000-0002-6412-9482

Conﬂict of Interest

The authors declare no potential conﬂicts of

interest.

Abbreviations

AMPK, AMP-activated protein kinase; APC,

antigen-presenting cell; DC, dendritic

cell; EAE, experimental autoimmune

encephalomyelitis; LCR, locus control

region; OXPHOS, oxidative phosphorylation;

Phd, prolyl-4-hydroxylase domain; pTreg,

regulatory T cell in the periphery; scRNA-seq,

single cell RNA sequencing; T, follicular

helper T; Tr1, type-1 regulatory T; tTreg,

regulatory T cell development in the thymus;

Vhl, von Hippel-Lindau.

Gap Ryol Lee

Department of Life Science, Sogang University, Seoul 04107, Korea

BRIEF HISTORY OF Th SUBSETS

For a long time, Th cells were regarded as cells that aid activation of B cells and macrophages

by producing cytokines. In 1986, Mosmann and colleagues (5) published a seminal work

showing that Th cells comprise 2 distinct cell types based on their cytokine proles, namely,

Th1 and Th2. The dierent cytokines produced by these cell types lead to mutually exclusive

functional properties (5). Th1 cells activate macrophages and mediate immune responses

against intracellular pathogens, while Th2 cells stimulate B cells (5). Identication of Th

cell heterogeneity and dierences in functional properties was a major conceptual advance

in the eld, and triggered further study of the molecular mechanisms that regulate the

fate-determining processes and functional properties. These eorts led to identication of

cytokines, signaling pathways, and transcription factors as determinants of Th1 and Th2

subset dierentiation. In 2005, Th17 cells were identied and proposed as an independent

subset (6-8). Th17 cells induce inammation and mediate immune responses against

extracellular bacteria and fungi (9,10). Another independent subset, called follicular helper

T (T) cells, was also identied (11-13). T cells reside in the germinal centers of lymphoid

tissue and interact with B cells to trigger maturation. It was thought that B cell stimulation

was the role of Th2 cells; however, this is now thought to be the role of T cells. In addition

to activator cells, repressor cell types have also been identied; these are termed Treg cells

(14-16). Many other subsets have also been identied, including IL-9-producing Th9 cells, IL-

22-producing Th22 cells, and IL-10 producing type-1 regulatory T (Tr1) cells. Th9, Th22, and

Tr1 cells will not be covered in this review because our understanding of their functions and

roles is still in its infancy.

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cytokine

receptor

CD28

TCR

nutrients

metabolism

Th1: IL-12, IFN-γ

Th2: IL-4

Th17: TGF-β + IL-6 (IL-21)

Treg: TGF-β

inducer

cytokines

STAT

or SMAD

master

Master TF

Activation/proliferation

differentiation

effctor

cytokine

Th1: STAT4, STAT1

Th2: STAT6

Th17: STAT3

Tfh: STAT3

Treg: SMAD2, SMAD3, STAT5

Th1: T-bet

Th2: GATA3

Th17: RORγt

Tfh: Bcl6

Treg: Foxp3

Th1: IFN-γ

Th2: IL-4, IL-5, IL-13

Th17: IL-17A, IL-17F, IL-22

Tfh: IL-21

Treg: IL-10

differentiation

Figure 1. Schematic diagram of Th cell diﬀerentiation. Th cell diﬀerentiation pathway is determined by inducer

cytokines that are present during T cell activation. The cytokine signaling activates STAT or SMAD, which in

turn induces master transcription factors. Master transcription factors drive the subset-speciﬁc diﬀerentiation

programs. Th cell proliferation and diﬀerentiation are also inﬂuenced by nutrient or metabolites.

Th CELL FUNCTIONS

Eector CD4 T cells either stimulate or repress other immune cells. The most important

mediators of these functions are cytokines (1-4). Th1 cells produce IFN-γ, which stimulates

macrophages and CTLs. Stimulated macrophages kill intracellular pathogens by activating

microbicidal mechanisms, including facilitation of fusion of phagosomes with lysosomes,

activation of NADPH oxidase, and generation of reactive oxygen species (17). Activation of CTL

requires the help of Th1 cells via secretion of IFN-γ and CD40L signaling, which trigger CTL

dierentiation. Th2 cells produce IL-4, IL-5, and IL-13, which mobilize or stimulate eosinophils,

basophils, and mast cells to combat helminthic parasites (18). IL-13 also stimulates intestinal

epithelial cells to increase cell turnover and movement, as well as acting on smooth muscle cells

in the intestine to increase contractility, all of which help to remove parasites from the gut. Th17

cells produce IL-17A, IL-17F, and IL-22, which stimulate epithelial cells, broblasts, and stromal

cells to produce GM-CSF, recruit neutrophils, and induce inammation, thereby mediating

immune responses against extracellular bacteria and fungi (9,10). T cells produce IL-21

and activate B cells in the germinal center to induce maturation via somatic hypermutation

and class-switch recombination (19). Treg cells inhibit eector T cell and B cell responses, as

well as innate immune responses (20). Treg cells produce IL-10, TGF-β, and IL-35 to inhibit

immune responses, although they can use other mechanisms too. Treg cells express high

amounts of IL2Rα on their surface, leading to deprivation of IL-2, which is required for growth

of eector CD4 T cells. Treg cells express coinhibitory receptors such as Ctla-4 and Lag3,

which interfere with the interaction with costimulatory molecules and MHC class II molecules

expressed by antigen-presenting cells (APCs), thereby preventing T cell activation (21,22).

Treg cells also kill CD4 T cells directly by secreting cytotoxins. They also express high amounts

of ectonucleotidases such as CD39 and CD73, leading to production of immunosuppressive

adenosine, which promotes T cell anergy (23). Although Treg development in the thymus

(tTreg) and Treg in the periphery (pTreg) cells have overlapping functions, tTregs are more

specialized at preventing autoimmune responses against self-antigens, whereas pTregs are

more specialized in inducing tolerance to foreign antigens.

Each CD4 T cell subset cross regulates other subsets. For example, Th1 cells inhibit Th2 and

Th17 cell dierentiation by suppressing expression of IL-4 and IL-17, respectively (7,24). Th2

cells repress Th1 dierentiation by inhibiting IL-12Rβ2 and IL-17 expression, respectively

(7,25). T cells inhibit dierentiation of other subsets via Bcl6-mediated repression of

Prdma1

(which encodes Blimp-1) expression (26,27).

In addition to the induction of protective immunity against infectious agents, Th cells are

responsible for the pathology of many other diseases caused by aberrant immune responses

(1-4). For example, Th1 and Th17 cells plays critical roles in rheumatoid arthritis, type I

diabetes, and multiple sclerosis; Th2 cells in allergic diseases such as asthma, rhinitis,

and allergic dermatitis; and Treg cells in autoimmune disease, allergy, and cancer. Thus,

regulating subset-specic functions is an eective strategy for treating these immune-related

diseases. Indeed, based on this knowledge, many therapeutic strategies have been, or are

being, developed (28,29).

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SIGNAL TRANSDUCTION AND TRANSCRIPTIONAL

REGULATION

Naïve CD4 T cells dierentiate into various subsets of eector cells when they recognize their

cognate antigens presented in conjunction with MHC II molecules on the surface of DCs in

lymphoid tissues (1-3). These subsets include Th1, Th2, Th17, T, and Treg cells (1-3). The

fates of these subsets are determined by the cytokines (produced mainly by APCs) present in

the environment at the time of activation (1-3). The cytokines bind to their cognate receptors

on naïve CD4 T cells and activate the STAT or SMAD pathway (1-3). Th1 cell dierentiation is

induced by IL-12 or IFN-γ, which activate Stat4 or Stat1, respectively (1-3). Th2 cell dierentiation

is induced by IL-4, which activates Stat6 (1-3). Th17 cell dierentiation is induced by TGF-β plus

IL-6 (or IL-21), which activate Stat3 (1-3). T cell dierentiation is induced by IL-21, which also

activates Stat3 (1-3). Treg cells are rather dierent in that they can be generated during T cell

development in the thymus (tTreg cells) or can dierentiate from naïve CD4 pTreg cells (1-3).

vitro

, Treg cells can be dierentiated by TGF-β, which activates Smad2 and Smad3 (1-3).

STAT proteins enter the nucleus and induce global epigenetic changes as well as expression

of master transcription factors which drive dierentiation programs (1-4). T-bet drives the

Th1 program, Gata-3 drives the Th2 program, Rorγt drives the Th17 program, Bcl6 drives

the T program, and Foxp3 drives the Treg program. These transcription factors induce

expression of a variety of genes required for dierentiation, and for the eector functions,

of each subset. Division of the roles played by STATs and the master transcription factors is

the focus of much interest. Although both factors are important for dierentiation, it seems

likely that STATs act as pioneering factors that shape global enhancer landscapes and gene

expression patterns, whereas master transcription factors regulate a limited set of genes that

is required for subset-specic dierentiation and function (30,31).

Although master transcription factors drive the dierentiation program in Th cells, many

other transcription factors play important roles in the process (32,33). In addition to the

master transcription factors, Irf1, Runx3, Hlx, Ets-1, Nfatc1, and Bhlhe40 play important roles

in Th1 cell dierentiation; c-Maf, Notch/CSL, IRF4, G-1, and Yy1 in Th2 dierentiation; Ifr4,

Batf, and Runx1 in Th17 dierentiation; and Runx1, Nr4a2, Foxo family members, Satb1, and

Helio in Treg dierentiation (32,33). These transcription factors form a coordinated network

that drives the dierentiation programs (34-36), even in the absence of master transcription

factors (36). The roles of these transcription factors depend on individual factors. In general,

these factors either enhance transcription, induce changes in epigenetic status, or promote

organization of the relevant genes or gene loci within the genome (32,33).

EPIGENETIC REGULATION

Along with transcription factors, the genetic loci of characteristic cytokine genes have been

investigated extensively with respect to their role in regulating Th cell dierentiation (37,38).

This is due in part to the fact that the dierentiation process is easily controlled

in vitro

cytokines, enabling investigation of cell fates within a short period of time. Such studies

have reported interesting ndings: 1) epigenetic changes accompany gene expression during

dierentiation; 2) poised state of cytokine loci even before dierentiation; 3) coordinate

expression of cytokine genes; 4) inter-chromosomal interactions between 2 opposing loci;

and 5) insulation of cytokine loci.

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Epigenetic changes during Th cell diﬀerentiation

Marked changes in gene expression occur during CD4 T cell activation and dierentiation.

Many of the genes expressed by activated T cells are required for cell division. Since naïve T

cells are in a quiescent state, transition to actively dividing cells requires systemic changes

in gene expression. Yet more genes are required for subset-specic eector functions; for

example, genes encoding cytokines that mediate the eector functions of a particular subset.

Not surprisingly, naïve CD4 T cells undergo epigenetic changes such as increased accessibility

by DNase I, histone acetylation, and DNA methylation (37,38).

Ifng

and the Th2 cytokine locus

(the

Il4-Il13-Il5

locus) show subset-specic epigenetic changes. During Th1 dierentiation,

the

Ifng

locus opens, but the Th2 cytokine locus remains closed. Likewise, the Th2 cytokine

locus opens during Th2 dierentiation, but the

Ifng

locus remains closed. This subset-specic

chromatin modication is inuenced by master transcription factors such as T-bet and Gata-3.

Thus, one of the roles of these transcription factors is to open the chromatin at the loci of

subset-specic genes to make it “competent” for expression of these genes.

Poised state of cytokine loci before diﬀerentiation

Certain genetic loci, such as the

Ifng

locus, are demethylated even before Th1 dierentiation.

This hypomethylation pattern seems to be established early during T cell development,

including that of naïve CD4 T cells, suggesting that these loci are predisposed to expression

(37,38). During Th1 dierentiation, this demethylation pattern at the

Ifng

locus is preserved;

however, the locus becomes methylated during Th2 dierentiation. Genome-wide studies

of the epigenetic status of chromatin and gene expression in CD4 T cells revealed that the

histone modication status of key cytokine and transcription factor loci correlates strongly

with gene expression patterns (39). Interestingly, this study also found that

Tbx21

and

Gata3

loci contain both active (H3K4me3) and suppressive (H3K27me3) histone marks (39). This

bivalent status with respect to histone marks also predispose gene expression, suggesting

that cells are preparing for rapid changes in gene expression that occur during activation and

dierentiation. The poised status of multiple key genetic loci facilitates easy conversion from

one dierentiated state to another.

Coordinate expression of cytokine genes

The

Ifng

locus and the Th2 cytokine locus are regulated by promoters, enhancers, and

silencers in the same manner as other genetic loci (37,38). Since the

Il4

Il13

, and

Il5

genes

are clustered, it is of interest to see whether they are coordinately regulated in Th2 cells.

One of the regulatory elements that exerts coordinate expression of clustered genes is the

locus control region (LCR), best exemplied by the β-globin LCR (40). The LCR is dened

as a sequence that enables copy number-dependent expression in transgenic mice (40). The

Th2 cytokine locus contains an LCR, called the Th2 LCR, which is interspersed in the intron

regions of the

Rad50

gene located between the

Il13

and

Il5

genes (41). The Th2 LCR undergoes

epigenetic changes during Th2 dierentiation by recruiting GATA3 and other transcription

factors such as Yy1, Stat6, Satb1, and Irf4 (42-44). This process is essential for Th2 cytokine

expression since deletion of the Th2 LCR or its components abolishes Th2 cytokine expression

(42,45,46). The Th2 LCR induces chromosomal looping to facilitate interactions with the

promoters of Th2 cytokine genes, thereby forming a basis for coordinated regulation (47).

Inter-chromosomal interactions between 2 opposing loci

Chromosomal interactions occur not only intra-chromosomally, but also inter-

chromosomally. Chromosomal interaction between 2 dierent chromosomes has been

suggested for long time, but it was proved experimentally in the study of

Ifng

and Th2 loci,

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shedding light on a novel mechanism of gene regulation (48). Although they are located

on dierent chromosomes,

Ifng

and RHS6, a component of the Th2 LCR, in naïve CD4 T

cells interact with each other. This physical interaction seems to maintain a poised state

for expression of each gene until the cell is activated, since this interaction is replaced with

intrachromosomal interactions upon Th2 cell dierentiation (48). In naïve CD4 T cells,

RHS6 also interacts with the

Il17

promoter through Oct-1 and CTCF, suggesting occurrence of

inter-chromosomal interactions among multiple chromosomes (49). When Oct-1, CTCF, or

the interacting genetic loci are decient, the interaction does not occur and expression of

Il17

increases (49), supporting its role in cytokine gene regulation.

Insulation of cytokine loci

The

Ifng

locus is located about 40 kb downstream of the

IL22

gene in humans and 245 kb

downstream of the

Il22

gene in mice. Since

Il22

is a Th17-specic gene, expression of

Ifng

and

Il22

should be regulated separately. Indeed, an insulator sequence between these gene loci was

identied by genome-wide screening of CTCF binding sites and cohesion binding sites (50,51).

The

Ifng

locus is insulated by −70 and +66 boundary elements, which contain CTCF and the

cohesion component Rad21 (50,51). Although recruitment of CTCF to the −70 site is conserved

in naïve, Th1, and Th2 cells, CTCF recruitment to the

Ifng

intronic region and +66 site is specic

to Th1 cells (50,51), suggesting that this insulator sequence, and its binding to CTCF, may play a

role in forming the intra-chromosomal loops needed for the

Ifng

gene transcription.

PLASTICITY AND HETEROGENEITY

I will provide a very brief overview of this topic as it is covered in depth by another article in

this issue. Initially, the status of Th1 and Th2 cells was thought to be stable and terminally

dierentiated; this is because reversal between subsets cannot be induced aer long-term

culture (52), and production of specic cytokines seems to be stably maintained (53).

However, later studies showed that Th subsets are not permanently dierentiated; rather

they can dierentiate into another subset under certain circumstances. This phenomenon

is called plasticity (54,55). Early

in vitro

studies suggested that forced expression of master

regulators by already dierentiated cells induces alternative dierentiation pathways (55).

For example, Th1 cells produce IL-4 upon transfection with Gata-3, and Th2 cells can express

IFN-γ upon transfection of T-bet. A fate-mapping study using a mouse model of experimental

autoimmune encephalomyelitis (EAE) revealed that most cells producing IFN-γ previously

expressed IL-17A (56). Studies in animal models of diabetes and cancer show that Th17 cells

acquire Th1-like properties (57,58).

In addition to plasticity, CD4 T cells show functional heterogeneity. Cells co-expressing

T-bet and RORγt are present in the gut and inamed brain tissue (56,59). These cells express

both IFN-γ and IL-17, and are thought to play an important role in immune responses to

Mycobacterium tuberculosis

(60). Treg cells oen co-express T-bet, Gata3, or Rorγt. T-bet-

expressing Treg cells inhibit type I immune responses (61). Gata3 is frequently co-expressed

with Foxp3 by Treg cells and is thought to help control Th2-type immune response (62).

Treg cells co-expressing Irf4 or Stat3 control Th2- and Th17-mediated immune responses,

respectively (63,64). Thus, co-expression of transcription factors by other subsets seems to

endow Treg cells with the ability to control specic eector functions.

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The plasticity and heterogeneity of Th subsets modies and expands the characteristics

of each subset. Plasticity is thought to be an adaptation to a changing environment or to

pathogens. Since some pathogens change their habitats (e.g., by infecting dierent cells/

organs) and their virulence strategy during their life cycle, the immune system needs to

change its defensive strategies to combat them.

METABOLIC REGULATION

When naïve CD4 T cells are activated and dierentiate into eector cells, they undergo

drastic changes in their metabolic programs (1,65-67). During this process, dierentiating

and proliferating cells require energy, as well as the building blocks to synthesize DNA,

RNA, proteins, and lipids. Eector subsets of Th cells, namely, Th1, Th2, and Th17 cells, rely

more heavily on glycolysis than Treg cells (1,34,65,66). Most cells generate ATP via oxidative

phosphorylation (OXPHOS), which occurs in the mitochondria. However, rapidly dividing

cells such as cancer cells use aerobic glycolysis instead of OXPHOS to generate ATP. Although

it generates less ATP than OXPHOS, aerobic glycolysis can provide many of the materials

required for biosynthesis in cells proliferating under hypoxic conditions. This phenomenon,

called the “Warburg eect,” also occurs in eector T cells, which are rapidly dividing cells. T

cells also show enhanced glycolysis, but cell-autonomous IL-2 inhibits the dierentiation and

metabolic programs of T cells. Th17 cells also use glutaminolysis and fatty acid synthesis

programs, whereas fatty acid synthesis antagonizes dierentiation of Treg cells. By contrast,

Treg cells rely on mitochondrial activity for their dierentiation, homeostasis, and function.

Since Th cell dierentiation requires metabolic reprogramming, factors that regulate this

process aect Th cell dierentiation (65). These factors include 1) nutrients/metabolites, 2)

transcription factors, and 3) signaling molecules.

Nutrients/metabolites

Glucose is a readily available nutrient that provides energy to activated and proliferating T

cells, as well as building blocks for nucleic acids, proteins, lipids, and carbohydrates. Glucose-

6-phosphate, the rst intermediate of glycolysis, can be converted to fructose-6-phosphate

to yield pyruvate, which is used to synthesize glycogen, or shuttled to synthesize purines

and pyrimidines via the pentose phosphate pathway. TCR and/or costimulatory signaling

through CD28 triggers rapid uptake of glucose by upregulating the glucose transporter Glut1

and by increasing glycolytic ux (1,65-67). Thus, activated CD4 T cells are highly dependent

on glucose uptake. Indeed, Th1 and Th17 cells have an increased glycolytic rate. Chemical or

genetic inhibition of specic steps within the glycolytic pathway leads to a marked reduction

in eector T cell proliferation and cytokine production. Deprivation of glucose impairs T cell

activation and survival, highlighting the importance of glucose for the T cell activation and

dierentiation processes (1,34,65,66). Glucose deprivation also inhibits IFN-γ production,

as well as reducing production of phosphoenolpyruvate, which supports Ca

/NFAT signaling

required for glycolytic reprogramming in T cells (1,65-67). Unlike eector T cell subsets,

Treg cells are not aected by Glut1 deciency (1,34,65,66). Blocking glycolysis with 2-DG,

an inhibitor of hexokinase (an enzyme that regulates the rate limiting step of glycolysis),

inhibits dierentiation of CD4 and CD8 T cells into eector cells (1,65-67). Deciency of

lactate dehydrogenase A, an enzyme that converts lactate to pyruvate, inhibits ATP generation

and PI3K/Akt activation, thereby repressing T cell activation (68). High levels of glycolytic

metabolism inhibit induction of Foxp3, instead inducing Th17 cell dierentiation (69).

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Fatty acid metabolism is a critical part of T cell dierentiation. Fatty acid oxidation is

essential for Treg cell generation and function, and for making membrane components by

proliferating eector T cells, including Th1, Th2, and Th17 cells (70). Proliferating activated

T cells show increased fatty acid synthesis, with a concomitant reduction in fatty acid

oxidation. Fatty acid metabolism is important for the balance between Th17 and Treg cells:

fatty acids aid the generation and function of Treg cells, while controlling the pathogenicity

of Th17 cells (70).

Transcription factors

Myc, induced by TCR stimulation, is essential for metabolic reprogramming of T cell activation

and function. Myc (71), which plays an essential role in glycolysis and glutaminolysis in activated

T cells, induces Glut1 and several glycolytic enzymes (71). When Myc is ablated, glycolysis

is impaired, resulting in decreased T cell activation and growth (72). Myc also stimulates

glutaminolysis by increasing expression of glutaminase and glutamine transporters (73).

HIF-1α plays an important role in Th cell dierentiation and function (74) by enhancing Th17

cell dierentiation via stimulation of

Il17

gene transcription while reducing Treg cell generation

via Foxp3 degradation (75). Conditional deletion of HIF-1α causes resistance to EAE, with

reduced inltration of the CNS by Th17 cells (69). In addition, HIF-1α induces glycolysis under

hypoxic conditions. The oxygen-sensing function is regulated by 2 proteins; the von Hippel-

Lindau (Vhl) complex supports and prolyl-4-hydroxylase domain (Phd) proteins oppose

the function. Upon activation,

Vhl

-decient T cells show augmented glycolysis but reduced

mitochondrial oxidative metabolism, leading to enhanced eector programming, eector/

memory cell dierentiation, and cell death (76,77).

Phd

-decient CD4 T cells, however, show

increased eector cell dierentiation but decreased Treg cell dierentiation (78).

Signaling molecules

The mTOR is a serine/threonine kinase that transmits signals from the PI3K-Akt pathway.

mTOR forms 2 functionally distinct complexes, mTORC1 and mTORC2, and is essential for

regulating glucose metabolism and upregulating glycolysis. It is also required for eector T

cell dierentiation; indeed, mTOR deciency causes failure of eector T cell dierentiation

and leads to Treg cell generation instead (79). mTORC1 and mTORC2 play distinct roles

during dierentiation of Th subsets. CD4 T cells lacking mTORC1 activity due to the absence

of one of its components (Rheb) failed to generate Th1 and Th17 cells (80), whereas those

lacking mTORC2 activity due to the absence of Rictor failed to generate Th2 cells (80,81).

Excessive mTOR activity caused by

Pten

-deciency reduces the stability of Treg cells, leading

to uncontrolled Th1 and T cell responses (82,83).

AMP-activated protein kinase (AMPK) is a glucose-sensitive metabolic sensor that regulates

mRNA translation and glutamine-dependent mitochondrial metabolism (84). AMPK

promotes catabolic processes but inhibits anabolic processes. It also promotes OXPHOS,

mitochondrial biogenesis, fatty acid oxidation, and autophagy, but inhibits glycolysis,

glutaminolysis, and glycogen and fatty acid synthesis (84). AMPK promotes energy

conservation by T cells, as well as maintaining T cell bioenergetics and viability (84). It also

negatively regulates eector Th cell dierentiation by inhibiting mTORC1 activity.

Ampka1

decient T cells show reduced metabolic plasticity and mitochondrial bioenergetic when

glucose is limited (84). Under glucose-limiting conditions, reduced expression of IFN-γ

mRNA and protein aects Th1 dierentiation. By contrast, AMPK induces dierentiation of

naïve CD4 T into Treg cells (85).

T Helper Cell Diﬀerentiation and Function

https://doi.org/10.4110/in.2023.23.e4

8/15

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CHALLENGES TO THE Th SUBSET PARADIGM

The paradigm of Th subsets has contributed greatly to our understanding of the immune

system. The paradigm divides immune responses into categories, and provides a simple

framework for understanding otherwise complex phenomena. Based on this paradigm,

many immune-related diseases have been categorized as type I, type II, and type III diseases.

This categorization is also applied to innate immune cells, such as innate lymphoid cells and

macrophages. The paradigm was originally proposed, and mainly studied, in the context of

vitro

-dierentiated CD4 T cells, generation of which requires excessive amounts of cytokines

and neutralizing antibodies.

The plasticity and heterogeneity of Th cells modies their functions rather elegantly without

necessarily challenging the classical view of Th cell subsets, which states that these subsets

are terminally dierentiated and stable. Recent technological advances such as single cell

RNA sequencing (scRNA-seq) and CyTOF have made it possible to analyze gene expression

at the single cell level. Studies using these technologies show that eector Th cells are much

more heterogeneous than the paradigm suggests.

scRNA-seq and computational analysis show that Th17 cells isolated from animal models

of autoimmune diseases are heterogeneous and show a spectrum of cellular states (86,87).

Tortola et al. (88) used CyTOF to analyze expression of subset-specic marker proteins

expressed by

in vitro

-dierentiated Th cells and by Th cells in animal disease models. They

found that eector Th cells are far more heterogeneous than expected, even under controlled

stimulation conditions. Kiner et al. (89) analyzed gut CD4 T cells using scRNA-seq and

ATAC-seq. Surprisingly, they found that CD4 T cells do not follow the classic subset-specic

gene expression pattern. IFN-γ and IL-17A are expressed by most eector cells, many of

which co-express both genes (89). Transcriptional and epigenetic patterns are determined

by infectious agents rather than by cytokine and master transcription factor signatures (89).

scRNA-seq followed by trajectory analysis revealed that Th1 cells and T cells bifurcate

during their dierentiation in malaria infection model (90). These results challenge the Th

subset paradigm, and suggest that Th cells exist in a heterogeneous state of dierentiation,

which may change according to the environment. Thus, more studies in this area are needed.

CONCLUSIONS AND PERSPECTIVES

Th cells play essential roles in coordinating immune responses to dierent pathogens.

They are also responsible for many immune pathologies. Thus, research on Th cells (either

the fundamental mechanisms underlying dierentiation and function, or therapeutic

applications based on the results of such studies) is ongoing. New technological advances

will speed up progress. For example, scRNA-seq can reveal detailed gene expression

patterns in individual cells within a large population, providing information about

possible dierentiation pathways. CRISPR/Cas9 technology enables functional study

of genes of interest on a large scale, either

in vitro

in vivo

. Introduction of the concept

of super-enhancers, which are dened as regulatory regions that are highly enriched in

transcription factors and activating/suppressive epigenetic marks, will facilitate more rapid

study of regulatory regions, since they can be identied by genome wide approaches and

bioinformatics analysis. These new technologies or concepts will enable comprehensive

analysis of the molecular mechanisms underlying these processes, while challenging the

T Helper Cell Diﬀerentiation and Function

https://doi.org/10.4110/in.2023.23.e4 9/15

https://immunenetwork.org

current paradigm. I anticipate and hope that more and more new and interesting ndings

about the underlying molecular mechanisms will come to light, and that we will make

progress in the treatment of dicult-to-cure diseases.

ACKNOWLEDGEMENTS

This work was supported by the National Research Foundation of Korea (NRF) grants

funded by the Korean government (2021K2A9A2A06048161, 2022R1A2B5B03001840,

2022R1A4A5032688 to GRL).

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