ACTIVATED T‐CELLS EXHIBIT DISTINCT GENE EXPRESSION SIGNATURES
Because there are a multitude of infectious agents, running the gamut from viruses, intracellular bacteria, large parasitic worms, extracellular bacteria, yeast, and other fungi, the reader will not be too surprised to learn that activated T‐cells become specialized towards dealing with the particular class of infectious agent that caused them to be woken from their slumber. This process, called T‐cell polarization, will be dealt with more fully in Chapter 8, but we will introduce it here because it is inextricably linked to T‐cell activation. Because of the diversity of intra‐and extracellular pathogens, activated T‐cells must differentiate into distinct types of effector T‐cells, specifically tailored to tackle a particular class of invader. As we have mentioned in previous chapters, activated T‐cells can undergo differentiation into at least three distinct subclasses: T‐helper (Th) cells, cytotoxic T‐cells (CTLs), and regulatory T‐cells (Treg). CD4+ T‐cells coordinate immune responsesby differentiating into distinct T‐helper subsets that tailor the immune response towards the particular infectious agent. T‐helper cells achieve this by releasing powerful inflammatory cytokines, which direct the subsequent responses of CD8+ T‐cells, B‐lymphocytes, and cells of the innate immune system such as macrophages. Recent studies have suggested that during the clonal expansion phase, the differentiation process starts as early as the second cell doubling, and in this context, activation and differentiation can be viewed as two halves of the same coin. Cumulatively, T‐cell activation and differentiation promotes the upregulation of a myriad of genes and we will now consider the most important of these (Figure 7.13).
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Figure 7.13 Gene expression analysis in T‐cells after TCR/co‐receptor engagement. CD4+ splenocytes were stimulated with anti‐CD3 alone or together with antibodies specific for various co‐stimulatory receptors. The heat map indicates fold
change over CD3 stimulation alone.
Integrated signals from the TCR, co‐receptors, and cytokines promote distinct gene expression programs
The classical type 1
response to infection with intracellular pathogens is driven by CD4+ Th1
cells, which secrete IFNγ to direct the activation of
CD8+ CTLs and phagocytic cells, such as macrophages (Figure 7.14). CD4+
Th2 cells secrete IL‐4, IL‐5, and IL‐13 to activate the B‐cell‐mediated antibody response
against multicellular parasites such as helminths, while CD4+
Th17 cells secrete IL‐17, required for effective neutrophil and B‐cell‐driven immune responses
against fungi and extracellular bacteria. Coordination of a particular T‐cell immune response is directed by signals from
the TCR/CD28 complex (i.e., signals 1 and
2), together with key exogenous cytokines (signal 3) supplied by APCs and
innate immune cells that have been activated by a particular pathogen.
Although TCR/CD28 stimulation provides signals to initiate and sustain T‐cell proliferation, it is
the accompanying innate immune cell‐delivered cytokines that direct T‐cell differentiation and thus shape the particular nature of the immune
response. Collectively, these powerful signaling events promote activation of a
number of key transcription factors, with associated expression of a myriad of
proinflammatory genes that shape the outcome of T‐cell activation (Figure
7.14).
Upon antigen stimulation,
TCR/CD28 stimulation promotes the activation of three transcription factors, NFκB, NFAT, and the AP‐1 complex, which promote
cell cycle entry, proliferation, and survival through activation of a host of
target genes. Transcription of IL‐2 is one of the key events in preventing the signaled T‐cell from lapsing into
anergy and is controlled by multiple binding sites for transcriptional factors
in the promoter region (Figure 7.11). Under
the influence of calcineurin, the cytoplasmic component of the nuclear factor
of activated T‐cells (NFATc)
becomes dephosphorylated and this permits its translocation to the nucleus
where it forms a binary complex with NFATn, its partner, which is
constitutively expressed in the nucleus. The NFAT complex binds to two
different IL‐2 regulatory sites (Figure 7.11). Note here that the calcineurin
effect is blocked by the anti‐T‐cell drugs cyclosporine and
tracrolimus (see Chapter 15). PKC‐and calcineurin‐dependent pathways synergize
in activating the multisubunit IκB kinase (IKK), which
phosphorylates the inhibitor IκB, thereby targeting it for ubiquitination and
subsequent degradation by the proteasome. Loss of IκB from the IκB–NFκB complex exposes the
nuclear localization signal on the NFκB transcription factor,
which then swiftly enters the nucleus. In addition, the ubiquitous
transcription factor Oct‐1 interacts with specific octamer‐binding sequence motifs. As
well as secreting IL‐2, activated T‐cells also increase expression of the IL‐2R to sustain IL‐2 signaling.
Differentiation of activated T‐cells
is controlled by different master regulators of transcription
Expression of T‐bet
directs polarization to Th1 cells
Although endogenous signals
such as IL‐2 expression initiate and
help to sustain proliferation, specific cytokines delivered by innate immune
cells direct differentiation of CD4+ T‐cells
into specific types of
effectors: Th1, Th2, and Th17 cells. In response to infection with virus or
intracellular bacteria, or by phagocytosing infected cells, macrophages and DCs
are activated and stimulated to secrete the Th1 polarizing cytokine IL‐12. Naive CD4+ T‐cells that recognize
pathogen‐derived peptide–MHC
complexes presented to them by these activated DCs will also be exposed to
copious amounts of IL‐12, which binds to and
activates the IL‐12R on T‐cell surfaces. Signal
transducer and activator of transcription (STAT) proteins play an essential
role in connecting signals from activated cell membrane cytokine receptors with
intracellular pathways leading to gene induction. Accordingly, IL‐12‐induced activation of STAT4
is important for the induction of the Th1 master regulator T‐bet. This transcription factor
activates T‐cell expression of the key Th1
cytokines IFNγ and TNFα, while simultaneously
upregulating cell surface expression of the IL‐12R, directing Th1 immune responses against
intracellular pathogens and reinforcing the Th1 phenotype (Figure 7.14).
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Figure 7.14 Regulation of T‐cell differentiation by transcription factors.
Specific T‐cell lineages are produced by the action of key
transcription factors, promoting differentiation and the secretion
of a specific set of cytokines that subsequently modulate the immune response.
Expression of GATA3 directs polarization to Th2 cells
In contrast, differentiation
of Th2 cells is initiated by IL‐4. Although the initial
source of IL‐4 is not entirely clear,
stimulation of naive T‐cells by this cytokine
triggers the activation of STAT6, which turns on the Th2 master
transcription factor GATA3, required to promote gene expression and
secretion of the Th2 cytokines IL‐4,
IL‐5, and IL‐13 from activated Th2 cells.
The role of GATA3 in Th2 cell differentiation is high lighted by the complete
failure of GATA3‐deficient mice to generate a
Th2 response. IL‐2 mediated STAT5 activation
also plays a major role in IL‐4 gene induction in Th2
cells, by binding to and enhancing expression at the IL‐4 gene locus. Activated Th2
cells subsequently coordinate the response to extracellular pathogens by
promoting IL‐4‐induced activation of B‐cellsto secrete IgE, IL‐5‐induced recruitment of
eosinophils, IL‐3‐ and IL‐4‐dependent activation of mast
cells, and the alternative activation of macrophages through IL‐4 and IL‐13. Interestingly, GATA3 can
also inhibit Th1 responses by down regulating expression of the IL‐12R, thereby reinforcing the
Th2 response.
Expression of Rorγt directs polarization to Th17 cells.
Th17 cells direct the immune
response against extracellular bacteria and fungi and are activated by IL‐6 and TGFβ, which in turn, promote
STAT3‐mediated activation of the master regulator
of IL‐17 differentiation, Rorγt. This transcription factor promotes expression of
the Th17 cytokines IL‐17A, IL17F, IL‐22,
and IL‐23 in Th17‐differentiated T‐cells, which in turn
activate many types of nonimmune cells, such as endothelial cells, to secrete
inflammatory mediators which recruit and activate neutrophils at sites of
infection. Additionally, STAT3 activation inhibits expression of the T‐regulatory cell (Treg)
master transcription factor Foxp3, thus sustaining
Th17 polarization over Treg generation.
Expression of Foxp3 directs polarization to
Treg cells.
Tregs are a distinct type of
T‐lymphocyte that play an
essential role in controlling the adaptive immune responses orchestrated by
effector T‐cells. While “natural” or thymic‐derived Tregs are thought to
be functionally differentiated cells that are released from the thymus,
inducible Tregs (iTregs) can be differentiated from naive T‐cells after antigen
stimulation. iTregs are induced by stimulation with TGFβ and IL‐2 and are characterized by
activation of Foxp3. Activation of this master transcription
factor promotes the expression of TGFβ and IL‐10 cytokines in Tregs, which
suppress effector T‐cell responses in particular
contexts (Figure 7.14).
CD8+ T‐cell
differentiation is under the control of T‐bet
CD8+ cytotoxic T‐cells (CTLs) play a central
role in the response to intracellular pathogens. These cells are differentiated
from naive CD8+ T‐cells after peptide:MHC
binding in the presence of a range of cytokines including IL‐2, IL‐12, IFNγ, IL‐27, and IL‐23. The concerted action of
TCR/co‐receptor triggering,
together with these cytokines, promotes the proliferation, differentiation, and
survival of CTLs together with the expression of the cytotoxic molecules perforin
and granzymes, which CTLs use to rapidly kill virus‐infected or tumorigenic
cells. Similar to Th1 cells, the master regulator, T‐bet, plays an important role
in CTL differentiation. Once an infection has been cleared, CTL numbers
contract by apoptosis, but a small percentage survive to differentiate into CD8+
memory T‐cells. Memory T‐cells are extremely long‐lived, providing
immunological memory perhaps as long as the life of the organism, and these
cells are characterized by IL‐7R expression, equipping
them to respond rapidly to reinfection after stimulation with IL‐7. What determines the
switch from CD8+ CTL to memory CD8+ T‐cell? Although CD8+
CTLs rely on T‐bet, memory CD8+ T‐cells preferentially express
a related master regulator, Eomes, which may be important in
driving the T‐memory phenotype. Genetic
ablation of Eomes had a profound effect on the generation of memory responses
to viral infection while having little impact on cytotoxic CTL numbers.
Although the picture we have portrayed here is a relatively linear one, recent developments
in single‐cell analysis have revealed
that T‐cell activation towards a
particular fate may be a relatively plastic process, with T‐cell subsets that were once thought of as terminally differentiated cell
types, retaining an ability to redifferentiate to a different phenotype
depending on the cytokine milieu and infection environment. We will delve
deeper into the topic of T‐cell effector generation in
Chapter 8. Although we have concentrated on a relatively small number of genes
that shape the outcome of T‐cell activation, more than
70 genes are newly expressed within 4 hours of activation, leading to
proliferation and the synthesis of several cytokines and their receptors (see
Chapter 8). In addition, TCR stimulation pro motes the expression of a range
of metabolic genes that drive a radical change in the metabolism of activated T‐cells, which we will address
more closely towards the end of this chapter. Although the triggering of TCR
complexes in response to cognate peptide:MHC binding may be the first step
towards T‐cell activation, it is clear
that signals from the TCR and co‐receptor complex, together
with pathogen‐specific information from
external cytokines, trigger a gene expression program in naive T‐cells that not only promotes
proliferation but also coordinately transforms the outcome of activation to
meet the challenges of a specific infection.
