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Interaction of the T cell receptor (TCR) with an MHC-antigenic peptide complex results in changes at the molecular and cellular levels in T cells. The outside environmental cues are translated into various signal transduction pathways within the cell, which mediate the activation of various genes with the help of specific transcription factors. These signaling networks propagate with the help of various effector enzymes, such as kinases, phosphatases, and phospholipases. Integration of these disparate signal transduction pathways is done with the help of adaptor proteins that are non-enzymatic in function and that serve as a scaffold for various protein–protein interactions. This process aids in connecting the proximal to distal signaling pathways, thereby contributing to the full activation of T cells. This review provides a comprehensive snapshot of the various molecules involved in regulating T cell receptor signaling, covering both enzymes and adaptors, and will discuss their role in human disease.
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REVIEW ARTICLE OPEN
T cell receptor (TCR) signaling in health and disease
Kinjal Shah
1,2
, Amr Al-Haidari
3,4
, Jianmin Sun
1,2,5
and Julhash U. Kazi
1,2
Interaction of the T cell receptor (TCR) with an MHC-antigenic peptide complex results in changes at the molecular and cellular
levels in T cells. The outside environmental cues are translated into various signal transduction pathways within the cell, which
mediate the activation of various genes with the help of specic transcription factors. These signaling networks propagate with the
help of various effector enzymes, such as kinases, phosphatases, and phospholipases. Integration of these disparate signal
transduction pathways is done with the help of adaptor proteins that are non-enzymatic in function and that serve as a scaffold for
various proteinprotein interactions. This process aids in connecting the proximal to distal signaling pathways, thereby contributing
to the full activation of T cells. This review provides a comprehensive snapshot of the various molecules involved in regulating T cell
receptor signaling, covering both enzymes and adaptors, and will discuss their role in human disease.
Signal Transduction and Targeted Therapy (2021) 6:412 ; https://doi.org/10.1038/s41392-021-00823-w
INTRODUCTION
T cells are key mediators in mounting an effective adaptive cell-
mediated immune response.
1,2
T cells continuously screen
lymphoid and peripheral tissues for antigens such as peptides or
lipids displayed by major histocompatibility complex (pMHC)
molecules of other cells. Normal T cell development in the thymus
undergoes a major developmental checkpoint in which T cell
receptor (TCR) signaling is involved. Thymocytes bearing TCR with
a high afnity for self-peptide MHC complexes undergo apoptosis
(negative selection), whereas those bearing low-afnity TCR
survive and differentiate into mature T cells (positive selection).
3
This ensures that only those T cells that are self-tolerant survive
while eliminating the self-reactive T cells.
4
These naïve single
positive mature T cells then leave the thymus and enter the
peripheral lymphoid organs, such as the spleen and lymph nodes,
where they get exposed to foreign peptides presented by the
MHC molecules of antigen-presenting cells (APCs), such as the
macrophages, dendritic cells, and B cells, during pathogenic
infection.
5
Upon engagement of TCR with the antigenic peptide,
T cells get activated, undergoing clonal expansion and differentia-
tion to perform their effector functions, due to a complex series of
molecular changes at the plasma membrane, cytoplasm, and
nucleus.
6,7
T cell signaling is thus important for efcient T cell
development, activation, and immune tolerance. TCR signaling
dysregulation can thus lead to anergy or autoimmunity.
8
In general, the transmission of external cues to the interior of the
cell occurs through binding of a ligand to the extracellular domain of
the receptor, leading to receptor aggregation or conformational
changes. Once this is accomplished, protein tyrosine kinases (PTKs)
phosphorylate various tyrosine residues present in the cytoplasmic
tail of the receptor, which serve as a docking site for various signaling
molecules containing specic phosphotyrosine recognition domains
such as the SRC homology 2 (SH2) and phosphotyrosine-binding
(PTB) domains.
6
This initiates proximal biochemical signals mediated
by the key effector enzymes, such as kinases, phosphatases, and
phospholipases, that culminate in distal signaling by activating
numerous transcription factors required for translating these events
into gene activation.
9
However, both the proximal and distal
signaling need to be integrated, and this is done by various adaptor
proteins whose main function is to form multiprotein complexes.
10
Adaptors are proteins that usually lack intrinsic enzymatic activity
and instead possess multiple binding domains for phosphotyrosine,
proline-rich region and lipid interactions, and sequence motifs that
in turn are involved in binding to such domains.
11,12
The
phosphorylated tyrosine residues on various adaptor proteins serve
as binding sites for many critical effector enzymes and other adaptor
proteins.
6
Thus, they behave as scaffolds, facilitating proteinprotein
interactions that aid in forming multiprotein complexes, thereby
integrating signaling cascades necessary for efcient T cell biology.
Apart from this, adaptors also interact with other adaptors present at
the plasma membrane microdomains and even play important roles
in the regulation of the cytoskeleton.
6,10,13
Hematopoietic-specic
adaptor proteins have led to a better understanding of T cell
signaling.
13
Those adaptor proteins can regulate signal transduction
both positively and negatively.
10,13
In this review, we discuss the role
of TCR signaling in human health and disease.
COMPONENTS AND STRUCTURE OF TCR COMPLEX
The core TCR complex consists of two TCR chains and six cluster of
differentiation 3 (CD3) chains. Several other components include
coreceptors, kinases, and ligands.
14,15
TCR-CD3 chains
The human genome expresses four TCR genes known as TCRα,
TCRβ, TCRγ, and TCRδ, which forms two distinct heterodimers:
Received: 28 February 2021 Revised: 2 November 2021 Accepted: 2 November 2021
1
Division of Translational Cancer Research, Department of Laboratory Medicine, Lund University, Lund, Sweden;
2
Lund Stem Cell Center, Department of Laboratory Medicine,
Lund University, Lund, Sweden;
3
Clinical Genetics and Pathology, Skåne University Hospital, Region Skåne, Lund, Sweden;
4
Clinical Sciences Department, Surgery Research Unit,
Lund University, Malmö, Sweden and
5
NHC Key Laboratory of Metabolic Cardiovascular Diseases Research, Science and Technology center, School of Basic Medical Sciences,
Ningxia Medical University, Yinchuan, China
Correspondence: Julhash U. Kazi (kazi.uddin@med.lu.se)
www.nature.com/sigtrans
Signal Transduction and Targeted Therapy
©The Author(s) 2021
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TCRα/TCRβor TCRγ/TCRδ.
1618
The majority of mature T cells
expresses TCRαand TCRβisoforms, generally referred to as T cells
(or αβ T cells), while a small portion (0.55%) of T lymphocytes (γδ
T cells) expresses TCRγand TCRδisoforms.
19
In this review, we will
focus on αβ T cells, and henceforth the nomenclature T cells will
refer to αβ T cells.
Both heterodimers form multiprotein complexes with CD3 δ,γ,
ε, and ζchains. TCR chains consist of an extracellular region,
transmembrane region, and a shorter cytoplasmic tail. The
extracellular region contains a variable immunoglobulin-like (V)
domain, a constant immunoglobulin-like (C) domain, and con-
necting peptide.
20
The RAG1 and RAG2 recombinases facilitate the
assembly of the V domain from gene segments that serve as the
antigen recognition site. The C domain is used for the interactions
with CD3 chains.
There are considerable structural differences between αβ and
γδ chains in terms of C domain and connecting peptide, which are
also reected in the assembly of the TCR complexes, surface
shape, and charge distribution.
2124
However, in both complexes,
three dimers of CD3 proteins, δε and γε heterodimers and ζζ
homodimers, are present.
23,25
These CD3 proteins associate with
TCR via non-covalent hydrophobic interactions and are required
for a complete TCR localization on the cell surface (Fig. 1a).
TCR co-receptors
Initial studies demonstrated that T cells expressing common TCRα/
TCRβheterodimer with distinct functionsfor example, cytotoxic
T cells that directly destroy infected cells and a subset of helper
T cells that help B cellsmay easily be distinguishable by the
expression of mutually exclusive cell surface molecules CD8 and
CD4.
2630
Later studies indicated that these two receptors may
play important roles in the association of MHC molecules and thus
are referred to as co-receptors.
3133
Both CD4 and CD8 molecules
play important roles during the development of T cells by helping
the TCR complex select a different class of MHC molecules.
34
Like
TCR molecules, both CD4 and CD8 molecules contain an
extracellular domain, transmembrane domain, and a short
intracellular tail.
35
Although both CD8 and CD4 act as coreceptors
with similar functionality, they share a minimal structural similarity.
The extracellular domain of CD4 contains two V domains (D1 and
D3) and two C domains (Fig. 1b). CD4 acts as a monomer on the T
cell surface where it uses the D1 domain for MHC recognition and
the cytoplasmic tail for interaction with non-receptor tyrosine
kinase LCK.
36
On the other hand, the CD8 extracellular domain
contains only a single V domain. However, two CD8 isoforms,
CD8αand CD8β, are expressed and can form homo- or
heterodimers (Fig. 1b). Most CD8-positive T cells express as a
heterodimer; some CD8-positive T cellsfor example, intraepithe-
lial lymphocytes and memory precursorsexpress as an αα
homodimer.
3739
TCR ligands
Ligands for T cells are divided into two classes: MHC class I (MHCI)
and MHC class II (MHCII) (Fig. 1c). Human MHCIs are complexes of
human leukocyte antigens (HLAs: HLA-A, HLA-B, and HLA-C) and
β2-microglobulin while MHCIIs are heterodimers of several HLAs
(HLA-DP, HLA-DQ, and HLA-DR).
40
Antigen peptide-bound MHCI
(pMHC-I) molecules can be presented on any nucleated cells
recognized by CD8+T cells. On the other hand, CD4+T cells
recognize antigen peptide-bound MHCII (pMHC-II) molecules that
are presented on the APCs, such as B cells, macrophages, and
dendritic cells.
40
Besides peptide presentation by MHC molecules,
lipid antigens present similarly structurally to MHCII molecules,
such as CD1 family proteins.
41,42
Lymphocyte-specic PTK (LCK)
LCK is a member of the SRC family kinase (SFK). The SFKs are a
family of ten structurally similar non-receptor PTKs which have
been implicated in various cellular functions.
4345
All SFKs contain
highly conserved regulatory domains (SH3 and SH2), a protein
tyrosine kinase domain (SH1), a C-terminal tail, and a poorly
conserved N-terminal region (SH4 domain). The SH4 domain
contains a myristoylation site by which SFKs anchor to the
membrane.
46
The SH3 domain, in general, recognizes proline-rich
motifs (PxxP), and the SH2 domain interacts with phosphotyrosine
residues.
47,48
However, the function of the SH4 domain cannot be
generalized for all SFKs except that it holds the myristoylation site.
The SH4 domain of some SFKs also contains a palmitoylation in
Fig. 1 TCR components. a TCRα/TCRβand TCRγ/TCRδheterodimers form complexes with the CD3 molecules. Heterodimers of CD3ε/CD3δ
and CD3γ/CD3ε, and a homodimer of CD3ζ/CD3ζform complexes with TCR dimers. TCR heterodimers contain intramolecular and
intermolecular disulde bonds. CD3 chains contain 10 ITAMs distributed in different CD3 molecules. The variable region (V) of TCR
heterodimers recognize the antigen peptide-loaded on MHC (pMHC). In the absence of pMHC, the intracellular part of the CD3 molecules
forms a close conformation in which ITAMs are inaccessible to the kinases for phosphorylation. bCoreceptor CD4 acts as a single molecule
while CD8αand CD8βcan form homodimers or heterodimers. cMCH-I consists of an α-chain containing three immunoglobulin domains (α
1
,
α
2
,α
3
) and β2-microglobulin (β2m). MCH-2 is the heterodimer of an αchain and a β-chain containing two immunoglobulin domains (α
1
,α
2,
and β
1
,β
2
) in each chain. dLCK-loaded CD4 molecules bind to the MHC-II bound TCR (TCRα/TCRβ) complex. This allows LCK to phosphorylate
two distinct sites on ITAMs. Then ZAP-70 interacts with the phosphotyrosine sites and mediates more tyrosine phosphorylation. CD4 and
MHC-II interaction is mediated through the membrane-proximal α
2
and β
2
domains of MHC-II and the membrane-distal D1 domain of CD4.
T cell receptor (TCR) signaling in health and disease
Shah et al.
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Signal Transduction and Targeted Therapy (2021) 6:412
addition to the myristoylation site. Likewise, other SFKsLCK
activity is tightly controlled by phosphorylation/dephosphoryla-
tion cycles (Fig. 1d). C-terminal SRC kinase (CSK) phosphorylates
LCK on Y
505
residue that interacts with the LCK-SH2 domain,
keeping it inactive.
49
The leukocyte common antigen (CD45), also
known as protein tyrosine phosphatase receptor type C (PTPRC),
removes the regulatory phosphotyrosine residue that releases the
kinase domain from autoinhibitory states (priming) and also
makes the SH2 domain available for interaction with other
proteins.
5052
This priming step results in autophosphorylation of
Y,
394
leading to full activation of LCK.
52
Besides a role in LCK
activation, several studies have pointed out that CD45 can
negatively regulate LCK function by removing tyrosine phosphor-
ylation from LCK-Y
394
.
53,54
T cells maintaining a certain level of
CD45 expression uphold appropriate LCK activation whereas low
levels of CD45 expression correlate with the lower TCR activation,
and higher CD45 expression reduces LCK activity by removing
tyrosine phosphorylation from LCK-Y
394
.
53
Thus, T cells can
regulate TCR activity by modulating CD45 expression.
55
Several
cytosolic phosphatases, including PTPN6(SHP-1), and PTPN22, also
control LCK activity by removing phosphate group from Y
394
.
56,57
Therefore, LCK activity is probably dynamically regulated by
cellular abundance and activity of CSK, CD45, PTPN6, and PTPN22.
TCR ACTIVATION AND PROXIMAL SIGNALING
Early T cell signaling takes place within a few seconds, and the rst
step is TCR activation.
58
An early event in the proximal signaling of
TCR is the involvement and activation of a set of PTKs.
6
Several
PTKs, such as LCK, FYN, and ZAP-70, are important signaling
components for T cell development and activation of TCR
signaling through tyrosine phosphorylation on CD3.
8,5961
The
cytosolic tail of the CD3 proteins contains a unique motif, the
immunoreceptor tyrosine-based activation motifs (ITAMs), that
consists of two tyrosine residues anked by leucine/isoleucine and
spaced by bulky aromatic amino acid, thus having a consensus
sequence of D/Ex
(0-2)
YxxI/Lx
(6-8)
YxxI/L.
6264
Each of the CD3δ,γ,
and εchains contain one ITAM each, whereas each CD3ζchains
contain three ITAMs, thus each TCR-CD3 complex contains ten
ITAMs.
6,65,66
For TCR activation, tyrosine residues in ITAMs need to
be phosphorylated, which is initiated by LCK and, to some degree,
by FYN.
6769
Although FYN can induce phosphorylation of ITAMs,
its role is dispensable for T cell development.
70,71
Thus, tyrosine
phosphorylation on CD3 ITAMs by LCK during T cell development
probably cannot be replaced by other tyrosine kinases.
72
LCK is known to be associated with several growth factor
receptors, including KIT, FLT3, and AXL, in a phosphorylation-
dependent manner.
7375
The interaction between LCK and growth
factor receptors is mediated via the SH2 domain of LCK that
interacts with the phosphotyrosine residue of the activated
receptor. Since the TCR-CD3 complex lacks intrinsic kinase activity,
the pMHC-loaded TCR-CD3 complex remains unphosphorylated,
and therefore LCK cannot directly interact with the inactive
complex through an SH2 domain. However, to facilitate the
phosphorylation of ITAMs, LCK needs to be localized to the cell
membrane. LCK can anchor to the cell membrane via its
myristoylation (serine 2) and palmitoylation (cysteine 3/5) sites
present in the SH4 domain or through the interaction with the
cytoplasmic tail of coreceptors CD4 and CD8.
69,7688
The
C-terminal tail of CD4 and CD8αcontains a conserved CxCP motif,
which is absent in CD8β, required for this interaction.
81,89,90
This
motif interacts with the CxxC motif present in the LCK SH4
domain, mediating the interaction in a zinc-ion-dependent
manner.
8994
Therefore, only the homodimer CD8α/CD8αand
heterodimeric CD8α/CD8βcan load LCK to the TCR complex, and
although a CD8β/CD8βhomodimer can be formed, it cannot
recruit LCK to the TCR complex and thereby does not play a role in
TCR signaling.
T cells usually express CD4 or CD8 coreceptors, and therefore
pMHC-bound TCR browses for LCK-loaded coreceptors where the
non-polymorphic part of pMHCs interacts with the distal mem-
brane part of the coreceptors.
7679
The membrane-distal D1
domain of CD4 associates with the membrane-proximal α
2
and
β
2
domains of MHC-II (Fig. 2), but it does not directly interact with
the TCR complex.
9598
Similar to the CD4MHC-II interaction,
binding with the CD8α/CD8αhomodimer or CD8α/CD8βhetero-
dimer to the MHC-I complex is mediated through the membrane-
distal D1 domain of CD8 and membrane-proximal β2M and α3of
the MHCI complex.
99
Additionally, the membrane distal α2 domain
of the MHC-I complex also participates in interaction with CD8.
99
Such interaction keeps TCR and coreceptors orthogonal, which is
likely important for the stability of the complex, LCK loading, and
TCR activation.
100103
Although both the CD8α/CD8αhomodimer
or CD8α/CD8βheterodimer binds with MHC-1 complex with a
similar afnity, the CD8β/CD8βhomodimer does not bind with
MHC-1.
104106
Interaction between coreceptors and LCK has two important
functions: it brings LCK in close proximity to the TCR complex and
it stabilizes coreceptors by preventing clathrin-mediated endo-
cytosis.
94
However, once LCK phosphorylates CD3 proteins, it
leaves coreceptors that result in the internalization of corecep-
tors.
107111
Nevertheless, the interaction between LCK-loaded
coreceptor and pMHC acts as a rate-limiting step to initiate TCR
signaling.
112
Initial studies suggest that coreceptor-bound LCK mediates
tyrosine phosphorylation on all four CD3 chains.
113116
However,
those studies have not suggested any site-specicity or whether
LCK loaded to the CD4 or CD8 makes any difference in CD3
tyrosine phosphorylation dynamics. To address the sequence of
CD3 tyrosine phosphorylation, several attempts have been taken
with a specic focus on CD3ζ, which has six tyrosine phosphoryla-
tion sites.
117,118
In resting T cells, at least two tyrosine residues in
second and third ITAMs of CD3ζremain to be phosphorylated.
117
When activated, N-terminal tyrosine residue in the third ITAM
displays dependency on the N-terminal tyrosine residue of the
rst ITAM, and C-terminal tyrosine residue in the second ITAM
needs the C-terminal tyrosine residue to be phosphorylated on
the rst ITAM.
117
Furthermore, LCK phosphorylates all six tyrosine
residues of recombinant CD3ζITAMs in a specic order, starting
from the N-terminal tyrosine residue of the rst ITAM.
118
These
studies suggest that tyrosine phosphorylation in ITAMs is a
controlled molecular event that has further been shown to be
regulated by TCR ligand.
117
LCK-induced phosphorylation of both tyrosine residues in ITAM
has been shown to be required for interaction with ZAP-70 where
ZAP-70 SH2 domains mediate the interactions.
67,119
This interac-
tion stabilizes tyrosine phosphorylation of CD3ζand induces ZAP-
70 tyrosine phosphorylation, which was independent of ZAP-70
kinase activity, suggesting that ZAP-70 does not play a role in
CD3ζtyrosine phosphorylation but that interaction with tyrosine
residues probably limits phosphatase access, protecting tyrosine
phosphorylation. Nevertheless, the interaction is important for
ZAP-70 activation and downstream signaling.
The mechanism through which CD3 ITAMs remain unpho-
sphorylated in an inactive TCR complex remains to be debated.
Using CD3εas a model, it has been depicted that the positively
charged cytoplasmic domain remains embedded in the nega-
tively charged inner membrane, which sequestrates tyrosine
residues.
120,121
Similarly, the cytoplasmic part of CD3ζremains
lipid-bound, preventing tyrosine phosphorylation.
122
However,
the role of the inner membrane in the prevention of tyrosine
phosphorylation was questioned by another study.
123
This study
demonstrated that removal of positively charged residues did not
enhance tyrosine phosphorylation but that pervanadate
enhanced CD3 tyrosine phosphorylation, concluding that phos-
phatase might be involved in the prevention of tyrosine
T cell receptor (TCR) signaling in health and disease
Shah et al.
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Signal Transduction and Targeted Therapy (2021) 6:412
phosphorylation.
123
Pervanadate prevents tyrosine phosphatases
by oxidizing the catalytic cysteine of phosphatase.
124
Those
claims were later contested by the fact that removal of positively
charged residues decreases LCK-mediated CD3 tyrosine phos-
phorylation, and pervanadate can prevent membrane association
of CD3 with lipid membranes.
125
Combining early studies, a model has been proposed suggest-
ing that interaction between the cytoplasmic chain and lipid
membrane prevents ITAM phosphorylation, and pMHC association
to the TCR induces structural changes that release CD3 from the
inner membrane.
126,127
This model has further been supported by
the fact that positively charged ions, such as Ca
2+
, can release
sequestered CD3 chains, facilitating tyrosine phosphorylation,
128
and that defects in Mg
2+
transport are linked to the defective T
cell activation due to impaired Ca
2+
inux in T cells.
129
Antigen
engagement increases Ca
2+
intake
86
as well as TCR proximal Ca
2+
concentration.
130
Although this model provides a simplied overview of TCR
activation, the model fails to explain how ITAMs in CD3δand CD3γ
remain protected from tyrosine phosphorylation, as they lack
membrane-binding residues,
126,131
and how a basal level of CD3ζ
tyrosine phosphorylation is maintained if they remain to be
sequestered in the plasma membrane.
60,117,120,132134
Thus,
complete sequestering to the plasma membrane might be an
unlikely event. Rather, dynamic switching between membrane
binding and cytosolic release might happen, and other forces may
also be involved.
135
Constitutive association of ZAP-70 to the CD3ζmight also go
against the model of complete sequestering, as ZAP-70 associa-
tion is mediated through the SH2 domain and phosphotyrosine
residues.
132
Furthermore, constitutively active LCK localized at the
cell membrane was detected in up to 40% of resting T cells.
53
Constitutively LCK activation is probably required for maintaining
basal tyrosine phosphorylation of CD3ζ, as T cells with reduced
constitutively LCK activation displayed undetectable levels of
CD3ζtyrosine phosphorylation.
53
But why is TCR signaling not
triggered in resting cells if free LCKs are available and apparently
are more active than coreceptor-bound LCKs
136
? Perhaps mem-
brane association CD3ζlimits the accessibility to the tyrosine sites
in ITAMs, and, if they are accessible in resting cells, CD3ζ
orientation allows phosphatases to remove tyrosine phosphoryla-
tion.
123
Therefore, the association of pMHC to the TCR complex
that mediates structural changes of the cytosolic part of CD3 is
important for TCR activation.
A two-stage kinetics of TCRpMHCCD8 interaction has been
suggested, where the rst TCR binds with the pMHC (within
<0.1 s), and then the tri-molecular complex is formed.
137
The tri-
molecular complex was affected by proximal signaling, as
pharmacological inhibition of SFKs or CD45 abolished initiation
of high-afnity binding.
137
LCK association to the coreceptor
stabilizes coreceptors by preventing endocytosis.
94
However, how
the kinase activity of LCK stabilizes the tri-molecular complex
remains to be determined. As an early event, CD8 interacts with
CD3ζ, which is independent of pMHC but LCK-dependent, and in
Fig. 2 TCR activation. In resting T cells, CD3ζand CD3εremain membrane-embedded. Perhaps membrane-bound CD3ζmight be released to
the cytosol, where free LCK induces tyrosine phosphorylation on at least two sites in ITAMs. This basal tyrosine phosphorylation creates
docking sites for ZAP-70 interaction. After antigen engagement, the TCR complex recruits coreceptor-bound LCK that phosphorylates ZAP-70
and interacts with it through the SH2 domain facilitating tyrosine phosphorylation on other residues on ITAMs.
T cell receptor (TCR) signaling in health and disease
Shah et al.
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Signal Transduction and Targeted Therapy (2021) 6:412
the later events, tri-molecular complex is required to maintain the
CD8CD3ζinteraction.
138
As LCK can be either free or CD8
bound,
138
CD3ζmight get a chance to meet either free LCK or
coreceptor bound LCK before ligand association, probably further
explaining CD3ζtyrosine phosphorylation in resting cells.
A single-molecule analysis suggests that the movement of LCK
during TCR activation is not directed but is rather a Brownian
movement.
139
Therefore, it would be challenging for a TCR
complex to nd LCK instantly unless LCK is already recruited to the
complex in resting cells. Free LCKs which are also membrane-
bound display higher mobility than coreceptor-associated LCKs,
probably due to the difference in molecular size.
136
This might
also explain why free LCKs are recruited in the early TCR
complexes.
138
Besides the ability to move faster, free LCKs display
higher catalytic activity as measured by tyrosine phosphorylation
(LCK-Y
394
).
136
However, in any case, coreceptor-bound LCK is
required for TCR activation, and the number of coreceptors-bound
LCK increases during the maturation process.
140
Finally, it has
been demonstrated that LCKs directly interact with CD3εin which
the interaction is ionic and is mediated through the juxtamem-
brane basic residue-rich sequence (BRS) of CD3εand the unique
domain (UD) of LCK.
141
The early steps of the TCR activation process seem to be highly
debated. Current models for TCR activation either considered CD3
membrane sequestering and ignored the basal level of CD3ζor
the other way around.
126,127,135
Perhaps both of the conditions
simultaneously occur, and therefore probably CD3ζholds its states
as membrane-embedded and outside the membrane, allowing
constitutively active LCK to phosphorylate tyrosine residues on
CD3ζITAMs (Fig. 2). Then ZAP-70 binds to the phosphotyrosine
residues in CD3 ITAMs. Once bound, ZAP-70 is phosphorylated by
LCK, which leads to its activation.
142
This results in the formation
of a multi-nucleated signaling complex as further phosphorylation
of ZAP-70 allows binding of additional proteins and adaptors,
thereby itself behaving as a scaffold.
142,143
Thus, the activated
state of TCR is characterized by phosphorylation of ITAMs,
followed by phosphorylation and activation of ZAP-70.
DISTAL TCR SIGNALING
Engagement of TCR with the MHC-antigenic peptide complex of
APCs triggers the formation of multi-molecular signalosomes at
TCR. This leads to the generation of proximal signaling, followed
by the activation of multiple distal signaling cascades, such as
Ca
2+
calcineurinNFAT, PKCθIKKNFκβ, RASGRP1RASERK1/2,
and TSC1/2mTOR, with the help of secondary messengers,
enzymes, and various adaptor proteins (Fig. 3). These signaling
cascades nally bring out the diverse phenotypic effects, as they
control many aspects of T cell biology.
1
Ca
2+
calcineurinNFAT pathway
Phospholipase Cγ1 (PLCγ1) is the main molecule connecting the
TCR proximal to distal signaling cascades.
144
The membrane-
bound phosphatidylinositol 4,5-bisphosphate (PIP
2
) gets hydro-
lyzed by activated PLCγ1 into diacylglycerol (DAG) and inositol-3-
phosphate (IP
3
).
145
Both of these essential secondary messengers
initiate a variety of distal signaling cascades important for T cell
activation. Membrane-bound DAG can activate PKCθ, RASGRP1,
and PDK1-mediated pathways. On the other hand, IP
3
triggers the
activation of a Ca
2+
-dependent calcineurin NFAT pathway.
145147
IP
3
generated from PIP2 binds to the Ca
2+
-permeable ion
channel receptors (IP3R) on the endoplasmic reticulum (ER),
thereby releasing ER Ca
2+
stores in the cytoplasm.
148
It has been
found that ERs can sense the intracellular Ca
2+
levels through the
constitutive expression of a transmembrane protein called stromal
interaction molecule (STIM). Depletion of intracellular Ca
2+
levels
thus triggers an inux of extracellular Ca
2+
into T cells from Orai1
type plasma membrane calcium-release activated calcium (CRAC)
channel.
149152
Increased intracellular Ca
2+
activates a protein
phosphatase, calcineurin, that dephosphorylates the nuclear
factor of activated T cells (NFAT), thereby causing its nuclear
translocation. Nuclear NFAT forms a complex with AP-1 transcrip-
tional factors (JUN/FOS) derived from the DAGRASMAPKERK1/
2 pathway. This transcriptional complex is responsible for inducing
the expression of various genes, like IL-2 and other effector
molecules, that are responsible for T cell activation. In contrast, in
the absence of AP-1, NFAT alone activates various genes, like
several ubiquitin ligases and diacylglycerol kinase α(DGKα), that
are responsible for T cell anergy, a state of T cell unresponsiveness,
one of the processes to induce immune tolerance.
153156
Thus,
two opposite T cell functionsactivation and anergyare
controlled by NFAT proteins.
153
In addition to calcineurin, Ca
2+
also activates a Ca
2+
/calmodu-
lin-dependent kinase (CaMK) that mediates T cell activation
through activation of transcription factors, such as cyclic-AMP-
responsive-element-binding protein and myocyte enhancer factor
Fig. 3 Positive regulation of T cell signaling. The gure depicts the activation of various enzymes and adaptor molecules upon engagement
of TCR with the MHC antigenic peptide complex. The phosphorylation events carried out are depicted as small, blue-colored circles. Black
lines with arrows indicate activation.
T cell receptor (TCR) signaling in health and disease
Shah et al.
5
Signal Transduction and Targeted Therapy (2021) 6:412
2.
157
A missense mutation in Orai1 can lead to impaired Ca
2+
signaling, which affects nuclear translocation of NFAT, and thereby
NFAT-induced cytokines production causes severe combined
immunodeciency (SCID) in humans.
158,159
Thus, the universal
secondary messenger Ca
2+
regulates several important functions,
including proliferation, differentiation, and cytokine production in
T cells.
160
PKCθIKKNF-κβ pathway
Protein kinase C (PKC) is a family of ten protein serine/threonine
kinases that plays numerous roles in physiological and patholo-
gical conditions.
161164
PKC family proteins are divided into three
subfamilies: classical (PKCα, PKCβ1, PKCβ2, and PKCγ), novel
(PKCδ, PKCε, PKCη, and PKCθ), and atypical (PKCζand PKCι). DAG
and Ca
2+
regulate activation of classical PKC isoforms, and novel
PKC isoforms are regulated by DAG while atypical PKC isoforms
are independent of DAG and Ca
2+
for activation.
161
Several PKC
isoforms have been implicated in T cell functions.
165
For example,
the novel isoform PKCθbinds to DAG through the PKC conserved
region 1 (C1) domain, which is required for its recruitment to the
lipid raft after TCR engagement. PKCθplays major non-redundant
roles in T cell activation, even though T cells express several other
PKCs.
166,167
Nuclear factor κβ (NF-κβ) is an evolutionarily conserved
transcription factor that plays important roles in regulating genes
involved in inammatory and immune responses, cell growth,
survival, and differentiation.
168
TCR-mediated T cell activation
involves both the canonical (classical) and the non-canonical
(alternative) NF-κβ pathway. An essential factor required for
complete T cell activation via the non-canonical NF-κβ pathway is
MAP3K14 (also known as NF-κβ-inducing kinase; NIK).
169
However,
more studies are required regarding this. On the other hand,
PKCθIKKβNF-κβ forms the canonical branch of the NF-κβ
pathway and is widely studied.
170
Once PKCθis activated following TCR stimulation, it triggers the
formation of a tri-molecular complex of adaptor proteins in
the cytoplasm called the CBM complex, which is composed of the
caspase recruitment domain-containing membrane-associated
guanylate kinase protein-1 (CARMA1), B cell lymphoma/leukemia
10 (BCL10), and mucosa-associated lymphoid tissue translocation
protein-1 (MALT1).
171
This is initiated by phosphorylation of
CARMA1 by activated PKCθ,
172
which is required for its
oligomerization and association with BCL10.
173
MALT1 then binds
to BCL10, and this association recruits an E3 ubiquitin ligase, called
the tumor necrosis factor receptor-associated factor 6 (TRAF6),
that polyubiquitinates and degrades IKKγ, or the NF-κβ essential
modier (NEMO), a regulatory protein of the IKK complex.
174,175
Consequently, the catalytic subunits of Iκβ kinases (IKK), αand β,
are no longer inhibited, and they phosphorylate Iκβ, thereby
inducing its ubiquitination and degradation. NF-κβ is thus
released from its inhibitory Iκβ complex in the cytoplasm, and it
translocates into the nucleus to regulate gene expression.
176178
This canonical PKCθIKKβNF-κβ pathway is extremely important
for T cell survival, homeostasis, activation, and effector function.
179
Deregulation of this pathway can cause defective T cell survival
and activation, autoimmunity, SCID, and lymphoma.
180182
RASGRP1RASERK1/2AP1 pathway
DAG from PIP2 induces the activation of another key molecule, a
RAS guanyl nucleotide-releasing protein (RASGRP1), and recruits it
to the plasma membrane.
183,184
RASGRP1 and Son of Sevenless
(Sos) are two known guanine nucleotide exchange factors (GEFs)
responsible for RAS activation in T cells.
1
RAS, a small G protein,
binds to GTP in the activated state and initiates the RAS-MAPK
cascade by activating the serine/threonine kinase Raf1.
183,185
Raf1,
a mitogen-activated protein kinase (MAPK) kinase kinase
(MAPKKK), then phosphorylates and activates MAPK kinases
(MAPKKs), such as MEK1/2, that further phosphorylate and activate
MAPK extracellular signal-regulated kinase-1 & 2 (ERK1/2).
6,185,186
T
cell development, differentiation, and TCR-induced signal strength
are all controlled by ERK1/2 signaling.
187189
Furthermore, ERKs
trigger the phosphorylation and activation of their downstream
target Elk, a transcription factor responsible for inducing the
expression of c-Fos transcription factor. The VAV1Rac pathway
induces the expression of Jun.
190
Thus, the formation and
activation of a dimeric complex, activator protein-1 (AP-1)
composed of Jun/Fos, that plays a critical role in immune
response, followed by IL-2 transcription, is sustained by the
DAGRAS pathway.
1,6
Further, the signal transducer and activator
of transcription (STAT3) and LCK get phosphorylated by
ERKs.
191,192
RASGRP1 is extremely important for the development of
conventional αβ T cells but not the meager population of γδ
T cells.
193195
However, it is important for the activation of both
types of T cell population as well as for the expression of IL-17.
195
RASGRP1 deciency can cause defects in the activation of various
signaling pathways, such as RASMAPKERK1/2, mTOR, and PI3K/
AKT.
193,196
Moreover, abnormal expression of both RASGRP1 and
RAS was described in T cells of systemic lupus erythematosus (SLE)
patients, thereby implicating the involvement of this pathway in
the generation of SLE.
197
p38 and JNK pathways
The other MAPKs such as p38 and JNK family proteins play
important roles in the proliferation, differentiation, and function of
different subsets of T cells.
198202
p38 is a family of four highly
structurally homolog proteins including p38α(MAPK14), p38β
(MAPK11), p38γ(MAPK12), and p38δ(MAPK13).
203
p38αis widely
known as p38 and is one of the most studied isoforms. On the
other hand, the JNK family is composed of three members which
include JNK1 (MAPK8), JNK2 (MAPK9), and JNK3 (MAPK10).
204
Three dual-specicity MAP2Ks including MKK3 (MAP2K3, MEK3),
MKK4 (MAP2K4, MEK4), and MKK6 (MAP2K6, MEK6) are involved in
p38 activation through phosphorylation on the conserved T180-X-
Y182 motif in the loop of the substrate recognition site.
203
Among
those three MAP2Ks, MKK3 and MKK6 display higher specicity to
p38 while MKK4 also activates JNKs.
203206
The conserved T180-X-
Y182 motif is required to be phosphorylated on both threonine
and tyrosine residues for p38 activation.
207
In mammalian cells,
the classical p38 pathway is regulated by ten different MAP3Ks
that allow for the integration of various signaling nodes. However,
in T cells, p38 activity is mediated by a non-classical pathway
which is downstream of proximal TCR signaling and probably
independent of MAPK cascades.
203
In such a case, activation of
TCR proximal signaling results in the phosphorylation of p38 at
Y323 residue by ZAP-70, which triggers autophosphorylation on
regulatory residues (T180-X-Y182) followed by p38 activation.
208
Additionally, activated p38 mediated phosphorylation of ZAP-70
on T293 residue may act as a negative feedback loop possibly by
limiting excessive TCR signaling.
209
JNK activation is likely
mediated through the activation of PKCθand CBM complex upon
the activation of TCR proximal signaling (reviewed in ref.
210
).
BCL10 oligomers in the CBM complex can recruit TAK1 (MAP3K7),
MKK7 (MAP2K7), and JNK2, leading to the activation of JNK.
210,211
TSC1/2mTOR pathway
Upon TCR engagement, both DAGRASGRP1RASERK1/2 and
PI3KAKT pathways induce the activation of mTORC1 and
mTORC2 (refs.
196,212
) that differentially regulate the generation
of CD4
+
helper effector T cell types (Th).
213
mTORC1 down-
regulates SOCS5 to promote STAT3 activity, thereby promoting
Th17 differentiation.
214
Resultingly, mice having Rheb or raptor-
decient T cells show defects in Th17 differentiation.
214,215
On the
other hand, mTORC2 phosphorylates AKT at S473 and PKCθat
S660/676 to induce Th1 and Th2 differentiation respectively.
215
Thus, mice having rictor-decient T cells show defects in
T cell receptor (TCR) signaling in health and disease
Shah et al.
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Signal Transduction and Targeted Therapy (2021) 6:412
differentiation of IFNγ-producing Th1 and IL-4-producing Th2
effector cells.
214,215
The activity of mTOR subsequently needs to
be tightly controlled, as it regulates T cell activation, differentia-
tion, and function.
216
As discussed above, the CBM complex plays a crucial role in TCR
signaling by recruiting key signaling mediators. Hamilton et al.
217
demonstrated that CARMA1 and MALT1 in CBM complex, but not
BCL10, are required for optimal activation of mTOR in T cells.
Furthermore, the CBM complex is involved in TCR-induced
glutamine uptake and the activation of mTOR pathways.
218
Nevertheless, mTOR regulates intracellular metabolic signaling
which links to biosynthetic and bioenergetic metabolisms
(reviewed in refs.
219221
).
SIGNALING BY THE CO-STIMULATORY MOLECULES
TCR engagement leads to activation of proximal and distal
signaling pathways. However, productive T cell activation also
involves the engagement of additional cell surface receptors, i.e.,
co-stimulatory molecules like CD28. This is required to avoid
anergy, a state of T cell unresponsiveness where T cells become
refractory to restimulation by IL-2. If the TCR signals are weak, it
results in cell death or anergy. These weak TCR signals are
amplied strongly by CD28 engagement, thereby resulting in cell
proliferation and differentiation. However, only CD28 engagement
results in the expression of a few genes transiently with no
biological consequences.
222
The key event coupling CD28 to several downstream signaling
pathways is the recruitment of phosphatidylinositol-3-kinase
(PI3K) to the phosphorylated cytoplasmic tail of CD28, which
converts PIP2 to PIP3. Once AKT is recruited to PIP3, it acts on
several substrates. AKT facilitates prolonged nuclear localization of
NFAT, and thus IL-2 transcription, by inactivating GSK-3. IL-2-
inducible T cell kinase (ITK) is also associated with PIP3, and this
kinase is important for phosphorylation and activation of
PLCγ1.
170
Apart from this, NF-κβ is one of the major signaling
pathways regulated by co-stimulation signaling in T cells. AKT
associates with CARMA1 and hence facilitates the formation of the
CBM complex, which enhances the nuclear translocation and
activation of NF-κβ. However, AKT is non-essential for NF-κβ
signaling in T cells.
223
The most important mediator of the NF-κβ
signaling pathway is phosphoinositide-dependent kinase-1
(PDK1), whose recruitment and phosphorylation enable its
efcient binding to both CARMA1 and PKCθ, thus inducing NF-
κβ activation.
224,225
Indeed, activation of NF-κβ and PKCθwas
found abrogated upon deletion of PDK1 in T cells.
224
VAV1 is a
GEF for small GTPases, such as Rac1, Rac2, and Rhog, where it
plays a crucial role in strongly amplifying CD28-mediated
activation of NFAT and NF-κβ signaling pathways.
226,227
Thus,
signaling by co-stimulatory molecules quanties the signals that
are already activated by TCR ligation, thereby strongly sustaining T
cell activation. These include the PI3KAKTmTOR, NFAT, NF-κβ,
and MAPK pathways (Fig. 3). While PI3K signaling is primarily
mediated by CD28, initial activation of PI3K results in upregulation
of phosphoinositide-3-kinase adaptor protein-1 (PIK3AP1, also
known as BCAP).
228
PIK3AP1 potentiates PI3K signaling in
response to CD3 engagement in CD8+T cells.
228
POSITIVE REGULATORS OF T CELL SIGNALING
Two of the most important adaptors that are phosphorylated by
activated ZAP-70 and play a critical role in positively regulating
TCR signaling are a transmembrane adaptor protein, linker for
activation of T cells (LAT), and a cytosolically localized SH2
domain-containing leukocyte phosphoprotein of 76 kDa (SLP-
76)
229,230
(Fig. 3). These adaptor proteins form the backbone of the
proximal signaling complex (proximal signalosome) that recruits
various other effector proteins,
231,232
along with phospholipase
Cγ1 (PLCγ1), which links the proximal with several distal signaling
pathways upon TCR engagement.
144
This results in a stable and
dynamic zone of contact between APCs and T cells, designated as
the immunological synapse (IS).
Lipid rafts are microdomains located within the plasma
membrane that are enriched with cholesterol, glycosphingoli-
pids, and sphingomyelin, and these rafts accumulate at the IS.
They are also known as glycolipid-enriched microdomains
(GEMs), detergent-resistant membranes (DRMs), or detergent-
insoluble glycolipid-enriched membranes (DIGs). Key compo-
nents of the TCR signaling pathway, such as LCK, LAT, RAS, CD4,
and FYN, along with some others, are located within the lipid
rafts.
6
Post-translational modication of lipids in these mole-
cules is very important for their localization in the lipid rafts.
RAS is both palmitoylated and farnesylated, whereas most of
the SRC PTKs, including the T cell-specic LCK, undergoes
myristoylation and palmitoylation, necessary for its localization
in the lipid rafts and subsequent targeting and phosphorylation
of the CD3 ζchain.
233235
TCR engagement and activation result
in its rapid association with the lipid rafts, and this localization is
important for the early tyrosine phosphorylation events of the
TCR subunits by the SRC family PTKs.
236,237
This can be achieved
by the PTK LCK that is present in the rafts, where its SH2 domain
binds to the phosphorylated tyrosine residues in activated ZAP-
70, thereby bringing TCRs bearing activated ZAP-70 to the lipid
rafts.
238,239
The GM-CSF/IL3/IL5 common β-chain-associated protein (CBAP)
is involved in the regulation of TCR downstream signaling, as
CBAP-decient cells display reduced phosphorylation of PLCγ1,
LAT, JNK1/2, and ZAP-70,
240
suggesting that it may play a role in
both proximal and distal signaling. Besides its role in normal
physiology, CBAP plays an important role in T cell acute
lymphoblastic leukemia (T-ALL) pathology. CBAP was found to
be highly expressed in T-ALL, and its expression enhanced T-ALL
cell growth.
241
Similar to TCR signaling, loss of CBAP decreased
ERK1/2, S6K, RSK, and TSC2 phosphorylation and thereby
decreased aerobic glycolysis and energy metabolism.
241
LAT
The rst adaptor essential for the successful transmission of TCR
signals is the LAT.
13
It is a transmembrane protein of 3638 kDa
242
consisting of a tyrosine-rich cytoplasmic tail and a short
extracellular region.
229
It requires palmitoylation on its two
cysteine residues (C26 and C29) for localization to the lipid rafts.
Mutation of C26 fully inhibited the localization of LAT to the lipid
rafts, whereas C29 mutation had a partial effect. Moreover, no
tyrosine phosphorylation of LAT was detected when C26 was
mutated, indicating that raft localization of LAT is vital for its
phosphorylation.
243
Several molecules have been proposed that
link TCR and LAT by binding to both of them. PLCγ1 binds to
phosphorylated tyrosine residue 132 of LAT
244
via its N-terminal
SH2 domain and to phosphorylated tyrosine residues on activated
ZAP-70 via its C-terminal SH2 domain.
245
An Abl-SH3 interacting
protein, 3BP2 has also been found to interact with both LAT and
ZAP-70 via its SH2 domain, probably in a multimeric form.
246
Another important molecule found to link TCR and LAT is a small
adaptor protein, Shb, that binds to the phosphorylated tyrosine
residues on the CD3 ζchain via its SH2 domain and also binds
phosphorylated LAT via its non-SH2 phosphotyrosine binding
domain.
247
TCR engagement results in rapid phosphorylation of
LAT on its tyrosine residues by ZAP-70.
248
Thus, LAT phosphoryla-
tion and distal signaling events were inhibited when mutant Shb
was expressed with a defective SH2 domain.
247
Once phosphory-
lated, LAT then binds to several proteins, such as enzymes and
adaptor molecules, via diverse binding sites as discussed below,
therein bringing them to the plasma membrane.
Overexpression of LAT did not augment TCR-mediated down-
stream signaling pathways.
249
Moreover, LAT-decient Jurkat cells
T cell receptor (TCR) signaling in health and disease
Shah et al.
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Signal Transduction and Targeted Therapy (2021) 6:412
also displayed TCR-induced receptor phosphorylation and ZAP-70
activation but were found to be defective in all steps distal from
this. There was no activation of PLCγ1 with reduction of Ca
2+
mobilization, ERK activation, NFAT activation, and reduced IL-2
gene transcription.
249,250
Reconstitution with LAT restored these
defects. Moreover, LAT-decient mice blocked thymic differentia-
tion at the pre-TCR stage, thereby showing no T cells in the lymph
nodes and spleen.
250
LAT, then, is important for TCR-mediated
signaling and intra-thymic development of T cells.
Growth factor receptor-bound protein 2 (GRB2) and GRB2-related
adaptor downstream of Shc (GADS)
GRB2 and GADS are cytosolic adaptor proteins, with the former
expressed ubiquitously and the latter expressed only in the
hematopoietic cells, playing an important role in hematopoietic
growth factor receptors signaling.
251,252
Activated/phosphorylated
LAT binds to both the GRB2 family proteins via their SH2 domains,
thereby translocating them to the plasma membrane, along with
their SH3 domain-associated proteins.
6
GRB2 is constitutively
associated with Sos, a dual-specic GEF for small GTPases such as
RAS and Rho. Upon TCR activation, Grb2-Sos complex associates
with LAT, leading to activation of RAS. However, Grb2 seems
insufcient for RAS activation in T cells, as LAT mutants failed to
induce complete RAS activation.
183,253
An additional small linker
molecule, Shc, was found to mediate the association of Sos with
GRB2 in T cells.
254
An E3 ubiquitin ligase, CBL (discussed later), is
another GRB2 associated protein that binds to phosphorylated
LAT in T cells.
255
Upon TCR stimulation, another member of the
GRB2 family, GADS not only binds to phosphorylated LAT but also
specically binds to a critical adaptor molecule of T cells, SLP-
76,
256
thereby associating LAT with SLP-76. GADS has also been
found to associate with a serine/threonine kinase, hematopoietic
progenitor kinase-1 (HPK1), involved in JNK pathway activa-
tion.
256,257
T cell development was found impaired, with specic
defects in both positive and negative selection of thymocytes, in
GADS-decient mice.
258
The three distal tyrosine residues of LAT (171, 191, and 226) are
involved in binding to Grb2, whereas Tyr 171 and 191 are involved
in binding to GADS.
244
Mutation in any one of the tyrosine
residues did not affect either Grb2 or GADS binding, whereas loss
of both 171 and 191 decreased GRB2 binding, and mutation of
both these residues completely abolished GADS binding. The
binding of GRB2 was only abolished when all three tyrosine
residues were mutated. Since these tyrosine sites might directly
interact with PLCγ1 through its C-terminal SH2 domain or
indirectly via GADSSLP-76PLCγ1 interaction, mutations of these
tyrosine residues also impacted PLCγ1 binding due to loss of SLP-
76 binding, with PLCγ1 activation completely inhibited, calcium
ux partially inhibited, and PLCγ1-LAT association being
undetected.
244
SH2 domain-containing leukocyte phosphoprotein of 76 kDa (SLP-
76)
SLP-76 is another crucial multidomain adaptor protein of 76 kDa,
localized in the cytoplasm
13,230
and expressed only in cells of the
hematopoietic system, such as thymocytes, mature T cells, natural
killer cells, megakaryocytes, and macrophages but not B cells.
259
It
plays a very important role by linking LAT, activating PLCγ1, and
other downstream signaling pathways.
13
The proline-rich region
of SLP-76 binds to the SH3 domain of PLCγ1,
260
leading to the
formation of the LATGADSSLP76PLCγ1 complex. Thus, the two
complexes described above, LAT-GADS-SLP-76 and LAT-PLCγ1,
interact with each other via binding of SLP-76 to PLCγ1.
261
SLP-76-
decient Jurkat cells subsequently displayed severe impairment of
PLCγ1 phosphorylation, resulting in decreased calcium ux and IL-
2 production, following TCR engagement.
262
Overexpression of
SLP-76 in Jurkat cells increased TCR-mediated NFAT activity
and IL-2 transcription as well as ERK activation, but calcium
ux remained unchanged.
263,264
Moreover, SLP-76-decient mice
showed an intra-thymic block at an early developmental stage of
T cells (double negative stage), thereby failing to generate normal,
peripheral T cells, displaying the same phenotype as the LAT-
decient mice.
265,266
Such ndings demonstrate that SLP-76, like
LAT, is important for TCR-mediated signaling and intra-thymic
development of T cells.
Upon TCR stimulation, SLP-76 gets phosphorylated by ZAP-70 at
its multiple tyrosine residues, which serve as binding sites for
various SH2 domain-containing proteins. These include proteins
involved in cytoskeletal rearrangements, such as VAV1, non-
catalytic tyrosine kinase (NCK), and the PTK ITK (described
below).
267269
On the other hand, the SH2 domain of SLP-76
associates with the phosphorylated tyrosine residues of a 130 kDa
multidomain adaptor protein, named SLP-76-associated phospho-
protein (SLAP)/FYN-binding protein (FYB)/Adhesion and degranu-
lation promoting adaptor protein (ADAP).
270,271
SLP-76 thus
regulates cytoskeletal changes in activated T cells by coordinated
and precise loading of the effector molecules VAV, NCK, and ADAP
into the complex, vital for the stability of the complex and its
optimal activation.
1
Connecting link PLCγ1
Since both LAT and SLP-76 form the backbone of the proximal
signaling complex, deciency of both the adaptors in Jurkat cells
and mouse models showed diminished activation of RAS signaling
due to impairment in formation of the proximal signalo-
some.
265,272
As the connecting link between proximal and distal
signaling pathways, PLCγ1 is the central signaling molecule in
T cells, and phosphorylation on its multiple tyrosine residues is
required for its full activation.
273
This is mediated by the TEC PTK
family members, such as IL-2 inducible T cell kinase (ITK) and
resting lymphocyte kinase (RLK)/TXK. Deletion of both ITK and RLK
exhibited complete loss of PLCγ1 activity along with defects in
calcium ux following TCR engagement.
274277
In contrast, over-
expression of RLK enhanced PLCγ1 phosphorylation and calcium
ux.
278
Plasma membrane localization of ITK and RLK/TXK is
possible via its N-terminal pleckstrin homology (PH) domain and
palmitoylation respectively. ITK interacts with many different
molecules via its various binding domains. The Tec homology (TH)
region of ITK is a proline-rich region that interacts with the SH3
domain of Grb2. The SH3 domain of ITK, in turn, interacts with the
proline-rich regions of PLCγ1.
260
LAT interactions with ITK have
also been reported; nevertheless, the exact mechanisms are still
unknown.
279
Moreover, the SH2 domain of ITK interacts with
tyrosine-phosphorylated SLP-76.
268,269
Thus, ITKs can interact with
the LAT-associated molecules via multiple mechanisms. ITKs in
turn are activated by both SLP-76 and LCK.
280,281
When ITK
associates with SLP-76, it is present in close proximity to its
substrate PLCγ1, and direct phosphorylation of ITK at Y511 by LCK
promotes its activation.
269,282
Moreover, apart from ITK, the
association of PLCγ1 with LAT, GADS, and SLP-76 is also required
for its optimal activation.
253
In response to TCR engagement,
PLCγ1 activation is thus regulated by the signaling complex
(signalosome) composed of LAT, GADS, SLP-76, PLCγ1, and ITK.
6
Signaling lymphocyte activation molecule (SLAM)-associated
protein (SAP)
Signals from both the TCR-CD3 complex and co-stimulatory
receptors, such as CD28, CD2, and the CD150/SLAM (signaling
lymphocyte activation molecule) family, are required for full
activation of T cells.
283
SAP is a small cytoplasmic protein of 128
amino acids
284
that associates via its SH2 domain with the
immunoreceptor tyrosine-based switch motifs (ITSMs) present in
the cytoplasmic tail of the SLAM family of receptors. Once bound
to a specic ITSM, it may prevent binding of the SH2 domain-
containing protein tyrosine phosphatase 2 (SHP-2) and thereby
compete with it. On the other hand, it may favor the recruitment
T cell receptor (TCR) signaling in health and disease
Shah et al.
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Signal Transduction and Targeted Therapy (2021) 6:412
of SH2 domain-containing inositol phosphatase (SHIP), causing
the switch between these two signaling pathways.
285,286
The
SLAM family consists of a number of transmembrane costimula-
tory receptors, such as CD150/SLAM, CD244/2B4, CD84, CD229/Ly-
9, CD319/CRACC, and NTB-A.
287
Thus, SAP can bind via its SH2
domain to the ITSMs of various SLAM families of receptors, and
this interaction plays a crucial role in mediating the costimulatory
signals necessary for T cell activation.
283
Moreover, SAP also exerts
its adaptor role by binding to various SH3 domain-containing
proteins, such as FYN, PKCθ,βPix, and NCK1,
288291
thus recruiting
them to the SLAM family of transmembrane receptors.
292
Along with SLAM, SAP has also been found to directly associate
with the rst ITAM (Y72-Y83) of the CD3ζchain in various T cell
lines and peripheral blood lymphocytes. Knockdown of SAP
resulted in a decrease of several canonical T cell signaling
pathways, such as AKT and ERK; reduced the recruitment of PLCγ1,
SLP76, and Grb2 to the phosphotyrosine containing complex; and
also reduced IL-2 and IL-4 mRNA induction. Through its direct
association with the CD3ζchain, SAP was found to play a central
role in T cell activation.
292
Indeed, mutations or deletions of the
SH2D1A gene encoding SAP resulted in X-linked lymphoprolifera-
tive syndrome-1 (XLP1), which is characterized by immunode-
ciency due to a specic defect in T cells (apoptosis resistance and
impaired interaction with B cell), reduced cytotoxicity of natural
killer cells, a decrease in B cell functions, and defective NKT cell
development.
293,294
SAP and NTB-A (SLAMF6) are essential proteins that potentiate
the strength of proximal TCR signals required for restimulation-
induced cell death (RICD).
295
RICD is an important consequence of
repeated TCR signaling essential for TCR-induced apoptosis in
thymocytes, mature T cells, T cell malignancies, and T cell
therapies (reviewed in refs.
296298
). Therefore, T cells with
impaired SAP function display resistance to RICD, which likely
explains severe CD8+T cell lymphoproliferation in XLP1
patients.
295
NEGATIVE REGULATORS OF T CELL SIGNALING
Inappropriate activation of T cells is prevented by the termination
of TCR signals, and this is mediated by certain proteins that
negatively regulate TCR signaling (Fig. 4).
Adaptors serving as negative regulators
Phosphoprotein associated with glycosphingolipid-enriched micro-
domains (PAG)/CSK-binding protein (CBP). An important trans-
membrane adaptor protein negatively regulating TCR signaling is
PAG/CBP which is found in the lipid rafts.
299,300
In the absence of
TCR engagement or in resting T cells, PAG is constitutively tyrosine
phosphorylated in its cytoplasmic tail. This serves as a docking site
for the SH2 domain of the major negative regulator of SRC kinases,
the tyrosine kinase c-terminal SRC kinase (CSK), thereby localizing
to the rafts and activating.
301303
Once activated, CSK phosphor-
ylates LCK at the C-terminal Y505 residue, which leads to its kinase
domain inactivation as it causes LCK to bind to its internal SH2
domain.
49,69,301303
Thus, CSK gets activated upon binding to PAG
in the lipid rafts, and it inhibits the activity of SRC family kinases.
However, upon TCR activation, tyrosine phosphatase CD45
transiently dephosphorylates PAG. This results in the dissociation
of CSK from the glycosphingolipid-enriched microdomains (GEMs),
relieving the inhibition of SRC kinases for signal transmission.
304
Moreover, the inhibitory Y505 residue of LCK also gets depho-
sphorylated by CD45 tyrosine phosphatase, which, furthermore,
slightly dephosphorylates positive regulatory autophosphoryla-
tion at Y394.
53,305
Thus, the PAG-CSK complex maintains T cell
quiescence by transmitting negative regulatory signals.
13
SH2 or SHP-2-interacting transmembrane adaptor protein (SIT).
Another transmembrane adaptor protein negatively regulating
TCR signaling is SIT expressed in lymphocytes.
306308
It associates
with the TCR complex as a disulde-linked homodimer.
306
The
cytoplasmic tail of SIT contains immunoreceptor tyrosine-based
inhibition motifs (ITIMs) that, upon tyrosine phosphorylation,
associate with SHP-2. SIT mediates its negative regulation of TCR
signaling through the inhibition of NFAT activity. That, however,
remains unaffected after mutation of the tyrosine residue within
the ITIM motif, which completely abrogates binding to SHP-2.
Thus, SIT-SHP-2 interaction seems unimportant for SI-mediated
negative regulation of T cell signaling.
306
GRB2 was also found to
be associated with SIT via two consensus YxN motifs whose
mutations abrogated the binding. This also had no effect on the
inhibitory function of SIT.
308
Moreover, the effector molecule that
might mediate the negative regulatory function of SIT was found
to be CSK via co-precipitation experiments.
308
However, the
Fig. 4 Negative regulation of T cell signaling. The gure depicts various adaptors and enzymes, like kinases and phosphatases, involved in
negatively regulating TCR signaling. The phosphorylation events carried out are depicted as small, blue-colored circles. Black lines with arrows
indicate activation. Dotted black lines with arrows indicate dephosphorylation events.
T cell receptor (TCR) signaling in health and disease
Shah et al.
9
Signal Transduction and Targeted Therapy (2021) 6:412
precise mechanism of SIT-mediated negative regulation of TCR
signaling needs to be elucidated,
13
as its role in lymphocyte
function seems to be more complex.
10
Enzymes serving as negative regulators
Enzymes such as phosphatases, kinases, and ligases also play
important roles in negatively regulating TCR signaling.
Phosphatases. Apart from CD45 and SHP-2 already mentioned
above, there are other tyrosine phosphatases that mediate
negative regulation. Adhesion molecules called carcinoembryonic
antigen-related cell adhesion molecule-1 (CEACAM1) are
expressed at later time points of TCR stimulation.
309
The SH2
domain-containing protein tyrosine phosphatase 1 (SHP-1) is
recruited to the phosphorylated ITIMs of CEACAM1, and it
dephosphorylates LCK at Y394, inactivating it
57,309,310
and thus
terminating TCR signaling. The binding of TCR to antagonists or
weak antigens induces LCK-mediated phosphorylation of SHP-1 at
Y564, thereby activating it, which, in turn, dephosphorylates and
inactivates LCK.
309,310
Moreover, SHP-1 binding to LCK is
prevented by ERK1/2-mediated phosphorylation of LCK at S59,
sustaining TCR signaling. SHP-1 activity is thus indirectly regulated
by ERK1/2.
311
Additional phosphatases that negatively regulate
TCR signaling include PTEN, which dephosphorylates PIP3, and
dual-specicity phosphatases, which dephosphorylate
MAPKs.
312,313
Diacylglycerol kinases. Subcellular levels of DAG are regulated by
lipid kinases, DGKs that phosphorylate DAG to produce phospha-
tidic acid (PA).
314,315
Consequently, the increase in DGK activity
attenuates RASMEKERKAP1 signaling induced by TCR-
mediated DAG activation.
316,317
Ten DGK isoforms are expressed
in mammals,
315
with DGK αand ζbeing expressed at high levels in
T cells.
318
Both isoforms negatively regulate the
DAGRASGRP1RASERK1/2 pathway and thus inhibit activation
of mTORC1 and mTORC2 complexes.
196
Indeed, the genetic
ablation of these isoforms resulted in increased activation of the
RASMEKERKAP1 pathway, mTOR signaling, and PKCθNF-κβ
pathway. This led to the loss of T cell anergy and increased T cell
hyperactivation.
145,155,156,319,320
Both DGK αand ζperform
redundant roles in T cells, as their deciency resulted in severe
T cell developmental blockade at the DP stage, which was partially
restored with the phosphatidic acid treatment.
321
DGK activity can
be regulated by SAP. Overexpression of SAP reduces DGKαactivity
which was shown to be dependent on the SH3-binding ability of
SAP in T cells, suggesting that SAP acts as a negative regulator of
DGKα.
322
As SAP-decient XLP1 displays resistance to RICD,
pharmacological inhibition of DGKαin SAP-decient cells can
restore RICD, indicating that XLP1 patients will likely benet from
DGKα-targeted therapy.
295,323,324
E3 ubiquitin ligases. E3 ubiquitin ligases are enzymes that
ubiquitinate different proteins and target them for proteasomal-
or lysosomal-mediated degradation.
325
Some of these ligases
regulate T cell tolerance, and T cells can become autoreactive
upon their deletion or mutation, leading to autoimmunity.
326
TRAF6, as mentioned before, is one such E3 ubiquitin ligase that
plays an important role in the activation of the NF-κβ signaling
pathway.
174,175
Itch is another ubiquitin ligase that not only
targets PLCγ1 and PKCθ
327,328
but also Jun, thereby causing
diminished activation of AP-1.
329
Itch thus regulates T cell anergy
by degrading certain components of TCR signaling.
330
Itch-
decient mouse models are therefore prone to autoimmune and
pro-inammatory phenotypes.
1
A well-studied E3 ligase that marks various proteins for
ubiquitin-mediated degradation is Casitas B cell lymphoma (CBLB).
This enzyme, along with c-CBL, another member of the CBL family,
negatively regulates TCR signaling.
331
In activated T cells, the CD3ζ
chain gets ubiquitinated by CBLB at its multiple lysine residues
and induces degradation of surface TCRs.
332335
Some other
targets for CBL-mediated ubiquitination and protein degradation
include members of the proximal signaling complex, SRC- and
Syk-family PTKs,
336,337
the regulatory p85 subunit of PI3K,
338
and
the adaptor molecule VAV1.
337
All these events result in the
attenuation of TCR signaling. In another mode of action, T cell
activation leads to dissociation of CBLB from Grb2, and it then
binds to CRKL, an adaptor molecule required for T cell adhesion
and migration. CRKL is in turn constitutively associated with C3G, a
GEF for the small GTPases such as RAP1, RAP2, and R-RAS.
339345
The CRKLC3GRAP1 signaling pathway increases the afnity of
β1-integrins to the extracellular matrix (ECM), regulating and
mediating the adherence of the hematopoietic cells to ECM and
stromal cells.
346,347
Cell proliferation, cytoskeletal reorganization,
and cell-to-cell contact are some of the most critical biological
effects of the CRKLC3GRap1 signaling pathway.
341,346,348
The
binding of CBL to CRKL results in the ubiquitination of CRKL, thus
disrupting the CRKLC3GRap1 signaling. On the other hand, an
increase in CRKLC3GRAP1 signaling, along with clustering of the
integrin, lymphocyte function-associated antigen 1 (LFA-1), was
observed in response to TCR engagement upon knockdown of
CBLB.
349,350
CBLB thus serves as the negative regulator of
CRKLC3GRAP1-mediated signaling events that promote T
lymphocyte adhesion, migration, and homing.
351
Both LCK and
FYN seem to be involved in TCR downregulation, as pharmaco-
logical inhibition of LCK and FYN led to stabilization of the TCR
complex.
352
ADAPTOR PROTEINS IN CYTOSKELETAL REORGANIZATION
Early events leading to T cell activation also involve cytoskeletal
changes required for lymphocyte migration and mediating cell-to-
cell adhesion.
353
Interaction of T cells and APCs results in T cell
activation, which involves supramolecular rearrangement of a
number of receptors at the contact zone, thus forming a synapse.
Initially, the integrin receptors of T cells and integrin receptor
ligands of APCs are present in the center surrounded by a ring of
MHC-peptide complexes. However, this pattern completely
reverses within a few minutes, and MHC-peptide complexes form
the central region known as the central supramolecular activation
cluster (cSMAC), surrounded by integrin receptors in the
periphery, forming the peripheral supramolecular activation
cluster (pSMAC).
354356
These structures are stable for a few hours
where specic molecules can be detected. For example, PKCθhas
been detected in cSMAC,
354
whereas CD45 is initially excluded
from cSMAC only to migrate back to it later.
357
These molecular
rearrangements are partly regulated by the cytoskeleton,
354,355
where a ring of polymerized actin accumulates in T cell-APC
conjugates or at the interface between T cells stimulated with
anti-TCR antibodies.
358
LAT and all its binding partners, such as
PLCγ1, GADS, and GRB2, are essential for efcient actin
polymerization, as the absence of LAT or mutation in binding
sites of either of the components inhibits polymerization of
actin.
358
SLP-76 is one of the molecular adaptors present in a
complex with LAT in activated T cells. It binds to several different
adaptors involved in regulating the cytoskeleton. Two such
adaptors, VAV and NCK, bind to the amino-terminal phosphotyr-
osine residues of SLP-76 (refs.
359,360
) to promote cytoskeletal
reorganization, whereas ADAP binds to the carboxyl-terminal
phosphotyrosine residues of SLP-76 to promote integrin signal-
ing
361
(Fig. 1).
VAV
VAV structurally is a multidomain adaptor protein and functionally
a GEF for the activation of Rac and Cdc42, members of the Rho/
Rac family of small GTPases.
362
Defects in IL-2 production and
partial blocks in calcium mobilization were seen upon targeted
T cell receptor (TCR) signaling in health and disease
Shah et al.
10
Signal Transduction and Targeted Therapy (2021) 6:412
disruption of VAV. VAV-decient T cells also showed defects in
cytoskeletal function,
363,364
along with impaired SMAC forma-
tion.
365
LAT-decient T cells would thus fail in the recruitment of
VAV via SLP-76, thereby decreasing the amount of activated Rac
and Cdc42. This could result in inadequate activation of
phosphatidylinositol 4-phosphate 5-kinase, an enzyme responsi-
ble for generating PIP2, a substrate of PLCγ1.
366
Moreover, there
could be inadequate activation of WiskottAldrich syndrome
protein (WASP) due to insufcient Rac activation.
6
WiskottAldrich syndrome protein
WASP, as the name suggests, was initially identied as a defective
protein from Wiskott-Aldrich syndrome patients.
367
Decreased IL-2
production, calcium ux, and defective actin polymerization were
seen in VAV/animals, and a similar phenotype was observed in
T cells from WASP patients and murine cells from WAS/
animals.
368,369
This could be explained by the fact that WASP
normally exists in an autoinhibited state in resting T cells, where
the GTPase-binding domain interacts with the C-terminus that
contains the Arp2/3 complex responsible for actin polymerization.
The effectors of VAV, RAC, and CDC42, along with PIP2, upon
activation, associate with WASP, thereby synergistically activating
it and stimulating actin polymerization through the Arp2/3
complex.
370,371
WASP is also associated with the NCK, which in
turn binds SLP-76.
372
Thus, SLP-76 might act as a scaffold by
binding to both the NCK and VAV, thereby bringing WASP, RAC,
and CDC42-GTP into close proximity so that they can interact with
each other.
373
NCK and T cell-specic adaptor protein (TSAd)
NCK is another adaptor protein known to regulate the actin
cytoskeleton that constitutively interacts with VAV1.
374
Both NCK
and VAV1 further interact with SLP-76 upon TCR engagement,
thereby forming a complex that associates with components of
the TCR-CD3 complex, leading to reorganization of actin at the T
cell-APC interface.
360,373,375
A T cell-specic adaptor protein, TSAd,
mediates the association of NCK with LCK and SLP-76 in T cells,
thus controlling actin polymerization events in activated T cells.
Both NCK and TSAd were found to co-localize in Jurkat cells,
where NCK, via its SH2 and SH3 domains, interacts with pTyr280
and pTyr305 and the proline-rich region (PRR) of TSAd respec-
tively. Further, increased polymerization of actin was observed in
Jurkat cells expressing TSAd, and this was due to the presence of
TSAd exon 7, which encodes interaction sites for both NCK and
LCK.
376
Moreover, many proteins tend to associate with NCK, as more
than 60 binding partners have been identied.
377,378
Thus, TSAd
may inuence the actin cytoskeleton by bringing LCK in the
vicinity of different NCK binding partners.
376
Moreover, CXCL-12-
induced migration and cytoskeletal rearrangements in T cells are
regulated via TSAd by promoting LCK-mediated tyrosine phos-
phorylation of ITK.
379
ITK not only binds to TSAd
281,379
but also
SLP-76,
375
thereby forming a multiprotein complex of NCK, TSAd,
LCK, SLP-76, and ITK that may interact with each other and several
other molecules in a cooperative manner.
376
NCK thus plays a vital
role in the regulation of the actin cytoskeleton, IS formation after
TCR engagement, and cell proliferation and migration.
377,380,381
Adhesion and degranulation promoting adaptor protein (ADAP)
Another component of the actin polymerization machinery in
T cells is the SLP-76-associated phosphoprotein of 130 kDa (SLAP-
130)/FYN-binding protein (FYB)/adhesion and degranulation
promoting adaptor protein (ADAP).
6
It is a multidomain adaptor
protein that, upon TCR engagement, gets phosphorylated by SRC
family kinases, such as FYN, enabling its binding to the SH2
domains of SLP-76 and FYN.
382,383
Peripheral T cells decient in
ADAP demonstrated defects in cell proliferation, cytokine
production, and clustering of the integrin LFA-1 upon TCR
stimulation,
270,271
whereas TCR-driven IL-2 transcription was
increased upon co-transfection of ADAP with SLP-76 and
FYN.
383,384
Integrin clustering with the help of ADAP facilitates T
cell migration in response to stromal cell-derived factor 1 alpha
(SDF1α)
385
and enhances T cell-APC conjugate formation.
386
ADAP
thus couples TCR-mediated actin cytoskeletal changes to integrin
activation.
13
ADAP associates with proteins of the Ena (enabled)/
VASP (vasodilator-stimulated phosphoprotein) family, important
for the regulation of actin dynamics and T cell polarization,
thereby regulating cell adhesion mediated by integrins.
387
ADAP
also interacts with a multiprotein complex composed of WASP,
Arp2/3, VAV, NCK, and SLP-76. TCR-mediated actin rearrangement
was inhibited when the binding between the WASP and Arp2/3
complex or ADAP and Ena/VASP proteins was hindered, suggest-
ing that TCR signaling is linked to cytoskeletal remodeling by
these interactions.
388
SRC kinase-associated phosphoprotein of 55 kDa (SKAP-55)
SKAP-55 or Scap1 is a T cell-specic adaptor protein that is
constitutively associated with ADAP.
389,390
It enhances cellular
adhesion by not only promoting the clustering of LFA-1 but also
enhancing its binding to intercellular adhesion molecule-1 (ICAM-
1) and bronectin. SKAP-55 also increases T cell/APC conjugate
formation, thereby inducing its translocation to the lipid rafts.
386
This brings it into close proximity with the SRC kinase FYN that
phosphorylates SKAP-55.
391
Both ADAP and SKAP-55 might
control the formation of SMACs since they enhance LFA-1-
mediated adhesion during T cell/APC interactions, which are
important for SMAC formation.
13
Mammalian CT10 (chicken tumor virus number 10) regulator of
kinase (CRK)
The CRK adaptor proteins are ubiquitously expressed and regulate
proliferation, differentiation, adhesion, migration, and apoptosis of
immune cells by integrating signals from various effector
molecules, such as ECM, growth factors, pathogens, and apoptotic
cells.
392396
There are three members belonging to the family of
CRK adaptor proteins, CRKI, CRKII, and CRK-like (CRKL), that
mediate various protein interactions through their SH2 and SH3
domains. Transient interactions with STAT5, ZAP-70, CBL, and
CASL (CRK-associated substrate lymphocyte type) are mediated by
the CRK SH2 domains, thus activating the lymphocytes. Cytokines
secreted upon TCR activation can induce STAT5 tyrosine
phosphorylation, possibly through Janus kinase 3 (JAK3), which
was shown to be required for T cell proliferation.
397
Furthermore,
the STAT5 function is required for amino acid biosynthesis.
398
Cell
adhesion of lymphocytes, their extravasation, and recruitment to
the sites of inammation are mediated by the constitutive
association of CRK with C3G via their SH3 domains. A detailed
function of CRKL-C3G is mentioned in the ubiquitin ligase section
of this paper, while the reader is also referred to the
comprehensive review on the role of CRK adaptor proteins in T
cell adhesion and migration.
399
TCR SIGNALING DYSREGULATION IN DISEASES
Dysregulation of TCR signaling can lead to the generation of
various diseases, given its importance in executing different
functions of T cell biology. Thus, defects in TCR signaling can lead
to immune deciency. On the other hand, its hyperactivation can
lead to autoimmune diseases. TCR signal transduction is thus
tightly regulated via multiple mechanisms by various enzymes
and non-enzymatic proteins that serve as scaffolds for efcient
signal transmission.
183
Mutations in any of these mediators can
contribute to the dysregulation of TCR signaling, leading to
various disorders.
Both immune deciency and autoimmunity have been
observed when tyrosine phosphatase CD45 is misexpressed. SCID
T cell receptor (TCR) signaling in health and disease
Shah et al.
11
Signal Transduction and Targeted Therapy (2021) 6:412
which is characterized by the absence or defective function of
T cells, displays deciency of CD45 expression,
400,401
whereas
multiple sclerosis (MS) is an end result of certain CD45
polymorphisms.
402
SCID was also generated due to mutations in
genes coding for CD3 δ,ε, and ζchains.
403
Both mice and humans
showed immunodeciency due to defective expression of
LCK.
404,405
Furthermore, a rare form of SCID was observed in
humans with functionally impaired CD4
+
T cells and the absence
of CD8
+
T cells due to deciency or mutation of ZAP-70.
406,407
In
contrast, the development of T cells at the CD4
+
CD8
+
double-
positive stage in mice was blocked due to deciency of ZAP-70,
thereby having a complete absence of single positive CD4
+
and
CD8
+
T cells.
1
On the other hand, autoimmune disorders can be
caused by an abnormal thymic selection of T cells or their
uncontrolled proliferation due to dysregulated TCR signaling.
Rheumatoid arthritis (RA) and SLE have been found to be
associated with reduced expression of CD3ζ.
408,409
Similar to
human RA, autoimmune arthritis has been observed in mice
having spontaneous mutations in the SH3 domain of ZAP-70.
410
Non-T cell activation linker (NTAL) is a transmembrane adaptor
molecule that enhances methylprednisolone and TCR-induced
apoptosis in T-ALL through increased ERK phosphorylation.
411
NTAL thus serves as a tumor suppressor in T-ALL, where its high
mRNA expression correlates with a good response to prednisone
and vice versa.
412
Accordingly, NTAL
/
mice displayed activated
T cells characteristic of an autoimmune syndrome.
411
Peripheral T cell lymphomas (PTCL) and T cell acute lympho-
blastic leukemias or lymphomas (T-ALL) both constitute different
groups of T cell malignancies; PTCL arises from post-thymic
mature T cells whereas T-ALL arises from thymic immature T cells
blocked at various stages of development.
413
Multiple molecular
aberrations have been described in genes involved in TCR
signaling in PTCL,
413
with 84% of Sezary syndrome samples
414
and 90% of adult T cell leukemia/lymphoma samples
415
showing
mutations in TCR signaling components. Upregulation of the LAT
adaptor along with frequent activating mutations (gain-of-
function alterations) in the adaptor CARD11 and PLCγ1 have
been observed in most cases of Sezary syndrome cutaneous T cell
lymphoma,
414
contributing to cell survival and proliferation and
disease progression. Thus, the TCR signaling in this context is
oncogenic. In contrast, translocations involving TCR genes have
been identied in T-ALL
416
with no recurrent mutations in any of
the TCR signaling components.
413
In a landmark study, Trinquand
et al. in 2016
417
identied that TCR engagement with an MHC-
restricted TCR-specic antigen or via CD3 stimulation with anti-
CD3 antibody OKT3 made TCR-positive T-ALL cells undergo
apoptosis in a similar transcriptional program as the thymic
negative selection. However, leukemia recurrence was observed in
TCR-positive T-ALL xenografts due to the presence and selection
of TCR-negative subclones as a mechanism of tumor relapse from
OKT3-mediated therapy. Nevertheless, it is quite encouraging to
see that mature T-ALL cells can be induced to undergo apoptosis
by TCR activation, using the gene signature for negative selection
that is reminiscent in these cells. Subsequently, when assessing
the potential of novel anticancer therapies, it is necessary to also
assess the importance of cell and disease, as TCR signaling
supports oncogenesis in PTCL whereas it appears to have an anti-
oncogenic effect in T-ALL.
413
APPLICATIONS OF TCR-BASED IMMUNOTHERAPY
The emergence of T cell-based immunotherapy has revolutionized
the understanding of the role of T cells in mitigating a wide variety
of diseases, including viral, autoimmune, and malignant diseases.
TCR engineering has provided a compelling approach to ght
cancer, disrupting the immuno-oncology research eld and
introducing a new class of impressive cancer immuno-
therapeutic strategies, including adoptive cellular therapy (ACT),
checkpoint blockade, tumor microenvironment (TME) regulation,
and cancer therapeutic vaccines.
Adoptive T cell transfer therapy
Experimental research in T cells adoptive transfer has revealed the
superior capabilities of T cells to identify tumor antigens and to
harness the immune system, contributing to anti-tumor activity.
418
This type of therapy was rst demonstrated clinically by Southam
et al.
419
in 1966 when patients with unresectable cancer displayed
tumor regression upon co-transplantation with patient-derived
leukocytes and autologous tumor cells. Although this strategy has
been successfully applied, adoptive T cell transfer has not been
generalized widely due to the fact that the number of inltrated
T cells was insufcient to exert a full potential of the anti-tumor
activity or to boost the bodys immune response against cancer. In
addition, the validated immune response in patients receiving this
type of therapy was found to be cancer-type and patient-
dependent.
420,421
Therefore, the engineering of T cells has
provided an effective alternative to activate and expand T cells
ex vivo with dened specicity against tumor antigens. In this
context, TCR-engineered lymphocytes have garnered considerable
attention over the past decade, offering signicant curative
outcomes in patients with cancer. Because tumor cells down-
regulate MHC molecules, also known as HLA, this posed a
challenge for proper T cell response directed against tumor
antigen presentation resulting in immune tolerance.
422
However,
the development of synthetic chimeric antigen receptors (CARs)
has overcome this challenge by redirecting T cell specicity to
recognize and lyse tumor antigens on the surface of the malignant
cell independently of MHC molecules.
423
To date, different types of ACTs have been developed, including
TCR engineered T cell therapy (TCR-T), tumor-inltrating lympho-
cytes (TILs), and CAR T therapy.
424427
These strategies allow the
fast entry of T cell-receptor-based immunotherapies to clinical
trials with encouraging clinical outcomes.
TILs therapy
TILs were the rst classical attempt for ACT in which inltrating
T cells are isolated from the tumor mass and then expanded
ex vivo, activated, and subsequently reinfused into the patient.
Several reports have shown that TILs therapy induced a signicant
durable response in melanoma, including in patients resistant to
immune-checkpoint blockade (ICB), as well as objective response
in different types of cancers, such as gastrointestinal, colon, and
breast cancers.
428432
However, this strategy has been hindered by
limited access to solid tumors localized at restricted areas or that
have non-resectable metastases as well as long ex vivo processing
time and insufcient anti-tumor immunity.
433
Completed clinical
trials which used TILs-based therapy are summarized brieyin
Table 1.
TCR-T therapy
TCR-T therapy was developed to overcome some drawbacks of
TILs therapy. This strategy utilizes the same principle as TILs but
with genetic modication through retroviral transduction of TCRs
to recognize tumor-specic antigens via MHC (Fig. 5). Despite the
success of this therapeutic approach, the specicity remains
challenging because tumors usually escape such attacks by
downregulating MHC. The rst clinical outcomes of TCR-T therapy
were reported in 2006 when Morgan et al.
434
demonstrated
durable response among patients with melanoma after transdu-
cing autologous T cells with a TCR recognizing the melanocyte
differentiation antigen (MART-1). Subsequent clinical trials in 2009
and 2014 conrmed TCR-mediated tumor regression in 30% and
69% of metastatic melanoma patients using MART-1 and gp100
TCR-engineered T cells, respectively.
435,436
Parallel clinical
responses were also documented using cancer-testis antigens,
such as MAGE-A3 and NY-ESO-1. Robbins et al.
437
observed that 6
T cell receptor (TCR) signaling in health and disease
Shah et al.
12
Signal Transduction and Targeted Therapy (2021) 6:412
out of 11 patients with synovial cell sarcoma and 5 out of 11
patients with melanoma treated with TCR targeting NY-ESO-1
antigen displayed objective responses. Moreover, targeting NY-
ESO-1 antigen in multiple myeloma (MM) using TCR-T therapy has
achieved similar robust clinical responses.
438
These clinical data
suggest that TCR-T therapy can potentially harness the immune
system to target and eliminate cancer cells. Although the most
commonly reported clinical outcomes were from clinical trials
targeting melanoma, other clinical trials have started to introduce
this type of therapy more frequently in other solid tumors. This is
based on the fact that melanoma incidence has been increasing
over the past few years more than other cancers.
439,440
Furthermore, melanoma lesions are relatively accessible compared
to other solid tumors; therefore, the means for ex vivo expansion
of T cells can be readily available, making melanoma one of the
best models for immuno-oncology not only in therapy but also in
research purposes. A list of current active clinical trials using TCR-T
is summarized in Table 2.
CAR T cell therapy
CAR T cell therapy is lauded as a major step in the development of
personalized cancer treatment. The patients own T cells are
collected by leukapheresis (or peripheral blood) and genetically
modied to express a synthetic receptor that binds a specic
tumor antigen. These cells are then activated and expanded
ex vivo and reinfused into the patient to target and attack cancer
cells (Fig. 5). Unlike traditional T cells, CAR T cells recognize
antigens independently of MHC presentation due to CARs unique
structure, containing a transmembrane region with antigen-
binding domain and intracellular signaling and co-signaling
domains, allowing MHC-independent CAR T cells to bind to their
target. Thus, CAR-T cell therapy can overcome cancer-mediated
immune tolerance response. Unprecedented clinical response
with a high remission rate has been observed using anti-CD19
CAR-T cell therapy in treating patients with B cell malignancies,
including B cell acute lymphoblastic leukemia (B-ALL), chronic
lymphocytic leukemia (CLL), and B cell non-Hodgkin lymphoma
(B-NHL).
441445
In a phase II multicenter clinical trial, anti-CD19
CAR-T therapy was conducted on patients with refractory B cell
lymphomas where 82% of patients displayed signicant tumor
regression and 54% showed complete response rate.
446
Moreover,
a systematic review and meta-analysis of all published clinical
trials conducted by Irbaz Bin Riaz et al. to study the efcacy and
safety of anti-CD19 and anti-CD20 CAR-T therapy for B cell
hematologic malignancies showed that, among 16 eligible studies,
the overall response rate was 61% with complete and partial
responses of 42% and 19% respectively. Another clinical trial
aimed at determining long-term follow-up of anti-CD19 CAR- T cell
therapy has reported the longest durable remission in patients
with B cell lymphomaup to 113 months after treatment
suggesting that anti-CD19 CAR T cells may be curative for B cell
lymphoma.
447
The success of anti-CD19 CAR-T therapy could be
related to the high expression of CD19 in some B cell malignancies
and its specicity to the B cell lineage. However, clinical studies
showed that the loss of CD19 antigen following treatment is a
common cause of disease relapse.
448
Thus, anti-CD22 CAR-T has
emerged as a potential alternative to anti-CD19 CAR-T therapy.
Clinical trials have shown that anti-CD22 CAR-T cells could
overcome resistance mediated by anti-CD19 CAR-T cell immu-
notherapy in patients with B-ALL.
449
B cell maturation antigen (BCMA/CD269) targeted therapy has
emerged as a promising target for CAR-T cell immunotherapy in
multiple myeloma (MM). Although this target is still under
investigation, clinical trials phase I exhibited parallel safety and
toxicity proles and suggest its clinical activity against MM.
450452
In addition, CAR-T has been shown to target prostate cancer
through directing CAR-T cells against prostate-specic membrane
antigen (PSMA) and displayed an acceptable safety and efcacy
Table 1. Completed clinical trials using TILs-based immunotherapy
Cancer type/conditions Study title Study type/phase Intervention/treatment Status NCT number
Metastatic ovarian cancer TIL therapy in combination with checkpoint inhibitors for
metastatic ovarian cancer
Interventional; Phase I and II TILs in combination with checkpoint inhibitors Completed NCT03287674
Metastatic melanoma Peginterferon and TIL therapy for metastatic melanoma Interventional; Phase I and II TILs infusion including lymphodepleting
chemotherapy and interleukin-2
Completed NCT02379195
Metastatic melanoma Vemurafenib and TIL therapy for metastatic melanoma Interventional; open-label
Phase I and II
T cell Therapy in combination with Vemurafenib Completed NCT02354690
Multiple myeloma Trial of activated marrow inltrating lymphocytes alone or in
conjunction with an allogeneic granulocyte macrophage colony-
stimulating factor (GM-CSF)-based myeloma cellular vaccine in
the autologous transplant setting in multiple myeloma
Interventional; Phase II Activated marrow inltrating lymphocytes alone or
in conjunction with an allogeneic GM-CSF vaccine
Completed NCT01045460
Multiple myeloma and
plasma cell neoplasm
Activated white blood cells with ASCT for newly diagnosed
multiple myeloma
Interventional; Phase I and II Activated marrow inltrating lymphocytes Completed NCT00566098
Melanoma Phase II study of short-term cultured anti-tumor autologous
lymphocytes after lymphocyte-depleting chemotherapy in
metastatic melanoma
Interventional; Phase II Cultured anti-tumor autologous lymphocytes
following a lymphocyte depletion
Completed NCT00513604
Melanoma Phase II study of metastatic melanoma with lymphodepleting
conditioning and infusion of anti-MART-1 F5 TCR-gene-
engineered lymphocytes
Interventional; Phase II Lymphodepletion followed by infusion of anti-
MART-1 F5 TCR-gene engineered lymphocytes
Completed NCT00509288
Melanoma neoplasm
metastasis
Lymphocyte re-infusion during immune suppression to treat
metastatic melanoma
Interventional; Phase II Lymphocyte re-infusion during immune
suppression
Completed NCT00001832
T cell receptor (TCR) signaling in health and disease
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Signal Transduction and Targeted Therapy (2021) 6:412
prole.
453
In a phase I clinical study, Yao Wang et al.
454
targeted
patients with CD133-positive and late-stage metastasis malignan-
cies by CAR-T cells in which three patients exhibited partial
remission and 14 achieved stable disease.
Despite the success of CAR-T therapy in hematological
malignancies, solid tumors have introduced a greater challenge
owing to their immunosuppressive microenvironment. While CAR-
T therapy has rendered the TME more immunogenic, CAR-T has
generated a signicant toxic prole; for example, cytokine release
syndrome, neurotoxicity, therapy-related mortality, and manufac-
turing issues have complicated CAR-T cell therapy for solid
tumors.
455458
Efforts to overcome these challenges to generate a
more favorable toxicity with CAR-T cell therapy are ongoing.
IMMUNE-CHECKPOINT BLOCKADE
A growing body of evidence indicates that peripheral T cell
tolerance is an essential factor of the specic immune response to
tumor cells. The low cytotoxic capabilities of T cells may be related
to the high expression levels of a number of inhibitory molecules
including Cytotoxic T lymphocyte antigen 4 (CTLA4) and
programmed cell death 1 (PD1). These evolutionarily conserved
negative T cell activation regulators act as checkpoint molecules.
CTLA4 and PD1 are highly expressed by various types of cancers,
and their binding to their respective ligands contribute to T cell
functional impairment, which fails to elicit the required immunity
against minimal residual disease, and thereby play an important
role in cancer recurrence.
459
The discovery of immune-check-
points role in cancer has changed the paradigm of cancer
therapeutics and added immunotherapy to the list of common
three cancer pillars including surgery, targeted therapy, radio-
therapy, and chemotherapy.
PD1 and CTLA4 are the most extensively studied immune-
checkpoint negative regulators due to their prominent role in ne-
tuning tumor-inltrating T cells. Targeting PD1 and its ligand
programmed death-ligand 1 (PD-L1), as well as CTLA4, have
gained immense attention after they had shown an unprece-
dented objective and durable responses across many clinical trials
in a subset of patients of metastatic and unresectable cancers
leading to different lines of FDA-approved immune-checkpoint
inhibitors, and thereby translating checkpoint blockade therapy
into an integral part of clinical standard therapy.
460466
For
example, PD-1/PDL-1 inhibitors are now considered as a rst-line
treatment for patients with melanoma after they have demon-
strated a signicant increase in overall survival compared to
dacarbazine chemotherapy.
467
PD-1 inhibitors such as nivolumab
and pembrolizumab have shown clinical efcacy in several lines of
solid and hematological neoplasms including non-small-cell lung
(NSCLC), bladder, pancreatic, follicular B cell, and non-Hodgkin
lymphoma.
468,469
In addition, it is worth noting that most of the
observed effects were correlated with the extent of tumor-
inltrating T cells. Furthermore, in NSCLC, pembrolizumab
displayed an improved objective response in patients harboring
a high nonsynonymous mutational burden due to a defect in the
DNA repair pathway, molecular smoking signature, and higher
neoantigen burden.
470
In a clinical study conducted to evaluate
the correlation between immune cell inltration and the clinical
outcomes in pancreatic ductal adenocarcinoma, with respect to
immune-checkpoint molecules, Rong Liu et al.
471
found that
increase inltration of PD-1-positive T cells is associated with
favorable patients prognosis and overall survival. Moreover,
patients with melanoma who respond to anti-PD-1 therapy
displayed increased intratumoral CD8+T cells which were
associated with tumor regression.
472
Caroline Robert et al.
473
reported signicantly longer overall survival in patients with
previously untreated metastatic melanoma using combination
therapy of ipilimumab and dacarbazine. Previous reports also
indicated that ipilimumab and nivolumab combination therapy
exhibited a signicant survival benet in patients with advanced
renal cell carcinoma and metastatic melanoma.
474,475
Although
this line of evidence supports the benecial role of checkpoint
blockade combination therapy, it increases the risk of drug-
induced toxicity and therefore should be evaluated with caution.
TARGETING THE TME
One of the most common challenges in TCR-based immunother-
apy is TME. The TME promotes an immunosuppressive nest
through: (1) tumor tissue remodeling by regulation of ECM and
inhibition of T cells migration
476
; (2) recruitment of tumor-
associated stromal cells, such as T regulatory cells (T reg),
myeloid-derived suppressor cells, and tumor-associated
Fig. 5 Schematic illustration of TCR-based immunotherapy. T cells are isolated from the patients cancer tissue or peripheral blood and
genetically modied by retroviral transduction to express antigen-specic TCR or CAR on T cells. Cells are then expanded ex vivo until
sufcient cell numbers are achieved and reinfused into the patients body, where they can ght cancer cells.
T cell receptor (TCR) signaling in health and disease
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