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Biochemical Society Transactions (2004) Volume 32, part 2
Phosphoinositide 3-kinase in T cell activation
and survival
K. Okkenhaug*1 , A. Bilancio†, J.L. Emery* and B. Vanhaesebroeck†‡
*Laboratory of Lymphocyte Signalling and Development, Molecular Immunology Programme, Babraham Institute, Cambridge CB2 4AT, U.K., †Cell Signalling
Group, Ludwig Institute for Cancer Research, 91 Riding House Street, London W1W 7BS, U.K., and ‡Department of Biochemistry & Molecular Biology,
University College London, Gower Street, London WC1E 6BT, U.K.
Abstract
PI3Ks (phosphoinositide 3-kinases) regulate diverse signalling pathways involved in growth, proliferation,
survival, differentiation and metabolism. In T cells, PI3Ks can be activated by a number of different receptors,
including the TcR (T cell receptor), co-stimulatory receptors, cytokine receptors and chemokine receptors.
However, the specific roles of PI3Ks downstream of these receptors vary. An inactivating mutation in the
leucocyte-specific PI3K isoform p110δ results in impaired TcR-dependent proliferation under circumstances
where CD28 co-stimulation is blocked or not required. Recruitment and activation of PI3K by CD28 promotes
survival by inducing increased expression of Bcl-XL . However, CD28 engages additional signals that regulate
proliferation and interleukin-2 production independently of PI3K. Thus a model emerges whereby PI3K is
involved in both TcR and CD28 signalling, but each receptor may only exploit a subset of the signalling
pathways potentially controlled by PI3K activation.
Introduction
The activation of T cells is initiated upon engagement of the
TcR (T cell receptor) by antigenic peptides presented by
MHC proteins. In the thymus, T cells with moderate affinity
for MHC–self-peptide complexes are selected for further
development, whereas T cells with too high or too low
affinity for MHC–self-peptide complexes are eliminated [1].
Immature T cells express both CD4 and CD8 receptors,
which bind MHC II and MHC I molecules respectively. T
cells selected by MHC class I molecules lose the expression
of CD4 to become CD8+ T cells, whereas those selected by
MHC II molecules lose the expression of CD8 to become
CD4+ T cells. The T cells that survive selection in the
thymus migrate to the lymph nodes and the spleen, where
they interact with specialized APCs (antigen-presenting cells)
known as dendritic cells [2]. Upon infection, these APCs
ingest and kill the pathogenic organism, digest its proteins
into small peptide fragments and present some of these in
the context of MHC molecules. For any given pathogenderived peptide presented, there will be a small subset of
T cells that can bind this peptide–MHC complex with an
affinity that is sufficiently high that the T cell can distinguish
it from self peptides expressed by APCs. The antigen-sensing
mechanism of the TcR thus needs to be exquisitely fine
tuned in order to distinguish relatively minor differences in
affinity and/or avidity. However, presentation of a foreign
peptide is not usually sufficient to activate T cells. Dendritic
Key words: CD28, co-stimulation, p110δ, phosphoinositide 3-kinase (PI3K), T cell receptor.
Abbreviations used: APC, antigen-presenting cell; GEF, guanine nucleotide exchange factor;
LAT, linker for activation of T cells; PI3K, phosphoinositide 3-kinase; PKB, protein kinase B;
PLC, phospholipase C; Pten, phosphatase and tensin homologue deleted on chromosome 10;
SH2, Src homology 2; TcR, T cell receptor; TRIM, TcR-interacting molecule.
1
To whom correspondence should be addressed (e-mail [email protected]).
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Biochemical Society
cells also express co-stimulatory ligands, such as CD80
and CD86, which bind CD28 on the T cell surface. The
expression of co-stimulatory ligands on the dendritic cells
is regulated by Toll-like receptors that recognize conserved
microbial molecules not expressed by the host and thus help
the immune system to distinguish infectious non-self from
non-infectious self [3]. The signals from the co-stimulatory
receptors are integrated by the T cell with those from the
TcR to initiate gene transcription, cell growth and mitosis.
To maintain homoeostasis and to prevent T cells with any
particular antigen specificity from dominating the repertoire,
the majority of T cells are programmed to die after several
rounds of division [4]. The few T cells that escape this
elimination by apoptosis can persist to provide lifelong
immunity against re-infection [5]. In this review, we describe
recent investigations into how the TcR and CD28 exploit class
IA PI3K (phosphoinositide 3-kinase) activation to influence
the response of T cells to antigen stimulation.
TcR signalling and the involvement of PI3K
TcR signalling is initiated by the cytosolic tyrosine kinases
Lck and Fyn, which phosphorylate ITAMs (immunoreceptor
tyrosine-based activation motifs) in the CD3 invariant
chains with which the TcR is associated on the plasma
membrane [6,7]. This leads to the SH2 (Src homology 2)
domain-mediated recruitment of ZAP-70 (ζ -associated protein, 70 kDa), which phosphorylates the transmembrane
adapter protein LAT (linker for activation of T cells) [8].
Grb2, GADS (Grb2-related adaptor downstream of Shc)
and PLCγ (phospholipase Cγ ) have SH2 domains that bind
phosphotyrosine residues in LAT, and phosphorylation of
these tyrosines is essential for the TcR to transmit signals to
the nucleus [9]. TcR engagement also leads to the activation
PI-3 Kinase in Signalling and Disease
of PI3K [10,11]. The p85 regulatory subunit of class IA
PI3Ks has SH2 domains that probably do not bind directly
to LAT, but which bind to a second transmembrane adapter
protein called TRIM (TcR-interacting molecule) [12]. It is
not yet known to what extent TRIM phosphorylation is required for TcR signals, nor is it known whether TRIM
phosphorylation is required for PI3K activation by the TcR.
The cytosolic adapter proteins Cbl and Gab2 also contain
tyrosine residues to which p85 can bind, but these interactions
seem more likely to negatively regulate PI3K signalling
[13,14]. The p85 subunit contains additional domains that
may contribute to the recruitment and regulation of PI3K
activity: Rac can bind the p85 Rho-GAP (GTPase-activating
protein) domain [15,16], and Lck and Fyn may interact
with proline-rich sequences in p85 via their SH3 domains
[17,18]. Upon recruitment of p85/p110 to the membrane,
Ras may bind directly to the Ras binding domain of p110 and
modulate PI3K activity further [19,20]. However, the precise
steps leading to PI3K activation after engagement by the TcR
have yet to be mapped out. In particular, it remains to be
determined whether several partially redundant mechanisms
work to activate PI3K, or whether this is a carefully orchestrated procedure with one or more essential upstream
components.
PI3Ks catalyse the phosphorylation of PtdIns(4,5)P2 on
the D3 hydroxy group to yield PtdIns(3,4,5)P3 , a second
messenger signalling molecule that remains tethered to the
lipid bilayer [21]. Canonical pathways downstream of PI3K
are regulated by the PtdIns(3,4,5)P3 -dependent recruitment
of PDK1 (phosphoinositide-dependent kinase 1) and PKB
(protein kinase B) to the plasma membrane [22]. These
enzymes phosphorylate other proteins on serine and threonine residues to regulate metabolism, cell growth, mitosis
and apoptosis. Itk is a Tec family tyrosine kinase which is
selectively expressed in T cells and which has been implicated in PI3K-dependent regulation of calcium signalling by
virtue of its capacity to phosphorylate PLCγ [23]. PI3Ks
may also regulate cytoskeletal changes by activating GEFs
(guanine nucleotide exchange factors), some of which have
PtdIns(3,4,5)P3 -specific pleckstrin homology domains [24].
GEFs catalyse the conversion of small GTPases, such as Rho,
Rac and Cdc42, from the inactive GDP-bound form into
the active GTP-bound form. IBP [IRF-4-binding protein;
also known as SLAT (SWAP17-like adaptor of T cells)] is
a recently described T cell-restricted GEF that regulates Rac
and Cdc42 in a PI3K- and Lck-dependent fashion [25]. Hence
PI3Ks have the potential to regulate numerous signalling
pathways in T cells.
Four class I PI3K isoforms
Of the three known isoforms of class IA PI3K catalytic
subunits, p110α and p110β are expressed in most cell types,
whereas the expression of p110δ is largely restricted to the
leucocyte lineages. Both p110α and p110β are essential for
embryonic development, which has thus far precluded direct
investigation of their role in T cell activation [26,27]. The class
IB PI3K isoform p110γ is regulated by G-protein-coupled re-
ceptors and is probably not directly involved in antigen
receptor signalling, although it may promote the survival
of T cells via other ligand/receptor systems, especially in
the thymus [28,29]. PI3K signalling is antagonized by the
lipid phosphatases Pten (phosphatase and tensin homologue
deleted on chromosome 10) and SHIP (SH2-containing inositol phosphatase), which dephosphorylate PtdIns(3,4,5)P3 on
the D3 and D5 positions respectively. Conditional knockout
of Pten expression in T cells results in a lethal CD4-mediated
lymphoproliferative disease as a consequence of impaired
T cell selection in the thymus and enhanced resistance of
peripheral T cells to apoptotic stimuli [30]. Thus PI3K is an
important regulator of T cell homoeostasis.
Role of p110δ in T cell activation
The leucocyte-restricted expression of p110δ suggested a
unique role in immune receptor signalling. To investigate
this possibility, a loss-of-function mutation (D910A) was
introduced in the p110δ gene [31]. Indeed, impaired TcR- and
CD28-dependent PKB phosphorylation in p110δ(D910A)
T cells indicated that p110δ is the main PI3K downstream
of these receptors ([31]; A. Bilancio, unpublished work).
T cell development in the thymus appeared to progress
normally, and both CD4+ and CD8+ T cells were found in
the spleens and lymph nodes of p110δ(D910A) mice, albeit
in reduced numbers. However, the T cells showed reduced
CD44 expression, which may be indicative of impaired
responsiveness to antigen in vivo. Consistent with this notion,
TcR transgenic p110δ(D910A) T cells showed reduced proliferation and interleukin 2 production in response to stimulation by APCs bearing peptide–MHC complexes in vitro
[31]. CD28 contributes to the recruitment of lipid rafts,
which are rich in associated signalling proteins, to the area
of contact between the TcR and the APC [32]. This process
is thought to help amplify signals through the TcR. Such raft
recruitment stimulated by anti-CD3- and anti-CD28-coated
beads was impaired in p110δ(D910A) T cells [31]. However,
while p110δ(D910A) T cells showed reduced proliferation in
response to stimulation with beads coated only with antiCD3, proliferation and interleukin 2 production in response
to anti-CD3- and anti-CD28-coated beads was unaffected, if
not enhanced [31]. It is possible that the high-affinity stimulus
provided by antibodies to CD3 and CD28 abrogated any need
for signal amplification via raft recruitment, but that such
raft recruitment plays a more important role in responses to
stimulation with peptide–MHC complexes. Together, these
results indicated that the requirement for PI3K activation
during T cell activation may be greatest in the context of
low-affinity stimuli provided by peptide–MHC complexes
in conjunction with sub-optimal co-stimulation. However, in
contrast with the LAT-associated signalling proteins, PI3K is
probably not an essential component of the TcR signalling
complex.
Role of PI3K in CD28 co-stimulation
Upon T cell activation, CD28 becomes tyrosine phosphorylated, providing a docking site for the SH2 domains
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Biochemical Society Transactions (2004) Volume 32, part 2
Figure 1 Regulation of PI3K signalling by the TcR and CD28
Although the TcR may regulate PI3K recruitment via phosphorylation of TRIM, it remains unclear precisely how the TcR
is coupled to PI3K activation. Available evidence suggests an important role for PI3K in transmitting signals that regulate
proliferation and differentiation upon antigen engagement of the TcR. In contrast, CD28 binds directly to the SH2 domains
of p85, but promotes proliferation and cytokine production independently of this association. However, CD28 activation of
PI3K promotes survival, contributing to the up-regulation of Bcl-XL expression. Experiments described in the text suggest that
the TcR exploits PI3K activation to regulate proliferation, whereas CD28 requires PI3K to promote survival. This differential
utilization PI3K signalling is indicated by thick compared with dotted lines, ZAP, ζ -associated protein; GADS, Grb2-related
protein downstream of Shc.
of p85. As such, CD28 had been thought to exploit, rather
than circumvent, PI3K signalling [10]. Is there a role for
PI3K in CD28 signalling despite the robust responses of
p110δ(D910A) T cells to CD28 co-stimulation? The immune
system of CD28 knockout mice is deficient in several important aspects. The T cells provide inadequate help to
the B cells after infection or immunization, such that the
B cells fail to form germinal centres and undergo immunoglobulin class switching [33]. As a consequence, while T cellindependent IgM responses are intact, T cell-dependent IgG
responses are impaired [34]. To evaluate the role of PI3K in
transmitting the CD28 co-stimulatory signal, CD28 knockout mice were reconstituted with a tyrosine-to-phenylalanine
(Y170F) mutant of CD28 that cannot bind PI3K [35]. The
CD28(Y170F) mutant restored T cell-dependent immune
responses, thus indicating that PI3K activation is not essential
for the CD28 co-stimulatory signal. Consistent with the
results obtained with p110δ(D910A) mice, CD28(Y170F)
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T cells showed unimpaired proliferation and interleukin 2
secretion in response to stimulation with anti-CD3 and antiCD28. However, T cells expressing the CD28(Y170F) mutant
were impaired in other important ways: the PI3K binding
site was required for the CD28-dependent induction of BclXL expression [35,36]. However, the activation of PI3K is
apparently not sufficient to drive Bcl-XL expression, because
ICOS (inducible co-stimulatory protein), a co-stimulatory
receptor that is related to CD28 and which appears to
bind PI3K with greater efficiency than does CD28, failed
to stimulate increased Bcl-XL expression [37]. Therefore,
both PI3K-dependent and PI3K-independent signals may
be integrated to promote Bcl-XL expression in response
to CD28 co-stimulation. It is feasible that even though
co-stimulation for primary immune responses occurred
independently of the CD28/PI3K/Bcl-XL pathway, the survival signals provided by Bcl-XL could play a role in the
long-term survival of memory cells. There is also evidence
PI-3 Kinase in Signalling and Disease
suggesting that CD28 regulates glucose uptake and glycolysis
via PI3K, with obvious implications for T cell homoeostasis
[38].
Concluding remarks
In conclusion, p110δ contributes to the response of T cells
to exposure to foreign antigen. However, CD28 provides
a signal that can surpass the requirement for p110δ to
promote T cell proliferation, although the capacity of CD28
to promote survival is PI3K dependent. These results indicate
that, despite forming part of the same signalling complex, the
TcR and CD28 differentially exploit PI3K activation to drive
cell proliferation and survival (Figure 1). Recent progress in
analysing PI3K signalling in T cells at the single-cell level
should facilitate investigation into the relative contributions
of different receptors to PI3K activation in response to antigen [39–42]. Moreover, although CD28 signalling can to
some extent overcome the requirement for PI3K signalling
as determined by proliferative responses, it is unlikely that
T cells activated independently of PI3K develop normally in
other aspects, or indeed are as long lived.
We thank Hanneke Okkenhaug for critical reading of the manuscript.
K.O. is the recipient of a BBSRC David Phillips Fellowship. Research
in the laboratory of B.V. is supported by the Ludwig Institute for
Cancer Research, Diabetes UK, the BBSRC and the European Union
Framework V Programme (QLG1-2001-02171). A.B. is supported in
part by the Fondazione Italiana per la Ricerca sul Cancro.
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