Immunological synapse: a multi-protein signalling cellular apparatus for controlling gene expression

Abstract

The interaction of T cells with antigen-presenting cells is the hallmark of adaptive immunity. In vitro studies have described the formation of an immunological synapse between these cells, and intra-vital imaging has described in great detail the dynamics of these interactions. The immunological synapse has become a paradigm to study signals exchanged between the two cells. A wealth of information has been amassed regarding the localization of signalling molecules, their kinetics and the transcription factors they activate. We continue to discover mechanisms that cause receptors and signalling molecules to compartmentalize in the cell; however, the emerging challenge lies in understanding how the immunological synapse contributes to differentiation. Here, we review some of the transcription factors activated downstream of T-cell receptor signalling and discuss mechanisms by which antigen dose and affinity may influence differentiation. Antigen affinity might change the kind of transcription factors that are activated whereas antigen dose is likely to influence the temporal dynamics of the transcription factors. The immunological synapse is therefore likely to influence differentiation by modulating the trafficking of transcription factors and by promoting asymmetric cell division, an emerging concept.

Introduction

The term immunological synapse was first proposed by Paul and Seder as a cognate interaction of a T cell and an antigen-presenting B cell which the T cell uses to secrete effector cytokines in the synaptic cleft to cause humoral responses.¹ Kupfer and colleagues were first to define the compartmentalization of interactions at the interface of T and B cells as the central accumulation of T-cell receptor–major histocompatibility complex–peptide (TCR-MHCp) interactions surrounded by a peripheral ring of adhesion molecule interactions. They called these zones central and peripheral supra-molecular activation clusters, respectively (c-SMAC or p-SMAC). In the context of the synapse they found that protein kinase C-θ (PKC-θ) was localized to the c-SMAC whereas Talin, a molecule known to modulate adhesion, was localized to the p-SMAC.²

The kinetics of synapse formation was first demonstrated by Grakoui et al.³ Using glass-supported planar lipid membranes incorporated with lipid-anchored peptide–MHC complexes and intercellular adhesion molecule 1, it was demonstrated that immediately after contact initiation TCR-MHCp interactions are largely in the periphery and the adhesion interactions are in the centre. Within a few minutes, there is a re-organization of these interactions to form the mature synapse. The impacts of antigen dose, affinity and the role of the co-receptor CD4 were also examined in these studies.³ The immunological synapse is also the site for signal initiation and integration.^4–6 This paradigm has been effective in conveying an understanding of the spatial and temporal dynamics of proximal signalling (see Fig. 1) components over short time-scales of minutes to an hour. Differentiation of T cells, however, takes place over days, and although several distinct environmental signals contribute to differentiation, TCR signals remain central to this differentiation process.⁷ Continuous TCR-MHCp interactions are required for days to commit cells to differentiation so the immunological synapse is likely to be important over the entire course of differentiation.⁸ T-cell differentiation occurs by a complex transcriptional programme initiated by TCR and environmental signals but it is also accompanied by epigenetic changes at specific loci.⁹ We first review the transcription factors that are activated downstream of TCR signalling and then explore certain principles that might operate in regulating them.

Figure 1.

Schematic representation of T-cell activation, proximal signalling complex and activation of transcription factors. Current model of signalling in T cells states that stimulation of T-cell receptor (TCR) with specific major histocompatibility complex–peptide complexes (MHC-p) leads to phosphorylation of tyrosine residues within the immune receptor tyrosine-based activation motifs (ITAMs) of the invariant CD3 chains by Src family tyrosine kinases (Lck and Fyn). This TCR triggering causes the formation of the multimolecular proximal signalling complex (PSC).⁷ Signalling at the PSC causes calcium signals, activation of the Ras–mitogen-activated protein kinase (MAPK) pathway and protein kinase C-θ-dependent formation of the Carma1-BCL10-MALT1 (CBM) complex. These signals activate nuclear factor of activated T cells (NFAT), activating protein 1 (AP-1) and nuclear factor-κB (NF-κB) transcription factors respectively.

Transcriptional programme downstream of TCR signalling

Signalling through the TCR activates at least three families of transcription factors: nuclear factor of activated T cells (NFAT), activating protein 1 (AP-1) and nuclear factor-κB (NF-κB) (see Fig. 1). Gene expression by these transcription factors is not restricted to T cells but rather is found in almost every cell type in the body. As a result, extensive biochemical analysis has been performed over the years describing the network of interacting proteins that activate them. We will briefly review the regulation of these factors in T cells.

Nuclear factor of activated T cells

The NFAT family consists of five members: NFAT1 (NFATp or NFAT c2), NFAT2 (NFATc or NFATc1), NFAT3 (NFATc4), NFAT4 (NFATc3) and NFAT5; NFAT3 is not expressed in immune cells. All NFAT proteins contain a conserved Rel homology domain (regulatory domain) and an NFAT homology domain (DNA-binding domain). All except NFAT5 are regulated by calcium.¹⁰ NFAT is a transcription factor that is normally resident in the cytoplasm and is de-phosphorylated by a calcium-dependent phosphatase, calcineurin. This de-phosphorylation activates it and causes its translocation into the nucleus.¹¹ Nuclear export of NFAT is mediated by phosphorylation. Glycogen synthase kinase 3 (GSK-3) is known to phosphorylate conserved serine residues necessary for nuclear export.¹² In peripheral lymphocytes, antigen receptor signalling leads to the rapid inactivation of GSK-3. Activators of PKA suppress interleukin-2 (IL-2) production and T-cell activation, consistent with the possibility that NFAT is a substrate for protein kinase A (PKA).¹² NFAT4 is known to be negatively regulated through phosphorylation by casein kinase 1 in the cytoplasm.¹³ Another mechanism of negative regulation of NFAT involves calcipressin, a target of NFAT that binds to and inhibits calcineurin.¹⁰ Members of the homer family have been shown to bind to NFAT and compete with calcineurin, hence negatively regulating NFAT activation.¹⁴ Nuclear retention of NFAT can also be achieved by sumoylation, adding another level of complexity in its regulation.¹⁵ Unlike NFATc2, which is constitutively transcribed in T cells, transcription of the NFATc1 gene in effector T cells is strongly induced within 3–4 hr of TCR and co-receptor stimulation.¹⁶

Members of the NFAT family are redundant, as the mice lacking individual NFAT proteins show mild alterations in immune function whereas more severe defects are observed when more than one member is knocked out.¹⁰ NFAT plays a crucial role in T-cell differentiation.¹⁷ Sustained NFAT signalling has been shown to promote T helper type 1 (Th1) -like differentiation; enhancing production of interferon-γ (IFN-γ).¹⁸ Chromatin immunoprecipitation experiments have shown binding of NFAT1 to promoter of IL-4 in Th2 cells but not in Th1 cells, suggesting chromatin remodelling as one of the mechanism that determines NFAT binding to its target genes.¹⁹ NFAT is also the major player in the ionomycin-induced anergy model.²⁰ Anergy is defined as a state of T cells where they are unresponsive to stimulation and fail to make IL-2 or proliferate.²¹ An anergic state is achieved when T cells are stimulated through the TCR in the absence of co-stimulation in vitro.²² Developed by Rao and colleagues, the ionomycin-induced anergic state is achieved by treating cells with the calcium ionophore for a period of about 12 hr subsequent to which cells become unresponsive to TCR stimulation and fail to make IL-2 or proliferate. This form of anergy is largely NFAT dependent because sustained high calcium levels cause cells to primarily activate NFAT. A constitutively active form of NFAT when expressed in T cells also leads to a similar state.²⁰

The NFAT rapidly translocates to the nucleus on a rise in intracellular calcium. Several studies indicate that NFAT translocation into the nucleus is more efficient if the calcium signal is oscillatory.^23,24 Within minutes of reducing the cytoplasmic calcium level, NFAT is rapidly exported out of the nucleus. In another cell type these kinetics were much slower.²⁵ Hence, the re-phosphorylation kinetics may differ from cell type to cell type. Because the formation of the immune synapse is preceded by calcium fluxes,^5,26 the transport of NFAT into the nucleus in T cells is presumably rapid.

Recently a novel regulation for NFAT-like proteins was described. Crz1 is a calcineurin-dependent transcription factor in yeast wherein it plays an important role in stress-induced apoptosis. Elowitz and colleagues monitored the real-time trafficking of Crz1 fused to green fluorescent protein in response to increasing extra-cellular calcium. They found that the amount of Crz1 translocated to the nucleus was not simply proportional to the concentration of extra-cellular calcium. Instead, Crz1 translocated into and out of the nucleus in oscillatory bursts. Neither the amplitude nor the duration of these bursts changed as extra-cellular calcium was increased; rather, the frequency of bursts increased. The authors further showed by mathematical modelling and experimental validation that the frequency-modulated trafficking of Crz1 was important for maintaining the same amount of relative gene expression across different Crz1 targets as the extra-cellular stimulus changed.²⁷ As NFAT is calcineurin dependent, it would be interesting to see if this form of regulation is valid for NFAT in mammalian cells.²⁸

Activating protein-1

Activating protein-1 is a large family of dimeric protein complexes mainly consisting of Fos (c-Fos, v-Fos, Fos B, Fra1, Fra2) and Jun (c-Jun, v-Jun, JunB, JunD).²⁹ These proteins, which belong to the bZIP group of DNA-binding proteins, have leucine zippers through which they associate to form a variety of homo- and hetero-dimers that bind to common AP-1 sites (TRE-TGAC/GTCA) or (CRE-TGACTCA) in DNA.³⁰ Both ATF (ATF2, ATF3, B-ATF, JDP1, JDP2) and Maf (c-MAF, MafA, MafB, Nr1) are also considered members of this family based on their dimerization potential with Fos or Jun.²⁹ Jun-proteins, but not Fos-proteins, are known to undergo homo-dimerization.³¹ Hetero-dimerization of Fos with Jun is crucial for nuclear-cytoplasmic shuttling.³² Monomeric Fos and Jun shuttle actively but hetero-dimerization of both proteins inhibits their cytoplasmic shuttling. Surprisingly, this retro-transport inhibition is not caused by the binding of the AP-1 complex to DNA.³²

Levels of Fos and Jun proteins in T cells are either low or absent and are generally induced on signalling.^33,34 Activity of AP-1 is regulated by mitogen-activated protein kinases (MAPK).^35,36 Extra-cellular signal-regulated kinase (ERK) activation causes c-Fos induction, which results in increased synthesis of c-Fos and translocation to the nucleus. In the nucleus it combines with pre-existing Jun proteins to form AP-1 dimers that are more stable than those formed by Jun proteins alone.³⁰ It has been shown that ERK-1 is associated with the synapse after TCR stimulation and prevents docking of Src homology-2 (SH2) domain-containing phosphatase -1 (SHP-1) phospha-tase.^37–39 Transcription of c-Fos is regulated by ternary complex factors (Elk-1, SAP-1 and SAP-2) of which Elk-1 is phosphorylated by ERK.^30,40 The c-Jun is expressed at low levels in unstimulated cells and its promoter is constitutively occupied by Jun-activating transcription factor 2 (ATF2) dimer.^41,42 Phosphorylation of c-Jun by Jun N-terminal kinases (JNKs) and of ATF2 by JNKs or p38MAPK stimulates their ability to activate transcription, thereby leading to c-Jun induction.³⁰ As part of their negative regulation, AP-1 proteins are degraded in both ubiquitin-dependent and ubiquitin-independent manners.^43–45 The GSK-3 can inhibit AP-1 transcriptional activity by producing inhibitory phosphorylation on Jun.^12,46 The MAPK are negatively regulated by MAPK phosphatases, which are known to interact with the cytoplasmic tail of CD28 and are regulated by CD28 signalling.^47,48

Mice lacking c-Jun die at mid-gestation, indicating that it is an essential factor required for development.⁴⁹ Mice lacking c-Fos are growth retarded and develop osteoporosis with a reduced number of B cells.^50,51 The function of peripheral T cells (including proliferation and production of cytokines), however, is not impaired in c-Fos knockout mice.⁵² This lack of impairment could be the result of degeneracy among Fos members. In T cells, AP-1 contributes significantly to the regulation of the IL-2 gene.⁵³ The main transcriptional partners of AP-1 are NFAT proteins. The AP-1 proteins have been shown to bind with NFAT at composite DNA elements having weak AP-1 binding sites.⁵⁴ Co-operative binding between NFAT and AP-1 induces the expression of IL-2, IFN-γ, granulocyte–macrophage colony-stimulating factor, tumour necrosis factor-α, IL-3, IL-4, IL-13, IL-5, Fas ligand and CD25.⁵⁴ The interaction between NFAT and AP-1 integrates calcium signalling as well as the Ras–MAPK pathway.⁷ The DNA-binding and transcriptional activity of AP-1 requires both TCR-mediated and co-stimulatory signals. In vivo and in vitro ligation of TCR induces JNK gene expression but its phosphorylation requires CD28 co-stimulation.⁵⁵ Whereas cFos and FosB of the Fos members contain transactivation domains, JunB and JunD of the Jun members lack these domains.⁵⁶ JunD^−/− T cells hyper-proliferate and produce higher amounts of both Th1 and Th2 cytokines.⁵⁷

Nuclear factor-κB

The NF-κB members are dimers of the Rel family of proteins. This family contains five members: RelA (p65), c-Rel, RelB, p50 and p52, all of which have a Rel homology domain responsible for DNA binding and dimerization.⁵⁸ p50 and p52 are the processed forms of p105 and p100 proteins, respectively. The transactivation domain is present only in RelA, c-Rel and RelB so homo-dimers of these members can positively regulate target genes.⁵⁸ The homo-dimers of p50 and p52 act as repressors of their target genes.⁵⁹ The most abundant NF-κB proteins in T cells are the p65-p50 hetero-dimers.⁶⁰ The NF-κB dimers are held in the cytoplasm in a complex with inhibitor of κB (IκB) proteins.^61,62 There are three typical IκB members: IκBα, IκBβ and IκBε. Other IκB members are IκBγ, Bcl-3, p100 and p105.⁶³ Binding of NF-κB dimers to any of the IκB protein masks the nuclear localization signal (NLS) while the nuclear export signal remains exposed⁶⁴

Upon signalling IκB kinases (IKK) phosphorylate the IκB proteins, which causes their subsequent degradation.⁶⁴ The IKK complex is a hetero-trimeric kinase complex consisting of two catalytic subunits – IKKα, IKKβ– and the regulatory subunit IKKγ (NEMO). Degradation of IκB releases NF-κB and causes its translocation into the nucleus where among other genes it transcribes the IκB genes.⁶⁵ Newly synthesized IκB proteins enter the nucleus by virtue of their nuclear import signal and bind to NF-κB dimers causing their inactivation and nuclear export.⁶⁶ These negative feedback loops have been shown to cause oscillations in NF-κB across the nucleus when continuous stimuli are present.^67,68 Proteosomal degradation of DNA-bound NF-κB proteins constitutes an additional negative regulation of NF-κB activity.⁶⁹

T-cell receptor stimulation causes activation of NF-κB by one of many pathways. Activation of TCR follows PKC-θ dependent formation of the CARMA1, BCL10 and MALT1 (CBM) complex, which promotes the K63-linked poly-ubiquitination and degradation of IKKγ, the inhibitory component of the IKK complex.⁷ The TCR- and PKC-dependent activation of Tak1 kinase appears to be responsible for phosphorylation of IKKα and IKKβ.⁷⁰ Both these events are necessary for the activation of the IKK complex and further activation of the NF-κB pathway; however, they may occur independently of each other.⁷⁰ Carma1, BCL10, MALT1, IKK components and Tak1 have been shown to localize to the immunological synapse.^71,72 An alternative pathway of NF-κB activation involves stabilization of NF-κB inducing kinase (NIK) owing to proteosomal degradation of tumour necrosis factor receptor-associated factor 3 following TCR stimulation. The NIK activates IKKα, which phosphorylates p100 leading to proteosomal processing of p100 to p52.⁶⁵ Proteosomal processing of the C-terminal half of p105 into p50 occurs constitutively in unstimulated cells.⁶⁴

Nuclear factor-κB is shown to regulate a number of genes involved in immunity, cell proliferation and apoptosis.^59,73,74 Which NF-κB dimers specifically target particular genes has not been resolved.⁶⁴ Studying the immune responses in mice deficient in NF-κB proteins has revealed that NF-κB plays a very important role in regulating immune responses. However, a specific role for NF-κB in regulating T-cell differentiation is not known. There are reports that suggest that NF-κB components may regulate both Th1 and Th2 responses. T cells lacking p50 failed to produce IL-4, IL-5 and IL-13 as a result of failure to induce GATA-3 under Th2-polarizing conditions and at the same time they have been shown to affect Th1 responses.^75,76 RelB-deficient T cells have defects in Th1 differentiation.⁷⁷ Deficiency of c-Rel in T cells has been shown to affect IFN-γ and IL-2 production, and so to affect Th1 responses.^78–81 c-Rel plays a role in autoimmunity and allogeneic transplants as revealed from studies on c-Rel-deficient mice.^78,82,83 Deficiency of p50 and c-Rel in CD4 T cells has revealed a role of these transcription factors in CD4 T-cell survival in vivo.^78,84 RelA-deficient T cells have reduced proliferation in response to TCR stimulation.⁸⁵ There is a general consensus that all NF-κB members affect TCR-induced proliferation of T cells to some extent.⁸⁶

Other transcription factors downstream of TCR

NFAT, AP-1 and NF-κB are not the only family of transcription factors that are activated downstream of TCR. Among the other transcription factor family members that are directly regulated by TCR signalling are the forkhead family of transcription factors Foxo1, Foxo3 and Foxo4.⁸⁷ Their nuclear export is regulated by phosphorylation by Akt, which is activated by phosphatidylinositol 3-kinase signalling known to occur downstream of TCR.⁸⁷ Mef2 is a transcription factor that is activated downstream of TCR by calcium signalling.⁴⁷ It is maintained in an inhibitory state in the cytoplasm in complex with a protein called cabin1 which is an inhibitor of calcineurin.⁸⁸ Intracellular calcium increase leads to dissociation of MEF2 from Cabin1 through competitive binding of calmodulin.⁸⁸ The Mef2 regulates apoptosis in T cells by regulating the expression of the Nur77 family of orphan nuclear receptors.⁸⁹ Many transcription factors are regulated as a result of cross-talk between cytokine receptors and TCR signalling pathways. Some examples of these are, but are not limited to, T-bet, GATA-3, interferon regulatory factor family and Foxp3.^90,91 These transcription factors play an important role in the differentiation of T cells, but are beyond the scope of this review.

Regulations of transcription factors

So far, we have reviewed the transcription factors that are activated downstream of TCR signalling and how components of the immunological synapse activate them. T cells can differentiate to perform various effector functions, be tolerized or be deleted. All these processes require engagement of TCRs by peptide–MHC complexes and happen over days. Tolerance induction can occur when TCR signals are delivered in the absence of co-stimulatory signals, whereas deletion can occur when high-affinity self-peptide interactions occur in the periphery.²¹ Effector T-cell differentiation occurs as a result of co-operation between TCR, co-stimulatory and cytokine signals.^92,93 Differentiation is also accompanied by epigenetic changes occurring at specific promoters, particularly in the promoters of cytokine genes.^9,94 Antigen dose and affinity, however, also play an important role in determining the differentiation state of effector T cells in the absence of polarizing cytokines. O’Garra and colleagues demonstrated that increasing antigen dose led to more IFN-γ production by T cells whereas very low or very high antigen doses caused cells to produce IL-4.⁹⁵ Another study, from Bottomly and colleagues, showed that a high dose led to IFN-γ-producing cells whereas stimulation with a lower antigen dose caused cells to produce IL-4.⁹⁶ A requirement for co-stimulation through CD28 was demonstrated in this system for Th2 responses by way of weak TCR signals.⁹⁷ Although peptide dose and affinity do show an impact on Th1 versus Th2 choices, Croft and colleagues demonstrated that the time of differentiation also played an important role in determining whether cells produced IL-4 or IFN-γ.⁹⁸ Bottomly and colleagues also demonstrated that antigen dose affected the balance of NFATp versus NFATc DNA-binding activity, with lower potency ligands favouring higher levels of nuclear NFATc and lower levels of NFATp conducive for IL-4 transcription.⁹⁹ More recently, Paul and colleagues have explored the mechanism by which high and low doses of peptide induce Th1 versus Th2 responses. They report that T cells stimulated by low peptide concentrations result in IL-2-dependent signal transducer and activator or transcription 5 (STAT5) phosphorylation, TCR-induced IL-4-independent early GATA-3 expression and IL-4 production. Stimulation with a higher concentration of peptide caused, by way of the ERK pathway, abrogation of GATA-3 expression and IL-2-dependent STAT5 phosphorylation and IL-4 production.¹⁰⁰ In an in vivo model of adaptive tolerance it was found that in the presence of high and low amounts of persistent antigen T cells maintained their unresponsiveness only in the presence of high amounts of antigen.¹⁰¹ In another in vivo model of T-cell tolerance by way of high-potency and low-potency TCR ligands, it was found that low-potency ligands could induce tolerance in a calcium-independent pathway.¹⁰² All these examples demonstrate that antigen concentration and affinity can qualitatively alter the activation of signalling pathways and impact the differentiated state.

Our view is that antigen dose may influence differentiation by modulating the nuclear–cytoplasmic shuttling kinetics of transcription factors. Target genes are often regulated by multiple transcription factors and co-operation between them is crucial for optimal gene expression. It is possible that in response to different antigen concentrations the transcription factors that can co-operate on target genes in the nucleus change. Antigen dose is also likely to influence the frequency of generation of second messengers such as calcium. It has been shown that the frequency of calcium oscillations may encode transcriptional specificity.²⁴ Continuous signals from the cell surface have been shown to cause oscillations in NF-κB nuclear–cytoplasmic shuttling.^67,68 Recent experiments explore the nuclear–cytoplasmic shuttling of NF-κB under conditions where the extra-cellular signal is pulsatile.¹⁰³ The authors find that lower frequency signals give rise to full amplitude oscillations of NF-κB shuttling whereas higher frequency signals mimic a continuous signal. Importantly, the NF-κB-dependent gene expression depended on the frequency of extra-cellular signals.¹⁰³ Antigen dose may therefore influence specific gene expression either by modulating the frequency of generation of second messengers or by changing the proportion of co-operating transcription factors.

Asymmetric cell division has been implicated in cell fate determination in development.¹⁰⁴ During such events, some proteins can be asymmetrically divided between parent and daughter cells and give rise to different fates. Proliferating T cells during an immune response undergo asymmetric cell division. This has been suggested as one of the mechanisms by which memory and effector cells can be generated.¹⁰⁵ Asymmetric cell division is a powerful concept that can quantitatively change the concentration of individual signalling molecules such that on signalling the subsequent nuclear–cytoplasmic shuttling of transcription factors could be altered between parent and daughter cells.

Disclosures

The authors have no competing financial interests or conflicts of interests to disclose.