CD28 Costimulation and Regulatory T Cells

CD28 Costimulation and Regulatory T Cells B Soskic and DM Sansom, UCL Institute of Immunity and Transplantation, London, UK r 2016 Elsevier Inc. All rights reserved.

Introduction

Central Tolerance

An important feature of a mammalian organism is its ability to protect itself against infectious agents such as viruses and bacteria present in the environment. Over millions of years of evolution, a highly specialized system consisting of different cells and molecules that enable immunity against harmful organisms has developed. Simultaneously, this requires mechanisms in order to prevent an immune response developing against host tissue. Innate immune cells like macrophages, are equipped with receptors which can recognize molecules that are not normally present in the mammalian organism (e.g., lipopolysaccharide (LPS) in bacterial walls), minimizing inappropriate activation. The limitation of this response is that it remains unaltered even in the face of repeated infections, responding in the same manner toward any microorganism that contains a particular recognized pattern. On the other hand, cells of the adaptive immune system such as T cells provide specific immunity, which is capable of longterm ‘memory’ and more vigorous and rapid responses in the case of reinfection. However, T cells recognize peptide fragments derived from proteins, which have a large, but nonetheless limited, range of possible amino acid configurations. Thus, although the nature of an antigen that T cell recognize enables a vigorous response to specific microorganisms, it also provides a constant threat to host tissues that may contain the same or similar amino acid sequences as part of their normal physiology. Therefore, both T cell development and T cell activation are tightly controlled in order to prevent autoimmune diseases, which can result from such recognition.

How Is the Self-Tolerance Established? The discrimination between host peptides and those derived from the pathogen proteins is not straightforward and presents a major challenge to adaptive immunity. To protect the host tissue from harmful T cell responses, T cells that recognize self-antigens with a high affinity are not released into the immune system. Instead, such cells are deleted during development in the thymus. This clonal deletion of highly self-reactive T cells represents the basis of central tolerance, steering the immune response away from self-antigens. However, although such developmental controls are important for establishing immune tolerance, they are not sufficient and T cells that survive this ‘negative selection’ are not completely purged of self-reactivity. In addition, not all host proteins in the periphery are expressed in thymus and T cells therefore emerge that can still recognize self-antigens albeit with the lower affinity. Finally, a large number of new harmless proteins are constantly introduced into our system (e.g., through food). In order to prevent unwanted T cell responses to such proteins a set of mechanisms has evolved to control T cell activation.

Encyclopedia of Cell Biology, Volume 3

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Using their highly variable T cell receptor (TCR), T cells recognize antigens in the context of major histocompatibility complex (MHC). Given that the number of potential antigens far exceeds the number of TCR genes in the human genome, the main problem is how to generate a TCR repertoire broad enough to recognize all conceivable antigens present in our environment (Sewell, 2012). Evolution has solved this problem by introducing random gene rearrangement of TCR genes during T cell development, which drives enormous variability. However, the random nature of the process introduces two major problems. To begin with, the majority of T cells that express randomly rearranged TCRs cannot recognize MHC molecules and are deleted in the thymus with only T cells that can bind to peptide–MHC complex being positively selected. Interestingly, a wide range of tissue-restricted proteins (e.g., insulin) are also processed and presented by thymic antigen-presenting cells (APCs) and T cells that recognize these antigens with high affinity also undergo cell death (negative selection). As such negative selection eliminates the most dangerous T cell clones preventing imminent tissue destruction. Whilst the detailed mechanisms of negative selection are still under investigation it is believed that strong TCR signals generated upon antigen recognition induce pro-apoptotic proteins that mediate cell suicide (Hogquist et al., 2005; Kyewski and Derbinski, 2004). Negative selection against ectopically expressed self-antigens undoubtedly has a major role in establishing tolerance and mutation in the transcription factor AIRE, which is responsible for driving the expression of tissue-restricted antigens in thymic epithelial cells, leads to a systemic autoimmunity (autoimmune polyendocrine syndrome) (Mathis and Benoist, 2009). However, it is equally clear that despite rigorous selection in the thymus, the peripheral T cell pool still contains T cells capable of reacting to our own tissues, which require additional specific mechanisms of control in a process generically termed peripheral tolerance.

Peripheral Tolerance Deleting every T cell that can recognize a self-antigen would result in an extremely limited repertoire and most likely would not be compatible with sufficient diversity of pathogen recognition. Therefore, a set of additional mechanisms has been established in the periphery in order to allow the existence of weakly self-reactive T cells that are potentially useful against pathogens (Walker and Abbas, 2002). In the last decade, a variety of molecules implicated in the peripheral regulation of T cell responses have been described. For instance, defects in FoxP3 (a master transcription factor in regulatory T cells), CTLA-4 (a negative regulator of CD28 pathway), TGF-β (T cell growth factor), and IL-10 and IL-2 (anti-inflammatory cytokines) all lead to a severe autoimmunity. Below we discuss in

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detail the CD28/CTLA-4 pathway as an essential component of peripheral T cell tolerance.

Responses to pathogens versus responses to self – the role of CD28 Since the T cell repertoire inevitably contains self-reactive T cells, the question arises: how are responses to self-proteins discriminated from the responses to foreign antigens? It is currently thought that activation of CD4 þ T cells needs at least two signals provided by specialized APCs, such as dendritic cells (DCs), macrophages, and B cells (Figure 1). The first signal is generated when TCR recognizes the antigen displayed by MHC II complex. The requirement for the second signal, known as costimulation, ensures that T cell activation does not occur in the absence of additional information. This is due to the fact that recognition of pathogens (e.g., via Toll-like receptors) or the presence of inflammation specifically upregulates the costimulatory ligands CD80 (B7-1) and CD86 (B7-2) which bind to CD28. Via their interaction with costimulatory receptor CD28, on T cells, these ligands provide signals necessary for a successful T cell activation, proliferation, and differentiation. In this way costimulatory signals are only triggered by the presence of inflammation or ‘danger’ associated with infection, limiting activation of potentially self-reactive T cells to such settings. Studies using TCR-transgenic mice that respond to myelin basic protein (MBP), demonstrated that mice housed in standard animal facilities develop experimental autoimmune encephalomyelitis (EAE). In contrast, the same TCR-transgenic mice housed in pathogen-free facilities fail to develop the same disease (Goverman et al., 1993). A simple interpretation of these results is that microbes in the environment are

important in driving the maturation of APCs, enhancing their ability to costimulate. Although these groups of mice have the same TCR specificity, the outcome of immune reaction is dependent on the CD28 engagement. These data along with numerous other studies have firmly established that, CD28 engagement presents a key immune checkpoint that influences self-tolerance: initiating T cell responses not based on the source of peptide antigen, but based on sensing the inflammatory context of recognition. Naturally, regulation of the CD28 pathway then serves as critical point in prevention of autoimmunity.

Regulatory T cells – a specialized cell type that regulate immune responses Regulatory T cells (Tregs) are a subset of CD4 þ T cells whose role is to regulate immune responses especially those against self-tissues. This is revealed by diseases such as IPEX (immune dysregulation, polyendocrinopathy, enteropathy, X linked) in humans and the Scurfy phenotype in mice due to loss of Treg function (Bennett et al., 2001; Brunkow et al., 2001). Tregs are characterized by high expression of IL-2 receptor α (CD25) and low level of IL-7 receptor α (CD127). Tregs are generated in thymus upon recognition of self-antigen (called thymusderived or natural Tregs), or in the periphery upon activation of naïve T cells under tolerogenic conditions (peripherally derived or induced Tregs). The similarities and differences between nTregs and iTregs have been extensively studied and they are reviewed elsewhere (Curotto de Lafaille and Lafaille, 2009; Bilate and Lafaille, 2012). After discovery of the suppressive activity of Tregs, a major hurdle was how to distinguish them from activated T cells due to the fact that they express high level of CD25, which is

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TCR Suppression Activation T cell Figure 1 CD28 costimulation controls T cell activation. The goal of T cell activation is to generate a large number of antigen-specific T cells with effector properties that can eliminate pathogens. It is currently thought that activation of CD4 þ T cells needs at least two signals provided by a specialized APC that captures, processes, and presents antigens as a part of MHC. Simultaneously, recognition of pathogen-related molecules or the presence of inflammation specifically upregulates the costimulatory ligands CD80 and CD86. The first signal is generated when TCR recognizes the antigen displayed by MHC complex. The requirement for the second signal, known as costimulation, ensures that T cell activation does not occur in the absence of additional inflammatory or ‘danger’ information. Via interaction with CD28, on T cells, CD80 and CD86 provide translate such danger signals to T cells and are therefore necessary for a successful T cell activation, proliferation, and differentiation. The second receptor for the CD80 and CD86 is cytotoxic T lymphocyte antigen 4 (CTLA-4). Expression of CTLA-4 is limited to activated conventional T cells and regulatory T cells (Tregs). CTLA-4 has higher affinity and avidity for both ligands, and serves as a negative regulator of T cell responses.

Cellular Immunology: Cell–Cell Interactions and the Immune System: CD28 Costimulation and Regulatory T Cells

also upregulated upon the activation of conventional T cells (Tconv). The study of Tregs was therefore greatly facilitated by the discovery of transcription factor FoxP3, which appears to be specific to this lineage (Fontenot et al., 2005). It has been demonstrated that this member of forkhead family of transcription factors is critical for Treg development (Hori et al., 2003; Fontenot et al., 2003). Loss of FoxP3 function causes fatal systemic autoimmune phenotype in both mice and humans characterized by uncontrolled proliferation of T cells, which leads to multiorgan infiltration and failure. Taken together, these studies indicate that Tregs are indispensible for immune homeostasis. Since the importance of Tregs has been discovered, much work has been performed to elucidate mechanisms by which Tregs regulate immune response. In short, various studies have demonstrated that Tregs can either directly suppress responder T cells, or indirectly modulate the stimulatory capacity of APCs (Shevach, 2009; Josefowicz et al., 2012). Direct mechanisms include production of anti-inflammatory cytokines (e.g., IL-10 or TGF-β), IL-2 consumption, granzyme-mediated apoptosis, and cell-cycle arrest. Of particular interest here is targeting the ability of APCs to provide efficient CD28 costimulation thereby preventing the initiation of T cell response. Evidence from both in vitro and in vivo experiments shows that Tregs express high levels of CTLA-4 (Takahashi et al., 2000), which is a nonredundant regulator of immune response and is tightly coupled with CD28 costimulation. CTLA-4 appears to be critical for Treg function (Wing et al., 2008; Schmidt et al., 2009) and the molecular basis of this process is described below.

The Biology of the CD28/CTLA-4 Pathway CD28 is 44 kDa glycoprotein constitutively expressed on the surface of the majority of T cells. It consists of a single extracellular Ig-V-like domain that forms a homodimer. Although other costimulatory pathways have been discovered (e.g., ICOS pathway), CD28 appears to be the apical costimulatory molecule governing T cell activation. As such, mice deficient in CD28 or its ligands CD80 and CD86 show defects in IL-2 production and T cell proliferation, as well as B cell functions such as germinal center formation, antibody levels, and immunoglobulin class switching. Upon the recognition of CD80 and CD86 ligands, CD28 relocates to the immune synapse where it initiates various costimulatory signaling events (Figure 1). The exact pathways involved in the signal transduction are still under investigation. To date the importance of various kinases in CD28 signaling has been demonstrated, such as PI3K, ITK, Lck, and PKC-Θ (Rudd et al., 2009). These molecules initiate a signaling cascade responsible for the activation of genes involved in survival and cell-cycle progression. However, the outcome and the importance of specific signaling pathways initiated upon CD28 engagement remains controversial. Recent data have indicated that CD28 costimulation may be important in actin reorganization within the T cells in order to facilitate activation signals, in a manner that may not be compensated by simply increasing TCR signals (Tan et al., 2014; Dustin and Davis, 2014).

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Functions of CD28 It is still unclear whether CD28 engagement causes unique changes in T cell signaling or simply amplifies TCR-based signals. Over years it has been established that costimulation serves to lower the threshold necessary to achieve the activation of T cells, particularly enabling responses to weak agonist TCR ligands. Data indicate that CD28 engagement ensures more rapid division and enhances production of different cytokines, particularly a T cell growth factor IL-2. In addition, it was demonstrated that CD28 increases the expression of the anti-apoptotic protein BCL-XL thus promoting the survival of T cells (Acuto and Michel, 2003). Furthermore, it has been suggested that CD28 signaling influences T cell differentiation. The most prominent defect in CD28 knockout mice is the lack of germinal centers and consequently the production of high-affinity antibodies is impaired (Ferguson et al., 1996; Shahinian et al., 1993). This could be due to an indispensable role of CD28 in driving inducible T cell costimulator (ICOS) upregulation that is necessary for the development of follicular helper T cell (Linterman et al., 2009). Moreover, there are indications that high-CD28 signaling in the context of weak-TCR signals drives the production of Th2 cytokines (Lenschow et al., 1996; Smeets et al., 2012), although CD28 does not seem to be absolutely required for Th2 differentiation (Brown et al., 1996). In addition to its role in conventional T cell activation, studies in CD28 knockout mice revealed that CD28 is also implicated in the generation and homeostasis of thymic regulatory T cells (Sansom and Walker, 2006). Surprisingly, it has been shown that CD28 is required for Treg generation in thymus independently of its role to stimulate IL-2 production. In addition, Gogishvili et al. (2013) have shown that CD28 deletion significantly lowered the number of Tregs, but not of conventional T cells in the periphery. More importantly, they showed that IL-2 is not able to compensate for the role of CD28 costimulation in maintaining the number of Tregs in the periphery. Therefore, it appears that in addition to IL-2, there is a nonredundant cell-intrinsic role of CD28 costimulation in peripheral Treg homeostasis (Sansom and Walker, 2013). A recent study showed slightly different results using a Treg-specific CD28 KO mice system (Zhang et al., 2013). Here, except in the thymus, the number of Tregs was fairly well preserved, however, their function was notably impaired. These Tregs had a significantly decreased expression of CTLA-4 and PD-1, leading to a robust autoimmune phenotype. Although a role for CD28 in Treg selection in the thymus is clear, the role of CD28 in peripheral Treg induction is still controversial. It has been reported that CD28 signaling negatively regulates differentiation of induced Tregs (Semple et al., 2011). However, others have shown that CD28 promotes differentiation of induced Tregs (Gabrysova et al., 2011). Thus, it is likely that the role of CD28 in Treg generation depends greatly on the other signals that T cell receives, particularly the strength of TCR, as well the cytokine milieu in which the activation occurs. Overall, the CD28 pathway is critical for T cells activation, differentiation, and function. Moreover, given that T cells interact with a number of immune cell types, regulation of

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Cellular Immunology: Cell–Cell Interactions and the Immune System: CD28 Costimulation and Regulatory T Cells

CD28 pathway is therefore crucial for successful B cell, macrophage, and cytotoxic T cell responses. This places CD28/ CTLA-4 pathway at the heart of immune regulation.

CTLA-4 – A Key Negative Regulator of T cell Activation The second receptor for the CD80 and CD86 is cytotoxic T lymphocyte antigen 4 (CTLA-4) (Figure 1). CTLA-4 is 34 kDa type 1 transmembrane glycoprotein expressed by T cells. Genes for CD28 and CTLA-4 are both located on the same region of the chromosome and they share around 30% of homology at the amino acid level indicating that CD28 and CTLA-4 have evolved from the same ancestral gene. Nonetheless, CD28 and CTLA-4 proteins have a very different expression pattern, biological properties, and effects upon T cell responses. Whereas CD28 is constitutively expressed on the surface of most T cells, CTLA-4 expression is limited to activated conventional T cells and more importantly, Tregs. Upon T cell activation, CTLA-4 is synthesized and directed toward the cell membrane, where it binds to CD80 and CD86 with a higher affinity than CD28. Also unlike CD28, CTLA-4 is a highly

endocytic molecule and the amount of time in which it is resident on the cell surface is very limited (Figure 2). Whereas CD28 engagement promotes T cell activation, CTLA-4 serves as a negative regulator of T cell responses. The importance of CTLA-4 has been widely demonstrated in CTLA4 KO mice (Tivol et al., 1995) and recently in humans with heterozygous nonsense/missense CTLA-4 mutations (Schubert et al., 2014). T cells from CTLA-4 KO mice exhibit a strong response to self-antigens, which leads to lymphoproliferative disease and these mice die after 3–4 weeks of age from multiorgan failure. In addition, various polymorphisms in CTLA-4 gene have been implicated in different autoimmune diseases in humans (Gough et al., 2005). These phenotypes provide strong evidence that CTLA-4 evolved to prevent autoimmunity, however, the molecular mechanisms used to achieve this have been controversial.

CTLA-4 and CD28 Bind Same Ligands with Different Affinities CD80 and CD86 are ligands for CD28 and CTLA-4 and both expressed by APCs (Figure 1). CD28 and CTLA-4 are both dimers and they share a conserved MYPPPY amino acid motif that forms the ligand binding domain (Yu et al., 2011).

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Figure 2 Cell biology of CTLA-4. CTLA-4 is synthesized on endoplasmic reticulum and matures in Golgi forming the functional homodimer. Upon T cell activation vesicles containing CTLA-4 proteins are targeted toward cell membrane. At the cell surface a YVKM motif in the cytoplasmic domain of CTLA-4 is recognized by the clathrin adaptor protein AP-2 leading to a rapid clathrin-mediated endocytosis. Following internalization, CTLA-4 either recycles back to the cell surface or is degraded in lysosomes. Therefore, CTLA-4 is a highly dynamic protein with short half-life, which recycles and is rapidly degraded in lysosomes.

Cellular Immunology: Cell–Cell Interactions and the Immune System: CD28 Costimulation and Regulatory T Cells

Importantly, CTLA-4 has higher affinity and avidity for both ligands, which allows CTLA-4 to out-compete CD28 for CD80/86 binding. Thus it has been proposed that CTLA-4 can act by trapping CD80 and CD86 and preventing their interactions with CD28 (Walker and Sansom, 2011). Surprisingly, although CD80 and CD86 share the same receptors, these two ligands have only around 25% of identity at the protein level. In addition, CD80 exists as a dimer while CD86 appears to be a monomer (Collins et al., 2002). Based on chromosomal location it seems likely that CD80 and CD86 have evolved from the same ancestral gene probably in the process of gene duplication (Zhu et al., 2014), and then acquired specific functions. However, the precise functional differences between CD80 and CD86 are not yet understood. In terms of interaction with their receptors, CD80 has a significantly higher affinities and avidities for both CD28 and CTLA-4 than CD86, thus suggesting that CD80 is a more potent ligand (Collins et al., 2002). However, studies in CD80 and CD86 knockout mice have revealed that CD86 is more important in T cell activation than CD80 (Borriello et al., 1997). The biology behind the superior capacity of CD86 to costimulate is still unclear and further studies are required to distinguish between effects of expression level and those of affinity and structure. More importantly these results raise another question: if CD86 alone is sufficient to costimulate what is then the role of CD80 in T cell differentiation and function? A more comprehensive understanding of exact roles of CD80 and CD86 during different phases of immune response is awaited and will almost certainly increase our knowledge of T cell biology and enable a more efficient use of immunomodulatory drugs that target CD28/CTLA-4 pathway.

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questionable. Moreover, the precise signaling mechanisms still remain unclear, for example, it has been shown that the interaction of CTLA-4 with PP2A is not necessary for the inhibition of T cell responses (Teft et al., 2009). Other pathways proposed to be initiated by CTLA-4 (i.e., PI3K and PKCθ pathway) are also implicated in CD28 signaling and generally are thought to have positive effect upon T cell activation. The fact that CTLA-4 is not well expressed on the cell surface and possesses a highly dynamic trafficking itinerary (see Section Cell biology of CTLA4), that limits amount of time in which it is expressed on cell surface is also not easily reconciled with a signaling function (Qureshi et al., 2012). The most persuasive argument to consider mechanisms other than intrinsic inhibitory signals comes from in vivo experiments with chimeric mice. These mice contain both CTLA-4 þ / þ and CTLA-4/ T cells and have challenged the cell-intrinsic view of CTLA-4 function. Unlike CTLA-4/ mice that die from lymphoproliferative disease, mice containing both CTLA-4/ and CTLA-4 þ / þ cells fail to develop this pathology (Bachmann et al., 1999). The most likely explanation for this finding is that CTLA-4 þ / þ cells are able to control expansion and effector functions of CTLA-4/ T cells. This led to the suggestion that CTLA-4 dominantly suppresses T cell activation via modifying phenotype and function of other cell types such as APCs, consistent with a function on a ‘suppressor’ cell such as a Treg. Until recently a molecular mechanism by which CTLA-4 can control T cell activation in cell-extrinsic manner has been difficult to elucidate (Walker and Sansom, 2011). However, we recently found that CTLA-4 was capable of carrying out a novel cellular process we term transendocytosis (Soskic et al., 2014; Qureshi et al., 2011).

Transendocytosis: A Molecular Mechanism of CTLA-4 Function

How Does CTLA-4 Function? The autoimmune phenotype in CTLA-4 deficient mice can be prevented by blocking CD80 and CD86 with CTLA4-Ig (Tivol et al., 1997) or by crossing CTLA-4 KO mice with mice deficient for both ligands (Mandelbrot et al., 1999). This clearly demonstrates that CTLA-4 functions as a regulator of CD28 signaling. Accordingly, CD28 independent T cell activation is not controllable by CTLA-4.

From a cell biology perspective, the most fundamental feature of the CTLA-4 protein is its highly endocytic nature (Figure 2). This is somewhat surprising giving that CTLA-4 operates on the cell surface by interacting with CD80 and CD86 expressed on APCs. However, the endocytic behavior of CTLA-4 appears to be crucial for CTLA-4 function. Here we explore cell biology of CTLA-4 and how endocytosis is employed as an effector pathway in order to control T cell responses.

Cell biology of CTLA-4 The Mechanism of CTLA-4-Mediated Suppression Is CellExtrinsic Numerous studies in CTLA-4 biology have suggested that upon ligation CTLA-4 delivers negative signal (Rudd et al., 2009) by recruiting protein phosphatases PP2A and SYP to TCR complex, which inhibit TCR signaling by turning off T cell activation and IL-2 production. Other studies have shown that CTLA-4 recruits PI3K or competes with CD28 for PKCθ binding. However, there is still debate as to whether CTLA-4 delivers an inhibitory signal, particularly in vivo. To begin with, the idea of negative signaling was predominantly based on experiments using agonistic antiCTLA-4 antibody, however, there is no natural ligand that solely binds CTLA-4 but not CD28, making such reagents

Upon TCR engagement, the level of CTLA-4 is rapidly increased (Egen and Allison, 2002) and vesicles containing CTLA-4 proteins are targeted toward immune synapse in ADPribosylation factor 1 (ARF1) and phospholipase D (PLD) dependant fashion (Mead et al., 2005). At the cell surface, a YVKM motif in the cytoplasmic domain of CTLA-4 is recognized by the clathrin adaptor protein AP-2 leading to a rapid clathrin-mediated endocytosis (Shiratori et al., 1997). Earlier studies have proposed that upon activation tyrosine in YVKM motif is phosphorylated, preventing binding of AP-2 (Shiratori et al., 1997). This would cause stabilization of CTLA-4 at the cell surface. Although evidence exists that in vitro phosphorylation of CTLA-4 or overexpression of Src kinase prevents AP-2 binding (Follows et al., 2001; Chuang et al., 1999), it is

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unclear whether this happens under physiological stimulation. Indeed our group has found that clathrin-mediated endocytosis generally persists during T cell activation, and at any given time approximately 90% of CTLA-4 molecules are expressed intracellularly (Qureshi et al., 2012). Therefore the increase of CTLA-4 at the cell surface following T cell activation is not due to the disruption of CTLA-4 internalization, but rather to the increased delivery of CTLA-4 containing vesicles to the cell surface resulting from the increased protein levels during T cell activation. Upon internalization, the fate of CTLA-4 is still rather unclear. Our lab has demonstrated that CTLA-4 co-localize with Rab11 (Qureshi et al., 2012), which is known to regulate recycling of endocytosed proteins and there is evidence that CTLA-4 can recycle back to the cell surface. Further precise studies are still required to elucidate motifs and molecular mechanisms involved in CTLA-4 recycling. Multiple studies have also shown that CTLA-4 is degraded in lysosomal compartments and neutralization of lysosomal pH using ammonium chloride (NH4Cl) increases the expression of CTLA-4 (Kaur et al., 2013; Egen and Allison, 2002). On the other hand, blocking protein synthesis with cycloheximide (CHX) results in the rapid decrease of CTLA-4 level giving a half-life of CTLA4 of around 2 h (Egen and Allison, 2002). The above data provide evidence that CTLA-4 is a highly dynamic protein with short half-life, which recycles and is rapidly degraded in lysosomes. Understanding the cell biology of CTLA-4 in the context of its function is therefore clearly important (Figure 2). Overall, although CTLA-4 shares the same ligands with CD28 it has strikingly different biology. The highly dynamic nature of CTLA-4 has presented a major conundrum in the field. Interestingly, it has been shown that cytoplasmic domain of CTLA-4 is highly conserved among all mammals, suggesting that there is a strong evolutionary pressure to maintain a complex CTLA-4 trafficking across species (Walker and Sansom, 2011). We have therefore taken the view that CTLA-4 trafficking is key to its function.

Transendocytosis: cell biology at the heart of immune regulation In recent studies we have observed that CTLA-4 effectively can act as a molecular hoover that removes CD80 and CD86 ligands from the surface of APCs, by ‘transendocytosis’ (Qureshi et al., 2011; Figure 3). By depleting ligands, CTLA-4 reduces the capacity of APCs to provide effective costimulation through CD28, thereby directly coupling the function of CTLA-4 to CD28 as observed in in vivo experiments. To elucidate the mechanism of CTLA-4-mediated depletion of ligands, we have generated cell lines that expressed CTLA-4 and CD80 or CD86 tagged with green fluorescent protein (GFP) (Figure 4). Perhaps most strikingly, we have observed that the entire receptor–ligand complex (including cytoplasmic domain of ligands tagged with GFP) gets internalized into CTLA-4 expressing cell. The complex is subsequently targeted to lysosomes where ligands are rapidly degraded while CTLA-4 may recycle back to the cell surface to capture more ligands (Figure 3). This latter point still requires more research but it is clear that ligands are degraded and that this is inhibited by agents such as NH4Cl. Further, to test the relevance of transendocytosis in vivo we generated mice that express CD86-GFP on the surface of their APCs. Strikingly, capture

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Figure 3 Transendocytosis – A molecular mechanism of CTLA-4 action. Upon activation CTLA-4 containing vesicles are targeted to the immune synapse (the site of T cell–APC interaction), where it binds CD80 or CD86. Subsequently, the entire receptor–ligand complex gets internalized into T cell. The process of CTLA-4 dependent CD80/86 internalization is termed transendocytosis. The complex is targeted to lysosomes where ligands are rapidly degraded while CTLA-4 may recycle back to the cell surface to capture more ligands. As such CTLA-4 expressed by Tregs can remove ligands from APC preventing the activation of all T cells that subsequently recognize antigens presented by that APC. This provides an effcient mechanism of immune suppression and controls the level of CD80 and CD86 expression.

of ligands was observed only in CD4 þ CD25 þ T cells (activated conventional T cells or Tregs) and CD4 þ CD25 þ T cells from CTLA-4/ mice did not acquire ligands demonstrating that the transfer of ligand is CTLA-4 dependent in vivo. More importantly, the transendocytosis occurred only upon peptide stimulation indicating that the process is antigen specific. These features are highly consistent with a role for CTLA-4 on regulatory T cells as an effector mechanism for reducing costimulation via CD28. Although the exact molecular players involved in CTLA-4 transendocytosis are currently unknown, the concept of intercellular exchange of membrane proteins has been observed elsewhere. A number of studies have shown that various cell types are capable of exchanging their membrane proteins via transendocytosis (Rechavi et al., 2009). A well-characterized example is Notch, which upon the interaction with its ligands is cleaved and internalized into the ligand-expressing cell (Klueg and Muskavitch, 1999). This process is necessary to initiate Notch receptor signaling which is implicated in cell differentiation during development. Several other examples of

Cellular Immunology: Cell–Cell Interactions and the Immune System: CD28 Costimulation and Regulatory T Cells

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Figure 4 Confocal microscopy of transendocytosis. Chinese hamster ovary (CHO) cells stably expressing CD86-GFP (green) or CTLA-4 (red) were co-incubated for 8 h. In areas of the coculture where CTLA4-expressing cells are absent (a) CD86-GFP is preserved at the plasma membrane. However, where ligand-expressing cells make close contact with CTLA-4 expressing cells CD86 ligand is taken up by the CTLA4-expressing cells and destroyed. Accordingly most of the GFP signal is lost (b). In the presence of lysosomal inhibitor NH4Cl (c) the degradation of CD86-GFP is blocked and the captured ligand accumulates in intracellular vesicles within the CTLA4-expressing cells.

transendocytosis have been described, including the bidirectional transfer of CD47–SHPS–1 complex (Kusakari et al., 2008), which controls T cell activation, and transendocytosis of ephrin B that regulates contact repulsion (Marston et al., 2003). While transendocytosis involves transfer of ligands directly into receptor-expressing cell, it has some distinctions from similar processes such as Trogocytosis (Daubeuf et al., 2010) which is common in the immune system (Davis, 2007). It has been identified that within the minutes of co-incubation of NK cells with their targets, NK cell receptor KIR is transferred to target cell in MHC I dependent fashion by trogocytosis. Similarly, several studies have shown that CD4 þ T cells can acquire MHC II, CD80, and CD86 from APCs. Interestingly, CD28-expressing cells can also acquire CD80 and CD86 ligands, however, CD28-mediated transfer is by trogocytosis. In our experience whilst trogocyosed proteins remain detectable on the cell surface after transfer, in transendocytosis transferred proteins are directly routed to an intracellular compartment, which precludes surface detection.

CD28/CTLA-4 and its ligands. The transendocytosis model therefore describes a form of competition between CD28 and CTLA-4 that is cell-extrinsic and where competition between CD28 and CTLA-4 can be temporally separated. As such CTLA4 expressed by Tregs can remove ligands from APC preventing the activation of all T cells that subsequently recognize antigens presented by the same APC. In transendocytosis, the ability of CTLA-4 to internalize CD80 and CD86 from APC membrane is dependent on both the number of ligands that are expressed by APC and number of CTLA-4 molecules. Therefore a key feature of CTLA-4-mediated immune control is that it is quantitative. As such, if the inflammatory environment (i.e., due to the presence of pathogen) drives increased expression of ligands, CTLA-4-mediated suppression has a diminished efficacy. In these settings, if the production of CD80/86 is greater than their downregulation, this allows the engagement with CD28. On the other hand, if the level of CD80/86 ligands is lower, CTLA-4 can act to keep the level below the threshold necessary for costimulation, thus preventing the activation of self-reactive T cells.

Transendocytosis as a mechanism of immune regulation A long-term conundrum in immunology has been why a key stimulatory protein (CD28) and a key inhibitory molecule (CTLA-4) would share the same ligands. If CTLA-4 is viewed as a receptor that delivers negative signal, then it is difficult to imagine the evolutionary logic behind controlling activation and inhibition of T cell responses with the same triggers. However, from transendocytosis perspective the core concept behind the model is that CTLA-4 must share the ligands with CD28 in order to function. Furthermore, in order for suppression to be efficient, CTLA-4 needs to be able to compete with CD28 for CD80 and CD86 binding, which is in agreement with biophysical characteristics of interaction between

CTLA-4: a key mechanism of Treg suppression Numerous studies have suggested that CTLA-4 plays a major role in Treg-mediated suppression of immune response (Wing et al., 2008; Schmidt et al., 2009; Onishi et al., 2008). Indeed, there is a remarkable similarity between CTLA-4 KO and Scurfy mice, suggesting that the Scurfy phenotype seen in FoxP3 deficient mice is at least partially a result of the loss of CTLA-4 due to absence of Tregs. Furthermore, removal of CD28 or injection of CTLA4-Ig significantly improves survival of Scurfy mice (Singh et al., 2007). Transendocytosis offers a simple explanation for CTLA-4 dependent Treg function. Upon the antigen recognition, the

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Antigen presenting cell

MHC II

CD80

CD86

Abatacept (CTLA4-lg)

CD28 Antagonist

Belatacept (CTLA4-lg variant)

CD28 superagonist

Anti-CTLA-4

CTLA-4

CD28 TCR

CD86

CD80

CD28 T cell

Figure 5 Manipulation of CD28/CTLA-4 pathway. Multiple therapeutic compounds have been designed to manipulate the CD28/CTLA-4 pathway. CTLA4-Ig binds to CD80 and CD86 expressed on APCs preventing their interaction with CD28 and therefore the initiation of T cell responses. CTLA4-Ig is clinically available in two forms: Abatacept and Belatacept. Compared to Abatacept, Belatacept has an increased affinity for CD80 and CD86, with an increased bias toward CD86 blockade. Both these compounds are used as immunosuppressive drugs. As an alternative approach to ligand blockade, CD28-blocking antibodies have been developed for induction of transplantation tolerance and treatment of autoimmunity. In contrast, CD28 superagonistic antibodies have been used to induce immune tolerance by preferential expansion of FoxP3 þ Tregs. Several CTLA4-blocking antibodies have also been developed and demonstrated antitumor effects. Thus the system is capable of being manipulated to promote immune tolerance or immune activation depending on the components that are targetted.

expression level and cycling of CTLA-4 dramatically increases in regulatory T cells. As functional consequence, CTLA-4 removes CD80 and CD86 ligands from the surface of APCs and efficiently prevents the activation of self-reactive T cells. In line with this, several studies have demonstrated that Tregs downregulate CD80 and CD86 from APCs in CTLA-4 dependent fashion (Oderup et al., 2006; Onishi et al., 2008; Wing et al., 2008). Another important concept that arises from this model is that any T cell that expresses CTLA-4 (activated conventional T cells and Tregs) has regulatory capacity (Corse and Allison, 2012; Wang et al., 2012). Indeed it was recently demonstrated that the combination of CTLA-4 expression and IL-2 suppression is sufficient to convert conventional T cells into regulatory T cells with potent immunoregulatory capacity (Yamaguchi et al., 2013).

Manipulation of CD28/CTLA-4 Pathway: Antagonist, Agonists, and Superagonists The last decade of research in immunology has taught us that the relationship between CD80- and CD86-CD28-CTLA-4 is

extremely well balanced and any disturbance may have a profound influence on the control of the immune response. Because of its central importance, several approaches have been used to manipulate T cell activation through CD28/ CTLA-4 pathway (Figure 5). Blocking CD80 and CD86 is widely used as a strategy for blocking CD28/CTLA-4 pathway. The higher affinity of CTLA4 for its ligands led to development of CTLA4-Ig fusion protein as an inhibitor of T cell responses. CTLA4-Ig binds to CD80 and CD86 expressed on APCs preventing their interaction with CD28 and accordingly the initiation of T cell responses. CTLA4-Ig is clinically available in two forms: Abatacept and Belatacept. Abatacept has been approved for the treatment of rheumatoid arthritis in patients who failed to respond to antitumor necrosis factor (TNF) therapy (Genovese et al., 2012) and polyarticular juvenile idiopathic arthritis (Goldzweig and Hashkes, 2011). However, a significant proportion of patients do not respond to Abatacept treatment leading to a second generation of artificially engineered CTLA4-Ig variant, LEA29Y (Belatacept). Two amino acid substitutions in Abatacept sequence increases the affinity for CD80 and CD86 with an increased bias toward CD86 blockade. Belatacept has now been approved for clinical use as an immunosuppressant

Cellular Immunology: Cell–Cell Interactions and the Immune System: CD28 Costimulation and Regulatory T Cells following kidney transplantation (Gardner et al., 2014; Melvin et al., 2012). Given the above discussions, it is interesting to reflect that removal of ligands by transendocytosis and blocking of ligands with Abatacept may ultimately be functionally equivalent strategies. Although ligand blockade has been widely accepted as a strategy for manipulating T cell responses, there are several issues that remain to be addressed. To begin with, it is widely appreciated that CD28 costimulation is necessary for generation and homeostasis of regulatory T cells. As such, reducing the number of Tregs by ligand blockade in patients with autoimmunity can be counter-productive. Therefore the impact on both Tregs and effector T cells may be significant for the outcome of the therapy. Another important issue is that CTLA4-Ig can only inhibit CD28-dependent responses and it is still unclear why some T cell responses appear to be less dependent on CD28 signaling (Gardner et al., 2014). As an alternative approach to ligand blockade CD28-blocking antibodies have been developed for induction of transplantation tolerance and treatment of autoimmunity (Poirier et al., 2012). The major challenge of using CD28 antibodies to suppress T cell activation is avoiding potential agonistic effects (Gardner et al., 2014), since CD28 agonists have had a somewhat chequered history. Initially, CD28 superagonistic antibodies were shown to induce immune tolerance by preferential expansion of FoxP3 þ Tregs in humanized mouse model of graft-versus-host disease (Kitazawa et al., 2009). This data along with numerous other studies have suggested that CD28 superagonistic antibodies may specifically target Treg cells in vivo and maintain their suppressive function. However in humans, CD28 superagonistic antibody (TGN1412) caused the rapid release of cytokines from effector memory T cells leading to multiorgan failure (Hunig, 2012). Despite this major setback, recent data again indicates that the selective expansion of Tregs may be possible by low dose of CD28 agonists (Tabares et al., 2014). Several CTLA-4-blocking antibodies have also been developed and efficacy as antitumor therapies has been tested with remarkable success. Here, the concept is that by blocking natural tolerance mechanisms to our own tissues it may be possible to unleash the immune system against tumors. Ipilimumab, which is the leading CTLA-4 antibody in its class, appears to be effective in treatment of melanoma with a small subset of patients demonstrating remarkable responses. Further understanding of the role of CTLA-4 on both Tregs and activated T cells in response to treatment will almost certainly lead to advancement of anti-CTLA-4 therapy (Callahan et al., 2010). The development of this CTLA4 blockade approach has now pioneered the rapid development of several tumor immunotherapy approaches (Pardoll, 2012).

Conclusions

•

CD28 signaling is essential for T cells activation, differentiation, and function. Moreover, given that T cells interact with a number of immune cell types, CD28 appears to be an apical control point for successful B cell, macrophage, and cytotoxic T cell responses.

• • • •

613

CTLA-4 functions as a direct regulator of CD28 signaling. CD28 and CTLA-4 bind the same ligands CD80 and CD86; CTLA-4 has higher affinity and avidity for both ligands. CTLA-4 is a highly endocytic protein and has a complex recycling behavior. CTLA-4 effectively can act as a ‘molecular hoover’ that removes CD80 and CD86 ligands from the surface of APCs, by ‘transendocytosis.’ By depleting ligands, CTLA-4 reduces the capacity of APCs to provide effective costimulation through CD28 and maintains immune tolerance.

See also: Cellular Immunology: Transcriptional Basis of B and T Cell Lineages and Memory Cells: T Follicular Helper Cells

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