CD28, CTLA‐4 and their ligands: who does what and to whom?

T-cell activation is a critical event in the organization of effective cellular and humoral immune responses. Activated T cells are essential for provision of T-cell help, promoting the development of high-affinity antibody production and the generation of cytotoxic T-cell responses. Accordingly, defects in proteins required for T-cell activation give rise to significant infectious pathology and malignancies. However, the decision to allow T-cell activation also has potentially dangerous consequences for the host and must therefore also be tightly controlled. Defects in proteins involved in regulating activated T-cell behaviour therefore tend to lead to autoimmunity. Thus, the major challenge faced in regulating T-cell responses is how to maintain a sufficiently large immune repertoire capable of recognizing all possible foreign antigens, whilst at the same time maintaining T cells in an unresponsive state towards self-antigens. In recent years significant progress has been made in our understanding of the mechanisms of self-tolerance. In the thymus it is clear that large numbers of potentially ‘self-reactive’ T cells are eliminated during negative selection in a process termed central tolerance. However, paradoxically, the process of positive selection that permits the expansion of T cells with low avidity for self–major histocompatibility complex (MHC) interactions must also lead to a degree of self-reactivity which is presumably tolerable in peripheral T cells. The question is how such T cells (albeit weakly self-reactive) can be ensured to remain non-reactive amongst a different array of self-antigens in the periphery. In the last few years a number of proteins have been identified that may serve the function of ‘quality controlling’ peripheral T-cell activation. This review focuses on two proteins, CD28 and cytotoxic T lymphocyte antigen-4 (CTLA-4), and explores how their interactions with their natural ligands may regulate the outcome of T-cell receptor engagement amongst peripheral T cells. CD28 and CTLA-4 (CD152) are transmembrane protein members of the immunoglobulin gene superfamily containing a single extracellular ‘V-like’ domain. 1–3 Both proteins are predominantly expressed by T cells and whilst CD28 is found in substantial amounts on the cell surface of the majority of resting T cells, in contrast CTLA-4 surface expression is much more limited. 4 The levels of CTLA-4 expression in most resting T cells are extremely low (or probably absent), and CTLA-4 predominantly appears following T-cell activation. However, despite maximal expression being reported at 48–72 hr post-activation, remarkably little stable surface CTLA-4 is found, although mRNA is equivalent to that of CD28.5,6 This lower level of cell-surface expression results from a motif in the cytoplasmic domain of CTLA-4 that facilitates its interaction with the clathrin pit adaptor complex (AP-50) causing its rapid internalization from the cell surface. 7–9 Consequently the majority of CTLA-4 is found in intracellular vesicles that may be then targeted to the cell surface at the site of T-cell receptor (TCR) contact. 10 It has been suggested that phosphorylation of the CTLA-4 cytoplasmic domain results in disengagement from the AP-50 internalization system and therefore stabilizes cell-surface expression. 8 The complexity of the CD28/CTLA-4 receptor interactions stems from the fact that there are two natural ligands CD80 (B7-1) and CD86 (B7-2) for these receptors. 11–17 Whilst these ligands can both interact with either receptor, they are only approximately 25% identical in sequence and it has therefore been attractive to speculate that they may serve different functions. Predictably, for co-stimulatory ligands, CD80 and CD86 are found on professional antigen-presenting cells such as dendritic cells, monocytes and activated B cells, although they can be induced on other cell types including T cells. 13,17–21 In general CD86 is the more abundant in terms of expression, and is increased more rapidly upon activation. In contrast CD80 is not generally found on resting antigen-presenting cells (APCs) and is induced more slowly upon cellular activation. A large variety of stimuli have been investigated in the control of CD80 and CD86 expression. Most of these, such as CD40, interferon-γ (IFN-γ), interferon-α (IFN-α), granulocyte–macrophage colony-stimulating factor (GM-CSF) and lipopolysaccharide (LPS) appear to result in increased expression 18,22–27 whereas others such as interlekin-10 (IL-10) and interleukin-4 (IL-4) may inhibit expression. 28–30 These expression studies, together with findings in CD80 and CD86 KO mice, 31–33 tend to indicate that CD86 is probably the major initial ligand for CD28 during T-cell activation, based mainly on its more rapid and abundant expression on APCs. However, functional data indicate that CD80 is probably the more potent CD28 ligand in terms of activation,34,35 which is consistent with affinity measurements. Whilst affinity estimates have varied, the interaction of CD80 with both CD28 and CTLA-4 (4 µm and 0·4 µm, respectively) appears substantially better than that of CD86 (approx. 15–40 µm and 4 µm, respectively), although overall these interactions are still relatively weak.36,37 An additional factor in these studies may relate to the fact that structural data indicate that CD80 is expressed as a dimer. 38 Thus in summary, CD28 can be considered a highly expressed but low-affinity receptor, whereas CTLA-4 is a low abundance but higher-affinity receptor where both receptors interact with CD80 and CD86. A diagram depicting these interactions in general is shown in Fig. 1. A schematic diagram of CD28 and CTLA-4 interactions with their ligands is depicted. CD86 is generally expressed at higher levels on antigen-presenting cells and is found more widely than CD80. CD80 is induced upon activation with a number of stimuli (see text) and is generally expressed at lower levels with later kinetics. Consequently, CD86 is the more likely primary ligand for CD28. On T cells CD28 expression is constitutive whereas CTLA-4 is not expressed by resting T cells. Both T-cell receptor stimulation and CD28 co-stimulation synergize to up-regulate CTLA-4, although CD28 stimulation is not essential. CTLA-4 expression is thought to be transient at the cell surface and rapidly re-internalized by a clathrin pit mechanism. It should be noted that both ligands can interact with both receptors. Current interest in the CD28 molecule stems from the concept that efficient activation of T cells requires signals from both the TCR and an additional co-stimulatory receptor. In the absence of this second signal, T cells either remain unresponsive or become actively tolerant to antigens. This concept was stimulated experimentally in a series of experiments by Jenkins et al., which involved the use of chemically modified peptide-pulsed APCs.39,40 These APCs were highly impaired in antigen presentation and T cells subsequently became unresponsive (T-cell anergy) to the same antigen. Anergy could also be prevented by provision of a co-stimulatory signal.41,42 Similar conclusions were reached in studies of transgenic expression of MHC molecules on non-APCs.43,44 Thus the concept of a ‘co-stimulatory signal’, which could rescue from anergy if provided at the same time as TCR engagement, began to emerge and was consistent with the functions of the newly identified CD28- ligand, CD80.12,25 In particular CD28 was shown to be important in enhancement of proliferation and cytokine production by T cells, as well as in the preventing T-cell anergy, thus identifying it as a key second signal for T-cell activation. 45–47 Whilst this two-signal model is likely to be an oversimplification (there are an increasing array of alternative co-stimulatory molecules) these studies provided impetus for the concept that T-cell tolerance in the periphery might be maintained by restricting the provision of co-stimulatory signals. This has resulted in the use of a recombinant molecule (CTLA-4–Ig) which acts as a high-affinity antagonist of both CD80 and CD86 co-stimulatory ligands. Results using this protein have demonstrated considerable potential for blocking CD28/CTLA-4 interaction with their ligands. 48–52 However, the mechanism by which CTLA-4–Ig works is not entirely clear, as both preventing T-cell activation and engineering tolerance are possible. In this regard several studies have indicated that CTLA-4 engagement may actually be necessary for tolerance induction (see below). 53–55 At first sight this appears to be at odds with data from CTLA-4–Ig treatment, which should theoretically remove the ability of CTLA-4 to interact with its only known ligands, CD80 and CD86. One possibility, is that the doses of CTLA-4–Ig used do not entirely blockade CD80/CD86 interactions, but selectively promotes CTLA-4–ligand interactions by restricting the amount of available ligand. Overall, in vivo studies have yielded impressive results in transplantation models and early results in human trials look encouraging. 56 Whatever the mechanisms, these studies have provided support for the view that one way to maintain peripheral tolerance is to limit the provision of co-stimulatory signals through CD80 and CD86. Despite considerable evidence that CD28 is critical in T-cell regulation, it is not entirely clear how its effects are mediated. The signalling pathways emerging from CD28 ligation have been studied in some detail, and have been reviewed elsewhere. 57 However, the absolute requirement for CD28 in vivo for T-cell proliferation has been brought into question by CD28KO mice. 58–60 Here, the response of T cells to antigen is not as severely impaired as might have been predicted. Nonetheless, there are substantial defects in the maintenance of responses and particularly in T-cell survival, which along with other studies supports a role for CD28 in maintaining T-cell responses. 61–64 Perhaps the most striking defect in vivo in CD28 KO mice is the lack of germinal centres, suggesting a gross defect in the ability of T cells to interact with B cells. This feature may well relate to the ability of T cells to express the chemokine receptor CXCR5 which is strongly influenced by CD28. 65 Thus CD28-deficient T cells may fail to migrate to appropriate sites of interaction with B cells. Mechanistically, there is evidence that CD28 may exert its co-stimulatory effects by lowering the threshold for T-cell activation, consistent with the presence of CD28 in lipid ‘rafts’ that are rich in signalling proteins.66,67 In addition, CD28 is also thought to exert effects on the cytoskeleton and promote its reorganization to the TCR contact site.68,69 One of the more interesting recent observations has been the suggestion that CD28 may be involved in the control of a population of CD25+ regulatory T cells. 70 Here, both CD28KO and CD80–CD86 double knockout (KO) mice crossed on to a non-obese diabetic (NOD) background demonstrated exacerbated diabetes that may be attributed to the lack of regulatory T cells. This study would suggest that CD28 co-stimulation may well be required for the proliferation and survival of this important T-cell subset. Overall, there is now an overwhelming body of data implicating CD28 as a critical molecule in the T-cell activation process and inhibition of CD28 functions can prevent or substantially decrease T-cell activation. However, some of these strategies are complicated by the fact that the same ligands also control the functions of CTLA-4, which appears to have opposite functions to CD28. Whilst studies on CD28 have demonstrated a co-stimulatory role in T-cell activation, the role of CTLA-4 has been more difficult to elucidate. It is now generally accepted that CTLA-4 plays a role in the inhibition of T-cell activation; 71–73 although there are some more controversial suggestions of a stimulatory role. 74 Nonetheless, several laboratories have shown that blocking CTLA-4 enhances T-cell proliferation whereas ligating CTLA-4 with agonistic antibodies suppresses T-cell proliferation, consistent with the function of a negative regulator. 75 However, the most compelling evidence for a regulatory function for CTLA-4 has come from CTLA-4 knockout mice that develop fatal lymphoproliferative disease at 3–4 weeks of age, suggesting a critical role for CTLA-4 in maintaining self-tolerance.76,77 This phenotype results from polyclonal activation of peripheral T cells that then infiltrate and cause multiorgan destruction. This disease can be effectively cured by preventing CD28 co-stimulation either using CD80 and CD86 double KO mice, CTLA-4–Ig78,79 or by crossing on to single TCR transgenic mice. 80–82 In addition the lymphoproliferation has been suggested to be CD4+ dependent. 83 Very recently, CTLA-4 blockade in normal mice has been shown to give rise to spontaneous autoimmunity. 84 Collectively, these studies suggest that one possible function of CTLA-4 may be to ‘threshold out’ weak TCR engagements that may exist for large numbers of potentially autoreactive circulating T cells. Thus in the absence of CTLA-4, B7 ligands provide unopposed stimulatory signals through CD28 that permit weakly self-reactive T cells to become fully activated. Whilst the importance of CTLA-4 is unquestioned, the nature of this inhibitory pathway is as yet poorly understood. Data from two laboratories indicate that CTLA-4 blocks T-cell function at a relatively early stage (within 24 hr), preventing up-regulation of activation markers, entry into cell cycle, and the generation of IL-2.72,73 However, most strikingly these functional effects are seen when surface levels of CTLA-4 are undetectable. Our own analysis of CTLA-4 expression in humans (unpublished data) support the view that resting T cells express little or no CTLA-4 but that CTLA-4 transcription can rapidly up-regulate the protein within 6 hr of activation. However, whether this up-regulation is sufficiently rapid to prevent activation, or whether expression is actually a reflection of activation, is not yet clear. Interestingly, where CTLA-4 protein is detected at later timepoints (24–72 hr) after activation, it is exclusively to T cells by are not through This the question as to whether CTLA-4 is highly efficient in extremely low amounts or whether there are alternative for this early inhibitory function (see and Fig. data using CTLA-4 antibodies it is important to that antibodies do not with in contrast with the natural ligands, and this may lead to inhibitory responses at lower levels of CTLA-4 expression. models of CTLA-4 function are The ligand model is This model requires that CTLA-4 is expressed at levels to ligands from CD28 thus preventing co-stimulatory signals from being This model not a CTLA-4 signalling and on the higher affinity of CTLA-4 for both ligands with CD28. In a CTLA-4 signalling model is CTLA-4 ligation results in signals that most likely inhibit T-cell receptor signalling in lack of activation signals. In the model a regulatory cell is stimulated CTLA-4 to exert other T cells. This of may be either by cell contact or inhibitory It should be noted that of these mechanisms is of the Whilst it is clear that natural ligands CTLA-4 function in the which CTLA-4 function have yet to be CTLA-4 regulate all types of T-cell stimulation or are its effects to of TCR the majority of T-cell experiments using ligands in have indicated that engagement of CD80/CD86 in the presence of effectively signals CD28 with relatively little evidence for functions these The data provide a model in which CD28 enhances and CTLA-4 T-cell responses yet both interact with the same ligands. This the question of do T cells using CD28 and The to this question to a large on which models of CTLA-4 function are being are it should be noted that of the models is and all of these may One of the most models of CTLA-4 is the concept that CTLA-4 may as a for the ligands required for CD28 activation Fig. Thus, by of its higher CTLA-4 should be capable of CD28 for ligand An of this is that interactions would be where the levels of ligands are low on resting the concept that low levels of ligand are to control of CTLA-4 has yet to be Our own experiments at this et al., do not generally support this Nonetheless, the is the of CD28 and CTLA-4 for their ligands. some to this is to have a significant in regulating the activation of resting T cells as these express levels of CTLA-4, highly at this initial In activated cells express more CTLA-4 and therefore ligand possible during stimulation of T cells with the expression of intracellular CTLA-4 at the 10 This concept is also by several studies that indicate more impressive CTLA-4 effects on In addition, support for ligand from studies of CTLA-4 KO mice that have been made transgenic for a CTLA-4 protein a cytoplasmic domain. Here, some but not all of the of CTLA-4 KO mice were suggesting that is a mechanism that for only some of the of CTLA-4 Whilst the control of CTLA-4 expression is in this it that for ligand can be an effective of CTLA-4 The most with the model is the fact that agonistic antibodies to CTLA-4 as potent of T-cell 75 as there are no ligands in this be the of CTLA-4 This support for a CTLA-4 signalling mechanism. support for a CTLA-4 signalling model Fig. most from studies with agonistic Whilst signalling studies have been by the low level of expression of this generally these indicate that CTLA-4 can with signals and early signalling The fact that CTLA-4 function can be in the absence of CD28 expression also supports the view that CTLA-4 may regulate TCR signals. One is that CTLA-4 a through a motif in the cytoplasmic domain. This interaction is then thought to be involved in the TCR signalling blocking early activation signals. However, in contrast to the models where in the CTLA-4 cytoplasmic domain are required for of signalling several recent studies have also shown that are not essential for CTLA-4 Thus at the nature of CTLA-4 inhibitory signalling mechanisms are still Whilst it is not a of the signalling the most of the signalling is that T cell a decision based on the in of CD28 CTLA-4 signals where CTLA-4 signals are a T cell not be activated to inhibition of TCR and CD28 activation However, this is not the only possible of CTLA-4 signalling as One possible of the effects of CTLA-4 when CTLA-4 expression levels are is that these effects are actually by a number of regulatory cells that express CTLA-4 Fig. This is by a number of recent findings and some of the a recent study by demonstrated that CTLA-4 functions were not T-cell Here, CTLA-4 KO was into mice either or with CTLA-4 Whilst fatal T-cell of could an ability of cells to regulate CTLA-4 negative cells. studies have also suggested a CTLA-4 and the cytokine This is consistent with KO mice and CTLA-4 KO mice, although this is still controversial at with this overall concept is the relatively recent of regulatory T cells that cause the development of when review recent studies, and our own suggest that that a relatively number of CD4+ CD25+ T cells may also express CTLA-4 Thus, the concept that important CTLA-4 functions may be by a of regulatory T cell that then the function of the majority of other T cells is now a that CD28 and CTLA-4 have such functions it is therefore that they should ligands. The most and attractive is that these ligands have and yet to this has been extremely difficult to there are studies that indicate these ligands. studies using no and were consistent with the that both ligands are capable of co-stimulation CD28. However, in CD80 appears to be the more potent stimulatory ligand. studies that have to the ligands have this has yielded In disease studies, blocking either molecule can disease or inhibit disease on the model These and other studies also provide evidence that CD80 or CD86 can towards T or T However, whilst there is no overall there are to suggest that these pathways do a role in T-cell One of the major in these studies is that a and highly of of of of at extremely It is therefore likely that in different are being which then the of the functions of CD80 and CD86. the different models for CTLA-4 then or all of these mechanisms may be by CD80 or and it is therefore necessary to which mechanism is being studied in to the role of CD80 and CD86 have been made using and in particular KO mice. 31–33 Here, the of CD80 and CD86 have been as and the more initial functions of CD86 have been These studies also the concept that either CD80 or CD86 is required for or cytokine However, a different with KO for CD28/CTLA-4 ligands in that by a ligand, both the stimulatory and inhibitory functions of that ligand are It is therefore theoretically possible that these two effects may for other and not the functions of a ligand. based on affinity CD80 is both the ligand for CD28 and it is that CD80 do not a CTLA-4 phenotype the interactions has also been Thus, CD80 and CD86 are an function of their interactions with both CD28 and CTLA-4, and therefore requires study of both pathways Whilst it is difficult to clear have been that could support a model where CD80 might be considered a not inhibitory ligand for the early expression of CD86 on APCs would generally promote the initial of T-cell whereas the later and generally more expression of an inhibitory CD80 molecule could then potentially regulate responses blocking antibodies to CD80 diabetes in mice. This model of disease is also known to be by CTLA-4 at an early stage and T-cell is exacerbated by CTLA-4 in a transgenic model of CD86 expression T-cell whilst CD80 In this if one that CD80 is a more potent this would for an additional role for CD80 in T-cell that is not seen in the CD86 in CD80 has been indicated to be the CTLA-4 ligand An interesting feature that these studies is that CTLA-4 function is being studied in the of spontaneous to or and that T-cell responses are not the result of an in the presence of as in some One is that these studies may all a consistent and of CTLA-4 function that may contrast with its role in the presence of or during activation of the immune structural data suggest that CD80 is as a which has not been shown for a factor that may its ability to signal 38 transgenic expression of CD80 and CD86 on T cells results in only in the CD86 but not in CD80. this would be consistent with a lack of regulatory ability within CD86. with this transgenic expression of CD80 has been shown to provide a negative regulatory our own recent data et al., CD80 and CD86 only CTLA-4 inhibition when it is to CD80 and not CD86. Whilst these are highly and alternative are they serve to that there is still little data that one ligand functions in to Whilst it is clear that both ligands can and do interact with both their mechanisms of are thus not the that several of CTLA-4 function it that models where the of T by CD28 or CTLA-4 is it to be difficult to CD80 and CD86. The CD28/CTLA-4 pathway important as for and into our ability to self-antigens. Whilst our understanding of these molecules has in the last few significant still remain two ligands receptors that have functions. The still in this are in of more models of the functions of of the in to this important