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Costimulation of T cells by OX40, 4-1BB, and CD27
Cytokine & Growth Factor Reviews 14 (2003) 265–273
Costimulation of T cells by OX40, 4-1BB, and CD27 Michael Croft Division of Molecular Immunology, La Jolla Institute for Allergy and Immunology, San Diego, CA 92121, USA
Abstract Costimulatory signals have been defined as signals brought about by ligation of membrane bound molecules that synergize with, or modify, signals provided when the T cell receptor engages peptide–MHC complexes. In large part, costimulatory signals are essential for many facets of a T cell response, and the general rule is that without these signals, a T cell is ineffective and may often succumb to death or become unresponsive. Until recently, costimulation has been dominated by studies of the Ig superfamily member, CD28, a constitutively expressed molecule that is required to initiate a majority of T cell responses. However, growing evidence over the past few years has now shown that several members of the TNFR family, OX40 (CD134), 4-1BB (CD137), and CD27, are equally important to the effective generation of many types of T cell response. In contrast to CD28, these molecules are either induced or highly upregulated on the T cell surface a number of hours or days after recognition of antigen, and appear to provide signals to allow continued cell division initially regulated by CD28 and/or to prevent excessive cell death several days into the response. An argument can be made that these molecules control the absolute number of effector T cells that are generated at the peak of the immune response and dictate the frequency of memory T cells that subsequently develop. The exact relationship between OX40, 4-1BB, and CD27, is at present unknown, including whether these molecules act together, or sequentially, or control differing types of T cell response. This review will focus on recent studies of these molecules and discuss their implications. © 2003 Elsevier Science Ltd. All rights reserved. Keywords: Costimulation; T cells; Ligation
Contents 1. Introduction: the role of costimulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Expression of OX40 (CD134) and OX40-ligand . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Expression of 4-1BB (CD137) and 4-1BB-ligand . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Expression of CD27 and CD27-ligand (CD70) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. Functional studies of OX40. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6. Functional studies of 4-1BB . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7. Functional studies of CD27. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8. Signals transmitted through OX40, 4-1BB, and CD27 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9. Spatio-temporal use of OX40, 4-1BB, and CD27 by CD4 and CD8 cells . . . . . . . . . . . . . . . . . . . . . 10. Spatio-temporal use of OX40, 4-1BB, and CD27 in Th1/Tc1 or Th2/Tc2 responses . . . . . . . . . . . 11. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Introduction: the role of costimulation The list of membrane bound molecules that can positively affect a T cell and enhance activation, division, survival, and cytokine secretion, has steadily been growing over the past 10 years. These molecules largely fall into three main groups, namely Ig superfamily members, TNFR superfamE-mail address: [email protected] (M. Croft).
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ily members, and cytokine receptors. CD28, ICOS, and CD2 typify costimulatory molecules of the Ig superfamily and recent reviews have detailed their importance in many immune responses driven by T cells. Cytokine receptors that can control T cell growth or survival in some situations are also numerous and include IL-2R, IL-7R, IL-15R, IL-1R, and IL-6R. Lastly, signals through a number of TNFR family members have also been shown to augment T cell responses in various settings and these include OX40 (CD134), 4-1BB
1359-6101/03/$ – see front matter © 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S1359-6101(03)00025-X
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Fig. 1. Members of the TNF/TNFR family implicated in costimulation of T cells. The Type I transmembrane proteins of the TNFR family are on the left with their characteristic cysteine-rich domains depicted. The ligands, the Type II transmembrane proteins of the TNF family, are on the right. The chromosomal location of the human genes are indicated. The interactions of CD30 with CD30L and HVEM with LIGHT can also costimulate T cells and may be functionally analogous to OX40, 4-1BB, and CD27.
(CD137), CD27, and CD30 and HVEM (Fig. 1). Because of this plethora of molecules that can be classified as costimulatory, a number of issues have arisen. Firstly, are all molecules required for every T cell response or does some degree of redundancy exist. Secondly, do all molecules perform similar functions, or can separate functions be ascribed to individual molecules or particular families of molecules. Thirdly, can a fully effective (protective or pathogenic) T cell response occur in the absence of signaling from one or a number of these molecules and if so, can we determine which are the relevant molecular interactions to target (positively or negatively) in terms of providing therapeutic benefit in varying disease scenarios. If there is little redundancy and most if not all of the above molecules are required for a T cell response that leads to effective and long-lived memory, several non-mutually exclusive models can be put forward to explain the need for multiple costimulatory signals. Firstly, a “simultaneous engagement” model would suggest that signals from two or more costimulatory molecules are required at the same time or within a brief time frame for maximal short-term response. Secondly, a “temporal engagement” model would include signaling from two or more molecules being required one after the other over an extended time frame thereby ensuring a long-lasting response. Thirdly, a “spatio-temporal engagement” model envisions that two or more molecules signal simultaneously or one after the other, but there is differential use between CD4 versus CD8 T cells, and/or depending on particular antigenic or inflammatory conditions that may favor expression of certain receptors on the T cell or expression of their membrane bound and soluble ligands. This review will not directly provide answers to all of these questions but will attempt to put some perspective on how OX40, 4-1BB, and CD27 may fit into these models.
2. Expression of OX40 (CD134) and OX40-ligand OX40 was originally identified in 1987 by an antibody that reacted with activated rat CD4 T cells [1]. It is often quoted that OX40 is primarily expressed on CD4 T cells, however CD8 T cells can also bear OX40 at least under certain conditions. Moreover, OX40 has now been visualized
on other more diverse cell types including B cells, dendritic cells, and eosinophils (Croft et al., unpublished observations), although as yet the physiological significance of this expression is unknown. OX40 is not expressed on resting T cells. It can be induced by TCR/CD3 signals in isolation and initially appears 12–24 h after stimulation of na¨ıve cells. Peak expression is seen after 2–3 days and then OX40 is downregulated, implying a delayed mode of action. Antigen-experienced effector/memory T cells can rapidly re-express OX40 within 4 h of reactivation [2]. OX40 has been visualized in vivo in T cell zones of spleen or lymph nodes several days after immunization with protein antigen, directly coinciding with the peak of the primary T cell response [3,4]. Activated T cells expressing OX40 have also been found in peripheral inflammatory sites in a number of disease scenarios such as in the CNS in mice undergoing EAE [5]; blood of mice and humans undergoing GVHD [6,7]; the sites of growth of a number of tumors [8]; synovial fluid from patients with chronic synovitis, and joints of mice undergoing rheumatoid arthritis [9,10]; lamina propria of mice undergoing colitis, and gastrointestinal tract samples from patients with Celiac and Crohn’s disease [11,12]; lymph nodes draining sites of growth of Leishmania major [13]; and lungs of mice with allergic asthma [14]. OX40-ligand is expressed on professional antigenpresenting cells (APCs) such as dendritic cells, B cells and macrophages many hours to days after activation [15–17]. Interestingly, it was first identified on the surface of HTLV-infected leukemic T cells [18]. Expression on non-transformed T cells has been seen but appears to be rare [19,20], although this observation still raises the possibility of a role in T cell–T cell interactions rather than T cell–APC interactions. In the case of dendritic cells and B cells, Toll-like receptor signals induced by LPS can promote OX40L expression in addition to contributions from Ig signals and CD40 signals [15–17]. Additionally, OX40L has been visualized on activated endothelial cells in vitro, and in tissues from patients with lupus nephritis and inflammatory bowel disease, implying a role in promoting migration of OX40-expressing T cells into inflamed tissues, or providing signals to T cells to augment their activity in these peripheral sites [21–23].
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3. Expression of 4-1BB (CD137) and 4-1BB-ligand 4-1BB was discovered in 1989 after screening cDNA libraries from activated CD4 and CD8 T cell clones [24]. 41BB is not constitutively expressed on resting T cells and like OX40 is induced within 24 h after activation, peaking several days after initial stimulation. Reagents that cross-link the TCR or CD3 are sufficient to induce 4-1BB on both CD4 and CD8 cells [25–28]. 4-1BB expression is not exclusive to T cells and has been detected on B cells, macrophages, dendritic cells, NK cells, and eosinophils, although the physiological significance on these cell types also remains to be resolved [26,29–31]. In vivo expression analyses of 4-1BB have been limited with 4-1BB visualized on T cells from peripheral blood of HIV-infected patients [32] and in T cells infiltrating cardiac allografts [33]. However, as described below a number of in vivo functional analyses suggest that 4-1BB is expressed in many responses involving T cells including those to tumor antigens, to alloantigens, and to viruses. Similar to OX40L, 4-1BB-ligand can be expressed on dendritic cells, B cells, and macrophages, and again is induced hours or days after activation, and can be regulated by LPS, Ig or CD40 signals [31,34,35]. Thus, both expression of OX40 and 4-1BB, and OX40L and 4-1BBL can parallel each other under certain conditions on activated T cells and activated APCs, suggesting that there may be similarities in their roles and in the responses in which they participate. 4. Expression of CD27 and CD27-ligand (CD70) CD27 was originally identified with antibodies that bound to a large proportion of peripheral blood CD4 and CD8 T cells [36,37]. As such this distinguishes CD27 from OX40 and 4-1BB in that the latter have to be induced rather than being present on resting T cells. However, similar to these molecules, CD27 is strongly upregulated by reagents that cross-link the TCR or CD3, although peak levels of expression may occur within 24 h, somewhat earlier than maximal expression of OX40 or 4-1BB [38,39]. CD27 can also be irreversibly lost on a subset of long-term repeatedly stimulated T cells, correlating with negative expression on small numbers of memory phenotype cells [38]. Whether similar subsets of cells exist that cannot re-express OX40 or 4-1BB has not been investigated, nor whether the CD27 negative T cells are OX40 or 4-1BB positive. This nevertheless remains an intriguing possibility, and suggests the possibility that segregation in use of these molecules may occur with long-term differentiation or after repeated encounters of T cells with antigen. The ligand for CD27 (CD27L or CD70) is also inducible on professional APC, similar to OX40L and 4-1BBL, although it may be predominantly expressed on B cells and not dendritic cells [40–42]. Similar to OX40L, CD70 has additionally been found on activated T cells, leading to the
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suggestion that CD27–CD70 interactions may also be involved in direct cellular communication between subsets of T cells as well as between T cells and APCs [43,44]. In summary, there appears to be much overlap in the expression patterns of OX40/OX40L, 4-1BB/4-1BBL, and CD27/CD70, with all receptor/ligand pairs expressed on activated T cells and activated APC at least under certain conditions. This raises the possibility of cooperation between these molecules and that each interaction may contribute to effective T cell priming. As each molecular pair are generally studied in isolation, it remains to be determined both in vitro, and in specific immune responses in vivo, whether there is direct overlap in expression or whether distinct subsets of T cells and APC can be visualized that express only one particular ligand or receptor. Ultimately, such studies will be essential to determining the physiological role of these molecules in T cell immunity and their relationship with one another.
5. Functional studies of OX40 A large number of in vitro studies that will not be detailed here, using either receptor specific antibodies or cells transfected with individual ligands, have shown that signals through OX40, 4-1BB, or CD27 can augment T cell responses, either in isolation or in combination with CD28 signals from B7. Although multiple activities have been described such as enhancing proliferation, cytokine secretion, and cell survival, these studies in minimalist systems do not necessarily convey the physiological function of the molecules when their signals are provided in the context of multiple signals from other cell surface receptors or from soluble cytokines. Recent studies of knockout animals, with antagonist and agonist reagents in vivo, or receptor-deficient T cells, have aided tremendously in not only defining the importance of these molecules in T cell responses, but also their physiological mode of action. Studies of OX40- and OX40L-deficient mice initially showed that CD4 responses to the viruses lymphocytic chriomenigitis virus (LCMV) and vesicular stomititis virus (VSV), to a common protein antigen, and in contact sensitivity reactions to haptens, were markedly reduced in vivo, and in vitro [17,45–47]. Further in vivo data in OX40-deficient mice provided the first indication of function, demonstrating a deficiency in numbers of antigen-specific CD4 T cells generated late in the primary response, and additionally after 5 weeks when the T cell memory population was formed [4]. Other data in which agonist anti-OX40 reagents were injected shortly after immunization augmented tremendously the number of antigen-reactive CD4 cells that accumulated over time [4,48]. Similarly, transgenic expression of OX40L on dendritic cells led to greater numbers of primed CD4 cells [49] and blocking OX40L in another model reduced accumulation of CD4 cells [50]. Collectively, these studies have suggested that a major role of OX40–OX40L
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Fig. 2. Temporal model of the roles of OX40, 4-1BB, and CD27 in T cell responses. Both OX40 and 4-1BB can provide anti-apoptotic signals several days after a na¨ıve T cell encounters antigen. These signals allow continued turnover of cells and provide survival signals to prevent excessive T cell death at the peak of the primary response. Both molecules appear to act in a temporal manner after CD28 signals are initially provided to the T cell. The role of CD27 is not as clear. As it is constitutively expressed on a na¨ıve T cell, it may participate in regulating initial cell division and clonal expansion as well as having the potential to promote survival late in the response.
interactions is to dictate the number of effector T cells that accumulate in primary immune responses, and consequently to govern the number of memory T cells that subsequently develop and survive. Further studies using antigen-specific TCR transgenic CD4 cells lacking OX40 have now provided greater insight into the mechanism of action [51]. These studies have shown that OX40 contributes little to the initial response of a CD4 cell, and minimally impacts cytokine production or early proliferation. This directly contrasts with the role of CD28, which is required for much of this early response. OX40-deficient T cells demonstrated reduced proliferation 4–5 days into the response, and a large proportion of these T cells could not survive over the long-term. The lack of survival was shown to be due to apoptotic cell death and could be rescued by inhibitors of the caspase cascade [51]. OX40 expression is not dependent on CD28 signals, but several systems have shown that CD28 can augment the level of OX40 expressed on a T cell [50,51], reinforcing the concept that the two molecules most likely co-operate together in a sequential manner. Therefore, to summarize, a model can be proposed whereby OX40 signals act in a temporal manner after CD28 signals, and allow effector T cells to survive and continue proliferating late in response, predominantly by transmitting anti-apoptotic signals that prevent excessive T cell death (Fig. 2).
signals additionally reduced the antigen-specific population that was generated. These studies have been supported by reports with either agonist anti-4-1BB antibodies or with tumor cells transfected with 4-1BBL. In all cases, greater CTL responses were revealed, and in some this was directly correlated with the absolute number of antigen-specific CD8 T cells being increased [55–58]. Of significance, in relation to studies of OX40, an agonist 4-1BB antibody was shown to prevent death and deletion of superantigen-stimulated CD8 cells [57], and a blocking 4-1BB–Fc fusion protein, which acts as a soluble decoy for the ligand, was shown to not affect the initial proliferative response of TCR transgenic CD8 cells responding to peptide in adjuvant, but inhibited the accumulation of effector T cells at the peak of the primary response [59]. The latter study additionally demonstrated
6. Functional studies of 4-1BB An understanding of the role of 4-1BB in T cell responses has largely centered on CD8 cells as opposed to CD4 cells for OX40. Initial reports of 4-1BBL-deficient mice responding to the viruses LCMV and influenza demonstrated that 2–10-fold fewer antigen-reactive CD8 cells were generated in primary responses, and fewer memory T cells developed weeks after priming [52–54]. Furthermore, preventing CD28
Fig. 3. Spatio-temporal model of regulation of CD4 and CD8 T cells by OX40, 4-1BB, and CD27. The majority of data indicates that OX40 is important for many CD4 T cell responses, whereas most data to date on 4-1BB has been directed to CD8 T cells. It is a possibility that differential use of OX40 and 4-1BB by CD4 and CD8 cells may be characteristic of many immune responses and explain their apparent overlap in function and potential redundancy. If CD27 largely regulates clonal expansion as opposed to survival, it could be equally involved in the initiation of both CD4 and CD8 responses.
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that increased levels of apoptotic CD8 cells resulted when 4-1BB signals were absent, and that apoptosis occurred in T cells that had already divided many times [59]. Although CD28 signals are also not required for 4-1BB expression, as for OX40, they have been shown to augment expression in several systems [27,60,61]. Moreover, direct comparison of influenza-specific CD8 responses in the absence of CD28 signals versus 4-1BB signals additionally implicated 4-1BB as acting in a temporal manner to promote survival and accumulation of T cells late in primary responses after CD28 signals regulated the initial phase of response [62]. Thus, an argument can be put forward that 4-1BB acts in a CD8 response, much like OX40 in a CD4 response, with both molecules being essential for the accumulation and survival of large numbers of antigen-primed T cells over time (Fig. 3).
7. Functional studies of CD27 Although a number of in vitro studies with agonist antibodies to CD27, or with CD70 transfectants, have demonstrated a strong costimulatory effect on T cell proliferation [44,63,64], in vivo studies have lagged behind those of OX40 or 4-1BB. Recently, CD27-deficient mice were generated and shown to be defective in both CD4 and CD8 responses to influenza virus, and similar to OX40 or 4-1BB, exhibited reduced numbers of antigen-specific memory T cells [65]. These observations have been supported in mice transgenic for CD70 expressed on B cells, which also have increased numbers of memory/effector phenotype T cells [66], and by studies of CD70-transfected tumor cells that led to augmented priming of both CD8 and CD4 cells [67–69]. At what stage in the T cell response CD27 acts is unclear, nor whether it is temporally regulated in comparison to CD28, nor whether its major effect is on controlling initial T cell proliferation, or also preventing apoptotic cell death as is the case for OX40 and 4-1BB. Because CD27 is constitutively expressed on a resting T cell and upregulated earlier than OX40 or 4-1BB, and as the majority of studies in vitro have shown strong effects on T cell proliferation, it is a possibility that CD27 functions either at the same time as CD28, or in a temporal manner between CD28 and OX40 or 4-1BB. If this is the case, the principle effect of CD27 costimulation could be to contribute to primary clonal division and expansion, rather than being essential for T cell survival after this expansion phase (Figs. 2 and 3). Future studies tracking the kinetics of antigen-specific T cells in vivo while blocking CD27–CD70 interactions compared to CD28/B7, or with CD27-deficient transgenic T cells, will be required to provide answers to these questions.
8. Signals transmitted through OX40, 4-1BB, and CD27 Although studies of signaling pathways induced by OX40, 4-1BB, and CD27, are in their infancy, a number of
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reports have revealed potential mechanisms of action and cross-regulation between these molecules, and enhanced the conclusions gained from in vivo studies. Each receptor can bind to several of the six known TNFR-associated factors (TRAFs), a family of RING finger proteins, however the data to date suggests that TRAF2 may be central to signal transduction by all three [70–74]. Whether this indicates a common functional action is not clear, but additionally all molecules have been shown to be capable of activating NF-B and JNK [70,72–74], suggesting that there is a strong potential for providing similar functional consequences. Moreover, recent data on the downstream targets of OX40 and 4-1BB have now added to the contention that these molecules may be functionally analogous. Initial studies of OX40 showed that OX40-deficient CD4 cells were incapable of maintaining high levels of the anti-apoptotic proteins Bcl-xL and Bcl-2 several days after activation [51], as well as another Bcl-2 family member, Bfl-1 (Song and Croft, unpublished). Similarly an agonist anti-OX40 augmented expression of these molecules in a CD28-independent fashion [51]. The results were additionally emphasized when defective survival of OX40-deficient T cells was completely prevented by retroviral transduction of these cells with either Bcl-xL or Bcl-2 genes. This study has now been followed recently by two reports of CD8 cells stimulated with agonist anti-4-1BB antibodies that also showed a strong upregulation of Bcl-xL and Bfl-1 expression, and a direct correlation with increased survival and reduced apoptosis [75,76]. Thus, both the molecular and cellular studies of OX40 and 4-1BB show similar if not identical effects on CD4 and CD8 cells respectively. To date, there is no information on whether CD27 also regulates the Bcl-2 family of proteins, although given the similarities in use of TRAF2, NF-B, and JNK, and some overlapping functional data, it is
Fig. 4. Signaling pathways induced by OX40, 4-1BB, and CD27. All three molecules can bind TRAF2 and functional data suggest that TRAF2 is responsible for many of the activities induced by ligating these receptors. NF-B and JNK can also be activated by all three receptors, although no data is available to show what are the functional consequences of activating these pathways. Both OX40 and 4-1BB can upregulate the expression of the anti-apoptotic members of the Bcl-2 family and block programmed cell death due to cytokine/antigen withdrawal. It is likely that NF-B will mediate these activities but awaits a direct demonstration. It is not clear if Fas- or TNFR1-induced death can be inhibited by OX40 or 4-1BB signals, but is a distinct possibility. No data is available on whether CD27 also positively regulates Bcl-xL, Bcl-2, or Bfl-1.
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highly likely that these proteins may also be targets of CD27 (Fig. 4).
9. Spatio-temporal use of OX40, 4-1BB, and CD27 by CD4 and CD8 cells Given the similarities in functional properties that have been reported for OX40, 4-1BB, and CD27 to date, several questions are raised. Firstly, do these molecules indeed have an identical role in T cell responses, and is it the sum of their signals that is critical to a T cell, rather than the individual signals themselves. If this is the case, an individual T cell should express all molecules either at the same time, or in a temporal fashion during the lifespan of the T cell. In vitro, we know that simultaneous expression of all three molecules can occur on an activated T cell, but whether or not there is really a kinetic difference in expression has not been addressed. Ultimately, however, this type of question can only be definitively answered by analyzing T cells in vivo in varying types of immune responses. Another question is whether the apparent overlap in function can be explained by differential usage by for example CD8 cells versus CD4 cells. As highlighted above, the majority of data on OX40 has been gathered on CD4 cells, and the majority of data on 4-1BB on CD8 cells, whereas that on CD27 is equally spread between CD4 and CD8. As CD27 is constitutively expressed on na¨ıve T cells at low levels, and assuming that CD27 is found to be important for initial clonal expansion in vivo as implied from in vitro studies, one model could then be a combination of temporal use and spatio-temporal use. For example, CD27 may equally contribute to the expansion phase of both na¨ıve CD4 and CD8 responses, and then OX40 is required for the subsequent survival of CD4 effector cells whereas 4-1BB is required for the survival of CD8 effectors (Fig. 3). The evidence from the viral studies with LCMV and influenza at least partially fit this model, i.e. defective CD4 and CD8 priming were seen in CD27 knockout animals [65], whereas only defective CD4 responses were seen in OX40-deficient animals [45] and only defective CD8 responses in 4-1BBL-deficient animals [53]. Although the model presented in Fig. 3 is attractive, there is not enough information from different systems to determine whether it can be applied to all CD4 and CD8 responses, and most likely it will not translate to every type of situation. Several studies have implied effects of OX40 on CD8 cells and 4-1BB on CD4 cells, but have fallen short of directly showing this. Data with agonist antibodies to OX40 in tumor models have shown enhancing effects on CTL generation [77,78], however it was not determined whether CD8 cells were direct targets or OX40 signals simply augmented CD4 cell help. Similarly, several tumor studies of 4-1BB have shown a requirement for CD4 cells during 4-1BB-induced anti-tumor responses [55,56], but no direct evidence has been provided as yet to show that
4-1BB was required by the CD4 cells themselves. In vitro, several reports have demonstrated that 4-1BBL can costimulate some CD4 responses [28,60] showing the potential of cross-regulation via 4-1BB, and a recent study of aged CD4 T cells in vivo also showed that their defective response could be restored with agonist anti-4-1BB [79]. However, to date only one study has presented more definitive evidence that 4-1BB can naturally contribute to a CD4 response. This data demonstrated that CD4 cells from 4-1BB-deficient animals were strongly impaired in their ability to mediate GVHD in adoptive transfer experiments [80]. At present, there is no published data on whether OX40 signals normally control CD8 cells, although another recent study showed that agonist anti-OX40 could strongly augment CTL priming induced by dendritic cells presenting only class I-restricted peptides [81]. This is suggestive evidence for an action on CD8 cells, although because of OX40 expression on non-T cells, definitive evidence will have to await the production and analysis of OX40-deficient CD8 T cells. In summary, there appears to be an overall bias for OX40 use by CD4 cells and 4-1BB use by CD8 cells, although it is unlikely that this will be absolute. Cross-regulation may be dictated by the inflammatory conditions encountered by CD4 and CD8 cells that could impact expression of receptors or ligands, rather than the T cell subtype that is responding.
10. Spatio-temporal use of OX40, 4-1BB, and CD27 in Th1/Tc1 or Th2/Tc2 responses Finally, a further question that needs to be addressed in future studies is whether the apparent overlap in function between OX40, 4-1BB, and CD27, can be explained by a differential requirement for these molecules during Type 1 versus Type 2 immune responses. Type 1 immune responses are dominated by the cytokines IFN-␥ and TNF whereas Type 2 responses involve the cytokines IL-4, IL-5, IL-9, and IL-13. Such distinct cytokine patterns can be produced by either CD4 cells (Th1 versus Th2) or CD8 cells (Tc1 versus Tc2). Although there are some reports in vitro that lean toward OX40, 4-1BB or CD27 regulating one response or another, these are again questions that ultimately need to be answered in studies of in vivo responses. Early data on OX40 in vitro implied that this molecule was preferentially involved in Th2 responses [82,83]. Studies in vivo in models of Leishmania in BALB/C mice [13] and in allergic asthma [14] have confirmed a major role for OX40 in at least these Th2 responses. However, more basic in vivo studies of OX40- and OX40L-deficient animals have shown reductions in both Th2 and Th1 cytokine responses [4,17], and applied studies of experimental autoimmune encephalitis (EAE) [84,85], rheumatoid arthritis [10], and colitis [11,86] have also shown that blocking OX40–OX40L interactions can strongly inhibit these diseases and their associated Th1 cytokines. Thus, OX40 appears to be involved in priming for both Th2 and Th1 responses, but a question
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still remains as to whether there may be a bias later in the response, for example when T cell memory is formed, or in situations where multiple encounters with antigen occur. Less is known about the participation of 4-1BB in Type 1 versus Type 2 responses. As most CD8 responses are Tc1-like, it can be concluded from the multiple studies described above that 4-1BB contributes to these responses. Whether 4-1BB also regulates many Th1 responses remains to be determined. In vitro experiments with human CD4 cells have indicated this potential [87], as has an in vivo study where anti-4-1BB augmented priming of aged T cells for Th1 cytokine production [79]. Few studies have attempted to determine if 4-1BB participates in either a Th2 or Tc2 response, although one report with anti-4-1BBL demonstrated no effect on a Leishmania T cell response in BALB/C mice in a situation where anti-OX40L completely blocked Th2 cytokines [13]. Thus, it is possible that 4-1BB may largely contribute to Th1/Tc1 responses, but many more studies are needed before any definitive conclusions can be drawn. Lastly, there is even less physiologic data to indicate whether CD27 could be differentially utilized in Type 1 versus Type 2 responses. Analyses of CD70 transgenic animals showed an increased production of IFN-␥ [66] suggestive of an effect on Type 1 responses. In vitro studies ligating CD27 on T cells have also leaned towards this conclusion with little effect on IL-4 production being seen [64,88], and exogenous IL-4 can negatively regulate CD70 expression on B and T cells in vitro [89,90]. Whether there is a true preference for CD27–CD70 interactions in Type 1 responses awaits analysis of in vivo Th2 or Tc2 responses.
11. Summary In conclusion, there is now extensive evidence that OX40, 4-1BB, and CD27 are integral to the effective development of a number of T cell responses. The majority of data suggest that the predominant role of these molecules is to regulate the absolute number of antigen-specific effector T cells that are generated late in primary T cell responses and to control the number of memory T cells that develop. Whether all three receptor systems are functionally equivalent still remains to be determined. However, growing evidence suggests that there is significant overlap in the activities and the roles of OX40, 4-1BB, and CD27 in T cell priming, and that the major mechanisms of action are either through promoting cell division or suppressing cell death. While there is now good evidence that OX40 and 4-1BB provide anti-apoptotic signals in vivo, and that the Bcl-2 family of proteins are some of their targets, it is not clear whether CD27 also plays a role in T cell survival. Moreover, questions still exist regarding whether OX40, 4-1BB, or CD27 contribute to augmenting T cell proliferation in vivo, and whether proteins involved in cell cycle regulation are also direct targets of their signals. Finally, given the apparent similarities in function, future studies are needed to define the relationship of OX40
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to 4-1BB, and these molecules to CD27. It is possible that each acts independently at different stages in the T cell response, or in different types of response mediated by CD4 or CD8 cells, or involving Type 1 or Type 2 cytokines. Alternatively, it is possible that OX40, 4-1BB, and CD27 act together, at the same time, or within a short time frame, and that a T cell is only signaled efficiently when all molecules are engaged. Answers to these questions will have to await expression studies in multiple types of T cell response, and the functional analysis of T cells made deficient in one or more molecule.
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Costimulation of T cells by OX40, 4-1BB, and CD27 Michael Croft Division of Molecular Immunology, La Jolla Institute for Allergy and Immunology, San Diego, CA 92121, USA
Abstract Costimulatory signals have been defined as signals brought about by ligation of membrane bound molecules that synergize with, or modify, signals provided when the T cell receptor engages peptide–MHC complexes. In large part, costimulatory signals are essential for many facets of a T cell response, and the general rule is that without these signals, a T cell is ineffective and may often succumb to death or become unresponsive. Until recently, costimulation has been dominated by studies of the Ig superfamily member, CD28, a constitutively expressed molecule that is required to initiate a majority of T cell responses. However, growing evidence over the past few years has now shown that several members of the TNFR family, OX40 (CD134), 4-1BB (CD137), and CD27, are equally important to the effective generation of many types of T cell response. In contrast to CD28, these molecules are either induced or highly upregulated on the T cell surface a number of hours or days after recognition of antigen, and appear to provide signals to allow continued cell division initially regulated by CD28 and/or to prevent excessive cell death several days into the response. An argument can be made that these molecules control the absolute number of effector T cells that are generated at the peak of the immune response and dictate the frequency of memory T cells that subsequently develop. The exact relationship between OX40, 4-1BB, and CD27, is at present unknown, including whether these molecules act together, or sequentially, or control differing types of T cell response. This review will focus on recent studies of these molecules and discuss their implications. © 2003 Elsevier Science Ltd. All rights reserved. Keywords: Costimulation; T cells; Ligation
Contents 1. Introduction: the role of costimulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Expression of OX40 (CD134) and OX40-ligand . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Expression of 4-1BB (CD137) and 4-1BB-ligand . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Expression of CD27 and CD27-ligand (CD70) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. Functional studies of OX40. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6. Functional studies of 4-1BB . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7. Functional studies of CD27. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8. Signals transmitted through OX40, 4-1BB, and CD27 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9. Spatio-temporal use of OX40, 4-1BB, and CD27 by CD4 and CD8 cells . . . . . . . . . . . . . . . . . . . . . 10. Spatio-temporal use of OX40, 4-1BB, and CD27 in Th1/Tc1 or Th2/Tc2 responses . . . . . . . . . . . 11. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Introduction: the role of costimulation The list of membrane bound molecules that can positively affect a T cell and enhance activation, division, survival, and cytokine secretion, has steadily been growing over the past 10 years. These molecules largely fall into three main groups, namely Ig superfamily members, TNFR superfamE-mail address: [email protected] (M. Croft).
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ily members, and cytokine receptors. CD28, ICOS, and CD2 typify costimulatory molecules of the Ig superfamily and recent reviews have detailed their importance in many immune responses driven by T cells. Cytokine receptors that can control T cell growth or survival in some situations are also numerous and include IL-2R, IL-7R, IL-15R, IL-1R, and IL-6R. Lastly, signals through a number of TNFR family members have also been shown to augment T cell responses in various settings and these include OX40 (CD134), 4-1BB
1359-6101/03/$ – see front matter © 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S1359-6101(03)00025-X
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Fig. 1. Members of the TNF/TNFR family implicated in costimulation of T cells. The Type I transmembrane proteins of the TNFR family are on the left with their characteristic cysteine-rich domains depicted. The ligands, the Type II transmembrane proteins of the TNF family, are on the right. The chromosomal location of the human genes are indicated. The interactions of CD30 with CD30L and HVEM with LIGHT can also costimulate T cells and may be functionally analogous to OX40, 4-1BB, and CD27.
(CD137), CD27, and CD30 and HVEM (Fig. 1). Because of this plethora of molecules that can be classified as costimulatory, a number of issues have arisen. Firstly, are all molecules required for every T cell response or does some degree of redundancy exist. Secondly, do all molecules perform similar functions, or can separate functions be ascribed to individual molecules or particular families of molecules. Thirdly, can a fully effective (protective or pathogenic) T cell response occur in the absence of signaling from one or a number of these molecules and if so, can we determine which are the relevant molecular interactions to target (positively or negatively) in terms of providing therapeutic benefit in varying disease scenarios. If there is little redundancy and most if not all of the above molecules are required for a T cell response that leads to effective and long-lived memory, several non-mutually exclusive models can be put forward to explain the need for multiple costimulatory signals. Firstly, a “simultaneous engagement” model would suggest that signals from two or more costimulatory molecules are required at the same time or within a brief time frame for maximal short-term response. Secondly, a “temporal engagement” model would include signaling from two or more molecules being required one after the other over an extended time frame thereby ensuring a long-lasting response. Thirdly, a “spatio-temporal engagement” model envisions that two or more molecules signal simultaneously or one after the other, but there is differential use between CD4 versus CD8 T cells, and/or depending on particular antigenic or inflammatory conditions that may favor expression of certain receptors on the T cell or expression of their membrane bound and soluble ligands. This review will not directly provide answers to all of these questions but will attempt to put some perspective on how OX40, 4-1BB, and CD27 may fit into these models.
2. Expression of OX40 (CD134) and OX40-ligand OX40 was originally identified in 1987 by an antibody that reacted with activated rat CD4 T cells [1]. It is often quoted that OX40 is primarily expressed on CD4 T cells, however CD8 T cells can also bear OX40 at least under certain conditions. Moreover, OX40 has now been visualized
on other more diverse cell types including B cells, dendritic cells, and eosinophils (Croft et al., unpublished observations), although as yet the physiological significance of this expression is unknown. OX40 is not expressed on resting T cells. It can be induced by TCR/CD3 signals in isolation and initially appears 12–24 h after stimulation of na¨ıve cells. Peak expression is seen after 2–3 days and then OX40 is downregulated, implying a delayed mode of action. Antigen-experienced effector/memory T cells can rapidly re-express OX40 within 4 h of reactivation [2]. OX40 has been visualized in vivo in T cell zones of spleen or lymph nodes several days after immunization with protein antigen, directly coinciding with the peak of the primary T cell response [3,4]. Activated T cells expressing OX40 have also been found in peripheral inflammatory sites in a number of disease scenarios such as in the CNS in mice undergoing EAE [5]; blood of mice and humans undergoing GVHD [6,7]; the sites of growth of a number of tumors [8]; synovial fluid from patients with chronic synovitis, and joints of mice undergoing rheumatoid arthritis [9,10]; lamina propria of mice undergoing colitis, and gastrointestinal tract samples from patients with Celiac and Crohn’s disease [11,12]; lymph nodes draining sites of growth of Leishmania major [13]; and lungs of mice with allergic asthma [14]. OX40-ligand is expressed on professional antigenpresenting cells (APCs) such as dendritic cells, B cells and macrophages many hours to days after activation [15–17]. Interestingly, it was first identified on the surface of HTLV-infected leukemic T cells [18]. Expression on non-transformed T cells has been seen but appears to be rare [19,20], although this observation still raises the possibility of a role in T cell–T cell interactions rather than T cell–APC interactions. In the case of dendritic cells and B cells, Toll-like receptor signals induced by LPS can promote OX40L expression in addition to contributions from Ig signals and CD40 signals [15–17]. Additionally, OX40L has been visualized on activated endothelial cells in vitro, and in tissues from patients with lupus nephritis and inflammatory bowel disease, implying a role in promoting migration of OX40-expressing T cells into inflamed tissues, or providing signals to T cells to augment their activity in these peripheral sites [21–23].
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3. Expression of 4-1BB (CD137) and 4-1BB-ligand 4-1BB was discovered in 1989 after screening cDNA libraries from activated CD4 and CD8 T cell clones [24]. 41BB is not constitutively expressed on resting T cells and like OX40 is induced within 24 h after activation, peaking several days after initial stimulation. Reagents that cross-link the TCR or CD3 are sufficient to induce 4-1BB on both CD4 and CD8 cells [25–28]. 4-1BB expression is not exclusive to T cells and has been detected on B cells, macrophages, dendritic cells, NK cells, and eosinophils, although the physiological significance on these cell types also remains to be resolved [26,29–31]. In vivo expression analyses of 4-1BB have been limited with 4-1BB visualized on T cells from peripheral blood of HIV-infected patients [32] and in T cells infiltrating cardiac allografts [33]. However, as described below a number of in vivo functional analyses suggest that 4-1BB is expressed in many responses involving T cells including those to tumor antigens, to alloantigens, and to viruses. Similar to OX40L, 4-1BB-ligand can be expressed on dendritic cells, B cells, and macrophages, and again is induced hours or days after activation, and can be regulated by LPS, Ig or CD40 signals [31,34,35]. Thus, both expression of OX40 and 4-1BB, and OX40L and 4-1BBL can parallel each other under certain conditions on activated T cells and activated APCs, suggesting that there may be similarities in their roles and in the responses in which they participate. 4. Expression of CD27 and CD27-ligand (CD70) CD27 was originally identified with antibodies that bound to a large proportion of peripheral blood CD4 and CD8 T cells [36,37]. As such this distinguishes CD27 from OX40 and 4-1BB in that the latter have to be induced rather than being present on resting T cells. However, similar to these molecules, CD27 is strongly upregulated by reagents that cross-link the TCR or CD3, although peak levels of expression may occur within 24 h, somewhat earlier than maximal expression of OX40 or 4-1BB [38,39]. CD27 can also be irreversibly lost on a subset of long-term repeatedly stimulated T cells, correlating with negative expression on small numbers of memory phenotype cells [38]. Whether similar subsets of cells exist that cannot re-express OX40 or 4-1BB has not been investigated, nor whether the CD27 negative T cells are OX40 or 4-1BB positive. This nevertheless remains an intriguing possibility, and suggests the possibility that segregation in use of these molecules may occur with long-term differentiation or after repeated encounters of T cells with antigen. The ligand for CD27 (CD27L or CD70) is also inducible on professional APC, similar to OX40L and 4-1BBL, although it may be predominantly expressed on B cells and not dendritic cells [40–42]. Similar to OX40L, CD70 has additionally been found on activated T cells, leading to the
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suggestion that CD27–CD70 interactions may also be involved in direct cellular communication between subsets of T cells as well as between T cells and APCs [43,44]. In summary, there appears to be much overlap in the expression patterns of OX40/OX40L, 4-1BB/4-1BBL, and CD27/CD70, with all receptor/ligand pairs expressed on activated T cells and activated APC at least under certain conditions. This raises the possibility of cooperation between these molecules and that each interaction may contribute to effective T cell priming. As each molecular pair are generally studied in isolation, it remains to be determined both in vitro, and in specific immune responses in vivo, whether there is direct overlap in expression or whether distinct subsets of T cells and APC can be visualized that express only one particular ligand or receptor. Ultimately, such studies will be essential to determining the physiological role of these molecules in T cell immunity and their relationship with one another.
5. Functional studies of OX40 A large number of in vitro studies that will not be detailed here, using either receptor specific antibodies or cells transfected with individual ligands, have shown that signals through OX40, 4-1BB, or CD27 can augment T cell responses, either in isolation or in combination with CD28 signals from B7. Although multiple activities have been described such as enhancing proliferation, cytokine secretion, and cell survival, these studies in minimalist systems do not necessarily convey the physiological function of the molecules when their signals are provided in the context of multiple signals from other cell surface receptors or from soluble cytokines. Recent studies of knockout animals, with antagonist and agonist reagents in vivo, or receptor-deficient T cells, have aided tremendously in not only defining the importance of these molecules in T cell responses, but also their physiological mode of action. Studies of OX40- and OX40L-deficient mice initially showed that CD4 responses to the viruses lymphocytic chriomenigitis virus (LCMV) and vesicular stomititis virus (VSV), to a common protein antigen, and in contact sensitivity reactions to haptens, were markedly reduced in vivo, and in vitro [17,45–47]. Further in vivo data in OX40-deficient mice provided the first indication of function, demonstrating a deficiency in numbers of antigen-specific CD4 T cells generated late in the primary response, and additionally after 5 weeks when the T cell memory population was formed [4]. Other data in which agonist anti-OX40 reagents were injected shortly after immunization augmented tremendously the number of antigen-reactive CD4 cells that accumulated over time [4,48]. Similarly, transgenic expression of OX40L on dendritic cells led to greater numbers of primed CD4 cells [49] and blocking OX40L in another model reduced accumulation of CD4 cells [50]. Collectively, these studies have suggested that a major role of OX40–OX40L
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Fig. 2. Temporal model of the roles of OX40, 4-1BB, and CD27 in T cell responses. Both OX40 and 4-1BB can provide anti-apoptotic signals several days after a na¨ıve T cell encounters antigen. These signals allow continued turnover of cells and provide survival signals to prevent excessive T cell death at the peak of the primary response. Both molecules appear to act in a temporal manner after CD28 signals are initially provided to the T cell. The role of CD27 is not as clear. As it is constitutively expressed on a na¨ıve T cell, it may participate in regulating initial cell division and clonal expansion as well as having the potential to promote survival late in the response.
interactions is to dictate the number of effector T cells that accumulate in primary immune responses, and consequently to govern the number of memory T cells that subsequently develop and survive. Further studies using antigen-specific TCR transgenic CD4 cells lacking OX40 have now provided greater insight into the mechanism of action [51]. These studies have shown that OX40 contributes little to the initial response of a CD4 cell, and minimally impacts cytokine production or early proliferation. This directly contrasts with the role of CD28, which is required for much of this early response. OX40-deficient T cells demonstrated reduced proliferation 4–5 days into the response, and a large proportion of these T cells could not survive over the long-term. The lack of survival was shown to be due to apoptotic cell death and could be rescued by inhibitors of the caspase cascade [51]. OX40 expression is not dependent on CD28 signals, but several systems have shown that CD28 can augment the level of OX40 expressed on a T cell [50,51], reinforcing the concept that the two molecules most likely co-operate together in a sequential manner. Therefore, to summarize, a model can be proposed whereby OX40 signals act in a temporal manner after CD28 signals, and allow effector T cells to survive and continue proliferating late in response, predominantly by transmitting anti-apoptotic signals that prevent excessive T cell death (Fig. 2).
signals additionally reduced the antigen-specific population that was generated. These studies have been supported by reports with either agonist anti-4-1BB antibodies or with tumor cells transfected with 4-1BBL. In all cases, greater CTL responses were revealed, and in some this was directly correlated with the absolute number of antigen-specific CD8 T cells being increased [55–58]. Of significance, in relation to studies of OX40, an agonist 4-1BB antibody was shown to prevent death and deletion of superantigen-stimulated CD8 cells [57], and a blocking 4-1BB–Fc fusion protein, which acts as a soluble decoy for the ligand, was shown to not affect the initial proliferative response of TCR transgenic CD8 cells responding to peptide in adjuvant, but inhibited the accumulation of effector T cells at the peak of the primary response [59]. The latter study additionally demonstrated
6. Functional studies of 4-1BB An understanding of the role of 4-1BB in T cell responses has largely centered on CD8 cells as opposed to CD4 cells for OX40. Initial reports of 4-1BBL-deficient mice responding to the viruses LCMV and influenza demonstrated that 2–10-fold fewer antigen-reactive CD8 cells were generated in primary responses, and fewer memory T cells developed weeks after priming [52–54]. Furthermore, preventing CD28
Fig. 3. Spatio-temporal model of regulation of CD4 and CD8 T cells by OX40, 4-1BB, and CD27. The majority of data indicates that OX40 is important for many CD4 T cell responses, whereas most data to date on 4-1BB has been directed to CD8 T cells. It is a possibility that differential use of OX40 and 4-1BB by CD4 and CD8 cells may be characteristic of many immune responses and explain their apparent overlap in function and potential redundancy. If CD27 largely regulates clonal expansion as opposed to survival, it could be equally involved in the initiation of both CD4 and CD8 responses.
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that increased levels of apoptotic CD8 cells resulted when 4-1BB signals were absent, and that apoptosis occurred in T cells that had already divided many times [59]. Although CD28 signals are also not required for 4-1BB expression, as for OX40, they have been shown to augment expression in several systems [27,60,61]. Moreover, direct comparison of influenza-specific CD8 responses in the absence of CD28 signals versus 4-1BB signals additionally implicated 4-1BB as acting in a temporal manner to promote survival and accumulation of T cells late in primary responses after CD28 signals regulated the initial phase of response [62]. Thus, an argument can be put forward that 4-1BB acts in a CD8 response, much like OX40 in a CD4 response, with both molecules being essential for the accumulation and survival of large numbers of antigen-primed T cells over time (Fig. 3).
7. Functional studies of CD27 Although a number of in vitro studies with agonist antibodies to CD27, or with CD70 transfectants, have demonstrated a strong costimulatory effect on T cell proliferation [44,63,64], in vivo studies have lagged behind those of OX40 or 4-1BB. Recently, CD27-deficient mice were generated and shown to be defective in both CD4 and CD8 responses to influenza virus, and similar to OX40 or 4-1BB, exhibited reduced numbers of antigen-specific memory T cells [65]. These observations have been supported in mice transgenic for CD70 expressed on B cells, which also have increased numbers of memory/effector phenotype T cells [66], and by studies of CD70-transfected tumor cells that led to augmented priming of both CD8 and CD4 cells [67–69]. At what stage in the T cell response CD27 acts is unclear, nor whether it is temporally regulated in comparison to CD28, nor whether its major effect is on controlling initial T cell proliferation, or also preventing apoptotic cell death as is the case for OX40 and 4-1BB. Because CD27 is constitutively expressed on a resting T cell and upregulated earlier than OX40 or 4-1BB, and as the majority of studies in vitro have shown strong effects on T cell proliferation, it is a possibility that CD27 functions either at the same time as CD28, or in a temporal manner between CD28 and OX40 or 4-1BB. If this is the case, the principle effect of CD27 costimulation could be to contribute to primary clonal division and expansion, rather than being essential for T cell survival after this expansion phase (Figs. 2 and 3). Future studies tracking the kinetics of antigen-specific T cells in vivo while blocking CD27–CD70 interactions compared to CD28/B7, or with CD27-deficient transgenic T cells, will be required to provide answers to these questions.
8. Signals transmitted through OX40, 4-1BB, and CD27 Although studies of signaling pathways induced by OX40, 4-1BB, and CD27, are in their infancy, a number of
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reports have revealed potential mechanisms of action and cross-regulation between these molecules, and enhanced the conclusions gained from in vivo studies. Each receptor can bind to several of the six known TNFR-associated factors (TRAFs), a family of RING finger proteins, however the data to date suggests that TRAF2 may be central to signal transduction by all three [70–74]. Whether this indicates a common functional action is not clear, but additionally all molecules have been shown to be capable of activating NF-B and JNK [70,72–74], suggesting that there is a strong potential for providing similar functional consequences. Moreover, recent data on the downstream targets of OX40 and 4-1BB have now added to the contention that these molecules may be functionally analogous. Initial studies of OX40 showed that OX40-deficient CD4 cells were incapable of maintaining high levels of the anti-apoptotic proteins Bcl-xL and Bcl-2 several days after activation [51], as well as another Bcl-2 family member, Bfl-1 (Song and Croft, unpublished). Similarly an agonist anti-OX40 augmented expression of these molecules in a CD28-independent fashion [51]. The results were additionally emphasized when defective survival of OX40-deficient T cells was completely prevented by retroviral transduction of these cells with either Bcl-xL or Bcl-2 genes. This study has now been followed recently by two reports of CD8 cells stimulated with agonist anti-4-1BB antibodies that also showed a strong upregulation of Bcl-xL and Bfl-1 expression, and a direct correlation with increased survival and reduced apoptosis [75,76]. Thus, both the molecular and cellular studies of OX40 and 4-1BB show similar if not identical effects on CD4 and CD8 cells respectively. To date, there is no information on whether CD27 also regulates the Bcl-2 family of proteins, although given the similarities in use of TRAF2, NF-B, and JNK, and some overlapping functional data, it is
Fig. 4. Signaling pathways induced by OX40, 4-1BB, and CD27. All three molecules can bind TRAF2 and functional data suggest that TRAF2 is responsible for many of the activities induced by ligating these receptors. NF-B and JNK can also be activated by all three receptors, although no data is available to show what are the functional consequences of activating these pathways. Both OX40 and 4-1BB can upregulate the expression of the anti-apoptotic members of the Bcl-2 family and block programmed cell death due to cytokine/antigen withdrawal. It is likely that NF-B will mediate these activities but awaits a direct demonstration. It is not clear if Fas- or TNFR1-induced death can be inhibited by OX40 or 4-1BB signals, but is a distinct possibility. No data is available on whether CD27 also positively regulates Bcl-xL, Bcl-2, or Bfl-1.
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highly likely that these proteins may also be targets of CD27 (Fig. 4).
9. Spatio-temporal use of OX40, 4-1BB, and CD27 by CD4 and CD8 cells Given the similarities in functional properties that have been reported for OX40, 4-1BB, and CD27 to date, several questions are raised. Firstly, do these molecules indeed have an identical role in T cell responses, and is it the sum of their signals that is critical to a T cell, rather than the individual signals themselves. If this is the case, an individual T cell should express all molecules either at the same time, or in a temporal fashion during the lifespan of the T cell. In vitro, we know that simultaneous expression of all three molecules can occur on an activated T cell, but whether or not there is really a kinetic difference in expression has not been addressed. Ultimately, however, this type of question can only be definitively answered by analyzing T cells in vivo in varying types of immune responses. Another question is whether the apparent overlap in function can be explained by differential usage by for example CD8 cells versus CD4 cells. As highlighted above, the majority of data on OX40 has been gathered on CD4 cells, and the majority of data on 4-1BB on CD8 cells, whereas that on CD27 is equally spread between CD4 and CD8. As CD27 is constitutively expressed on na¨ıve T cells at low levels, and assuming that CD27 is found to be important for initial clonal expansion in vivo as implied from in vitro studies, one model could then be a combination of temporal use and spatio-temporal use. For example, CD27 may equally contribute to the expansion phase of both na¨ıve CD4 and CD8 responses, and then OX40 is required for the subsequent survival of CD4 effector cells whereas 4-1BB is required for the survival of CD8 effectors (Fig. 3). The evidence from the viral studies with LCMV and influenza at least partially fit this model, i.e. defective CD4 and CD8 priming were seen in CD27 knockout animals [65], whereas only defective CD4 responses were seen in OX40-deficient animals [45] and only defective CD8 responses in 4-1BBL-deficient animals [53]. Although the model presented in Fig. 3 is attractive, there is not enough information from different systems to determine whether it can be applied to all CD4 and CD8 responses, and most likely it will not translate to every type of situation. Several studies have implied effects of OX40 on CD8 cells and 4-1BB on CD4 cells, but have fallen short of directly showing this. Data with agonist antibodies to OX40 in tumor models have shown enhancing effects on CTL generation [77,78], however it was not determined whether CD8 cells were direct targets or OX40 signals simply augmented CD4 cell help. Similarly, several tumor studies of 4-1BB have shown a requirement for CD4 cells during 4-1BB-induced anti-tumor responses [55,56], but no direct evidence has been provided as yet to show that
4-1BB was required by the CD4 cells themselves. In vitro, several reports have demonstrated that 4-1BBL can costimulate some CD4 responses [28,60] showing the potential of cross-regulation via 4-1BB, and a recent study of aged CD4 T cells in vivo also showed that their defective response could be restored with agonist anti-4-1BB [79]. However, to date only one study has presented more definitive evidence that 4-1BB can naturally contribute to a CD4 response. This data demonstrated that CD4 cells from 4-1BB-deficient animals were strongly impaired in their ability to mediate GVHD in adoptive transfer experiments [80]. At present, there is no published data on whether OX40 signals normally control CD8 cells, although another recent study showed that agonist anti-OX40 could strongly augment CTL priming induced by dendritic cells presenting only class I-restricted peptides [81]. This is suggestive evidence for an action on CD8 cells, although because of OX40 expression on non-T cells, definitive evidence will have to await the production and analysis of OX40-deficient CD8 T cells. In summary, there appears to be an overall bias for OX40 use by CD4 cells and 4-1BB use by CD8 cells, although it is unlikely that this will be absolute. Cross-regulation may be dictated by the inflammatory conditions encountered by CD4 and CD8 cells that could impact expression of receptors or ligands, rather than the T cell subtype that is responding.
10. Spatio-temporal use of OX40, 4-1BB, and CD27 in Th1/Tc1 or Th2/Tc2 responses Finally, a further question that needs to be addressed in future studies is whether the apparent overlap in function between OX40, 4-1BB, and CD27, can be explained by a differential requirement for these molecules during Type 1 versus Type 2 immune responses. Type 1 immune responses are dominated by the cytokines IFN-␥ and TNF whereas Type 2 responses involve the cytokines IL-4, IL-5, IL-9, and IL-13. Such distinct cytokine patterns can be produced by either CD4 cells (Th1 versus Th2) or CD8 cells (Tc1 versus Tc2). Although there are some reports in vitro that lean toward OX40, 4-1BB or CD27 regulating one response or another, these are again questions that ultimately need to be answered in studies of in vivo responses. Early data on OX40 in vitro implied that this molecule was preferentially involved in Th2 responses [82,83]. Studies in vivo in models of Leishmania in BALB/C mice [13] and in allergic asthma [14] have confirmed a major role for OX40 in at least these Th2 responses. However, more basic in vivo studies of OX40- and OX40L-deficient animals have shown reductions in both Th2 and Th1 cytokine responses [4,17], and applied studies of experimental autoimmune encephalitis (EAE) [84,85], rheumatoid arthritis [10], and colitis [11,86] have also shown that blocking OX40–OX40L interactions can strongly inhibit these diseases and their associated Th1 cytokines. Thus, OX40 appears to be involved in priming for both Th2 and Th1 responses, but a question
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still remains as to whether there may be a bias later in the response, for example when T cell memory is formed, or in situations where multiple encounters with antigen occur. Less is known about the participation of 4-1BB in Type 1 versus Type 2 responses. As most CD8 responses are Tc1-like, it can be concluded from the multiple studies described above that 4-1BB contributes to these responses. Whether 4-1BB also regulates many Th1 responses remains to be determined. In vitro experiments with human CD4 cells have indicated this potential [87], as has an in vivo study where anti-4-1BB augmented priming of aged T cells for Th1 cytokine production [79]. Few studies have attempted to determine if 4-1BB participates in either a Th2 or Tc2 response, although one report with anti-4-1BBL demonstrated no effect on a Leishmania T cell response in BALB/C mice in a situation where anti-OX40L completely blocked Th2 cytokines [13]. Thus, it is possible that 4-1BB may largely contribute to Th1/Tc1 responses, but many more studies are needed before any definitive conclusions can be drawn. Lastly, there is even less physiologic data to indicate whether CD27 could be differentially utilized in Type 1 versus Type 2 responses. Analyses of CD70 transgenic animals showed an increased production of IFN-␥ [66] suggestive of an effect on Type 1 responses. In vitro studies ligating CD27 on T cells have also leaned towards this conclusion with little effect on IL-4 production being seen [64,88], and exogenous IL-4 can negatively regulate CD70 expression on B and T cells in vitro [89,90]. Whether there is a true preference for CD27–CD70 interactions in Type 1 responses awaits analysis of in vivo Th2 or Tc2 responses.
11. Summary In conclusion, there is now extensive evidence that OX40, 4-1BB, and CD27 are integral to the effective development of a number of T cell responses. The majority of data suggest that the predominant role of these molecules is to regulate the absolute number of antigen-specific effector T cells that are generated late in primary T cell responses and to control the number of memory T cells that develop. Whether all three receptor systems are functionally equivalent still remains to be determined. However, growing evidence suggests that there is significant overlap in the activities and the roles of OX40, 4-1BB, and CD27 in T cell priming, and that the major mechanisms of action are either through promoting cell division or suppressing cell death. While there is now good evidence that OX40 and 4-1BB provide anti-apoptotic signals in vivo, and that the Bcl-2 family of proteins are some of their targets, it is not clear whether CD27 also plays a role in T cell survival. Moreover, questions still exist regarding whether OX40, 4-1BB, or CD27 contribute to augmenting T cell proliferation in vivo, and whether proteins involved in cell cycle regulation are also direct targets of their signals. Finally, given the apparent similarities in function, future studies are needed to define the relationship of OX40
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to 4-1BB, and these molecules to CD27. It is possible that each acts independently at different stages in the T cell response, or in different types of response mediated by CD4 or CD8 cells, or involving Type 1 or Type 2 cytokines. Alternatively, it is possible that OX40, 4-1BB, and CD27 act together, at the same time, or within a short time frame, and that a T cell is only signaled efficiently when all molecules are engaged. Answers to these questions will have to await expression studies in multiple types of T cell response, and the functional analysis of T cells made deficient in one or more molecule.
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