CD40, a 50kDa transmembrane member of the tumor-necrosis factor (TNF) receptor (TNFR) superfamily of molecules, plays a key role in adaptive immune responses. This includes contact-mediated signals to B cells from activated T lymphocytes, as well as costimulatory interactions between T cells and other antigen-presenting cells (APC), such as dendritic cells (DC) and macrophages. CD40-mediated interactions between B and T cells make important contributions to the development of an optimal humoral memory response (Bishop 2009).
CD40 lacks intrinsic enzymatic activity and therefore depends on cytoplasmic (CY) adaptor molecules referred to as TNFR-associated factors (TRAFs) for delivery of signals to the cytoplasm. CD40 engagement on B cells stimulates the binding of TRAFs to the CD40 CY domain. This in turn stimulates kinase activity and gene expression pathways that lead to a variety of responses, including B cell survival and expansion, immunoglobulin (Ig) production, Ig isotype switching and affinity maturation, B cell humoral memory, and the production of various cytokines and chemokines (Bishop 2009).
CD40 was identified in 1984 as an antigen associated with transitional cell carcinoma of the human urinary bladder, and its importance in B cell activation was first suggested by the agonistic activity of CD40-specific mAbs upon human B cells (Bishop 2012). The first signaling role of the CD40 receptor was suggested by experiments in which an anti-human CD40 agonistic mAb induced in human B cells the tyrosine phosphorylation of cellular proteins, activation of phospholipase Cγ2, and activation of specific Ser/Thr protein kinases (Uckun et al. 1991). The natural ligand for CD40, CD40L/CD154, was discovered via the complementary methods of in vitro mouse cell experiments and the study of a human immunodeficiency disease, X-linked hyper IgM syndrome (HIGM), caused by loss-of-function mutations in the gene encoding CD154. Experiments with mouse cells showed that plasma membrane fractions from activated T helper (Th) cells induce B cell cycle entry. Ultimately, the identity of the Th membrane protein responsible was elucidated by cloning of CD154 in 1992 (previously known as CD40L, gp39, TRAP, or TRAM) (Schönbeck and Libby 2001).
It was subsequently discovered that defects in CD154 expression or its CD40 binding site cause HIGM, in which patients are susceptible to recurrent extracellular bacterial infections as well as certain intracellular pathogens. HIGM does not affect T cell-independent humoral responses, but features a decrease in the production of “switched” isotypes of IgG, IgA, and IgE, as well as defects in antigen presentation. CD40- and CD154-deficient mice display defects in T cell-mediated immunity, including immune responses to parasites, due to defects in activation of T cells by APC. CD40 signals to APC play important roles in inducing upregulation of costimulatory molecules, and production of cytokines needed to activate T cells. CD40- and CD154-deficient mice also display defective germinal center (GC) formation in secondary lymphoid organs and in B cell memory development in response to antigenic challenge. Together, these findings show that CD40-CD154 interactions are needed for successful T-dependent B cell activation, promoting B cell proliferation, antibody production, isotype switching, GC formation, B cell memory, upregulation of costimulatory and adhesion molecules, and cytokine production (Bishop and Hostager 2003).
Although CD40-CD154 interactions are important for adaptive immune responses, recent studies reveal more diverse roles of this receptor in physiological and pathological processes. While constitutive CD40 expression is generally restricted to B lymphocytes and myeloid cells (DC, monocytes, and macrophages), it is also expressed under specific conditions by epithelial cells, basophils, eosinophils, T cells, vascular endothelium, neuronal cells, and smooth muscle cells. Additional studies have demonstrated CD40 expression on fibroblasts, keratinocytes, and platelets (Bishop 2009).
This unique receptor-ligand pair has been implicated in chronic inflammation and immune responses implicated in transplantation rejection and diseases including cancer (both in tumor-promoting and growth-inhibitory roles), Grave’s disease, type 1 and obesity-associated diabetes, atherosclerosis, neuroinflammatory disease, systemic lupus erythematosus, psoriasis, arthritis, and inflammatory bowel disease (Schönbeck and Libby 2001; Peters et al. 2009; Yi and Bishop 2014). Therefore, a detailed understanding of CD40 signaling pathways in normal immunity and disease is important for possible manipulation of this important receptor for therapeutic benefits.
CD40 and TRAFs
Although CD40 itself lacks intrinsic enzymatic activity, it delivers a variety of important signals to cells. Due to space constraints, this review will focus upon studies performed in B lymphocytes, the cell type in which CD40 signaling has been explored in the greatest detail to date. Human CD40’s short CY domain (62 amino acids in human; 73 in mouse) has a highly conserved protein sequence, very similar in the two species and identical for a 37-amino acid span. The human CD40 CY domain does not contain tyrosines, and thus is devoid of tyrosine-kinase or phosphatase-binding motifs common to many other immune receptors. The CY domain does contain serine and threonines, and structure-function studies determined that T234 (human) or T254 (mouse) in the CD40 CY domain plays a regulatory role in CD40 signals, possibly by altering the avidity of TRAF2 association (Bishop and Hostager 2003). However, phosphorylation of the CY domain of CD40 has not been shown to have a major functional role. Rather, proximal signaling events appear largely dependent upon specific sequences in this domain that mediate binding to TRAF adapter proteins (Bishop et al. 2007).
CD40, like most other members of the TNFR superfamily (TNFRSF), utilizes various TRAFs to transduce intracellular signals. TRAF3 was initially isolated via its association with both mouse and human CD40 and initially called CD40-binding protein. CD40 also directly binds TRAFs 2, 3, 5, and 6 and recruits TRAF1 primarily via a heterotypic interaction with TRAF2. TRAFs 1, 2, and 6 play important positive roles in CD40-mediated signaling, while TRAF3 plays an inhibitory role, and TRAF5 appears to play a relatively modest role (Bishop et al. 2007; Hostager 2007; Hildebrand et al. 2011).
CD40 has three known CY TRAF binding sites: (a) a membrane proximal region for direct TRAF6 binding, (b) a more medial site for overlapping binding of TRAFs 1, 2, 3, and 5, and (c) a second distal TRAF2-binding region (Graham et al. 2010).
To fully understand the complex details of CD40-mediated signaling pathways in B cells, the specific contributions of each TRAF, and their interactive functions in CD40 signaling, must be elucidated. A variety of experimental approaches have been applied to this question. The study of mutant TRAF molecules which can still associate with CD40, but no longer initiate downstream signals (“dominant negative” TRAFs) provided hints about the biological roles of their full-length counterparts in CD40 signaling. A similar alternative approach is the use of CD40 molecules that contain mutations in the CY domain, in TRAF binding sites. Application of these approaches in cell lines and transgenic mouse models yielded useful initial information about TRAF association and CD40 functions. However, mutant versions of TRAFs can alter the binding of other TRAFs, due to the overlapping nature of the TRAF 1, 2, 3, and 5 binding site of CD40, a concern that also arises for mutations of the CD40 CY domain in this region (Bishop 2004). Another caveat to many early studies of TRAF-CD40 interactions is their performance primarily or exclusively in the transformed epithelial cell line HEK293, in which both CD40 and wild-type or mutant TRAFs were transiently overexpressed. In addition to concerns about the artificial stoichiometry of the CD40 signaling complex in such systems, CD40 association with TRAFs may have cell-type-specific contextual features as well. Examples are the failure of the CD40 CY domain mutant T234A to associate with TRAF3 when overexpressed in HEK293 cells, while TRAF3 associates normally with this mutant in B cells (Haxhinasto et al. 2002), as well as the preferential usage of TRAF6 rather than TRAF2 for CD40-mediated activating signals in macrophages (Mukundan et al. 2005).
The application of gene targeting by homologous recombination, in which the expression of specific gene products has been disrupted, facilitated the creation of mice deficient in various signaling proteins, including each of the TRAFs. However, because TRAFs also mediate multiple pathways involved in normal development and physiology, their removal from the entire animal results in early lethality of many of these strains, as is the case for mice deficient in TRAFs 2, 3, and 6. Advances in gene targeting techniques led to the production of “conditional” knockout mice, in which a gene flanked by bacterial recombinase recognition sequences is removed from specific cell types by breeding with a strain expressing the recombinase behind a promoter specific for expression in the desired cell type(s). This technology has allowed new insights into TRAF function; however, it can be difficult to identify recombinase-expressing mice that target TRAF deletion to a single specific cell type or developmental stage, which can complicate data interpretation.
To overcome these obstacles, we created TRAF-deficient mature B cell lines by targeting various TRAF genes via homologous recombination. This system allows stable transfection of the cells with mutant CD40 molecules as well as wild-type and mutant TRAFs and avoids the interpretation complications of earlier approaches described above. This approach revealed that TRAFs 2 and 6 are critical for overlapping but distinct early signaling events mediated by CD40, while TRAF3 competitively inhibits TRAF2-mediated CD40 signals. TRAF1 enhances TRAF2-mediated signaling (Bishop 2004; Rowland et al. 2007). Through information gained from the various complementary approaches discussed above, a more detailed picture of CD40-mediated molecular signaling events is emerging.
TRAF Recruitment, Ubiquitination, and Degradation
Following ligation, CD40 redistributes into membrane lipid rafts and recruits TRAFs to the membrane signaling complex. The Zn-binding RING domains of TRAFs 2 and 3 are required for optimal raft recruitment, as their removal or incubation with a membrane-permeable Zn-chelating agent inhibits this process (Bishop et al. 2002). Following recruitment to CD40, a complex of TRAF3, TRAF2, and cellular inhibitors of apoptosis (cIAPs) leads to the poly-ubiquitination of TRAFs 2 and 3 and their proteasomal degradation (Lin et al. 2015). It has also been shown that TRAF3 can be modified by the small ubuitin-related modifier (SUMO) following CD40 signaling (Miliara et al. 2013). CD40 association does not mediate degradation of TRAFs 1 or 6.
The second phase of CD40-mediated signaling regulated by ubiquitination events targets TRAFs 2 and 3 for proteasomal degradation by chains of ubiquitin molecules linked through lysine at position 48 (K48). After cIAP1/2 are activated by K63-linked ubiquitination as described above, they may act as E3 ligases for TRAF3, promoting its K48-linked poly-ubiquitination and proteasomal degradation. More recent work indicates that another E3 ubiquitin ligase, NEDD4, also ubiquitinates TRAF3 in CD40 signaling; the action of NEDD4 appears particularly important for CD40-induced Akt activation (Fang et al. 2014). TRAF3 is a negative regulator of CD40-mediated signals, including NF-κB and MAPK activation (Yi et al. 2014; Lin et al. 2015). TRAF3 degradation is also thought to release signaling complexes into the cytosol where MAPKKKs can undergo autophosphorylation and activate downstream events (Häcker et al. 2011).
CD40 signals need to be tightly regulated because CD40 plays a central role in activation of multiple facets of immune responses. CD40-mediated TRAF degradation is important in limiting downstream signaling activation, as dysregulation of CD40 or CD40-like signals can contribute to autoimmunity and/or malignancies. The Epstein Barr virus transforming protein latent membrane protein 1 (LMP1) is a CD40 mimic that provides amplified and sustained signals associated with lymphomagenesis and, like CD40, associates via its CY C-terminal domain with TRAFs 1, 2, 3, 5, and 6. Unlike CD40 however, association of TRAFs 2 and 3 with LMP1 does not initiate their ubiquitination nor degradation, which is likely a major factor in the abnormally amplified and sustained signaling mediated by LMP1 (Graham et al. 2010).
CD40-Mediated Kinase Activation
Clustering of CD40 upon engagement leads to TRAF aggregation and the subsequent activation of downstream kinase cascades, including a variety of proteins in MAPK pathways. The major kinases activated by CD40 are illustrated in Fig. 1.
CD40-mediated activation of JNK and p38 has been attributed to various upstream kinases including members of the MAPKKK family. Mechanisms confirmed in B cells include interactions of germinal-center kinases (GCK) with the TRAF domain of TRAF2, which contributes to the induction of JNK activation. TRAF2 may also activate the MAPKKK, apoptosis-signaling kinase 1, which is also upstream of JNK and p38 (Bishop and Hostager 2001a). Other MAPKKKs associated with JNK and p38 activation in B cells include MEKK1 and the TRAF6-dependent kinase TAK1 (Hostager 2007; Arcipowski and Bishop 2012). MAPKKs demonstrated to participate in CD40-mediated activation of JNK and p38 include MKK4 and MKK3/6, respectively (Bishop and Hostager 2001a). Another MAPK regulated by CD40-mediated signaling is the extracellular signal-regulated kinase (ERK). There are many factors upstream of ERK activation, including MEK1/2 phosphorylation (Bishop et al. 2007). CD40 signals can also activate the Src-family kinase Lyn and the phosphorylation of both phosphatidylinositol 3-kinase and Akt (Bishop 2004).
Transcriptional Regulation Mediated by CD40 Signals
The most well-studied transcriptional pathways activated by CD40 signaling involve members of the NF-κB family. CD40 activates both the canonical (NF-κB1) and noncanonical (NF-κB2) pathways, in multiple cell types (Zarnegar et al. 2004; Hostager and Bishop 2013). Direct TRAF-protein interactions involved in NF-κB1 activation include the TRAF6-dependent activation of TAK1 (Arcipowski and Bishop 2012). TAK1 mediates activation of the downstream IKK complex, which phosphorylates IκB proteins. This leads to IκB poly-ubiquitination and proteasome-mediated degradation, followed by release and nuclear translocation of NF-κB1 subunits (p50/RelA) to activate transcription.
Unlike the rapid response of the canonical NF-κB1 pathway, the noncanonical NF-κB2 pathway requires new protein synthesis and is activated with much slower kinetics (Hostager and Bishop 2013). The precursor protein p100 is processed to p52, which complexes with RelB and translocates to the nucleus. It is thought that the first kinase downstream of TRAF-mediated NF-κB2 activation is NF-κB-inducing kinase (NIK) a serine/threonine MAPKKK. NIK activates the IKK complex, similarly to TAK1 (Fig. 1).
Basal levels of NF-κB1 subunits in the nucleus, together with induced activation of the transcription factors AP-1 and C/EBP-β, are required for maximal CD40-incuded IL-6 promoter activity as well as maximal IL-6 protein production in B cells. In addition to regulating IL-6, AP-1 and C/EBP-β transcription factors play a role in CD40-mediated B cell IgM secretion and CD80 surface expression (Baccam et al. 2003).
Additional transcription factors contributing to CD40-dependent functions include the B cell-specific activator protein and Stat6. In B cells, these factors play roles in transcription of the germ line Igε gene that precedes class switch recombination to IgE. Furthermore, Stat6 and NF-κB can interact, with a potential contribution towards the synergy between CD40 and IL-4 signals in the induction of germline Igε transcription. NFAT, Stat5, and E2F are also activated in B cells following CD40 ligation. However, the causative roles of each of these factors in B cell functions have not been fully explored. Both the germline Igε and CD23 promoters may contain yet unidentified regulatory elements specific to CD40-mediated gene expression (Bishop and Hostager 2001b).
CD40-Induced Effector Functions
CD40-mediated signaling to B cells causes increased production of surface molecules, cytokines, and Igs and plays a critical role in the activation of Ig isotype switching. Upstream of these events, CD40 can affect transcriptional regulation of the involved genes (Bishop and Hostager 2003). NF-κB-mediated transcriptional regulation participates in a large number and variety of cellular functions, so studies of mice deficient in the various proteins associated with NF-κB transcription (RelA, RelB, p52, p50) cannot provide clearly interpretable information on the role of NF-κB in CD40-mediated effects. B cells from these mice develop in an altered environment and are not ideal models. An inducibly expressed mutant form of the inhibitory protein IκBα that cannot be phosphorylated and degraded in mouse B cell lines revealed that CD40-mediated NF-κB1 activation is critical for some, but not all, CD40 effector functions. Functions dependent on NF-κB1 include upregulation of the costimulatory CD80 molecule, Pim-1 kinase activation, IgM secretion, and isotype switching to IgE. There is also a partial dependence upon NF-κB1 for upregulation of CD23, CD95, and CD54. NF-κB1 independent functions include upregulation of LFA-1 and CD11a, and JNK activation (Hsing and Bishop 1999).
The production of cytokines and chemokines is also an important function of APCs. These factors help regulate many CD40-mediated processes, including Ig switching, and antigen presentation. CD40 signaling has been shown to induce the production of various cytokines and chemokines including IL-2, IL-6, IL-7, IL-10, IL-12, IL-15, IL-17, IFNγ, lymphotoxin-α (TNF-β), and TNF-α (Graham et al. 2010). As discussed above, CD40-mediated signals play an important role in antigen presentation, a process that requires the upregulation of the co-stimulatory molecules CD80, CD86, and MHCII. The CD40-mediated upregulation of adhesion molecules including CD23, CD30, CD54, Fas, ICAM, and LFA-1 is also important for enhancing T-B-cell interactions during an immune response (Graham et al. 2010).
CD40 engagement results in downstream signaling events that include the activation of MAPK/SAPK kinase cascades and many transcription factors that enhance and regulate a variety of biological processes. CD40 signaling pathways play a key role in mediating adaptive immune responses and are implicated in the pathogenesis and progression of many inflammatory and autoimmune diseases, as well as malignancies. An accurate and detailed understanding of CD40-mediating molecular signaling may offer many new molecular targets for the development of new vaccines and new molecular therapies for the treatment of immune-mediated disorders. However, much remains to be learned, including how CD40 regulates transcriptional activation of a variety of genes. Other questions include the specific contributions of each of the known TRAFs and the specific mechanisms mediating their regulatory interactions with CD40. TRAFs can initiate signaling cascade via directly interacting with receptors, or alternatively, with intracellular signaling proteins to regulate downstream pathways. The activities of the TRAF proteins involved in CD40 signaling are, as yet, only partially defined. It is also quite likely that additional signaling molecules both directly and indirectly associate with the CY domain of CD40; CD40-mediated signaling cascades can also interact with other receptors’ signaling pathways. Addressing these questions will lead to more detailed understanding of CD40 functions and also provide important principles governing the regulation of signaling by other TRAF-utilizing receptors.
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