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CD40 and CD40 Ligand Deficiencies 

CD40 and CD40 Ligand Deficiencies
Chapter:
CD40 and CD40 Ligand Deficiencies
Author(s):

Luigi D. Notarangelo

, Silvia Giliani

, and Alessandro Plebani

DOI:
10.1093/med/9780195389838.003.0026
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Introduction

The primary B-cell repertoire is generated in the bone marrow by means of antigen-independent stochastic rearrangement and assembly of Variable (V), Diversity (D), and Joining (J) gene elements of immunoglobulin heavy and light chain genes. In the periphery, two key events mark maturation of the antibody response upon antigen encounter: class-switch recombination (CSR) and somatic hypermutation (SHM). During CSR, the mu heavy chain is replaced by a downstream constant heavy chain gene element. This process results from deletional recombination between characteristic repetitive sequences (switch regions) located 5′ of the Cµ gene and of each CH gene, except Cδ‎. Replacement of the μ‎ heavy chain with other constant heavy chain elements has a significant impact on the biological properties of the immunoglobulin that is produced. SHM, on the other hand, consists of the introduction of nucleotide substitutions in the immunoglobulin V regions and may thus cause changes in the amino acid sequence and in the affinity of the immunoglobulin for the antigen. As a result of this process, B-cells expressing surface immunoglobulins with higher affinity for the antigen are positively selected during the germinal center reaction.

A variety of defects of CSR are known in humans and are characterized by recurrent infections and very low levels of serum IgG, IgA, and IgE, with normal or elevated IgM (Notarangelo et al., 2006). Both primary and acquired forms of the disease have been reported. Among primary CSR defects, X-linked, autosomal recessive, and (less frequently) autosomal dominant forms have been reported, reflecting genetic heterogeneity. Defects of CSR are often associated with abnormalities of SHM and of generation of memory B-cells. Based on the underlying cellular pathophysiology, CSR defects can be divided into B-cell–intrinsic and B-cell–extrinsic disorders. The latter group includes an X-linked variant of the disease, due to mutations of the CD40LG gene, that encodes for CD40 ligand (also known as CD154), a molecule predominantly expressed by activated CD4+ T lymphocytes. CD40LG interacts with CD40, which is constitutively expressed by B-cells. CD40LG/CD40 interaction elicits B-lymphocyte intracellular signaling, with induction of the NF-κ‎B signaling pathway and expression of activation-induced cytidine deaminase (AICD) and uracil N-glycosylase (UNG), two B-cell–specific enzymes that play a key role in CSR and SHM (Notarangelo et al., 2006). In humans, mutations of CD40LG, CD40, IKBKG (encoding for IKK-γ‎/ΝΕΜΟ‎, a regulator of the NF-κ‎B signaling pathway), and AICD and of the UNG genes are all associated with defective CSR. In this chapter, we will focus on CD40LG and CD40 defects. Deficiencies of AICD, UNG, and IKK-γ‎/NEMO will be discussed in Chapters 27 and 36, respectively.

Biology of CD40 and CD40 Ligand

Expression and Biological Role of CD40

CD40, a 50kDa glycoprotein, is a member of the tumor necrosis factor receptor (TNFR) family of surface molecules. The gene, CD40 (*109535), encodes a Cys-rich type I transmembrane protein of 277 amino acids (a.a.) (Stamenkovic et al., 1989). CD40 is constitutively expressed on all B-cells but also on other cell types, including dendritic cells (DCs), monocytes, and macrophages (Brouty-Boye et al., 2000; van Kooten and Banchereau, 2000). Expression of CD40 has been demonstrated also on CD34+ hematopoietic precursor cells (Pyrovolaki et al/, 2009), T lymphocytes (Munroe et al., 2007; Vaitaitis et al., 2008), and platelets (Inwald et al., 2003). Finally, CD40 is also expressed by nonhematopoietic cell types such as thymic epithelial cells, some other epithelial and endothelial cells, and neurons (van Kooten and Banchereau, 2000).

CD40 plays an important role in B-cell survival, growth, and differentiation. CD40 ligation on the surface of B-cells in the presence of interleukin-4 (IL-4) initiates proliferation and growth and induces homotypic cell adhesion and upregulation of the expression of CD23, CD54, CD80, CD86, CD95 (Fas), and lymphotoxin-α‎. CD40-mediated upregulation of Fas expression on B-cells makes them susceptible to killing by Fas ligand+ (FasL+)-activated T-cells (Banchereau et al., 1994; van Kooten and Banchereau, 2000). This may play a role in the CD40-mediated apoptosis of tumor B-cells (Georgopoulos et al., 2006) and in peripheral purging of self-reactive B lymphocytes, as indicated by the increased frequency of self-reactive B-cells in patients with CD40LG deficiency (Hervé et al., 2007). However, CD40 also delivers an antiapoptotic signal to B-cells by inducing antiapoptotic genes that include Bcl-xL and A20 (Ishida et al., 1995; Sarma et al., 1995). It has been shown that C4 binding protein (C4BP), a regulator component of the classical complement pathway, may also bind to CD40 on human B-cells at a site that differs from that used by CD40LG. Engagement of CD40 by C4BP triggers signaling pathways similar to those triggered by CD40LG, as demonstrated by the fact that this interaction induces proliferation, upregulation of CD54 and CD86 expression, and IL-4–dependent IgE isotype switching in normal B-cells (Brodeur et al., 2003). IL-4 and IL-13, the switch factors for IgE, induce the transcription of a 1.8 kb ε‎ germline mRNA that initiates 5′ of the Sε‎ region. This transcript is sterile, as it is not translated into a functional protein. Induction of a mature 2.0 kb ε‎ mRNA and of IgE protein synthesis requires a second signal, provided by T-cells, via CD40LG/CD40 interactions (Oettgen, 2000). Cd40–/– mice fail to undergo T-cell–dependent isotype switching and fail to develop germinal centers following immunization with T-cell–dependent antigens (Castigli et al., 1994; Kawabe et al., 1994). Similar results were obtained in mice with disrupted CD40LG genes (Borrow et al., 1996; Xu et al., 1994). Cross-linking of CD40 in the presence of IL-4 induces the expression of AICD. AID has a critical role in CSR, as demonstrated by the fact that B-cells from AID–/– mice and from patients with AID deficiency are unable to undergo isotype switching (Muramatsu et al., 2000; Revy et al., 2000). Several molecules that play a role in DNA repair have been found to be important or essential for isotype switching; these include Ku70, Ku80, DNA-PK, Msh2, Pms2, and others (Stavnezer et al., 2008).

CD40/CD40LG interaction is also important for the maturation of myeloid DCs (marked by upregulation of CD80, CD83, CD86, and CD203) and for IL-12 production (Cella et al., 1996; Fontana et al., 2003). CD40LG-expressing CD8+ T-cells activate CD8α‎+ DC for IL-12 p70 production during antigen-specific T-cell responses and favor cross-presentation and cross-priming of cytotoxic T-cells (Wong et al., 2008). In addition, engagement of CD40 on the surface of plasmocytoid DCs drives a potent Th1 polarization and promotes interferon (IFN)-α‎ secretion in response to viral infections (Cella et al., 2000). In keeping with these observations, both maturation and activation of myeloid and plasmocytoid DCs are severely impaired in patients with CD40 deficiency (Fontana et al., 2003).

CD40 plays also a critical role in the induction of inflammatory responses and in thrombosis. Both CD40 and CD40LG are expressed on the surface of platelets. CD40LG/CD40-mediated platelet–platelet and platelet–lymphocyte interactions favor recruitment of leukocytes to sites of thrombosis or inflammation (Li, 2008). It has recently been shown that CD40 may also be expressed by T-cells, in particular activated CD4+ T lymphocytes, and that it can act as a co-stimulatory molecule and synergize with CD28 during TCR-mediated activation (Munroe et al., 2007).

CD40 plays an important role also in establishing the thymic medullary microenvironment and self-tolerance. In postnatal life, cooperation between CD40- and RANK-mediated signaling was found to be required to support differentiation and maintenance of thymic medulla in mice through a TNF-associated factor 6 (TRAF-6)-, NF-κ‎B inducing kinase (NIK)-, and Iκ‎B kinase β‎ (IKKβ‎)-dependent manner (Akiyama et al., 2008). In this process, CD40 on the surface of medullary thymic epithelial cells (mTECs) is engaged by CD40LG expressed by positively selected CD4+ thymocytes that bear self-reactive TCR-recognizing self-antigens expressed by mTECs in the context of major histocompatibility complex (MHC) class II molecules (Irla et al., 2008). Furthermore, negative selection of endogenously expressed antigens and superantigens is blocked by the administration of antibodies to CD40 ligand (Foy et al., 1995). Altogether, these findings establish a critical role for CD40 in central tolerance.

Regulation of CD40 Expression and Significance of CD40 Variants

In humans, transcription of the CD40 gene results in production of a 277-a.a. protein, which contains of a 22-a.a. signal sequence, a 171-a.a. extracellular domain, a 22-a.a. transmembrane domain, and a 62-a.a. cytoplasmic domain. Murine CD40 is a 289-a.a. protein and includes a C-terminal 11-a.a. sequence that is not present in human CD40. Regulation of CD40 expression and function is driven by posttranscriptional and posttranslational mechanisms.

CD40 expression is regulated at the transcriptional level by the AT-hook transcription factor AKNA. This factor is known to bind A/T-rich regulatory elements of the promoter DNA and to change their architecture, increasing promoter accessibility. This transcription factor coordinately regulates CD40LG expression as well and thus is important to promote homotypic cell interactions (Siddiqa et al., 2001).

Regulation of CD40 expression is achieved also at the posttranscriptional level through alternative splicing. Modulation of CD40 mRNA isoform expression appears to play an important role in the regulation of CD40LG-mediated signaling. Five different CD40 splicing variants are differentially expressed in activated murine macrophages and DCs (Tone et al., 2001). The type I isoform corresponds to the full-length, functional mRNA and is also the most abundant splicing variant. A C-terminal truncated CD40 mRNA isoform (type II) represents the major alternative splicing variant and encodes for a protein unable to deliver intracellular signaling. Type III and IV mRNA products also encode for nonfunctional protein and may act through a dominant negative mechanism by forming nonfunctional trimers with type I CD40 protein. Differential expression of CD40 mRNA splicing variants during brain development has been implied to play an important role in modulating neuronal differentiation, and CD40-deficient mice have aberrant neuron morphology and gross brain abnormalities (Hou et al., 2008).

In humans, various alternative CD40 splice variants have been also described. In particular, three splice variants have been identified that differ from full-length CD40 mRNA because they lack exon 5, exon 6, or both exons 5 and 6. Using computational biology tools, the first two of these variants were predicted to encode for soluble decoy receptors because of the lack of the transmembrane domain. Indeed, soluble forms of CD40 have been detected in the serum of uremic patients, but only the isoform lacking the a.a. encoded by exon 6 has the ability to bind CD40LG and may thus serve as a true decoy receptor (Eshel et al., 2008).

Several variants have been identified in the CD40 gene, and some of these have been associated with an increased risk to develop certain diseases. Association of CD40 single nucleotide polymorphisms (SNPs) with autoantibody-positive rheumatoid arthritis (possibly involving increased NF-κ‎B-mediated signaling) has been reported (Raychaudhuri et al., 2008). This association suggests that CD40 signaling could mediate rheumatoid pathogenesis through NF-κ‎B activation. Contradictory data are available on the association between CD40 known variants (particularly one in the Kozak fragment) and increased susceptibility to Graves disease, with some evidence that increased thyroidal expression of mutated CD40 may contribute to this disease specificity (Jacobson et al., 2007; Kurylowicz et al., 2005). The same intronic variant has been associated with an increased risk of follicular lymphoma associated with lower expression of CD40 molecule (Skibola et al., 2008).

Some of these SNPs have been characterized from the functional point of view. The P227A CD40 variant resides in the cytoplasmic domain, has been found only in heterozygosity, and has a frequency of 29 percent in South American–descent subjects and less than 1 percent in all the other ethnic groups. This variant has been found to result in increased IgM production, augmented secretion of IL-6 and TNF-alpha, and phosphorylation of the JNK (MAPK8) target, c-Jun, even if binding affinity for TRAF molecules is similar to that reported for wild-type CD40 (Peters et al., 2008).

A single nucleotide change, resulting in an a.a. substitution (p.H78Q) in the second cysteine-rich extramembrane region of CD40, has been described in a multiple myeloma cell line (U266) and in freshly isolated tumor cells. This mutant protein has unique functional properties, including decreased binding affinity for CD40LG and constitutive association with TRAF6 in the absence of CD40LG stimulation. The overall survival rate of patients with multiple myeloma carrying this CD40 mutation was significantly poorer than those expressing wild-type CD40, suggesting that the mutated protein has a role in tumor invasion and metastasis and that CD40/CD40LG interaction may modulate tumor pathogenesis and/or progression (Qi et al., 2009).

CD40-Mediated Signaling

A schematic representation of CD40-mediated signaling pathways is shown in Figure 26.1. CD40 lacks intrinsic catalytic activity, but its cytoplasmic domain contains binding motifs for several signal-transducing molecules, thus permitting CD40-mediated regulation of humoral and cellular immunity processes (Elgueta et al., 2009). In particular, CD40 binds Jak3 in its proline-rich Box 1 membrane proximal region (a.a. 222–229) and has one additional binding site for TRAF-6 and one for TRAF-2/3/5 proteins (Hanissian and Geha, 1997). Recently, another TRAF-2 binding site has been identified, which is also involved in mediating B-cell activation, proliferation, and differentiation (Lu et al., 2007). CD40 ligation by membrane-bound trimeric CD40LG causes a higher degree of oligomerization and, most importantly, a conformational change that results in translocation of CD40 and associated TRAF proteins to lipid rafts (Xia et al., 2007). When TRAF proteins bind to the cytoplasmic tail of CD40, various signaling cascades are activated, including JAK3-STAT5, NF-κ‎B, c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein (MAP) kinase pathways (Jabara et al., 2009; Jalukar et al., 2000; Revy et al., 1999; Zarnegar et al., 2004). In B lymphocytes, CD40-induced activation of TRAF-6 is required for IgM production and isotype switching, whereas CD40 interaction with TRAF-2/3/5 promotes CD80/CD86 upregulation and protection from B-cell-antigen-receptor–mediated growth arrest (Ahonen, 2002).

Figure 26.1 Hypothetical model of CD40 signaling. CD40 ligation causes enhanced association of CD40 with tumor necrosis factor receptor-associated factor (TRAF) proteins, resulting in activation of various signaling pathways. In B lymphocytes CD40-induced activation results in transcription of critical genes involved in B-cell proliferation and terminal B-cell differentiation. TRAF2 and TRAF6 interact and activate both the NF-κ‎B and the JNK/p38 pathways. NF-κ‎B activation is regulated via the binding with the RIP protein. Various signaling pathways induce phosphorylation of the IKK complex, leading to nuclear translocation of NF-κ‎B and transcription of target genes.

Figure 26.1
Hypothetical model of CD40 signaling. CD40 ligation causes enhanced association of CD40 with tumor necrosis factor receptor-associated factor (TRAF) proteins, resulting in activation of various signaling pathways. In B lymphocytes CD40-induced activation results in transcription of critical genes involved in B-cell proliferation and terminal B-cell differentiation. TRAF2 and TRAF6 interact and activate both the NF-κ‎B and the JNK/p38 pathways. NF-κ‎B activation is regulated via the binding with the RIP protein. Various signaling pathways induce phosphorylation of the IKK complex, leading to nuclear translocation of NF-κ‎B and transcription of target genes.

TRAF2 and TRAF6 play an important physiological role in CD40 signaling also in nonhematopoietic cells, and their role is mutually linked, as TRAF6 regulates CD40 signal transduction not only through its direct binding to CD40 but also indirectly via its association with TRAF2 (Davies et al., 2005). A truncated derivative of TRAF2 lacking an amino-terminal RING finger domain is a dominant-negative inhibitor of NF-κ‎B activation mediated by CD40 (Rothe et al., 1995). An 11-a.a motif in the intracellular domain of CD40 that spans the core Px-QxT TRAF2,3 binding sequence was found to be sufficient for the activation of Jun, p38, and NF-κ‎B. A CD40 mutant that binds TRAF2 but not TRAF3 (or TRAF5) was shown to activate NF-κ‎B. This finding, together with the inability of TRAF1 to activate NF-κ‎B, suggests that TRAF2 is the most important element in CD40-mediated activation of NF-κ‎B. TRAF2 has also been found to bind to the kinase NIK, which can also phosphorylate and activate the IKK complex. TRAF2 also binds to RIP, a protein that is central to NF-κ‎B activation by TNFR1 and to the protection of cells from TNF-mediated death. RIP binds to MEKK3, which then phosphorylates and activates IKK (Wang et al., 2001). It has been shown that TRAF6 functions as an E3 ubiquitin ligase to catalyze, together with Ubc13/Uev1A, the synthesis of polyubiquitin chains linked through lysine-63 (K63) of ubiquitin and the activation of a TAK1–TAB1/2 complex, which phosphorylates and activates IKK (Deng et al., 2000; Shuto et al., 2001; Yang et al., 2000).

Data from knockout mice and from patients with IKKγ‎/NEMO mutations (Döffinger et al., 2001; Jain et al., 2001; Zonana et al., 2000) strongly suggest that NF-κ‎B plays an important role in CD40-mediated isotype switching. Traf2–/– mice die perinatally, but Traf2–/–Tnfr1–/– and Traf2–/– Tnf–/– double-mutant mice are viable (Yeh et al., 1997). Isotype switching to IgG is impaired in Traf2–/– Tnfr1–/– mice. This finding further supports the idea that a pathway consisting of TRAF2-mediated activation of NF-κ‎B in CD40-activated B-cells is critical for isotype switching. Two different NF-κ‎B activation pathways have been described: type 1 (p50-dependent) and type 2 (p52-dependent). In resting B-cells, the alternative NK-κ‎B signaling pathway is inhibited by TRAF2, TRAF3, cIAP1, and cIAP2 by targeting and ubiquitin-dependent degradation of NIK (NF-κ‎B-inducing kinase), thus preventing cleavage of p100 precursors. CD40 and B-cell activating factor receptor (BAFF-R) engagement results in TRAF3 degradation, which blocks association of NIK with the cIAP1–cIAP2–TRAF2 ubiquitin ligase complex and p100 processing and hence enables activation of the type 2 NF-κ‎B signaling pathway (Vallabhapurapu et al., 2008). CD40 and lipopolysaccharide (LPS) can both mediate activation of the type 1 NF-κ‎B signaling pathway. The observation that CD40LG/CD40 interaction promotes homotypic cell adhesion, whereas neither LPS nor BAFF is proficient in this process, indicates that simultaneous activation of type 1 and type 2 NF-κ‎B signaling pathways is required to induce homotypic B-cell adhesion (Zarnegar et al., 2004). Finally, the redox factor APE/Ref-1 has been reported to act as a key signaling intermediate in response to CD40-mediated B-cell activation. Upon CD40 cross-linking, TRAF2 adapter is involved in APE/Ref-1 translocation from the cytoplasm to the nucleus, where it modulates the DNA-binding activity of the Pax5 and EBF transcription factors. APE/Ref-1 appears to be required for CD40-mediated Pax5 activation, as the repression of APE/Ref-1 protein production is able to block CD40-induced Pax5 binding activity (Merluzzi et al., 2008).

CD40 Ligand

The murine CD40LG has been identified and the gene cloned as an EL4 thymoma cell surface molecule that binds to soluble CD40 (Armitage et al., 1992). Subsequently, the human CD40LG gene (*300386), encoding for CD40LG (CD154), was cloned from activated T-cells (Graf et al., 1992; Hollenbaugh et al., 1992; Spriggs et al., 1992). Independently, Lederman et al. identified a subclone of Jurkat T-cells that is able to provide contact-dependent helper function to B-cells (Lederman et al., 1992); the molecule responsible for this helper activity was cloned and confirmed to be CD40LG. The human cDNA has an open reading frame of 783 base pairs (bp) that codes for a type II membrane protein 261 a.a. long. The extracellular domain is 215 a.a. long, the transmembrane domain is 24 a.a. in length, and the intracellular domain is 22 a.a. long. The presence of a proteolytic cleavage site (His-Arg-Arg-Leu) immediately proximal to the transmembrane domain allows production of soluble forms of CD40LG. Platelets represent the major source of soluble CD40LG in the blood, and recent data indicate that the matrix metalloproteinase-2 (MMP-2) might be the protease primarily responsible for CD40LG cleavage from platelet surface (Reinboldt et al., 2009). Molecular modeling of CD40LG based on the crystal structure of TNF indicates that a.a. residues 189 to 209 are critical for the binding of CD40LG to CD40 (Peitsch and Jongeneel, 1993).

Although de novo synthesis of CD40LG occurs following antigen recognition by T lymphocytes, surface mobilization of preformed, intracellular CD40LG has been also observed in both effector and memory CD4+ T-cells following antigen recognition. Intracellular CD40LG is stored in secretory lysosomes and co-localizes more strongly with Fas ligand than with CTLA-4, two other molecules that are delivered to the cell surface following antigen recognition (Koguchi et al., 2007).

Detailed organization of the murine (Tsitsikov et al., 1994) and human (Shimadzu et al., 1995; Villa et al., 1994) CD40LG genes has been reported. The gene consists of five exons. Exon 1 encodes for the 5′ untranslated region and the first 52 a.a., including 22 a.a. of the intracytoplasmic tail, 24 a.a. of the transmembrane domain, and 6 a.a. of the extracellular domain. Exons 2, 3, and 4 encode a.a. 53 to 96, a.a. 97 to 114, and a.a. 115 to 135, respectively. Exon 5 encodes the rest of the protein (a.a. 136–260) and the 3′ untranslated region. There is no homology in the position of the exons between CD40LG and any of the known genes from the TNF family, but in all these genes the sequence encoding the receptor binding domain is located in the last exon.

Several studies have illustrated the importance of CD40 and its ligand, CD40LG, in humoral immunity. Recombinant CD40LG mimics the action of CD40 monoclonal antibody (MAb) and is able to stimulate B-cell proliferation in the presence of phorbol myristate acetate (PMA) and to induce immunoglobulin synthesis (Lane et al., 1993; Spriggs et al., 1992). Immunoglobulin secretion by B-cells is highly modulated by cytokines. In the presence of recombinant CD40LG, IL-2 and IL-10 induce specifically the secretion of IgM, IgG1, and IgA (Durandy et al., 1993), whereas IL-4 is necessary for the secretion of IgG4 and IgE. Antibody to CD40LG can block T-cell–dependent B-cell activation (Noelle et al., 1992a, 1992b). Immunization of mice with KLH (a thymus-dependent antigen) or DNP-Ficoll (a thymus-independent antigen) induces the expression of CD40LG by T helper (Th) cells, and this induction coincides with the development of cytokine-producing cells. Through the use of immunocytochemical techniques, it was elegantly demonstrated that CD40LG cells and cytokine-producing cells are juxtaposed in the lymphoid organs (Van den Eertwegh et al., 1993). Short-term treatment with anti-CD40LG antibody suppressed the immune response against these antigens, but adoptive transfer of cells from anti-CD40LG–treated mice could fully reconstitute Th-cell function in irradiated recipient mice, showing that immune suppression does not involve clonal anergy or deletion (Foy et al., 1993). Thus it appears that CD40LG expression is essential for both thymus-dependent and thymus-independent antigens. However, in mice with targeted disruption of the Cd40 or Cd40lg gene, only the antibody response against the T-dependent antigen is impaired. This finding suggests that stimulation via CD40/CD40LG may not be critical for antibody responses to T-independent antigens.

As reported above, CD40LG is also essential for the maturation of myeloid DCs and the activation of plasmacytoid DCs. In addition, it promotes macrophage effector function, as shown by the fact that Cd40lg–/– mice are less effective in stimulating allogenic macrophages to produce inflammatory cytokines and reactive nitrogen intermediates (Stout et al., 1996). CD40LG/CD40-dependent interaction between activated CD4+ T-cells and DCs is also essential for efficient T-cell priming and licensing of cytotoxic CD8+ T lymphocytes. On the other hand, recent data have shown that stimulation of TLR3 and TLR9 with agonists and certain viruses (such as influenza virus) can induce CD40LG expression of the surface of DCs and may promote efficient priming of cytotoxic CD8+ T-cells, even in the absence of CD4+ T lymphocytes (Johnson et al., 2009). Finally, CD40LG+ T-cells that are generated under strong antigenic stimulation, in concert with certain microbial stimuli, synergistically increase DC IL-6 production and Th17 polarization. CD40-deficient DCs exhibit reduced cytokine release and fail to drive Th17 development in vitro (Iezzi et al., 2009).

Regulation of CD40 Ligand Expression

CD40LG is a highly inducible gene and is mainly expressed by CD4+ T-cells, although it can also be expressed by other cell types, including CD8+ cells, monocytes, natural killer (NK) cells, and megakaryocytes (Cocks et al., 1993; Crist et al., 2008). In addition, masT-cells, basophils (Gauchat et al., 1993b), and eosinophils (Gauchat et al., 1995) can also express functional CD40LG. The T-cell expression of CD40LG is well regulated; resting T lymphocytes do not express CD40LG. In α‎/β‎ T-cells, CD40LG mRNA becomes detectable as early as 1 hour after stimulation with PMA and ionomycin, peaks at 3 hours, and disappears by 24 hours. Surface CD40LG is detected as early as 3 hours after stimulation with PMA and ionomycin, peaks at 6 hours after stimulation, starts to decline by 8 hours, and is barely detectable by 16 hours. However, activation with anti-CD3 results in sustained expression of CD40LG for 24 hours after stimulation (Castle et al., 1993; Roy et al., 1993). Simultaneous engagement of the T-cell co-stimulatory molecule CD28 by mAb enhances CD40LG expression (Ding et al., 1995; Klaus et al., 1994). Highly purified peripheral blood γδ‎ T-cells also express CD40LG after stimulation (Horner et al., 1995). The kinetics of mRNA synthesis and surface expression closely resembles that of αβ‎ T-cells, but the levels are much lower. Prolonged Cd40lg mRNA half-life has been observed following activation of antigen-primed T-cells as compared to naïve T-cells, indicating that posttranscriptional mechanisms play an important role in controlling CD40LG expression during immune responses. In particular, it has been demonstrated that a CU-rich site within the 3′-untranslated region of CD40LG mRNA binds to a polypyrimidine tract-binding protein-containing complex (mComplex I) that is activation dependent (Vavassori et al., 2009).

CD40LG expression is developmentally regulated. Newborn T-cells were found to be deficient in CD40LG expression and in their ability to induce isotype switching in B-cells (Brugnoni et al., 1994; Durandy et al., 1995; Fuleihan et al., 1994; Nonoyama et al., 1995), and reduced CD40LG expression persists for the first 6 months of age (Gilmour et al., 2003).

Cyclosporin A (CsA) inhibits the surface and mRNA expression of CD40LG in human (Fuleihan et al., 1994) and murine (Roy et al., 1993) T-cells. CsA is a naturally occurring immunosuppressant that binds to its cellular receptor(s), cyclophilin, forming a complex that inhibits the activity of the phosphoprotein phosphatase calcineurin. Calcineurin dephosphorylates the cytoplasmic subunit of the transcription factor, NFAT, which translocates into the nucleus to form the functional NFAT complex and regulates the expression of the gene encoding IL-2 (Jain et al., 1993). The transcription of CD40LG mRNA and the surface expression of the protein are inhibited by pretreatment of T-cells with CsA in a dose-dependent manner. The ability of CsA analogs to inhibit CD40LG expression correlated with the affinity of the cyclophilin–drug complex to calcineurin and not with the affinity of the drug to cyclophilin. These results suggest that transcription factors activated by calcineurin, such as NFAT, regulate the transcription of the CD40LG gene, and this has been experimentally confirmed (see below).

Hydrocortisone upregulates CD40LG mRNA and protein expression in peripheral blood mononuclear cells (PBMCs) and induces IgE synthesis in IL-4–stimulated normal human B-cells. Disruption of CD40LG–CD40 interaction by soluble CD40-Ig fusion protein or anti-CD40LG mAb blocks the capacity of hydrocortisone to induce IgE synthesis in normal B-cells. Upregulation of CD40LG mRNA and induction of IgE synthesis by hydrocortisone were inhibited by the steroid hormone receptor antagonist RU-486 (Jabara et al., 2001). These results indicate that ligand-mediated activation of the glucocorticoid receptor (GR) upregulates CD40LG expression in human lymphocytes. It is possible that hydrocortisone acts by inducing GR binding to glucocorticoid-responsive elements present in the CD40LG promoter.

The organization of the murine and human CD40LG genes shows remarkable conservation even in the 5′ upstream regulatory sequences. The transcription originates from a G residue 68 bp upstream from the A of the initiation codon in both species (Schubert et al., 1995; Tsitsikov et al., 1994). Sequences up to 1.5 kb upstream from the murine gene lack the TATAA or CCAAT boxes but have an Sp1 sequence, six NFAT-like sequences, and one OAP-like site. Three of the NFAT-like consensus sequences (including the two most proximal ones) are conserved in the human CD40LG gene. The most proximal NFAT-binding motifs of the murine gene form two complexes as detected by electrophoretic mobility shift analysis (EMSA). Both complexes contain NFATc and NFATp and are sensitive to CsA (Tsytsykova et al., 1996). Similar findings have been reported for the human CD40LG promoter (Schubert et al., 1995).

A T-cell–specific CD40LG transcriptional enhancer is located upstream of the promoter. This enhancer binds NFAT1 and the Th2 transcription factor GATA-3 (Brunner et al., 2008). The 3′ untranslated region of the murine gene has two adjacent microsatellite repeats (a 50-bp-long CT and a 90-bp-long CA repeat) as well as two ATTTA elements that are putatively responsible for the stability of mRNA.

Clinical and Pathological Manifestations of CD40 Ligand Deficiency

CD40LG deficiency is characterized by an X-linked pattern of inheritance and clinical features that are suggestive of a combined immunodeficiency. Most patients with CD40LG deficiency present in infancy with recurrent upper and lower respiratory tract infections and have a unique predisposition to Pneumocystis jiroveci pneumonia, which may even mark the clinical onset of the disease (Levy et al., 1997; Marshall et al., 1964; Ochs and Wedgwood, 1989; Winkelstein et al., 2003). The frequency of P. jiroveci pneumonia in CD40LG deficiency varies from 31.7 to 48.1 percent in the European and U.S. series of patients (Winkelstein et al., 2003; L. Notarangelo et al., unpublished observation). Lung infections due to cytomegalovirus, respiratory syncytial virus, cryptococcus, and mycobacteria, including bacillus Calmette-Guérin, have been also reported and may lead to disseminated disease (Banatvala et al., 1994; Fremerey et al., 2009; Hostoffer et al., 1994; Iseki et al., 1994; Kyong et al., 1978; Levy et al., 1997; Tabone et al., 1994; Tu et al., 1991). An increased risk for paracoccidioidomycosis and other unsual infections has been recently reported (Cabral-Marques et al., 2012).

Diarrhea occurs in over 50 percent of CD40LG-deficient patients (Levy et al., 1997; Notarangelo et al., 1992; Winkelstein et al., 2003) and may become chronic and require total parenteral nutrition. Chronic watery diarrhea is often associated with Cryptosporidium infection (Stiehm et al., 1986), which may also contribute to sclerosing cholangitis, a severe and often fatal complication (Banatvala et al., 1994; DiPalma et al., 1986; Hayward et al., 1997; Levy et al., 1997; Winkelstein et al., 2003). The incidence of liver and biliary tract disease increases with age (Hayward et al., 1997).

Oral ulcers and proctitis are common manifestations (Banatvala et al., 1994; Benkerrou et al., 1990; Hong et al., 1962; Kyong et al., 1978; Macchi et al., 1995; Notarangelo et al., 1992; Rieger et al., 1980) and are usually associated with neutropenia, either chronic (Aruffo et al., 1993; Levy et al., 1997) or cyclic (Notarangelo et al., 1992; Shimadzu et al., 1995; Wang et al., 1994).

CD40LG-deficient patients have osteopenia, with lower bone mineral density and elevated levels of N-terminal telopeptides of type I collagen, a urinary marker indicative of osteoclast activity. Osteoclast differentiation of myeloid cells is induced by RANKL, and osteoclastic activity is modulated by IFN-γ‎. CD4+ CD40LG-deficient human T-cells have normal expression of RANKL and promote marked osteoclastogenesis of myeloid cells; however, this activity cannot be properly modulated because of impaired T-cell priming and reduced IFN-γ‎ production (Lopez-Granados et al., 2007).

Patients with CD40LG mutations are also at increased risk for lymphomas (Filipovich et al., 1994) and liver/biliary tract and gastrointestinal tumors (including peripheral neuroectodermal tumors), which are rarely observed in other primary immunodeficiencies (Erdos et al., 2008; Facchetti et al., 1995; Hayward et al., 1997).

Autoimmune manifestations (arthritis, thrombocytopenia, hemolytic anemia, hypoparathyroidism, immune complex–mediated nephritis, and retinal pigment epithelium hypersensitivity) may occur in CD40LG deficiency, although they are less common than in autosomal forms of CSR defects (Benkerrou et al., 1990; Hollenbaugh et al., 1994, Pascual-Salcedo et al., 1983; Schuster et al., 2005). Anemia may also be secondary to chronic infections or to parvovirus B19-induced red blood cell aplasia. The latter may even be the only manifestation of disease in patients with a mild phenotype (Blaeser et al., 2005; Seyama et al., 1998a).

Severe neurological involvement has also been reported. Meningoencephalitis due to enterovirus infection may occur, despite regular administration of intravenous immunoglobulins (IVIG) (Cunningham et al., 1999; Halliday et al., 2003). Cerebral toxoplasmosis and progressive multifocal leukoencephalopathy, in some cases caused by reactivation of the human neurotropic JC virus, have been reported as the first sign of disease in adults with hypomorphic CD40LG mutations (Aschermann et al., 2007; Suzuki et al., 2006; Yong et al., 2008).

Lymph nodes of CD40LG-deficient patients lack germinal centers (Facchetti et al., 1995; Hong et al., 1962; Rosen et al., 1961; Stiehm and Fudenberg, 1966) (Color Plate 26.I). This is the consequence of ineffective CD40–CD40LG interaction in the extrafollicular areas, resulting in poor recruitment of germinal-center precursors. In addition, severe depletion and phenotypic abnormalities of follicular DCs have been reported that may contribute to poor antigen trapping and result in inefficient rescue of the few germinal-center B-cells from apoptosis (Facchetti et al., 1995). Bone marrow examination in patients with concurrent neutropenia often reveals a block of myeloid differentiation at the myelocyte/promyelocyte stage (Benkerrou et al., 1990; Hong et al., 1962; Kyong et al., 1978; Notarangelo et al., 1992). By contrast, serum levels of granulocyte colony-stimulating factor (G-CSF) in neutropenic CD40LG-deficient patients are normal or elevated (Wang et al., 1994).

Plate 26.1 Pathology of lymph nodes in X-linked form of hyper-IgM (XHIM). (A) Morphology of a normal lymph node is shown, with presence of secondary follicles within germinal centres. (B) A lymph node from an XHIM patient shows primary follicles with no evidence of germinal centers. Sections were stained with hematoxylin-eosin. (Courtesy of Prof. F. Facchetti, Department of Pathology, University of Brescia, Italy.)

Plate 26.1
Pathology of lymph nodes in X-linked form of hyper-IgM (XHIM). (A) Morphology of a normal lymph node is shown, with presence of secondary follicles within germinal centres. (B) A lymph node from an XHIM patient shows primary follicles with no evidence of germinal centers. Sections were stained with hematoxylin-eosin. (Courtesy of Prof. F. Facchetti, Department of Pathology, University of Brescia, Italy.)

A European registry of CD40LG-deficient patients has been organized that includes clinical, immunological, and molecular data (Notarangelo et al., 1996). The registry is accessible through at http://bioinf.uta.fi/CD40Lbase. A similar registry has been set up in the United States by USIDnet.

Laboratory Findings

Immunoglobulin Levels

Like all forms of impaired CSR, CD40LG deficiency is characterized by markedly reduced serum IgG, IgA, and IgE with variable IgM levels and a normal number of circulating B-cells (Geha et al., 1979; Levy et al., 1987; Notarangelo et al., 1992; Winkelstein et al., 2003). Variability of IgM serum levels has been reported among affected members of the same family, indicating that increased IgM may reflect chronic antigenic stimulation rather than the direct effect of a molecular defect (Kroczek et al., 1994). In a series of 56 CD40LG-deficient patients, the majority (53 percent) had normal IgM serum levels at the time of diagnosis (Levy et al., 1997). In another study, however, as many as one quarter of patients with confirmed CD40LG deficiency had low concentrations of serum IgM, indicating that even low serum IgM levels should not preclude testing for CD40LG deficiency (Gilmour et al., 2003). Altogether, the variability of IgM serum levels and the broad range of clinical manifestations, which go beyond abnormalities of humoral immunity, have led researchers to abandon the older designation of “X-linked hyper-IgM” that was previously used to indicate CD40LG deficiency. Although serum levels of IgG, IgA, and IgE are normally very low in patients with CD40LG deficiency, exceptional cases with elevated IgA, IgE, or even IgG have been observed (Levy et al., 1997; L. Notarangelo, unpublished observation), indicating that environmental and/or other genetic factors may have an impact on CSR. In particular, signaling through Toll-like receptors may permit CD40LG/CD40-independent CSR (Glaum et al., 2009; Pasare et al., 2005; Xu et al., 2008).

Serum isohemagglutinins are usually normal (Benkerrou et al., 1990; Kyong et al., 1978; Rosen et al., 1961). In contrast, immunization with T-dependent antigens (e.g., bacteriophage φ‎x174) leads to reduced primary and secondary IgM antibody responses and little or no production of IgG-specific antibodies following recall immunization (Benkerrou et al., 1990; Nonoyama et al., 1993; Stiehm and Fudenberg, 1966). In addition, analysis of VH gene segments in patients with CD40LG mutations has revealed a lower frequency of somatic mutations in IgM-expressing B-cells than that in controls (Chu et al., 1995; Razanajaona et al., 1996). This defect is particularly pronounced at the hypermutable G in the RGYW motif, which is a typical target of AICD. Along with the defect in frequency, SHM in patients with CD40LG deficiency is characterized by an increase in transitions versus transversions, reminiscent of decreased UNG activity (Longo et al., 2009). These data are in keeping with the notion that both AICD and UNG are transcriptional targets of CD40LG.

Defects of CSR and SHM are a direct consequence of the underlying genetic defect. Because of the inability to express functional CD40LG trimers, activated T-cells are unable to provide a key helper signal for terminal B-cell differentiation. The functional integrity of their B-cells, by contrast, was initially suggested by the experiments performed by Mayer et al. (1986), who demonstrated that B-cells from these patients can be driven to secrete immunoglobulins of various isotypes in the presence of pokeweed mitogen when co-cultured with “helper T lymphoblasts” from a patient with a Sézary-like syndrome. Subsequently, it was shown that co-culture of patient-derived PBMCs with anti-CD40 mAb (or soluble CD40LG) and appropriate cytokines, such as IL-4 and IL-10, results in normal CSR and in vitro production of IgG, IgA, and IgE (Allen et al., 1993a; Aruffo et al., 1993; Callard et al., 1994; Durandy et al., 1993; Fuleihan et al., 1993a; Korthauer et al., 1993; Saiki et al., 1995).

B and T Lymphocytes

Subjects with CD40LG deficiency have a normal number of circulating B lymphocytes, and this allows differentiation from X-linked agammaglobulinemia. However, circulating B-cells express IgM and/or IgD, but not other isotypes (Benkerrou et al., 1990; Levitt et al., 1983). Furthermore, since CD40LG–CD40 interaction is essential for memory B-cell generation, the number of circulating IgDCD27+ switched memory B-cells is strongly diminished in patients with CD40LG mutations (Agematsu et al., 1998).

In spite of the T-cell nature of the defect in CD40LG deficiency, the number and distribution of T-cell subsets are normal, although the proportion of CD45R0+ primed T-cells is reduced (Jain et al., 1999). In vitro proliferative response to mitogens is normal, but T-cell proliferation to antigens is often reduced (Ameratunga et al., 1997; Levy et al., 1997). A defect in TH1 responses has been reported, with reduced secretion of IFN-γ‎ and failure to induce antigen-presenting cells to synthesize IL-12 (Jain et al., 1999; Subauste et al., 1999). A summary of laboratory features typically observed in CD40LG deficiency is shown in Table 26.1.

Table 26.1 Synopsis of Laboratory Findings in Patients with CD40LG Deficiency

Parameter

Typical Phenotype

Percent of CD40LG-Deficient Patients Showing Phenotype* Phenotype*

Immunological Features

Serum IgG

<2 SD below normal range

99–100

Serum IgA (mg/dL)

Undetectable

76–93

Serum IgM (mg/dL)

Normal

53–59 (at diagnosis)

Elevated

32–47 (at diagnosis)

Antibody response to T-dependent antigens (ϕ‎ × 174)

Lack of specific IgG production

100

B-cell count

Normal

90

CD4+ cell count

Normal

94

CD8+ cell count

Normal

98

Proliferative response to PHA

Normal (>50,000 cpm)

93

Proliferative response to T-dependent antigens

Often reduced (<5,000 cpm)

37

CD40LG expression**

As assessed with CD40-Ig

Absent

Nearly 100††

As assessed with mAb

Usually absent

95

As assessed with polyclonal antiserum

Detectable in some cases

23

Hematological Features

Neutropenia

Generally present (mostly chronic)

63–68

Anemia

Often present

15–32

* Unless differently specified, data are from Levy et al. (1997) and from Winkelstein et al. (2003).

Elevated levels of IgM are detected in about 70% of patients during follow-up.

** From a series of 22 patients analyzed at the Department of Pediatrics, University of Brescia, Italy.

†† Activated CD4+ T-cells from patients with missense mutations in intracytoplasmic or transmembrane domains of CD40LG may occasionally react with CD40-Ig (Seyama et al. 1998b).

Molecular Basis of CD40 Ligand Deficiency

The gene causing X-linked hyper-IgM (as CD40LG deficiency was originally known) was mapped to Xq26.3–27 by linkage analysis (Mensink et al., 1987; Padayachee et al., 1992, 1993). Cloning of the human CD40LG gene and coincidental mapping to the same region of the X chromosome (Graf et al., 1992) were soon followed by the recognition that CD40LG mutations account for the disease (Allen et al., 1993a; Aruffo et al., 1993; DiSanto et al., 1993; Fuleihan et al., 1993b; Korthauer et al., 1993) and by definition of the gene organization (Shimadzu et al., 1995; Villa et al., 1994). The human CD40LG gene encompasses about 13 kb of genomic DNA and is organized in five exons and four introns. Definition of the exon–intron boundaries has enabled a search for mutations at the genomic level (Lin et al., 1996; Villa et al., 1994).

CD40LG Mutations

Since 1993, a long list of unique CD40LG gene mutations have been identified in patients with CD40LG deficiency. Figure 26.2 illustrates the mutations identified in 188 patients included in the European CD40LGbase Registry or the Human Gene Mutation Database (http://www.hgmd.cf.ac.uk/ac/index.php). Although mutations may affect the entire gene, they are unequally distributed and the majority are located in exon 5, which contains most of the TNF-homology domain (Hollenbaugh et al., 1992).

Figure 26.2 Description of CD40LG mutations identified in 188 patients from independent families enrolled in the European CD40Lbase Registry or reported to the Human Gene Mutation Database (http://www.hgmd.cf.ac.uk/ac/index.php). The various mutations are shown with different symbols, as indicated in the figure, and aligned along the CD40LG protein. Correspondence between the protein and the five exons of the CD40LG gene is also shown.

Figure 26.2
Description of CD40LG mutations identified in 188 patients from independent families enrolled in the European CD40Lbase Registry or reported to the Human Gene Mutation Database (http://www.hgmd.cf.ac.uk/ac/index.php). The various mutations are shown with different symbols, as indicated in the figure, and aligned along the CD40LG protein. Correspondence between the protein and the five exons of the CD40LG gene is also shown.

Missense mutations are the most common cause of the disease. Mutational hot-spots (leading to missense or nonsense mutations) have been identified at codons 140, 155, and 254; none of these involves the presence of CpG dinucleotides. In some cases, for example the Trp140stop mutation, premature termination is compatible with expression of a truncated molecule at the cell surface, as shown by staining with polyclonal anti-CD40LG antibody (Korthauer et al., 1993, Seyama et al., 1998b). Small insertions or deletions in the CD40LG gene are also common; these may be due to polymerase slippage, occurring at sites of nucleotide duplications or tandem repeats in the CD40LG sequence (Macchi et al., 1995). A few patients with two different mutations have been described (Aruffo et al., 1993; Grammer et al., 1995; Lin et al., 1996).

Investigation of the effect of amino acid substitutions on CD40LG expression and function, studied through several approaches, has contributed to definition of the role that single residues play in determining folding, assembling, and CD40-binding properties of CD40LG. For some naturally occurring mutations, transfectants that express surface membrane or soluble forms of mutant CD40LG molecules have been generated. The mutagenized recombinants fail to bind CD40-Ig or to induce B-cell proliferation and immunoglobulin secretion in the presence of IL-4 (Allen et al., 1993a; Aruffo et al., 1993).

Functional Aspects of CD40LG Deficiency

Human CD40LG belongs to the TNF family (Hollenbaugh et al., 1992). As mentioned above, mutations cluster predominantly in the extracellular TNF-homology domain. Despite the rather limited sequence identity of the CD40LG TNF-like domain with TNF (27.3 percent), the homology is sufficient to allow computer modeling based on the crystal structures available for TNF and for the TNF/TNFR complex (Banner et al., 1993; Eck and Sprang, 1991; Jones et al., 1989). Thus, models have been generated for murine (Peitsch and Jongeneel, 1993) and human (Bajorath et al., 1995a, 1995b; Notarangelo et al., 1996) CD40LG, and an X-ray structure of the extracellular portion of human CD40LG has been produced (Karpusas et al., 1995). Analogous to TNF, CD40LG forms a trimer and exhibits a remarkably similar overall fold, with the extracellular portion assuming the shape of a truncated pyramid (Karpusas et al., 1995). A disulfide bond between Cys178 and Cys218 stabilizes the top of the molecule. The CD40 binding site consists of a shallow groove formed between two monomers. Buried and solvent-accessible residues of the CD40LG molecule have been identified (Bajorath et al., 1995a, 1995b; Karpusas et al., 1995). Interestingly, several residues (Gly144, Lys143, Tyr145, Arg203, and Gln220) that are predicted to be directly involved in CD40 binding (Bajorath et al., 1995a, 1995b) were found to be mutated in patients with CD40LG deficiency. The crystal structure of the extracellular portion of CD40LG has shown that both hydrophobic and hydrophilic residues form the surface of the CD40 binding site (Karpusas et al., 1995). In addition, a number of buried residues appear to be important for proper monomer folding and trimer formation. By applying structural and bioinformatics tools, the consequences of the missense CD40LG mutations have been analyzed in detail in several studies. In one recent study, 40 percent of the missense mutants were predicted to cause major structural abnormalities, and 37 percent affect residues that are important for trimerization or for ligand binding (Thusberg and Vihinen, 2007). In particular, Ser128 and Glu129 (which have both been reported to be mutated) lie close to Lys143 in the three-dimensional structure. Since Lys143 is involved in CD40 binding, it is likely that mutations at codons 128 and 129 disturb ligand–receptor interaction (Bajorath et al., 1996; Karpusas et al., 1995); furthermore, they were shown to cause clashes with other side chains of the molecule (Thusberg and Vihinen, 2007). Similarly, Val126 and Leu 155 (also mutated in patients) participate in the formation of a hydrophobic core that is very close to residues 143–145, which are involved in CD40 binding; it is predicted that these mutations also affect the CD40 binding property of CD40LG, and replacement of Leu155 was also found to have an impact on protein structure. Finally, three additional mutations (Thr147Asn, Thr211Asp, Gly250Ala) involve residues that are either buried (Thr147) or exposed (Thr211, Gly250) and are located at the interface between monomers; they participate in the formation of three equivalent CD40 binding sites, one per interface between monomers. Mutation at these residues should also affect CD40 binding (Bajorath et al., 1996).

However, the majority of missense mutations reported in patients do not involve the CD40 binding site. The amino acids affected may participate in the generation of the hydrophobic core, so mutations at these residues compromise core packing and folding of the monomer (this is the case for Trp140Arg, Trp140Cys, Trp140Gly, Leu232Ser, Ala235Pro, Val237Glu, Thr254Met, and Leu258Ser) (Bajorath et al., 1996). Alternatively, they may involve buried residues at the interface between monomers; mutations at these sites are likely to disturb trimer formation, as predicted for Ala123Glu, Tyr170Cys, Tyr172His, and Gly227Val (Bajorath et al., 1996; Karpusas et al., 1995). Finally, the amino acid substitutions Met36Arg and Gly38Arg in the transmembrane domain of CD40LG introduce a polar residue in a very hydrophobic sequence, causing severe problems for the insertion of the molecule in the membrane. Indeed, these mutations are associated with a marked reduction in cell surface expression (Garber et al., 1999). By analogy with similar mutations in other proteins, it has been speculated that these mutations promote retention of the mutant in the endoplasmic reticulum and degradation (Thusberg and Vihinen, 2007).

Strategy for Diagnosis

Along with typical clinical and immunological features that distinguish CD40LG deficiency from XLA, a positive X-linked family history is important in the diagnosis of CD40LG deficiency; however, sporadic occurrence in males is not rare. For this reason, diagnosis of CD40LG deficiency is usually accomplished by demonstrating in vitro the inability of activated patient CD4+ T-cells to express functional CD40LG (CD154) molecules, as assessed by binding of soluble CD40-Ig chimeric constructs or of anti-CD40LG mAbs. However, false-negative results may be obtained with use of mAbs, because occasionally they may recognize mutant forms of CD40LG expressed at the cell surface. This phenomenon is even more common when polyclonal antisera to CD40LG are used in the staining procedure, as they may recognize even truncated forms of the protein (Seyama et al., 1998b). Therefore, polyclonal antisera to CD40LG should not be used for diagnosis. However, rarely even the chimeric CD40-Ig molecule may bind to mutant CD40LG, particularly in patients with missense mutations in the transmembrane or cytoplasmic domains, which are permissive for membrane protein expression (Lee et al., 2005; Seyama et al., 1998a, 1998b). Ultimately, mutation analysis at the CD40LG locus may be required for a definitive diagnosis.

A number of critical factors should be considered when performing diagnostic assays for CD40LG deficiency. First, appropriate controls for T-cell activation (e.g., expression of CD69) should be included. This is particularly important to distinguish CD40LG deficiency from common variable immunodeficiencies (CVIDs); a subgroup of CVIDs patients have defective CD40LG expression in the context of a broader T-cell activation defect (Farrington et al., 1994). Second, because CD40LG is preferentially expressed by activated CD4+ cells (Lane et al., 1992), any condition characterized by CD4 lymphopenia would also result in a low proportion of CD40LG-expressing cells upon activation in vitro. In particular, it has been recognized that activated T-cells from patients with MHC class II deficiency fail to express CD40LG because of the markedly reduced proportion of CD4+ T lymphocytes (Callard et al., 1994). Third, the age of the proband has to be considered. There is ample evidence showing that expression of CD40LG by activated neonatal T-cells is physiologically reduced (Brugnoni et al., 1994; Durandy et al., 1995; Fuleihan et al., 1994; Nonoyama et al., 1995). As mentioned above, definitive confirmation of CD40LG deficiency can be achieved through CD40LG gene mutation analysis.

Carrier Detection and Prenatal Diagnosis

In contrast to carriers of other X-linked immunodeficiencies (e.g., X-linked agammaglobulinemia [XLA]; X-linked severe combined immunodeficiency [X-SCID]; Wiskott-Aldrich syndrome [WAS]), carrier females of CD40LG deficiency exhibit a random pattern of X inactivation and are indeed mosaics for two populations of circulating T lymphocytes (one expressing the wild-type CD40LG allele and the other expressing the mutated one) (Hollenbaugh et al., 1994). In carriers with skewed lyonization, no clinical or immunological abnormalities are found, indicating that limited CD40LG expression is sufficient to induce isotype switching and normal generation of memory B-cells (Callard et al., 1994; Hollenbaugh et al., 1994). However, extreme lyonization with selective expression of the mutant form of CD40LG may exceptionally result in an overt clinical phenotype (de Saint Basile et al., 1999).

Carrier detection is best achieved with CD40LG gene molecular analysis. In families with clear X-linked inheritance, linkage analysis can be performed, taking advantage of two sets of hypervariable microsatellites at the 3′ untranslated region of the CD40LG gene (Allen et al., 1993b; DiSanto et al., 1993, 1994; Gauchat et al., 1993a; Ramesh et al., 1994; Shimadzu et al., 1995). Whenever the mutation is known, DNA sequencing, with a search for heterozygosity for the specific mutation, is the most simple and direct way to attempt carrier detection (Lin et al., 1996; Shimadzu et al., 1995; Villa et al., 1994). Heterozygosity for the mutation (e.g., carrier detection) can also be investigated at the cDNA level, as for splice-site mutations that cause exon skipping (DiSanto et al., 1993; Hollenbaugh et al., 1994).

Knowledge of the specific CD40LG gene defect also allows prenatal diagnosis on chorionic villi DNA by 10 to 11 weeks of gestation (Villa et al., 1994). Segregation analysis with hypervariable microsatellites at the 3′ untranslated region of the CD40LG gene has been also successfully used for this purpose (DiSanto et al., 1994).

Despite the reduced ability of activated T-cells from neonates to express CD40LG (Brugnoni et al., 1994; Durandy et al., 1995; Fuleihan et al., 1994; Nonoyama et al., 1995), surface membrane CD40LG was detected on activated T-cells from 19- to 28-week-old fetuses (Durandy et al., 1995). However, because of the obviously complex developmental control of CD40LG expression, staining for CD40LG on fetal cord blood T lymphocytes should not be used as the sole technique for prenatal diagnosis of CD40LG deficiency.

Treatment and Prognosis

The long-term prognosis of CD40LG deficiency appears to be worse than that in other forms of congenital hypogammaglobulinemia—for example, XLA. Data from the European registry on 128 patients with CD40LG deficiency indicated that survival at 25 years of age was only 40 percent. In addition, 30 of the 128 patients had died. Mortality was somewhat lower in the U.S. registry, in which 8 of 79 subjects had died. This difference in mortality between the European and the U.S. series may reflect a lower incidence of Cryptosporidium infection, sclerosing cholangitis, and irreversible liver damage in the latter (Winkelstein et al., 2003). The main causes of death in patients with CD40LG deficiency include infections early in life and, later on, severe liver disease and malignant tumors (Hayward et al., 1997; Levy et al., 1997).

The complex array of clinical manifestations and the increased risk of opportunistic infections and chronic neutropenia require multiple therapeutic approaches. Regular infusion of IVIG (400–600 mg/kg every 21–28 days) is the most important form of treatment. It significantly reduces the severity and frequency of infections (Levy et al., 1997) and may occasionally correct neutropenia (Banatvala et al., 1994; Levy et al., 1997). In addition, prophylaxis with co-trimoxazole is necessary to prevent P. jiroveci pneumonia (Banatvala et al., 1994; Levy et al., 1997; Notarangelo et al., 1992). Long-term treatment with amphotericin B and flucytosine has been used in patients who have developed cryptococcosis (Iseki et al., 1994; Tabone et al., 1994). Patients with severe neutropenia may benefit from treatment with recombinant G-CSF; in some cases, this approach has caused a change from chronic to cyclic neutropenia (Shimadzu et al., 1995; Wang et al., 1994). Total parenteral nutrition may be necessary in patients with protracted diarrhea and malabsorption, particularly if these are due to Cryptosporidium (Benkerrou et al., 1990). Cryptosporidium infection should be prevented by avoiding risk factors (such as swimming in lakes), but use of filtered or sterile water at home has been also suggested. Early detection of Cryptosporidium infection is best achieved by PCR-based amplification of stool DNA and microscopy of bile fluid (McLauchlin et al., 2003). Acute infection has been treated with azithromycin or nitazoxanide, but results have been often unsatisfactory.

Because of the rather dismal prognosis, more radical forms of treatment have been proposed, in particular hematopoietic cell transplantation (HCT), which at the moment remains the only strategy that can result in permanent cure of the disease (Thomas et al., 1995). In a series of 38 European CD40LG-deficient patients treated by HCT, 58 percent were cured; among those without preexisting liver disease the survival rate was 72 percent. However, the overall mortality rate was high (31.6 percent) (Gennery et al., 2004), and in all cases, death was from infections. Preexisting lung disease was associated with a poor outcome. Promising results have been obtained using a nonmyeloablative conditioning regimen in CD40LG-deficient patients with severe liver disease (Jacobsohn et al., 2004; Kikuta et al., 2006). Importantly, lack of a strict genotype–phenotype correlation in CD40LG deficiency prevents selection of high-risk patients who might benefit from HCT early in life.

Attempts to treat severe liver disease (sclerosing cholangitis, cirrhosis) with liver transplantation usually fail because of relapse of the disease in the transplanted organ (Hayward et al., 1997; Levy et al., 1997). Combined bone marrow and cadaveric orthotopic liver transplantation has resulted in disease correction in one patient (Hadzic et al., 2000); however, additional severely affected CD40LG-deficient patients treated with HCT and liver transplantation have failed to survive (Notarangelo, unpublished).

The recognition that expression of the CD40LG gene is under tight regulatory control makes gene therapy a less viable option, particularly since deregulated expression of CD40LG in transgenic mice has been shown to result in tumor development (Brown et al., 1998; Sacco et al., 2000). Use of lentiviral vectors might enable insertion of autologous regulatory gene elements (Barry et al., 2000), thus making gene therapy–based treatment with acceptable risks possible in the future. However, the finding that mutant forms of CD40LG interact with wild-type molecules and prevent expression of functional trimers (Seyama et al., 1999; Su et al., 2001) raises further doubts that gene therapy could become an effective form of treatment for CD40LG deficiency. These problems may be circumvented by trans-splicing (a process by which two different pre-mRNAs are joined by the cellular splicing apparatus), which allows complementation of the gene defect while preserving the natural regulation and cell specificity of CD40LG expression. Indeed, this strategy has been successfully used to correct a murine model of CD40LG deficiency (Tahara et al., 2004).

Clinical and Pathological Manifestations of CD40 Deficiency

Defective expression of CD40 by B lymphocytes (CD40 deficiency, MIM *606843) has been detected in five children from four unrelated families from the Mediterranean region (Ferrari et al., 2001; Kutukculer et al., 2003; Lougaris et al., 2005; Mazzolari et al., 2007). Twelve additional patients with molecularly proven CD40 deficiency have been identified in Saudi Arabia (El-Ghonaium, personal communication). Clinical and immunological data at presentation were very similar to those observed in CD40LG deficiency, but autosomal recessive inheritance was indicated by parental consanguinity and by the fact that four of the five reported patients were female. Patient 1, an 8-year-old Italian girl, suffered from P. jiroveci pneumonia at 4 months of age and had another episode of pneumonia at age 2, when she was found to be panhypogammaglobulinemic and was started on IVIG treatment (Lougaris et al., 2005). At the age of 7 years, she developed persistent eosinophilia (800–13,500/μ‎L) and a mild increase of liver enzymes. Liver biopsy showed a severe pattern of sclerosing cholangitis, and microscopic examination of bile specimens revealed the presence of Cryptosporidium oocysts. The patient died at the age of 9 years from liver insufficiency. Patients 2 and 3 are first cousins from a multiply related Arabian family. Patient 2 is a 5-year-old boy who suffered from recurrent pneumonia, hypogammaglobulinemia with elevated IgM, and neutropenia. Patient 3 is a 7-year-old girl who also experienced recurrent lower respiratory tract infections. She was diagnosed with immunodeficiency with hyper-IgM at 8 months of age, and substitution treatment with IVIG was started. At 3 years of age, she was admitted to a pediatric intensive care unit for severe interstitial pneumonia. Patient 4, a 12-month-old Turkish girl born to consanguineous parents, was hospitalized for respiratory distress. She developed necrotizing pneumonia caused by Pseudomonas aeruginosa and chronic watery diarrhea and disseminated Cryptosporidium parvum infection. She received a matched-sibling stem cell transplantation but died of cardiorespiratory arrest at day 16 posttransplant. Patient 5 was hospitalized at 2 years of age because of recurrent pneumonia, otitis, and skin infections and was diagnosed with a hyper-IgM phenotype associated with impaired CD40 expression. She developed neutropenia that required treatment with G-CSF. At 3 years of age, she received HCT from her HLA-identical healthy carrier sibling. She is now fully reconstituted at 4 years after HCT.

All five children had very low levels of IgG and IgA, and three of them had increased serum IgM levels. Lymphocyte numbers and subset distributions were normal, as were in vitro proliferative responses to mitogens. However, CD40 expression on the surface of B lymphocytes and monocytes was abrogated or severely reduced. Importantly, in contrast to what observed in CD40LG deficiency, B-cells from CD40-deficient patients could not be induced to secrete IgG and IgA upon in vitro activation with anti-CD40 mAb and IL-10, indicating a B-cell intrinsic defect.

Molecular and Immunological Features of CD40 Deficiency

Patients with CD40 deficiency are clinically and immunologically undistinguishable from those with CD40LG deficiency. However, circulating B-cells and monocytes lack surface membrane CD40, whereas expression of CD40LG on the surface of in vitro activated CD4+ T-cells is preserved. Western-blot analysis of lymphoblastoid cell lines showed that the CD40 protein was also undetectable intracellularly in patient 1, whereas an aberrant pattern of migration of the CD40 protein was detected in patients 2, 4, and 5. Mutation analysis at the CD40 gene showed that all of the patients carried homozygous mutations (Fig. 26.3). In particular, patient 1 was homozygous for a silent mutation (A-to-T substitution at nucleotide 408, corresponding to the fifth nucleotide of exon 5), which involves and disrupts an exonic splicing enhancer, thus preventing incorporation of exon 5 in the mRNA. Consequently, cDNA from this patient lacked 94 nucleotides (matching exon 5), resulting in frameshift and premature termination. Patients 2 and 3 were both homozygous for a C-to-T change at nucleotide 247, resulting in a nonconserved Cys83-to-Arg amino acid substitution. The mutation in patient 4 occurred at position 2 of the acceptor site of intron 3; use of a cryptic splice site in this patient resulted in a 2-a.a. deletion (Δ‎N86_L87) and replacement of the next a.a. (G88R). Patient 5 was homozygous for a 3-nucleotide deletion in exon 2, resulting in one a.a. deletion (del I33) in the extracellular domain of CD40. This was also the only patient who had residual, though markedly reduced, CD40 expression at the cell surface.

Figure 26.3 Description of CD40 mutations in affected individuals from four unrelated families with CD40 deficiency. The different types of mutations identified are indicated with different symbols and are aligned along the structure of the CD40 protein with its extracellular, transmembrane, and intracytoplasmic domains. Correspondence between the protein and the nine exons of the gene is also shown.

Figure 26.3
Description of CD40 mutations in affected individuals from four unrelated families with CD40 deficiency. The different types of mutations identified are indicated with different symbols and are aligned along the structure of the CD40 protein with its extracellular, transmembrane, and intracytoplasmic domains. Correspondence between the protein and the nine exons of the gene is also shown.

Investigation of the intracellular fate and biochemical properties of CD40 mutants carrying amino acid substitutions has revealed that the mutant proteins are synthesized but retained in the endoplasmic reticulum (ER). Moreover, accumulation of the C83R is associated with ER stress and activation of the unfolded protein response. By contrast, the Δ‎N86_L87;G88R mutant is efficiently disposed of by the ER-degradation pathway, whereas the Δ‎I33 partially negotiates transport to the cell membrane and is competent for CD40LG binding and intracellular signaling (Lanzi et al., 2010). Further investigation of the functional consequences of CD40 deficiency showed that both memory B-cell generation and somatic mutation were affected. DCs, cultured with TNF-α‎ or with lipopolysaccharide (LPS) combined with IFN-γ‎, displayed a consistent defect in their ability to induce proliferation of allogeneic T-cells and secretion of IFN-γ‎. The defective co-stimulatory activity of DCs derived from patients with CD40 deficiency was associated with lower cell surface levels of MHC class II antigen and with a decreased release of IL-12. These findings support the notion that CD40 deficiency is not an exclusive defect of humoral immunity but should be considered a combined defect of B- and T-cell compartments (Fontana et al., 2003).

The diagnosis of CD40 deficiency is usually made by demonstrating the inability of peripheral blood B-cells to express CD40, as assessed by flow cytometry, in patients with clinical features and an immunoglobulin profile suggestive of impaired CSR. However, the possibility of false-negative results, due to recognition of mutant CD40 molecules by the monoclonal antibody, exists. Furthermore, because CD40 is expressed as a trimer, is it theoretically possible that heterozygous mutations that allow expression of CD40 but affect its function may result in an HIGM phenotype through a dominant-negative effect. Ultimately, diagnosis of CD40 deficiency requires mutation analysis at the CD40 locus.

Treatment and Prognosis

Management of patients with CD40 deficiency is similar to that outlined for patients with CD40LG deficiency. Treatment includes regular infusions of IVIG, Pneumocystis pneumonia prophylaxis with co-trimoxazole, measures to prevent Cryptosporidium infection, and monitoring of liver status by ultrasound scanning and biochemical analysis. A persistent increase of liver enzymes and/or eosinophils requires a careful investigation for the presence of sclerosing cholangitis and/or Cryptosporidium infection, as these events seem to be highly correlated. For this purpose, microscopic examination of the stools may not be sensitive enough, and more sensitive methods should be applied, such as PCR-based amplification of stool DNA and microscopy of bile fluid. Although CD40 expression is not restricted to hematopoietic cells, successful experience with HCT in one patient with CD40 deficiency indicates that correction of the defect on hematopoietic cells is sufficient to cure the disease.

Animals Models of CD40 and CD40LG Deficiency

Gene Disruption

Mice with Disruption of the CD40LG Gene

Cd40lg–/– mice exhibit normal percentages of B- and T-cell subpopulations but display selective deficiencies in humoral immunity. Basal serum immunoglobulin isotype levels are significantly lower than in normal mice, and IgE is undetectable. Furthermore, cd40lg–/– mice fail to mount secondary antigen-specific responses to immunization with T-dependent antigens. By contrast, they produce antigen-specific antibody of all isotypes except IgE in response to thymus-independent antigens. These results underscore the requirement of CD40LG for T-cell–dependent antibody responses (Renshaw et al., 1994; Xu et al., 1994). Moreover, Ig class switching to isotypes other than IgE can occur in vivo in the absence of CD40LG, a phenomenon supporting the notion that alternative B-cell signaling pathways (such as TLR-mediated B-cell activation) regulate responses to thymus-independent antigens.

CD4+ T-cells from cd40lg–/– mice were fourfold less effective than normal T-cells in activating the nitric oxide response in allogeneic macrophages. Cd40lg–/– T-cells fixed with paraformaldehyde after a 6-hour activation period, a time point at which CD40LG dominates the macrophage-activating capability of T-cells, failed to activate the production of inflammatory cytokines (TNF-α‎) or the generation of reactive nitrogen intermediates. After 24 hours of activation, however, both Cd40lg–/– and normal T-cells could induce similar but weak responses from activated macrophages (Stout et al., 1996). These studies demonstrate that Cd40lg–/– mice have a deficient T-cell–dependent macrophage-mediated immune response. However, Cd40lg–/– mice are able to generate normal primary cytotoxic T-cell responses (in spite of a defective humoral response) to a viral infection (Whitmire et al., 1996).

Cd40lg–/– mice are susceptible to P. jiroveci infection (as are CD40–/– mice). Treatment of wild-type mice with soluble CD40LG-fusion protein evokes a pulmonary inflammatory response that is not observed in identically treated Cd40–/– mice (Wiley et al., 1997). This finding supports evidence that ligation of CD40 results in inflammatory responses and that soluble CD40LG is a potent inflammagen that may be important for protection against P. jiroveci infection.

Finally, when injected with Cryptococcus neoformans, Cd40lg–/– mice show increased fungal growth in the brain, associated with reduced production of IL-12, IFN-γ‎, and nitrites (Pietrella et al., 2004).

Mice with Disruption of the Cd40 Gene

Cd40–/– mice have normal numbers of T and B-cells, indicating that CD40 is not essential for B-cell development. Their B-cells fail to proliferate and undergo isotype switching in response to soluble CD40 ligand (sCD40LG) and IL-4 but respond normally to LPS in the presence of IL-4. Cd40–/– mice completely fail to mount an antigen-specific antibody response or to develop germinal centers following immunization with T-cell–dependent antigens, but they respond normally to the T-cell–independent antigens. The most noticeable alteration in the serum immunoglobulin levels of young Cd40–/– animals is absence of IgE and a severe decrease of IgG1 and IgG2a (Castigli et al., 1994; Kawabe et al., 1994). These results indicate an essential role of CD40–CD40LG interactions in the antibody response to T-cell–dependent antigens and in isotype switching.

B-cells deficient in CD40 expression are unable to elicit the proliferation of allogeneic T-cells in vitro. More importantly, mice immunized with Cd40–/– B-cells become tolerant to allogeneic MHC antigens as measured by a mixed lymphocyte reaction and cytotoxic T-cell assay. The failure of Cd40–/– B-cells to serve as antigen-presenting cells in vitro is corrected by the addition of anti-CD28 mAb. Moreover, LPS stimulation, which upregulates CD80/CD86 expression, reverses the inability of Cd40–/– B-cells to stimulate an alloresponse in vitro and abrogates the capacity of these B-cells to induce tolerance in vivo (Hollander et al., 1996). These results suggest that CD40 engagement by CD40 ligand expressed on antigen-activated T-cells is critical for the upregulation of CD80/CD86 molecules on antigen-presenting B-cells that subsequently deliver the co-stimulatory signals necessary for T-cell proliferation and differentiation.

In addition to susceptibility to P. jiroveci infection, CD40-deficient mice are also prone to Mycobacterium avium infection (Florido et al., 2004). Furthermore, when inoculated with the defective murine leukemia retrovirus LP-BM5def, Cd40–/– mice become infected and show virus expression similar to that in wild-type mice. However, unlike the wild-type mice, CD40-deficient mice do not develop symptoms of immunodeficiency, lymphoproliferation, and the typical histological changes in the lymphoid tissue (Yu et al., 1999). These results show that the CD40–CD40LG interaction in vivo is essential for anergy induction and the subsequent development of immunodeficiency and pathological expansion of lymphocytes.

In keeping with similar in vitro observations, mice deficient in CD40LG or in CD40 expression show impaired cross-talk between activated T-cells and DCs, with failure to expand IL-17–producing cells, and are resistant to the development of experimental autoimmune encephalitis (Iezzi et al., 2009).

Cd40–/– adult mice develop neuronal cell dysfunction and gross central nervous system abnormalities with age. These findings suggest that CD40 signaling plays an important role in normal neuronal cell maintenance and confers resistance to aging-induced stress (Tan et al., 2002).

Conclusion

Detailed clinical, molecular, and immunological analysis of patients with CD40LG or with CD40 deficiency has been instrumental in unraveling the complex effects that CD40LG (CD154) exerts in vivo upon interaction with CD40, and that extend beyond promoting B-cell activation and differentiation. The broad range of cellular effects that CD40LG/CD40 interaction promotes also account for the complexity of immune defects that characterize deficiency of CD40LG or of CD40. Accordingly, these disorders are currently classified among human combined immunodeficiencies (Notarangelo et al., 2009), and use of more aggressive forms of treatment (such as HCT) than immunoglobulin replacement therapy alone has been advocated. At the same time, involvement of CD40LG/CD40 signaling in inflammation and in tumor cell killing has prompted use of biological modifiers that interfere with the CD40-signaling pathway in autoimmune, inflammatory, and neoplastic diseases and in prevention of graft rejection and graft-versus-host disease (Durie et al., 1994).

Acknowledgments

This work was partially supported by the Manton Foundation (to L.D.N.), by European Union FP7 grant 201549 and by the “Camillo Golgi” Foundation (to A.P.), and by the “Angelo Nocivelli” Foundation (to S.G. and A.P.).

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