CD40-CD40 Ligand: A Multifunctional Receptor-Ligand Pair

Cees Van Kooten1; Jacques Banchereau2    Laboratory for Immunological Research, Schering-Plough, 69571 Dardilly, France
1 Present address: Department of Nephrology, AZL Building 1 C3P, Rijnsburgerweg 10, 2333 AA Leiden, the Netherlands.
2 To whom correspondence should be addressed at Schering-Plough, Laboratory for Immunological Research, 27 chemin des Peupliers, BP 11, 69571 Dardilly, France. Fax: (33) 78.35.47.50.

I Introduction

A decade has passed since monoclonal antibodies allowed the identification of the CD40 antigen. Meanwhile it has become clear that, in addition to soluble factors (cytokines), direct cellular interactions play a pivotal role in the regulation of immune responses. The CD40 antigen was independently identified in 1985 and 1986 by monoclonal antibodies reacting with carcinomas and B cells (antibody S2C6, antigen p50 (Paulie et al., 1985)) and showing costimulatory effects for B lymphocyte proliferation (antibody G28-5, antigen Bp50 (Clark and Ledbetter, 1986)). This antigen was designated as CDw40 at the Third International Workshop on Leukocyte Antigens in Oxford in 1986, and CD40 at the Fourth Workshop in Vienna in 1989. A cDNA encoding CD40 was isolated in 1989 (Stamenkovic et al., 1989) whose sequence demonstrated a relationship with the human low-affinity nerve growth factor receptor (LNGFR). These molecules are now considered as part of the tumor necrosis factor receptor superfamily. Cross-linking of CD40, in conjunction with IL-4, was then found to induce B cells to undergo long-term B cell growth, as well as isotype switching (Banchereau et al., 1991; Banchereau and Rousset, 1991; Jabara et al., 1990). In 1992, expression cloning using CD40-Fc fusion protein allowed the isolation of a CD40 ligand (CD40-L) expressed on activated T cells (Armitage et al., 1992a), an observation which led to the demonstration of the key role of CD40-CD40-L interactions in T-cell-dependent B cell activation by many groups (Noelle et al., 1992a). The CD40-L is now one of the members of the recently identified tumor necrosis factor superfamily. In 1993, it was demonstrated that a genetic alteration of the CD40-L was responsible for the X-linked hyper-IgM syndrome which is characterized by the lack of circulating IgG and IgA and the absence of germinal centers (Allen et al., 1993; Aruffo et al., 1993; DiSanto et al., 1993; Fuleihan et al., 1993; Korth-uer et al., 1993). These phenotypes have been confirmed in murine "knockout" experiments where either the CD40 or the CD40-L was inactivated by homologous recombination (Kawabe et al., 1994; Xu et al., 1994).

While the function of CD40 has principally been studied on mature B lymphocytes, more recent studies have shown the presence of biologically functional CD40 on other cell types, such as monocytes/macrophages (Alderson et al., 1993), dendritic cells (Caux et al., 1994), hematopoietic progenitors (Saeland et al., 1992), epithelial cells (Galy and Spits, 1992), and endothelial cells (Hollenbaugh et al., 1995;Karmann et al., 1995). Many aspects of CD40-CD40-L biochemistry and biology have been reviewed extensively in recent papers (Armitage and Alderson, 1995; Banchereau et al., 1994; Bonnefoy and Noelle, 1994; Callard et al., 1993; Clark and Ledbetter, 1994; Hollenbaugh et al., 1994) and the reader is referred to these articles for additional details. In the present review, we will concentrate on the most recent developments, including (i) those concerning CD40 signal transduction, (ii) a comparison with other members of the superfamily, (iii) a functional expression of CD40 on cell types other than B cells, (iv) the in vivo relevance of CD40-CD40-L interactions.

II CD40 as a Member of the TNF-Receptor Superfamily

A CLONING AND STRUCTURE OF CD40

The CD40 antigen is a phosphorylated glycoprotein which migrates in SDS Polyacrylamide gel electrophoresis as a 48-kDa polypeptide under both reducing and nonreducing conditions. It is a hydrophobic molecule with an acidic Pi of 3.2. A significant proportion of CD40 from the Burkitt lymphoma Raji cells and normal B cells is in a dimeric form whereas such dimers are virtually absent from carcinoma lines or EBV-transformed cells (Braesch-Andersen et al., 1989; Paulie et al., 1989).

A cDNA encoding CD40 was isolated by expression cloning from a library of the Burkitt lymphoma Raji (Stamenkovic et al., 1989). The mature molecule is composed of 277 amino acids (AA) with a 193-AA extracellular domain including a 21-AA leader sequence, a 22-AA transmembrane domain, and a 62-AA intracellular tail. Therefore, CD40 has the structure of a typical type I transmembrane protein. The human CD40 gene is expressed as a single 1.5-kb mRNA species. The extracellular segment of CD40 displayed significant similarity (34.5% in the extracellular region) to the analogous protein domain of the p75 LNGFR (Johnson et al., 1986). In particular, the 22 cysteine residues in CD40, which form four predicted protein domains of about 40 AA with 6 cysteine residues each (Fig. 1), are homologous to LNGFR and the other members of what is now called the TNF-R family (see Section II,B). This is in contrast to the intracellular chain of CD40 which does not betray a close relationship to any other characterized molecule.

The murine cDNA was cloned from LPS + IL-4-stimulated murine B cells by cross-hybridization with the human cDNA probe (Torres and Clark, 1992). The mouse CD40 protein is composed of 305 AA with a 193-AA extracellular region including a 21-AA leader sequence, a 22-AA transmembrane domain, and a 90-AA intracellular region. Human and murine CD40 molecules share 62% amino acid identity in the complete coding sequence and 78% identity in the intracellular extensions. The last 32 C-terminal AA of human CD40 are completely conserved in the mouse sequence. In addition 22/22 extracellular cysteine residues are conserved, suggesting that both mouse and human CD40 fold into the same protein domains. The mouse CD40 gene is expressed as two mRNA species of 1.7 and 1.4 kb. These two forms are generated by alternative polyadenylation using two different polyadenylation sites in the 3' untranslated sequence and thus result in the same coding sequence. Activation of mouse B cells seems to give a specific up-regulation of the smaller 1.4-kb transcript (Torres and Clark, 1992).

The CD40 murine gene consists of nine exons and spans a total region of 16.3 kb genomic DNA (Grimaldi et al., 1992). The schematic representation of the mouse CD40 gene organization, shown in Fig. 1, clearly indicates that the cysteine repeat domains are not coded for by separate exons. In contrast, cysteine repeats are mostly divided by introns at positions which are conserved by other members of the TNF-R family (Birkeland et al., 1995). This is especially true for the type 1 introns (intercutting codons after the first nucleotide base) in the first two N-terminal cysteine repeats. For murine CD40, the signal peptide, the transmembrane region, and the cytoplasmic tail are coded for by exons separate from the cysteine repeats, but this is not a general rule for all TNFR family members.

The mouse CD40 gene is located on the distal region of chromosome 2 which is syntenic to human chromosome 20q11-q13 (Grimaldi et al., 1992). Accordingly, the human CD40 gene was mapped to chromosome 20 by using human-rodent somatic cell hybrids (Ramesh et al., 1993b) and to 20 q11-20q13-2 by in situ hybridization (Lafage-Pochitaloff et al., 1994). Deletions of this part of chromosome 20 are observed frequently in myeloid malignancies, but the significance of this observation remains to be established.

B RELATIONSHIP WITH OTHER TNF-R SUPERFAMILY MEMBERS

As stated above, CD40 was found to be homologous to the previously described p75 low-affinity nerve growth factor receptor. The p75 LNGFR can therefore be seen as the founding father of a superfamily of receptorlike molecules that share a common binding domain composed of tandemly repeated cysteine-rich modules (Mallett and Barclay, 1991). However, since interaction of LNGFR with its natural ligands is not typical for the rest of the receptor family, nowadays the family is more appropriately named after two later additions of this family: the TNF-R superfamily. The family is composed of at least 10 genes described in human and mouse (LNGFR, TNF-R1, TNF-R2, TNF-Rrp, CD40, CD27, CD30,...