annurev.iy.12.040194.004313] j. banchereau; f. bazan; d. blanchard; f. briè; j. p. galizzi; -- the...

Annu. Rev. Immunol. 12:881-922 Copyright 1994 by Annual Reviews Inc. All rights reserved

THE CD40 ANTIGEN AND ITS

LIGAND

J. Banchereau, F. Bazan', D. Blanchard, F. Briere, J.P. Galizzi, C. van Kooten, Y.l. Liu, F. Rousset, S. Saeland

Schering-Plough, Laboratory for Immunological Research, Dardilly, France; ** DNAX Research Institute, Palo Alto, California

KEY WORDS: CD40, CD40-ligand, lymphocyte activation, B cell(T cell interactions, hyper IgM syndrome

Abstract CD40 is an integral membrane protein found on the surface of B lymphocytes, dendritic cells, follicular dendritic cells, hematopoietic progenitor cells, epithelial cells, and carcinomas. It is a 45-50 kDa glycoprotein of 277 aa, which is a member of the tumor necrosis factor receptor superfamily. The CD40 gene maps to human chromosome 20q 1 1 -2-q 1 3-2. CD40 binds to a ligand (CD40-L) which is an 35 kDa glycoprotein of 261 aa, a member of the tumor necrosis factor superfamily. The CD40-L gene maps to human chromosome Xq24. This CD40-L is expressed on activated T cells, mostly CD4 + but also some CD8 + as well as basophils/mast cells. The CD40-L is defective in the X-linked hyper- IgM syndrome.

Cross-linking of CD40 with immobilized anti-CD40 or cells expressing CD40-L induces B cells to proliferate strongly, and addition of I L-4 or IL-1 3 allows the generation of factor-dependent long-term normal human B cell lines and the secretion of IgE following isotype switching. Addition of IL- l O results in very high immunoglobulin production with limited cell proliferation. IL- IO induces naive B cells to produce IgG3, IgGI, and IgAl , and further addition ofTGFfJ permits the secretion ofIgA2. Several evidences suggest that CD40-dependent activation of B cells is important for the generation of memory B cells within the germinal centers: (i) CD40 activated germinal center B cells cultured in the presence of IL-4 acquire a memory B cell phenotype, (ii) CD40 activated B cells can undergo isotype switching, (iii) the deficit of CD40-L results in the hyper-IgM syndrome characterized by lack of germinal centers in secondary lymphoid organ

88 1 0732-0582/94/0410-088 I $05 .00

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882 BANCHEREAU ET AL

follicles and lack of IgG, IgA, and IgE, and (iv) CD40-L positive T cells are present in secondary follicles. Thymic epithelial cells, activated monocytes, and dendritic cells express CD40 antigen which may be involved in an enhanced cytokine production by these cells, allowing an amplification of T cell proliferation. Finally, as other members of the tumor necrosis factor receptor family have been shown to bind several l igands, it is possible that CD40 may bind other l igands that may trigger CD40 on d ifferent cell types such as hematopoietic cells or epithelial cells.

INTRODUCTION The CD40 antigen was independently identified in 1985 and 1 986 by monoclonal antibodies reacting with carcinomas and B cells (antibody S2C6, antigen p50) ( 1 ) and showing costimulatory effects for B lymphocyte (antibody G28-5, antigen Bp50) (2). This antigen was designated as CDw40 at the Third International Workshop on leukocyte antigens in Oxford in 1 986, and as CD40 at the Fourth Workshop in Vienna in 1 989. A cDNA encoding CD40 was isolated in 1 989 (3), and this 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 (4-6). In 1 992, expression cloning using CD40 Fc fusion protein allowed the isolation of a CD40-ligand (CD40-L) expressed on activated T cells (7), an observation which led to the demonstration of the key role o f CD40-L/CD40 interactions in T cell-dependent B cell activation by many groups (8). The CD40-L is one of the members of the recently identified tumor necrosis factor superfamily. In 1 993, a genetic alteration of the CD40-L was shown to be 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 (9). While the function of CD40 has principally been studied on mature B lymphocytes, more recent studies show the presence of b iologically functional CD40 on other cell types, such as epithelial cells ( 10), monocytesj macro phages ( 1 1 ), and hematopoietic progenitors ( 1 2, l 3) .

CD40 ANTIGEN Modular Structural Design and Evolutionary Relationship of CD40 The CD40 antigen is a phosphorylated glycoprotein which migrates in SDS polyacrylamide gel electrophoresis as a 48 kDa polypeptide under

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CD40 AND ITS LIGAND 883

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 ( 14, 1 5) .

A cDNA encoding CD40 was isolated by expression cloning from a library of the Burkitt lymphoma Raji (3) . The mature molecule is composed of 277 aa with a 1 93 aa extracellular domain, a 22 aa transmembrane domain, and a 62 aa intracellular tail. While the extracellular segment of CD40 displayed significant similarity to the analogous protein domain of the p75 low-affinity, nerve growth factor receptor (LNGFR) ( 1 6), the intracellular chain did not betray a relationship to any other characterized molecule. Human and murine CD 40 molecules share 62% amino acid identity in the extracellular domains and 78% identity in the intracellu lar extensions ( 1 7) .

The p75 LNGFR is the founding father of a superfamily of receptorlike molecules that share a common binding domain composed oftandemly repeated cysteine rich modules ( 1 8) (Figure I, Figure 2, Table 1) . It is remarkable that this nascent group of receptors is more appropriately named after two later additions: the p55 and p75 receptors for tumor necrosis factor (TNFR I and TNFR2, respectively) ( 19-2 1 ) that bind the related cytokines tumor necrosis factor-ex (TNF-ex; also known as cachectin) and lymphotoxin (L T; also known as TNF-f3) (22, 23). LNGFR acts principally to recruit a number of neurotrophin ligands, including NGF, to the cell surface, aiding in the formation of the signaling complex formed by dimeric trk receptors that possess intracellular tyrosine kinase domains (24, 25). LNGFR does not apparently play a role in signal transduction (26). In contrast, the two TNFRs are both active signalling receptors and bind ligands with a "TNF fold" that is unrelated to the neurotrophin structure (27). Subsequent additions to the TNFR superfamily display conserved interactions to TNF-like cytokine ligands (28), cementing the TNFR moniker for the cognate receptors ; perhaps the LNGFR molecule represents an "escaped" TNFR analogous to the tissue factor cell surface tether for coagulation Factor VII that is surprisingly related to the hematopoietic cytokine receptor superfamily (29).

In addition to the LNGFR, CD40, and the two TNF receptors, the TNFR superfamily currently includes CD27, a molecule expressed on T cells and activated B cells (30, 3 1) ; CD30, an activation molecule ofT cells and B cells initially detected on Reed Sternberg cells (32); OX40, a rat activated T cell antigen (33); 4-1BB, another T cell molecule (34); Fas/Apol, a lymphocyte antigen whose triggering induces apoptosis (35-37); and a receptor-like protein from an expressed open reading frame

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rt Ii..NGFf( ntl.JlfQFR rltLNGm r1 hrNFA I rlmTN'FAl n 1'I1NFFtO r1mTNmU

rll\TNfRh

t1 hC040 n mC(:)t1l rt tX.(l) r1 n4tH

r1 m.HSS n h'til n mFIU n hCot'1 n mCOOl r1 ncOlo

r3" hctn(t r1 mC:tO t1 my,v 111 "dV 12 n cplVermS nG4R (1 utI/AS!

rI ru

r tLJr4GR't

r3n.kGFR

t:) d .NGf'R rJ, hTNFff 1 r3mTNFRI

r31'1'tNffln

rl mTNFAIl

t$hTNffth rl h04o fl mC04Q-1'31'0.40 (3 h44&e rt m4-1Se ,3 hfu rlmf'a. t3 !'ICOn 11 t'rIC027 ,3 hCD:Jo r1 hCOao t3 meet!) t:J myxV Tt r3 lilY 12 r3 eplV frn8 tllr.U'V G4R f3 ciiCJI1

r

uperfalllily ytoki"e

uperfamily Rceptors

.'J

".-1.ii! .. ..',. q,. ,.. ..,.,., '111_... .. -. . ... ... -.. ... .. ........ .... ,-J

.... -I1ltf""I+I."'''''''''-1rr''[}-P-St .......

Figure 2_ Schematic organization of receptor and cytokine superfamilies: The receptor supergroup comprises twelve molecules, ten from mammalian cells: LNGRF, TNFR1 and TNFR2, the TNFR homolog from human chromosome 12 (TNFRh), C040, OX-40, 4-1 SS, Fas, C02? and C030 antigens. A single, representative poxvirus TNFR homolog chain is noted (from myxoma virus: ref). The small, single-repeat fungal TNFR homolog is ECP1 protein from Cladosporium fulvum. Repeats are drawn as diamonds (red, purple, yellow and cyan denote repeats 1-4, respectively) that are divided into two halves (see box of the TNFR Cys-repeat fold) that likely form disulfide linked loops. Ser-Thr-Pro rich chain segments that typically link the repeat domains to the transmembrane segment are drawn as wavy lines-note that the poxvirus and fungal receptor homologs are soluble proteins. Two types of cytoplasmic domain homologies are noted by blue or orange colored boxes. The TNFR2 chain does not resemble either motif. The known cytokine ligands of the TNFR1, TNFR2, C040, C02? and C030 are drawn as folded sheets with opposing red and grey faces. A separate box shows the -strand construction of the fold-the grey (inner) sheet is formed by strands 6"-8-I-O-G, while the red (outer) sheet comprises strands 8'-C'C-H-E-F. Note that all ligands, save LT, are membrane anchored by N-terminal hydrophobic segments.

The bottom box shows a decomposition of structural features in the receptor and cytokine chains. A composite receptor is shown divided into four repeats each of which show a variation {)f the Cys-repeat pattern: disulfide links are shown above. Repeats are also colored (red, purple, yellow and cyan) as in the receptor superfamily figure above. Intron positions are mapped to the receptor chain and show some correlation to repeat and subrepeat structure. The composite ligand shows the position of the eleven -strands with the few identical amino acids in the alignment of Fig. 5 noted above the chain-all of these contribute to the hydrophobic core. Orange and green stars note the location of residues in contact with two distinct receptor chains in the trimer complex of L T and TNFR1.

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00 00 0'\

Table 1 Members of the TNF receptor and cytokine superfamily analyzed in the present work

t:C M olecule Chain length (aa) Gene structure Chromosome location References :> Z

(") :r:

Receptors tr1 ;;c LNGFR 399; 396; 397 (h; r; c) 6 exons (h) 17q21-22 (h) (16) tr1

:> TNFR I 434; 433 (h; m) 10 exons 12pI 3; 6 (h; m) (19, 20, 61, 62) c:: TNFR II 493; 452 (h; m) 1; 4 (h; m) (21) tr1 ..., TNFR h 408 (h) 12p (h) (38) :> r CD40 258; 286 (h; m) 9 exons 20qlI-q13; 2 (h; m) (3, 17, 63, 66, 67) OX-40 262 (r) (33) 4-1 BB 229; 229 (h; m) 1; 4 (h; m] (34, 39, 139a) Fas 319; 306 (h; m) partial (m) IOq24.1; 19 (h; m) (35-37) CD27 240; 230 (h; m) 12q I 3 (h) (30, 31, 64) CD30 577; 480 (h; m) I p36 (h) (30, 40) Poxvirus Rh 310; 309; 337; 330 single viral orfs (41, 43-46)

(myx; sf; cpx; var) ECP 1 65 3 exons (cf) (47)

Cytokines TNFo: 233; 235 (h; m) 4 exons (h; m) 6 (131) LT 205; 202 (h; m) 4 exons (h; m) 6 (132) LT-f3 244 (h) 4 exons (h) 6 (137) CD40-L 260; 261 (h; m) 4 exons (h) Xq26-q27 (h) (7, 117, 119, 127-129) CD27-L 193 (h) 19p I 3.3 (h) (138) CD30-L 234; 239 (h; m) 9q33; 4 (h; m) (139) 4-1 BB-L 309 (m) 17 (m) (I39a) A

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branch of the TNFR superfamily include the Variola virus G4R (45) and Cowpox virus crmB (46) gene products.

The most intriguing member of the TNFR superfamily is the fungal pathogenic protein ECP I isolated from Cladosporium julvum, a tomato pathogen (47). ECPI is a secreted protein of 65 residues that appears to contain a single, characteristic TNFR-like cysteine repeat of 46 amino acid length; ECP l is suggested to play a role in suppressing the tomato plant defense response by b inding plant cytokine-like molecules secreted in the face of fungal attack (48). This economical molecule may represent the minimal binding structure of a TNFR homolog and is a functional analog of the poxvirus T2 factors.

It is instructive to examine the modular protein architecture of the TNFR superfamily from the vantage point of the recently described xray crystal structure of the complex formed by soluble b inding domains of p55 TNFR I and a LT trimer (49). Early comparisons ofTNFR superfamily members highlighted the striking division of the extracellular binding domains into three or four imperfect repeats of ,.... 40 residues, anchored by a superimposable pattern of six cysteines ( 1 6, 1 8-2 1 , 30, 32, 33). While the disulfide bridging pattern was unknown, each repeat was assumed to form an independently folding entity with three main intrachain disulfide bridges-covariant loss of Cys 3 and 5 in several receptor modules led to the suggestion that these were likely linked ( 1 8) . The protein fold of the TNFR repeat module is shown to be a tandem arrangement of "tethered loops, " where the N-terminal loop is fixed by a Cys l-Cys2 link, and the C-terminal loop features an analogous Cys4-Cys6 bridge (49) (see Figure 2). The non-essential Cys3-Cys5 link ties the stalk connecting the two loop structures to the second loop (49) . A structural subdivision nevertheless does seem to indicate that the TNFR modular fold arose from the union of two smaller folding units (48). This finding explains the occasional, puzzling truncations of exact half-repeats in several superfamily receptors: TNFRI repeat 4, OX-40 repeat 3, human CD30 repeat 4, and CD27 repeat 3-a half-repeat corresponds to either the N- or C-terminal loop structure that is presumably capable of correct folding. The comparative alignment of available TNFR modules ( 1 8 ; see also Figure 1) also h ighlights the conservation of additional residues, notably an aromatic amino acid located 5 residues after Cys I that in the crystal structure is shown to be important for the interaction of N- and C-terminal loops (49). The stacked repeat modules form an elongated, s lightly bent rod that intercalates in the groove between two L T subunits in the packed trimer. In addition, the key receptor-ligand interactions involve residues in repeats 2 and 3 (49). Together, the three receptor subunits encage the LT trimer and scrupulously avoid. contact between each other.

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A notable superstructural motif in the TNFRI rods is a spiralling "staircase " formed by the successive stacking of disulfide links perpendicular to the rod axis (49). The gross spatial architecture of the CD40/CD40 ligand interaction is schematically shown in Figure 9 and should help in the following structural discussion on CD40 and its ligand. This energetically favorable packing arrangement places the disulfide bridges in a preferred hydrophobic core location (50); it has been observed in other small, disulfide rich proteins like toxins and defensins (5 1-53), and the cystine-knot family of NGF/TGFP/pDGF folds (28). These disulfide staircases may appear again in the binding domains of other cytokine receptors in spite of distinct folding topologies; notable candidates include cysteine-rich modules present in the EGF and insulin receptor extracellular segments (54) and the TGF-p family signalling receptors (55).

The cysteine repeat pattern of TN FRs resembles an eight-cysteine motif in laminin-like modules (33, 56). These latter laminin repeats are believed to fold in a manner similar to EGF, with an "extra " disulfide-bridged loop at the C-terminus (57). An X-ray crystallographic structural analysis of a representative fragment of laminin containing a three-repeat segment of chain that binds the protein nidogen appears to be in progress (58) and may soon resolve the matter.

The modular organization of the TNFR binding structures invites the question of an underlying genetic organization-do the repeats, or halfrepeats, correspond to exon-encoded folding units as observed for other protein modules (59) ? Several gene structures have been elucidated for superfamily members: the mouse LNGFR gene (60), the human and mouse TNFRI genes (61 , 62), the mouse CD40 (63), human CD27 (64), the fungal ECP l (47), and a fragment of the mouse Fas gene (65). The CD40 murine gene is fairly typical of this collection with nine exons that span 1 6.3 kb of genomic D NA (63). A diagrammatic i llustration of exon boundaries is presented in Figure 2. While there is no consistent subdivision of repeats into exons for any given gene, the composite picture that emerges suggests that type 1 introns (which intercut codons after the first nucleotide base) typically map to the dividing boundaries of repeats, or half-repeats. Type 1 modules are fairly common in the diverse collection of small protein motifs found in mosaic cell surface molecules (57).

The mouse CD40 gene is located on the distal region of chromosome 2 which is syntenic to human chromosome 20q I l -q13. Accordingly the human CD40 gene was mapped to chromosome 20 by using human-rodent somatic cell hybrids (66) and to 20 q ll-20q1 3-2 by in situ hybridization (67). It is obvious that superfamily receptor genes have been dispersed throughout the genome. Table 1 shows the chromosomal localization of other receptor genes.

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Cells Expressing CD40


B LYMPHOCYTE C D40 was early considered as a pan B cell antigen (68). CD40 is detected on all B cells isolated from adult and cord blood, tonsils and spleens (Figure 3A) . It is expressed on resting virgin sIgD+ sIgM+ B cells in the primary follicles and the mantle zone of secondary follicles within the peripheral lymphoid organs (Figure 4). The density of CD40 is identical on naive B cells centroblasts and centrocytes, composing the germinal centers of secondary follicles, and on the CD38 - sIgD - memory B cells (69).

Plasmablasts isolated from tonsils express CD40 (P Merville et aI, manuscript in preparation), whereas roughly half of the antibody secreting cells circulating in the blood eight days after vaccination have lost CD40 (70). The fully differentiated plasma cells of mucosal lamina propria and bone marrow do not express C D40. Polyclonal activators such as anti-IgM, anti-CD20, or anti-Bgp95 antibodies or phorbol esters slightly upregulate CD40 expression on B cells (3, 7 1) . IFNy (3) and IL-4 (72) are also able to increase CD40 levels on B cells. The human C D40 gene is expressed as a single 1 .4 kb species in B cells. The murine C D40 gene is expressed in B lymphocytes as two mRNA species, a predominant one of l .7 kb and a minor one of 1 .4 kb, which are generated by alternative usage of polyadenylation signals in the 3' untranslated region. The activation of murine B lymphocytes preferentially increases the level of the smaller transcripts (17). Anti-human C D40 antibodies react with the CD40 antigen from macaques and baboons (73, 74).

Virtually all chronic lymphocytic leukemia B cells and non Hodgkin's lymphoma cells express C D40. Burkitt lymphoma cell lines and EBVtransformed B cell lines all display CD40 ( 1 ) . The plasmacytoma cell line RPMI 8226 displays low levels of C D40 while U266 cells do not. Most IL-6-dependent myeloma cell lines are CD40 positive (B Klein, personal communication).

Numerous EBV-transformed B cell lines release soluble CD40 (sCD40) spontaneously, as detected with a specific ELISA (75). Supernatants of Staphylococcus aureus strain Cowan I-activated normal B cells cultured in the presence of IL-4 or IL-2 also contain sCD40. This is in line with the described soluble forms of the other members of the TNF -R family, which include sTNFR l and sTNFR2 (76, 77), sLNGFR (78), and sCD27 (79). Although the mechanisms of sCD40 release have not yet been studied, it is possible that these molecules originate from proteolytic cleavage as shown for sCD27 (80), and as indicated by the lack of alternatively spliced C D40 mRNA. sCD40 of EBV cell line supernatants is able to bind to the C D40-L expressed on activated T cells.

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CD40

A

CD40

B

CD40

c

CD19 sIgM sIgD

CDlO CDl9 sIgM

CD34 CDIO CDl9

1\

Figure 3 Expression of CD40 on B lineage cells and hematopoietic progenitor cells. Total B cells isolated from tonsils (panel A), B cell precursors (CD I 9 + CDl O + sIg - ) obtained from mid-term fetal bone marrow (232) (panel B), and CD34+ progenitors separated from full-term umbilical cord blood (233) (panel C), were stained with the anti-CD40 mAb 89 (72). In parallel, and as indicated, cells were monitored for expression of CD19, CDlO, CD34, sIgM, and sIgD. Histograms represent log of FITC-fiuorescence analyzed on a fiowcytometer. Dotted lines correspond to negative control staining with an unrelated murine mAb.

HEMATOPOIETIC PROGENITORS In human fetal and adult bone marrow, CD40 is detectable on the majority of B cell precursors (BCP), which express CDl9 and CDl O but lack surface Ig (Figure 3B; 1 3 ,8 1-83). CD40

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CD40 AND ITS LIGAND 89 1

is acquired early in B cell ontogeny, as it is present on BCP expressing the CD34 progenitor cell antigen ( 1 3) and has been reported on fetal liver CDI9+ cells prior to rearrangements at the IgH locus (pro-B cells) (8 1 ).

CD40 is also expressed on malignant BCP in B lineage acute lymphoblastic leukemias at various maturation stages, ranging from pro-B cells to pre-B cells expressing cytoplasmic J1 chain (8 1 , 84). About 28 to 44% of BCP-ALL cases have been shown to express CD40 (81, 82). Among the positive BCP-ALL, CD40 is present only on a proportion of the leukemic cells, a feature that has been associated with clonogenic capacity (81).

Finally, CD40 is not restricted to early B lineage cells in normal hematopoiesis; it is found on most cord blood CD34 + progenitors, which lack a CDI9+ CDIO+ subset (Figure 3C), and on the majority of bone marrow CD34 + cells ( 1 3). In this context, CD40 expression is more heterogeneous on CD34 + progenitors than on mature B cells (Figure 3A and 3C). CD40 expression is lost during myeloid development in cultures of CD34+ cells ( 13). It is important to further define the CD34+ CD40+ population, and to investigate whether the most primitive nonlineage committed CD34+ cells, characterized by lack of CD38 antigen (85), express CD40.

DENDRITIC CELLS AND MONOCYTES Immunohistological analysis (68) on tonsil and spleen sections has shown high levels of CD40 on interdigitating dendritic cells in the T cell-rich areas of secondary lymphoid organs (Figure 4). These cells derive from skin/mucosal Langerhans cells which only weakly express CD40 (86). However, following culture, Langerhans cells express CD40 at high levels. Dendritic cells can be generated in vitro by culturing CD34 + hematopoietic progenitor cells in the presence of GMCSF /IL-3 and TNFIX (87). These cells, which resemble Langerhans cells as they express CDl a and display Birbeck granules, express CD40 at high levels and may thus represent cells at a stage of differentiation between Langerhans cells and interdigitating dendritic cells. In fact, dendritic cells isolated from peripheral blood also express CD40 and may represent Langerhans cells homing towards secondary lymphoid organs (88).

Primary human monocytes freshly isolated or cultured for 48 hr show low but detectable CD40 surface protein ( 1 1 ) . GM-CSF, IL-3, and IFNy strongly upregulate their CD40 expression.

OTHER CELL TYPES CD40 is expressed in the CD45-negative stromal cell population of human thymus ( 10). Immunohistology shows CD40 expression on cortical and medullary thymic epithelial cells, as well as thymic interdigitating cells and B cells (Figure 4). Expression of CD40 is specifically maintained on cultured thymic epithelial cells but not on thymic

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CD40-L in NK cells and purified monocytes (1 27). Northern blot analysis of the B cell line Daudi and the histiocytic lymphoma U937 have shown specific hybridization signals at 3.7 and 1 .7 kb, suggesting expression of CD40-L related molecules in B cells and monocytes (1 29).

CD40-Ligand Is a Member of an Emerging Cytokine Family

The initial characterization of the mouse CD40-L did not easily yield i ts chain similarity to the TNF superfamily (7). Subsequent analyses showed that an 200 aa domain which formed the major extracellular domain of CD40-L could be aligned with available TNF-O( and LT sequences in a structurally sound manner (27, 1 1 9, 1 29) (Figure 2, Figure 5). The region in question comprises the bioactive, receptor-binding, globular portion of TNF-like molecules that folds, by example of TNF-O( ( 1 30, 1 3 1) and LT (49, 132), into a barrel-like structure reminiscent of viral capsid proteins. This distinctive TNF fold consists of two packed sheets of eight major antiparallel f3-strands linked in a "f3-jellyroll" topology with an N-terminal loop insertion that contains two additional, short f3-strands (1 3 1 , 1 32, 1 33). In this manner, and following a s tandard nomenclature for viral capsid proteins ( 1 30, 1 33), the "inner " sheet which is primarily involved in trimer contacts is formed by s trands B '-B-I-D-G in correct spatial order, and the "outer" sheet analogously consists of s trands C' -C-H-E-F (see Figure 2). While there is no detectable sequence similarity with viral capsid proteins, detailed structural comparisons u tilizing the TNF three-dimensional coordinates show excellent backbone superposition, similar types of sidechain contacts, conserved amino acids, and various geometric matches with available fj-jellyroll folds ( 1 34).

The concept of structural conservation in spi te of amino acid divergence has prompted the construction of detailed CD40-L models ( 1 35, 1 36) utilizing homology modeling techniques and the available protein frameworks of TNF-O( and L T. These models show that the sparse amino acid matches between CD40-L and TNF-O( or LT are important for the construction of the fJ- jellyroll core fold, and also predict that CD40-L is capable of trimerization. Gaps in the protein alignment partition to variable loops in the model and two cysteines (residues X and Y) are suggested to form a disulfide link analogous to the Cys l 45-Cys 1 77 link in TNF-a ( 1 35).

Recent additions to the TNF cytokine superfamily have been a L T-fj molecule capable of forming heterotrimers with L T (1 37), and the cytokine ligands for CD27 ( 1 38), CD30 ( 1 39), and 4- 1 BB (139a). It is interesting to note that all TNF superfamily cytokines, with the exception of LT, are produced as type II membrane-tethered molecules ( 1 39). Secreted LT has an alternative membrane-bound form when complexed with a p33 factor

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Spleen

Tonsil Thymus

Figure 4. Expression of CD40 antigen on frozen sections of human spleen, tonsil and thymus: Expression of CD40 was analysed with anti-CD40 antibody G28.5 (a kind gift of professor E. A. Clark) using the APAAP method. Spleen: CD40 staining on B cells in follicle (F) and marginal zone (MZ). Very strong CD40 staining is observed on interdigitating cells in the periarteriolar lymphocytic sheath (PALS) around the central arteriole (CA). Tonsil: CD40 staining on follicular mantle (FM) B cells, germinal center (GC) B cells and follicular dendritic cells. Again, very strong CD40 staining is seen on interdigitating cells in the extrafollicular area. Thymus: CD40 staining on thymic epithelial cells both in the cortex (CT) and the medulla (M). Very strong CD40 staining is observed on dendritic cells in the medulla.

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fibroblasts. IL-IO(, TNFO(, and IFNy signi ficantly upregulate CD40 levels and 1 .4 kb CD40 transcripts on cultured epithelial cells.

CD40 is also expressed on follicular dendritic cells (FDC) of secondary lymphoid organs, as shown by immunohistology analysis on tissue sections (Figure 4) and by flow cytometry on freshly i solated FDC (89), and on FDC cultured in the presence of GM-CSF (90).

Immunohistological analysis performed on many different tissues (9 1 ) has also indicated staining of various cell types with the anti-CD40 mAb G28.5 : endothelial cells (mixed pattern of reactivity = + / - ), smooth muscle cells (+ / - ), cardiac myocytes (+ ), epidermis (weakly + ), gastrointestinal mucosa (+ ), gallbladder mucosa (+), bronchus mucosa (+ ), salivary gland ductal and acinar cells ( + ), pancreas ductal cells, mammary ductal and acinar cells (+ / - ), sweat gland (weakly +), prostate gland cells (weakly + ), thyroid gland ( + ), parathyroid gland ( + ) .

Basophils isolated from the blood of chronic myeloid leukemia patients have been reported to express CD40, shown by the binding ofmAb B-El O (92).

The CD40 antigen was initially identified with the mAb S2C6, which was generated from mice immunized with a urinary bladder carcinoma (93). Subsequently, CD40 antigen has been identified on carcinomas of other origins such as colon, prostate, breast, and lung, as well as on melanomas (94). CD40 has also been detected on T cell lines transformed with HTLV I and II (95). Accordingly baboon T cell lymphomas, which display HTLV I, are CD40 positive (74). Thus, CD40 is expressed on cells with high proliferation potential such as hematopoietic progenitors, B lymphocytes, and epithelial cells, and cells able to present antigen such as dendritic cells, activated monocytes, B lymphocytes, and follicular dendritic cells.

Signal Transduction Through CD40

The TNFR superfamily is primarily defined by common structural motifs in the extracellular binding domains-there is no equivalent organizing principle for the diverse cytoplasmic extensions ( 1 8) . This is an important distinction: perhaps the TNFR superfamily is a grouping of composite receptors with related binding folds grafted onto a diverse collection of intracellular signalling domains. After all, most growth factor/cytokine receptors operate by a common signalling paradigm irrespective of their cytoplasmic mass. Receptors rely on the ligand-induced association of extracellular domains to drive the concommitant association of intracellular structures, independently of whether these cytoplasmic domains are enzymes (i.e. tyrosine or serine kinases) or not (96, 97) . The TNFR intracellular domains clearly fall into the latter category--by analogy to

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the T cell antigen receptor complex (9S) or the hematopoietic superfamily receptors (99, 100), there may be a reduced number of protein motifs that act as binding epitopes for intracellular signalling molecules like kinases.

Perhaps a cytoplasmic grouping is possible: it has been noticed that the cytoplasmic domains of Fas, TNFR1, and LNGFR show a distant but significant similarity (35, 37). The more chain-economical domain ofCD40 has also been proposed to belong to this group (35, 37), but further comparison suggests a fortuitous alignment. The structural integrity of the intracellular domain of Fas is required for apoptosis as assayed by both site-directed mutagenesis and deletion analysis (101), or by naturally occurring variants involved in murine lymphoproliferative disorders ( 1 02). The Fas gene in mice is also the structural gene for lymphoproliferation (ipr) mutation (102, 103 , 104). While the key ipr mutation causes a rearrangement in gene intron 2 (and no viable mRNA is detectable), the allelic iprcg defect involves a single amino acid change in the Fas cytoplasmic domain, converting Ile225 Asn (102-104). The Fas-similar domain in TNFRI mediates TNF-directed cell cytotoxic activity ( l05). Most recently, t he homologous domain in the LNGFR was shown to be also involved in the induction of apoptosis (103). Together, these observations suggest that a subgroup of TNFR superfamily receptors share a common signalling domain. That CD40 is not included in this group makes some biological sense: while TNFRI and Fas require triggering by antibodies or ligands to induce cell death, CD40 acts instead as a survival factor (4) . Some functional analogy between CD40 and LNGFR was observed in that LNGFR constitutively induces cell death unless triggered by NGF or a monoclonal antibody (106) .

Of the remaining TNFR superfamily members, a cytoplasmic domain homology has been noticed only between CD27 and 4-1 BB (31). Interestingly, this region of 4-1BB displays a short sequence bracketed by two cysteines that resembles the kinase binding motifs of CD4 and CDS ( 107, l OS). Indeed, Kim et al (109) have recently shown that the mouse T cell antigen 4-1 BB directly associates with the tyrosine kinase p56 \Ckl through these motifs. CD40, OX-40, CD30, and the TNFR homolog from human chromosome 12 in turn show a short stretch of sequence similarity centered on a Glu-(AspjGlu)-Gly-Lys motif at the C-terminal end of their respective cytoplasmic domains. TNFR2 remains unclassified in this organizational scheme. Further dissection of this multitude of domains may uncover their role in TNFR superfamily signal transduction (110).

The importance of the CD40 intracellular domain in signal transduction has been directly inferred by the deleterious nature of a Thr234 Ala mutation (1 11). Cross-linking of CD40 on nonresting human B lymphocytes i nduces tyrosine phosphorylation of four distinct substrates (S4).

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Activation of protein tyrosine kinases (PTK) appears important for the transduction of CD40 signals because PTK inhibitors block B cell aggregation (112). As the CD40 cytoplasmic segment does not contain any enzymatic domain, it is likely that the CD40 mediated protein tyrosine kinase activity (113) occurs through activation of separate kinases. Accordingly, engagement of CD40 on the Daudi cell line induces activation of the src type kinase, iyn, following its increased phosphorylation (113). In contrast, anti-CD40 alters neither the phosphorylation nor the activity of jyn, another kinase of B lymphocytes. CD40 engagement results in phosphorylation of the 85 kDa subunit of phosphatidyl inositol 3 kinase (PI3K), while it does not affect its 110 kDa subunit. CD40 cross-linking induces an increased activity ofPI3K which catalyzes the phosphorylation of phosphoinositols on the 3' moiety and which plays a key role in mitogenesis. Finally, CD40 ligation induces within one minute increased phosphorylation ofPLCy2 (phospholipase C). PLCyl , which is present in lower amounts in B cells (114) does not appear to be phosphorylated in response to CD40 ligation. Phosphorylation of PLCy2 is consistent with anti-CD40 induced IP3 production (84).

CD40-LIGAND

Expression of CD40-Ligand on Activated T Cells

Fusion proteins made from the extracellular domain of human CD40 and the Fc region of human IgGI have been used to identify CD40-L. CD40-L is expressed on activated EL-4 thymoma cells, activated mature T cells but not on resting T cells (7, 115-123, 123a). CD40-L can be detected on T cells very early (1-2 hr) after activation. The expression of CD40-L is primarily on CD4+ T cells, although a small population of CD8+ cells also acquires CD40-L. The CD40-L can be induced on THO, THI and TH2 cells. The functional expression of CD40-L, as detected with the CD40-chimera is inhibited after co-culture with B cells (120). This effect can be explained by a downregulation of CD40-L mRNA and by the release of sCD40 which binds to CD40-L. As a consequence, T cells stain positive with a polyclonal anti-CD40 antiserum (75). The capacity of B cells to release CD40 in cultures was confirmed by a specific ELISA. Studies performed with monoclonal antibodies specific for the CD40-L (122, 124), isolated for their ability to block T cell-dependent B cell activation, confirm the expression of CD40-L observed with the CD40 fusion protein. Immunohistochemistry demonstrates that human CD40-L is expressed on CD3 + CD4 + T lymphocytes of the mantIe zone and germinal center light zone of secondary follicles in all peripheral lymphoid tissues. CD40-L positive cells can be identified within the interfolIicular T

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cell rich areas of secon dary lymphoi d tissues and the medulla and cortex of normal thymus.

Immunohistochemistry on murine spleen i solated three to four days, following immunization with the thymus-dependent antigen KLH, demonstrates an increase of CD4 + CD40-L + T cells in and around the terminal arterioles, and on the periphery of the outer peri arteriolar lymphoid sheath. Double immunohistochemical analysis reveals that the B cells producing the specific antibodies are juxtaposed to CD40-L + T cells ( 1 25). The latter study failed to show the presence of CD40-L + T cells in germinal centers. This difference with human data may be linked either to species or to the reagents as the anti-murine CD40-L may not detect CD40-L saturated with sCD40.

Lung mast cells and blood basophils also express CD40-L which i s functional as shown by the ability of basophils to induce B cells to secrete IgE ( l 25a).

Characterization of a CD40-Ligand cDNA

Murine EL-4 thymoma cells can induce the proliferation of resting human B lymphocytes ( 1 26). A cDNA that encodes CD40-L (7) was i solated from the EL-4 line following enrichment of cells binding to a CD40-Fc fusion protein. The murine cDNA predicts a polypeptide which has 260 aa consisting of a 22 aa cytoplasmic domain, a 24 aa transmembrane domain, and a 214 aa extracellular domain with four cysteines. Murine CD40-L i s a type I I membrane protein which has an extracellular carboxy-terminus.

A human CD40-L cDNA has been i solated by screening stimulated human blood T cell libraries with the murine CD40-L probe ( 1 17 , 1 1 9,1 27, 128). Another group had independently isolated a TNF-related activation protein (TRAP) from activated human T cells, which turned out to be the human homolog to murine CD40-L ( 1 29). The cDNA for human CD40-L encodes a polypeptide of 261 aa. The human CD40-L has a 22 aa cytoplasmic domain, a 24 aa transmembrane domain, and a 2 1 5 aa extracellular domain with five cysteines. The murine and human CD40-L display a conserved N-linked glycosylation site in the extracellular domain and the human CD40-L displays an additional, but probably not utilized, glycosylation site in the cytoplasmic domain. The two sequences exhibit 78% aa identity. There is 75% identity in the extracellular domain, 96% between the transmembrane region, and 81 % between the cytoplasmic domains.

Northern blot analysis of activated T cells demonstrates the presence of two mRNA species of 2 .1 and 1.4 kb (117, 127) that differ in the length of the 3' untranslated ends. CD40-L mRNA has been detected in activated CD4+ and CD8+T cells. RT-PCR analysis also indicates the presence of

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R __ IU m ... I ..... U m_ hUb 13 .. S me ........ 108 h(lD.H ,."' ..... $7 mCD3O-< .. "'498-1. lOG --

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Figure 5 Sequence and structural alignment ofTNF superfamily cytokines. The known X- ray structures ofTNF-a, LT and the model construction of CD40-L (135, 136) were used to accurately align the available sequences of human (h) and mouse (m) TNF-like cytokines. J3-strands derived from the TNF-(l( and LT structures are boxed and labeled according to standard viral capsid nomenclature: B- C- D-E-F-G-H-I, in such a manner that strands B-I- D-G and C-H-E-F form two opposing J3-sheets of a flattened barrel structure. Note a small insertion of sequence (large boxed region) between strands B and C that forms threc short J3-strands labeled 8', B, and C. The line beneath the sequences denotes the residue environment from TNF-a and LT X-ray structures: d marks highly exposed (i.e. solvent accessible) residues, are moderately exposed amino acids, *residues are buried in the hydrophobic core of the fold while t denotes residues buried in the trimer interface. Above the sequences are noted residues potentially in contact with receptors by homology to the determined L T- TNFR I receptor complex: & and $ symbols separate these interactions to the two receptor subunits that contact each ligand subunit in the trimer.

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( 140) that was revealed to be LT-f3 ( 1 37). These membrane-tethered homoor heteromeric cytokines bind to specific TNFR superfamily receptors that are displayed on the surface of neighboring cells, signaling the direct, contact-mediated immunoregulation of one cell type by another. However, as aptly demonstrated by the X-ray complex of LT and TNFRI proteins, soluble forms of these cytokines are perfectly capable of inducing receptor association, and subsequent intracellular signalling.

A comprehensive alignment of representative TNF-C( and LT molecules with the LT -13 chain and the ligands for CD27, CD30, and CD40 illustrates the great divergence of amino acid sequences within the TNF superfamily and is a graphic measure of the plasticity of the TNF fold (Figure 2). How can we decide which sequence variations are covariantly absorbed by the core fold, and which contribute to changes in receptor specificity? Several groups have tried to answer this question by exhaustive mutagenic studies of the structure and function of TNF-C( and LT ( 14 1-144) prior to the determination of the complex structure. Van Ostade et al ( 145) have remarkably arrived at the identification of a single residue change in human TNF-C( (Arg32 ---t Trp) that drastically lowers the binding affinity for TNFR2 but retains wild-type binding to TNFR I . As each receptor has distinct signalling pathways (23), this finding has important therapeutic potential ( 146). In addition, these studies may reveal the structural basis of receptor promiscuity for TNF-like ligands: clearly the distantly related LT and LT-f3 molecules can bind to the same receptor subunits ( 1 37, 1 40). Similar situations may exist with other TNF-like cytokines, reminiscent of the multitude of promiscuous couplings that are found between hematopoietic cytokines and their receptors ( 147, 148).

FUNCTIONAL CONSEQUENCES OF CD40 ENGAGEMENT Mature B Lymphocytes For simplifying the description of the effects ofCD40 ligation on human B cells, we use the classical model where B cells, following antigen encounter, undergo activation, proliferation, and differentiation in a stepwise fashion ( 149).

ACny A nON AND PROLIFERA nON OF B LYMPHOCYTES Resting B cells cultured in the presence of anti-CD40 antibody increase their size (72) and form homotypic aggregates ( 1 1 2, 1 50-1 53). Studies with antibodies have shown that this aggregation involves not only LFAI-ICAMl , but also the recently identified pair CD23/CD21 ( 1 54, 1 55). This latter interaction is inhibited by the anti-CD23 antibody MHM6 and the anti-CD2 1 antibody

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BU32, while antibodies directed to other epitopes of CD23 and CD2 1 are inefficient. Interestingly, fucose-2-P04 also blocks CD40 induced homotypic aggregation in accordance with the lectin-like nature of CD23. Activation of B lymphocytes through CD40 also stimulates their adhesiveness to endothelial cells in a VLA-4 dependent fashion ( 1 56). Soluble antiCD40 ( 1 57) and CD40-L transfected cells ( 1 58) are able to prevent apopto tic death of germinal center B cells. Triggering B cell CD40, either by cross-linking with monoclonal anti-CD40 antibody presented by a murine fibroblastic Ltk-cell iine transfected with FcyRIIjCDw32 (CD40 system) (4, 5) or with CD40-L transfected cells, results in an increased expression of CD23, class II antigens and B7jBB l ( 1 2 1 , 1 24, 1 59-161 ) . CD38+ CD44-germinal center B cells cultured in the CD40 system (CDw32 + L cells + anti-CD40 antibody) in the presence of IL-4, acquire the phenotype of memory CD3S- CD44+ B cells (YJ Liu et aI, manuscript in preparation). Finally, CD40 cross-linking induces B cells to produce IL-6 and IL- l O ( 162, 1 63).

Anti-CD40 antibodies have been isolated for their ability to co stimulate with either anti-IgM antibodies or phorbol esters (2, 7 1 , 72). In a soluble form, some anti-CD40 antibodies can induce DNA replication in resting B cells although this does not result in sustained proliferation ( 1 50, 1 64, 1 65). B cells cultured with soluble CD40-1igand in a monomeric form enter into limited DNA synthesis, but a trimeric form of soluble CD40 ligand obtained through various constructions results in quite significant DNA synthesis particularly in combination with anti-Ig ( 1 1 5, 1 1 9, 1 66). Culture of resting B cells in the CD40 system results in strong and long lasting B cell DNA replication (4). At least 70-80% of B cells enter into the G 1 phase of cycle and 50--60% into the S phase, permitting B cell numbers to increase by three- to four-fold over two weeks. These culture conditions allow the proliferation of various B cell subpopulations including mantle zone sIgD+ sIgM + B cells, sIgD- sIgM + /- B cells, CD5+ and CD5- B cells ( 167). Furthermore, quite significant DNA synthesis is observed in leukemic B cells, such as non-Hodgkin B cell lymphomas and chronic lymphocytic leukemia B cells ( 1 68). Transient expression of transfected murine and human CD40-L into various cell lines allows short-term proliferation of murine and human B lymphocytes ( 1 1 7, 1 19, 1 2 1 , 1 27, 1 28, 1 59, 1 61) . L cells stably expressing human CD40-L can induce a proliferation of B lymphocytes at least as important as that obtained using CDw32 L cells and anti-CD40 (Figure 6).

DNA synthesis of B cells in response to soluble anti-CD40 antibodies and either anti-IgM or phorbol esters is further boosted by IL-4 (72, 1 50, 1 69). Addition of IL-4 to B cells cultured in the CD40 system results in their sustained proliferation (4), and the total B cell population can expand

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A

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CDw32 L-celJs

none IL2 IL4 IUO none IL2 IL4 IL 1 0 mAb89

CD40-ligand L-celJs

none IL2 IL4 IL 10 none IL2 IL4 IL 1 0 mAb89

Figure 6 B cell proliferation induced either by CDw32-L cells and anti-CD40 mAb 89 (CD40 system) or by CD40-Ligand-L cells. Purified total tonsil B cells (20 x 1 031200 III well) were cultured with irradiated L cells (8000 Rad; 4 x 103lwell) which were stably-transfected with either FcyRIIICDw32 (Panel A) or with CD40-L (Panel B). Cultures were performed without or with IL-2 (40 Vlml), IL-4 ( 100 Vlml) or IL- IO ( 1 00 nglml) in the absence or presence of anti-CD40 antibody mAb89, as indicated. B cell proliferation was determined at day 5 of culture by the addition of 3H-thymidine, present during the last 16 hr of the culture period. Indicated is the mean cpm observed in triplicate cultures. Note that the Mab89, in a soluble form, is able to inhibit CD40-L induced B cell proliferation while it is a powerful stimulator in an immobilized form.

up to lOOO-fold. This results in the generation of factor-dependent longterm normal B cell lines which are negative for Epstein-Barr viral infection. B cell clones can be generated that contain several hundred cells. IL-4 also strongly enhances the proliferation of B cells cultured in the presence of cells expressing CD40-L (Figure 6) ( 1 1 7, 1 59) . In recent studies, we have been able to show that L cells stably expressing CD40-L are able to induce

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CD40 AND ITS LIGAND 90 1

the multiplication of B cells over two weeks provided IL-4 is added to cultures. IL- 1 3, a cytokine which shares homology with IL-4 ( 1 70, 1 7 1 ) can also induce a strong and long lasting proliferation o f B cells stimulated in the CD40 system or with CD40-L transfected cells (1 27).

IL- l and IFNy enhance the DNA synthesis observed in the CD40 system or with CD40-L transfected cells, with or without IL-4 ( 1 59, 1 72). In our hands, IL-2 poorly enhances the proliferation of B cells cultured in the CD40 system or with CD40-L transfected cells, while another study indicates that IL-2 is able to stimulate the proliferation of B cells activated with CD40-L transfected cells ( 1 59). Cells cultured in the CD40 system with IL-4 express CD19 , CD20, CD40, sIg, high levels of CD23, and HLA class II antigens. Surprisingly, a quite significant proportion of cells cultured for three weeks still express sIgD ( l 72a). Addition of anti-IgM antibody or SAC particles, which enhance DNA synthesis ( 173), does not down-regulate sIgD expression (our unpublished results).

Both viral and human IL- l O enhance the proliferation ofB cells cultured in the CD40 system or with CD40-L transfected cells, as determined both by tritiated thymidine incorporation and increased viable cell numbers ( 1 59, 1 74) (Figure 6). IL- l 0 appears to be almost as efficient as IL-4 during the first week of culture, but proliferation slows down thereafter and eventually ceases after 1 4 days. The combination of IL-4 and IL- l O is additive and results in a 60-100 fold expansion of viable B cells over two weeks. IL- IO upregulates the expression of CD25jTac on anti-CD40 activated B cells, and accordingly addition of IL-2 strongly enhances B cell proliferation ( 1 75). In fact, the production of IL-I 0 by CD40-L transfected CVl cells could explain the response of B cells to IL-2 ( 1 59) which is only marginal in our studies. B cells cultured in IL- IO differ microscopically from those cultured in IL-4 in that loose aggregates are observed early on, which then yield cultures mostly composed of single large cells.

DIFFERENTIATION OF B LYMPHOCYTES Human and murine B cells cultured with anti-CD40 antibody, with or without CDw32 L cells or with CD40-L transfected cells, produce marginal amounts of immunoglobulins ( 1 2 1 , 1 59, 1 6 1 , 1 72). However, coculture o f human B cells with SAC particles, anti-CD40 antibody, and CDw32 L cells results in the production of very large amounts of IgM, IgG, and IgA without IgE ( 1 76). Mantle zone sIgD + sIgM + B cells secrete only IgM, whereas sIgD - sIgM + j - B cells secrete IgG and IgA and lower amounts of IgM. This indicates that the concommitant triggering of sIg and CD40 results in differentiation of human B lymphocytes independently of exogenous cytokines.

Addition of IL-4 to cells cultured in the CD40 system only results in a

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slight increase in the production of IgM and IgG and in the secretion of large amounts of IgE ( 1 72). In fact, IgE production results from isotype switching as highly purified naive slgD+ B cells produce as much IgE as isotype committed slgD - B cells. In contrast to long-term B cell proliferation, the production of IgE does not require the presence of CDw32 L cells (6, 1 77-1 79). Cells transfected with CD40-L can also induce murine and human B lymphocytes to secrete IgE in response to IL-4 ( 1 17 , 1 2 1 , 1 59, 1 6 1) . Addition of lFNy or IFNa: to CD40 activated B cells fails to inhibit IL-4-induced IgE production. Indeed the inhibitory effects of interferons on IL-4-induced IgE production by mononuclear cells ( 1 80) may to be due to downregulation of CD40-L on IL-4 activated T cells ( 1 23, 1 28). TGFp and TNF!X are able respectively to block and to stimulate the production of IgE by CD40-activated B cells ( 1 8 1 ) . B cells stimulated through their CD40 antigen secrete IgE and IgG4 in response to IL- 1 3 , as a result of isotype switching ( 1 27, 1 82).

Addition of lL- l O to CD40-activated B lymphocytes results in the production of considerable amounts of IgM, IgG, and IgA without any IgE ( 1 59, 1 74). In fact, cells cultured in the CD40 system in the presence of lL-10 differentiate into plasma cells expressing large amounts of intracytoplasmic immunoglobulins. IL- l O induces anti-CD40 activated tonsil B cells to secrete IgG I , IgG2, and IgG3. Purified human B lymphocytes cultured in the CD40 system in the presence of IL- I 0 produce IgG and IgM antibodies able to bind to antigens such as tetanus toxoid (Table 2). The combination of soluble anti-CD40 or soluble CD40-L and IL- l O is, however, insufficient to allow production of bacteriophage-specific antibody by memory slgD- B cells from immunized individuals (I 82a). Specific antibody can however be detected provided bacteriophages are added to cultures, a finding consistent with a preferential expansion of antigen specific B cells and reminiscent of the comito genic effect of anti-CD40 and anti-Ig antibody (2, 72, 1 73). Interestingly, CD40 activated slgD+ JslgM+ B cells were found essentially to secrete IgM but also IgG 1 and IgG3 in response to IL- l O, indicating that this cytokine may act as a switch factor for certain IgG subclasses ( 1 83a). CD40 activated naive slgD+, slgM+ B cells cultured with IL- l O also produce low levels of lgA l , and addition of TGFp induces large amounts of both IgAI and IgA2 subtypes, while inhibiting IgM and IgG production ( 1 76) (F. Briere, manuscript in preparation). In contrast, TGFfl inhibits the production of lgM, IgG, and IgA by isotype committed sIgD - B cells stimulated by IL- l O in the CD40 system. This strongly suggests that TGFfl may represent an IgA-switch factor both in human and in mouse ( 1 83, 1 84). In line with these findings, recent studies have indicated that TGFfl is able to induce !X I and !X2 germline transcripts in activated human B cells ( 1 85).

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Table 2 Production of tetanus toxoid specific antibody by B lymphocytes activated in the CD40 system with cytokines'

% Wells with IgG anti-tetanus toxoid

5000 B cells per well 500 B cells per well

No cytokine 0 0 IL2 0 0 IL4 3 0 ILIO 9 0 ILIO + IL4 19 8 ILIO+ IL2 29 1 1

' Purified tonsillar B lymphocytes were cultured over irradiated CDw32-L cells (8000 Rad; 4 x I O'/well) with 0.5 Jig/ml anti-CD40 Mab 89. Cultures were performed without or with IL2 (40 V/ml) or ILIO (100 ng/ml) or their combination. Wells were harvested after 10 days and the presence of antitentanus toxoid antibody was determined by standard ELISA. Positive wells yielded an optical density equal to at least three times the background observed without addition of culture supernatants.

IL-5 acts synergistically with IL-4 to induce CD40 activated murine B cells to secrete IgG I and IgE, and to promote IgM and IgG3 secretion. Surprisingly, the induction of antigen specific antibody responses of CD40-L activated murine B cells requires the presence of antigen and IL-2, while IL-4 and IL-5 are only poorly efficient ( 1 2 1 ). This suggests that triggering of the antigen receptor may skew the cytokine response of CD40 activated B cells. Thus the engagement of CD40 on B cells turns on their isotype switching machinery, the specificity of which is subsequently provided by cytokines.

ROLE OF CD40 IN T CELL-DEPENDENT B CELL ACTIVATION Many different culture systems have been set up to elucidate the cell surface and soluble molecules involved in the growth and differentiation of B lymphocytes. Activated T cells can induce resting B cells to proliferate and differentiate into Ig secreting cells ( 1 86, for a review). In particular, the mouse thymoma EL-4 can activate resting human B cells to proliferate ( 1 26), an observation which allowed the cloning of the ligand for CD40 (7). T cells activated with immobilized anti-CD3 antibodies, which mimic T cell receptor engagement, are able to induce normal B cells to proliferate and secrete Igs in the complete absence of accessory cells or lectins that might favor cellular interactions ( 1 87, 1 88). Cytokines produced by activated T cells are involved in the growth and differentiation of B cells, but the cell contact cannot be replaced by T cell supernatants. In contrast, fixed activated T cells or their membrane enriched fraction can induce B cell proliferation, and addition of cytokines can further enhance growth and can induce Ig

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secretion ( 1 88-195). The CD40-Fc fusion protein inhibits B cell stimulation induced by activated T cells and their membranes ( 1 1 6, 1 2 1) , as well as IL-4-induced IgE production by mononuclear cells ( 1 96). Monoclonal antibodies blocking T cell---dependent B cell activation against murine ( 1 1 6) and human (1 24) activated T cells were in fact specific for CD40-L. In line with these findings, anti-CD40 antibody strongly block both proliferation and Ig secretion of tonsillar B cells induced by T cells stimulated with immobilized anti-CD3 ( 197, 1 98). Interestingly, activation of naive sIgD+ B cells is significantly less inhibited by anti-CD40 than that of sIgD - B cells, suggesting that the differentiation of naive B cells may also occur independently of CD40-CD40-L interactions. Accordingly, patients with defective CD40-L display increased circulating IgM levels (see below).

B Lymphocyte Precursors Soluble anti-CD40 antibodies neither stimulate the proliferation of normal B cell precursors (BCP) nor alter the effect of known growth signals for such cells ( 12, 82). However, they enhance tyrosine phosphorylation and inositol 1 ,4,5,-trisphosphate production in fetal liver pro-B cells (84). CD I9+ CDlO+ BCP can grow in the CD40 system provided IL-3 is added ( 12). This proliferation is further potentiated by IL-7 and IL- IO. However, at variance with mature B cells, IL-4 does not induce proliferation of BCP cultured in the CD40 system. BCP can also be induced to proliferate by triggering their CD40 with CD40-L present on activated CD4+ T cell clones ( l 98a). The signal provided by CD40-L is essential, as is demonstrated by the blocking of T cell---dependent BCP proliferation with the anti-CD40 mAb 89 and the lack of stimulatory effect of T cell clones lacking functional CD40-L obtained from a hyper-IgM patient. Activation of BCP through CD40, either in the CD40 system or in the presence of T cells, does not promote differentiation into mature sIg+ B cells. A small proportion of immature sIgM+ cells emerge in CD40-dependent cultures, but cells bearing other isotypes are not observed ( 1 2) . Of interest, triggering of CD40 can induce high-level surface membrane expression of CD23 on BCP ( 1 2). As CD23 is involved in myelopoiesis ( 1 99) and T cell ontogeny (200), it appears of interest to evaluate the role of this molecule in B lymphopoiesis.

Other Cell Types

Recent studies indicate that CD40 is functional on cells other than B lineage cells. Thymic epithelial cells are induced to secrete GM-CSF when triggered with soluble anti-CD40 in conjunction with IL- l and most notably IL- l and IFNI', the latter upregulating CD40 expression ( 10). This effect occurs in the absence of cell proliferation.

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Monocytes stimulated by CD40-L transfected cells secrete low amounts of IL-6 and IL-8 ( 1 1 ) . Addition of GM-CSF, IL-3, or IFNy, which upregulate CD40 expression on monocytes, further boosts CD40-L-induced secretion of IL-6 and IL-8 and allows secretion ofTNFcx. Cross-linking of monocyte CD40 using CD40-L transfected cells also results in the activation of tumoricidal activity against a melanoma cell line.

T lymphocytes also appear to respond to CD40-L (20 1) . CD40-L, on transfected cells or in a soluble trimeric form, induces (i) resting T cells to express CD25/Tac and CD40-L; (ii) activated T cells to secrete IFNy, TNFa, and IL-2, and (iii) activated T cells to proliferate. Both CD4 + and CD8+ T cells appear to proliferate in an IL-2-independent fashion. It is proposed that CD40-L binds to CD40 antigen which is expressed at very low density on T cells (201 ).

ROLE OF CD40 IN ANTIGEN-DRIVEN IMMUNE RESPONSES Mutated CD40-Ligand in the X-Linked Hyper-IgM Syndrome Several immunodeficiencies mapped to loci distributed throughout the X chromosome include: X-linked agammaglobulinemia, X-linked severe combined immunodeficiency, Wiskott-Aldrich syndrome, X-linked lymphoproliferative syndrome, and X-linked hyper IgM syndrome (202, 203, for a review) . Males affected with hyper-IgM syndrome are susceptible to various infections (204). These patients do not make antibodies to exogenous antigens but make a variety of autoantibodies. Their serum has slightly or vastly elevated concentrations of polyclonal IgM and IgD, but no detectable IgA or 19B and very low levels of IgG. The secondary lymphoid organs of these patients display no germinal centers although they have normal levels of circulating B cells and plasma cells producing IgM and IgD in lymphoid tissues and the gastrointestinal tract. Immunization of these patients with bacteriophage 1 74, a thymus-dependent antigen, results in a poor humoral immune response that is restricted to the IgM isotype (205). These features indicate a defect in the generation of memory B cells. It is important to note that these patients often also suffer from neutropenia.

The gene mutated in the hyper-IgM syndrome was mapped to chromosomal region Xq24-27 close to HPRT (206). The localization of the CD40-L gene in region Xq26-3-Xq27- 1 ( 129, 1 36, 207) led to the demonstration that the defect in the hyper-IgM syndrome is due to point mutations or deletions in the gene encoding the CD40-L (9, 1 36, 207-2 10) . Activated T cells from these patients do not bind CD40 fusion proteins, although cells from some of these patients stain with polyclonal antibody

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specific for the CD40-L. This suggests either expression of the truncated CD40-L on the cell surface or expression of a conformationally altered protein unable to bind CD40. mRNA transcripts for the CD40-L have been sequenced in 1 3 patients, and 1 2 displayed either point mutations (9 cases) or deletions (3 cases) (Figure 7). Of note, one of the patients displayed a CD40-L cDNA without any nucleotide change within the coding region (207). One study on four patients showed that the CD40-L of three of the patients' mothers had the same mutation, while a fourth patient appeared to have had a de novo alteration (21 0) . Expressed mutated CD40-L are unable to activate B lymphocytes from normal individuals (1 36, 207), whereas the B lymphocytes from hyper-IgM patients can be stimulated either with activated T cells (2 1 1 ) orwith anti-CD40 and cytokines ( l 36, 207-209, 2 1 2, 2 1 3). In line with the Ig status of hyper-1gM patients, mice develop an hyper-IgM response to the thymus-dependent antigen DNP-Ovalbumin when CD40-CD40-L interactions are blocked in vivo by injecting CD40-Fc chimeric molecules (D Gray, personal communication).

A Tentative Synthesis on Where, When, and How CD40 Is Triggered

The key role of CD40 for T cell-dependent B cell activation in vitro was firmly supported by the in vivo finding that mutations in CD40-L gene result in hyper-lgM syndrome. Based on the kinetics and in vivo expression of CD40 and CD40-L, the following model for the timing and sites of

Figure 7 Schematic representation of the coding region of CD40-Ligand and localization

of the mutations, deletions found in 12 hyper-IgM patients. Patients A.T., B.W., T.G. (209),

P I-P4 (210), P5-P7 (207) and CD., J.W. ( 1 36). IC, intracellular domain; TM, transmembrane

domain; EC, extracellular domain.

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CD40/CD40-L interactions during T cell-dependent immune responses is proposed (Figure 8), based on earlier versions (21 4-2 1 8) . The entry of pathogen/antigen into the mammalian organism elicits specific and nonspecific immune responses. Dendritic cells from skin or mucosa

( differentiation) memory

B cell

l -, - t follicular dendritic apoptosisl cell (FOC)

'-c-.o-na-I-ex-p-a-ns-;o-n-, , ? somatic mutations

centroblast ,

-m-ig-ra-ti-on-in-to- - - - - - - t - - - -primary follicle naive or , . "., ,

/-0. interdigitating

APICAL LIGHT ZONE

BASAL UGHT ZONE

DARK ZONE

SECONDARY LYMPHOID QBM!'

PARACORTICAL . AREA

_ eric:I - -t- ,- '- _ - _

II --.... Slillfl M!,/,!;QA

dendritic I Langerhans cell

Figure 8 Model for CD40/CD40-Ligand interactions in antigen dependent immune responses (See section: A tentative synthesis on where, when and how CD40 is triggered), = Antigen,


initiate the specific reaction by capturing antigen and migrating into the paracortical T cell-rich areas of secondary lymphoid organs such as lymph nodes. In these sites, these APC are called interdigitating dendritic cells (IDC). During the antigen loading and migration phases, dendritic cells acquire surface CD40, possibly following interaction with GM-CSF released by various cell types (keratinocytes, neutrophils, mast cells) at the site of antigen entry. In the T cell-rich areas, the IDCs present antigenderived peptides bound to MHC class II antigens, to naive or memory antigen-specific T cells initiating the extrafollicular reaction. Cross-linking of the T cell receptor readily turns on CD40-L expression (Figure 9). Meanwhile various sets of adhesion molecules strengthen the interactions between T cells and the IDe. The cross-linking of CD40 on IDC by T cell CD40-L enhances IDC cytokine production which subsequently potentiates T cell activation, proliferation and differentiation. In a mirror fashion (Figure 9), the cross-linking of CD28/CTLA4 on T cells by B7 antigen on IDC results in increased cytokine production by T cells which may then act (i) in an autocrine fashion as T cell growth and differentiation factors, (ii) to further activate the IDC, and (iii) to induce the proliferation and differentiation of recruited antigen-specific B cells. It is possible that IDC present antigen to B cells. The antigen activated B cells interact with T cells through various surface molecules. In particular, CD40 cross-linking boosts B cell activation and differentiation and may represent a key signal for the migration of B cells into primary follicles composed of a network of follicular dendritic cells (FDC) (2 19, 220). Some antigen-specific activated T cells may also migrate at that time. These interactions result in massive B cell proliferation which starts the germinal center (GC) reaction (221 , 222). When a full germinal center is developed, proliferating centroblasts can be identified in the dark zone. This is the anatomic site where high-rate somatic mutations occur within the variable Ig regions (223, 224). There is no evidence, at the present time, that CD40/CD40-L interactions are operating at that stage. The centro blasts then mature into nonproliferating centrocytes that compose the light zone. In the basal light zone, the somatic mutants undergo selection based on their ability to bind to antigen deposited on FOCs in the form of immune complexes. Antigen receptor triggering will permit the survival of these cells while others die from apoptosis. Subsequently, in the apical light zone, the antigen on FDC will be processed by selected B cells and presented to antigen-specific activated T cells that produce IL-2, IL-4, and IL- l 0 (225, 226). It is possible that the CD40 on FDC may engage the CD40-L on activated T cells. At that stage, C040/C040-L interactions may play a key role in (i) inducing multiplication of the rare selected B cells, (ii) turning on the isotype switch-

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ing machinery, and (iii) inducing the differentiation of selected B cells either into memory cells which will recirculate or into plasmablasts that will leave the germinal centers to migrate either to the bone marrow or to the mucosal lamina propria where they become plasma cells.

The clinical and biological status of patients suffering from the hyperIgM syndrome permits us to conclude that the role of CD40 on APC to boost T cell responses is either of secondary importance or can be replaced by other molecular pairs as suggested by normal T cell numbers and lack of viral infections. However, the susceptibility of these patients to opportunistic agents such as Pneumocystis yet indicate a partially altered repertoire of T cell responses. The presence of normal or elevated circulating IgM levels of polyclonal origin indicates that primary B cell

Figure 9 CD40/CD40-Ligand in cellular interactions. T cells recognize peptide presented by MHC Class II on antigen presenting cells (APC) (dendritic cells or B cells). Adhesion molecules strengthen the interaction. This results in upregulation of CD40-ligand on T cells and B7/BBI on APC. Triggering of CD40 on APC permits activation of B cells and/or cytokine production by B cells or dendritic cells. The produced cytokines further activate T cells and allow their proliferation. The increased B7/BB I expression on APCfB cells triggers CD28 or CTLA-4 on T cells which then secrete cytokines which will either further activate the proliferation/differentiation of B cells or act as autocrine T cell growth factor. Thus, the interaction between CD40/CD40-L signals B cells and APC, while the triggering of CD28/CTLA-4 signals T cells to produce cytokines, which results in T cell proliferation, B cell growth and differentiation, and APC activation.

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9 1 0 BANCHEREAU ET AL

reactions are not affected by lack of CD40jCD40-L interactions either because this interaction is not involved in this process or because other molecules can substitute at that level. In this respect, the B cell stimulatory effect of membrane TNF on activated T cells may play a significant role (227, 228). The lack of germinal centers in the secondary lymphoid organs of hyper-IgM patients suggest either an altered migration of activated T and B cells into primary follicles or an altered proliferation of centroblasts. The mechanisms leading to the proliferation of centro blasts still remain unclear and may be CD40-L independent, because no CD40-L + T cells can be detected in the GC dark zone and because B cells do not appear to undergo somatic mutations at a high rate when cultured in the CD40 system in the presence of IL-4 (229).

PERSPECTIVES

As CD40 belongs to a family of molecules that bind several ligands as exemplified by the two TNFRs, the LNGFR, and 4-1 BB, it is plausible that CD40 may bind to other presently uncharacterized ligands. Indeed, as 4- l BB binds both a TNF-like molecule ( l 39a) and extracellular matrix proteins (56), it is possible that CD40 may also bind to such a matrix. This would be in agreement with the high levels of binding of soluble CD40 observed on murine B cells (229a). The function of CD40 on cells other than B cells and monocytes remains to be determined. In particular the broad expression of CD40 on CD34 + progenitors raises the possibility that CD40 plays an important role in hematopoiesis. CD40 activation could represent a pathway through which CD40-L + T cells may regulate hematopoietic production levels in the bone marrow during acute situation. Such a regulatory function would be compatible with the fact that hyper-IgM patients have a normal output of newly formed B cells despite alteration of their CD40-L. However, CD40 may also participate in constitutive hematopoiesis, as might be suggested from the frequent neutropenia, as well as the less common anemia and thrombocytopenia observed in hyper-IgM patients. Also, the existence of an alternative ligand for CD40, as evoked above, should be considered within the bone marrow, as it could point to a more central function of CD40 in constitutive hematopoiesis. Obviously, further studies are necessary to define the role of CD40 in hematopoietic development. In this context, evaluation of mice in which the CD40 gene has been disrupted by homologous recombination appears of importance.

The apparently crucial role of CD40 triggering in isotype switch will permit us to dissect the mechanisms leading to the intracellular assembly

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CD40 AND ITS LIGAND 9 1 1

of a functional isotype switch machinery. The elucidation of these intracellular pathways may ultimately allow the construction of antagonists blocking isotype switch. Finally, the lack of memory cells in patients suffering from CD40-L mutations suggests that pharmacological targeting of the CD40/CD40-L interactions may make it possible to down-regulate undesired humoral responses such as antibody-mediated autoimmune diseases. The administration of anti-CD40-L antibody to animals was recently found to interfere with the development of primary and secondary humoral immune responses (230). Furthermore, such antibody treatment also prevented the arthritis induced in mice by immunization with type II collagen (23 1) . The available structural information on CD40 and CD40-L will be refined in the near future and will facilitate the construction of mutants acting as antagonists.

ACKNOWLEDGMENTS

The authors wish to thank Nicole Courbiere for her invaluable editorial assistance and Drs. R. Armitage, R. Geha, J. Gordon, D. Gray and M. Howard for communicating preprints before publication.

Any Annual Review chapter, as well as any article cited in an Annual Review chapter, may be purchased from the Annual Reviews Preprints and Reprints service.

1-800-347-8007; 415-259-5017; email: [email protected]

Literature Cited

1 . Paulie S, Ehlin-Henriksson B, Mellstedt H, Koho H, Ben-Aissa H, Perlmann P. 1985. A p50 surface antigen restricted to human urinary bladder carcinomas and B lymphocytes. Cancer Immunol. Immunother. 20: 23-28

2. Clark EA, Ledbetter JA. 1986. Activation of human B cells mediated through two distinct cell surface differentiation antigens, Bp35 and Bp50. Proc. Natl. Acad. Sci. USA 83: 4494-98

3. Stamenkovic I, Clark EA, Seed B. 1989. A B-Iymphocyte activation molecule related to the nerve growth factor receptor and induced by cytokines in carcinomas. EMBO J. 8: 1403-10

4. Banchereau J, de Paoli P, Valle A, Garcia E, Rousset F. 199 1. Long term human B cell lines dependent on interleukin 4 and anti-CD40. Science 251: 70-72

5. Banchereau J, Rousset F. 1991. Growing human B lymphocytes in the CD40 system. Nature 353: 678-79

6. Jabara HH, Fu SM, Geha RS, Vercelli D. 1990. CD40 and IgE: synergism between anti-CD40 monoclonal antibody and interleukin 4 in the induction of IgE synthesis by highly purified human B cells. J. Exp. Med. 172: 1861-64

7. Armitage RJ, Fanslow WC, Strockbine L,

annurev.iy.12.040194.004313] j. banchereau; f. bazan; d. blanchard; f. briè; j. p. galizzi; -- the...

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