Defective Expression of CD40 Ligand on T Cells Causes "X-Linked Immunodeficiency with Hyper-IgM...

Immunological fieviens 1994, No. 138Printed in Denmark . All rights reserved

No part may be reproduced by any process without writtenpermission from the author(s)

Copyright © Munksgaard 1994

Irnmunological ReviewsISSN 0/05-2896

Defective Expression of CD40 Ligand on

T Cells Causes "X-Linked

Immunodeficiency with Hyper-IgM

(HIGMl)"

RTCHARD A. KROCZEK'*, DANIEL GRAF', DUILIO BRUGNONI^ SILVIA GILIANI^

ULF KORTHAUER', ALBERTO UGAZIO^ GABRIELE SENGER\ HANS W. MAGES',

ANNA VILLA*" & LUIGI D. NOTARANGELO^

X-LINKED IMMUNODEFICIENCY WITH HYPER-IgM (HIGMl)

Clinical features of HIGMl

X-linked immunodeficiency with hyper-IgM (HIGMl) is a rare disorder, char-acterized by recurrent infections associated with very low or absent IgG and IgA,and normal to increased IgM serum levels (Rosen et al. 1961, Notarangelo et al.1992). Affected males experience early-onset infections, usually within the 1styear of life, when levels of maternally-derived antibodies decline. Although mostinfections are of bacterial origin, HIGMl patients are also unusually susceptibleto infections with opportunistic pathogens and often suffer from Pneumocystiscarinii pneumonia and Cryptosporidium intestinal infection - disease entities oftenobserved with T-cell immunodeficiencies but not with other forms of hypogamma-globulinemia. Hematological disturbances (such as anemia, thrombocytopenia,and particularly neutropenia) are common in HIGMl patients. Because of neutro-

'Molecular Immunology, Robert Koch-Institute. Berlin, Germany, "Department of Pedi-atrics, Institute of Chemistry, and Clinical Immunology Service, University of Brescia,Italy, 'Human Cytogenetics Laboratory, Imperial Cancer Research Fund, London, U.K.,M.T.B.A., C.N.R.. Milan, Italy.•Corresponding author: Richard A. Kroczek, Molecular Immunology, Robert Koch-Insti-tute, Nordufer 20, L^353 Berlin, Germany.This work was supported by the Deutsche Forschungsgemeinschaft (grant 827/10-1 loR.A.K.) and partially by Progetto Finalizzato Ingegneria Genetica (SP4), C.N.R., and byTelethon (grant A14).

40 KROCZEK ET AL.

petiia, affected males frequently suffer from recurrent oral ulcers. Fitially, autoim-mune manifestations and iticreased susceptibility to neoplasms are also welldocumented in HIGMl. Although no data are available oti the overall early-mortality rate iti HIGMl, this figure appears to be aroutid 10% from a surveyof 67 patients with primary hyper-IgM immunodeficiencies (Notarangelo et al.1992). In spite of intravenous immunoglubulin and antibiotic prophylaxis HIGMlpatietits often suffer from life-threatening infections. Bone marrow transplan-tation is therefore now considered a potential form of treatment, if HLA-identicaldonors are available. HIGMl has to be distinguished from the very rare auto-somal dominant or autosomal recessive primary immunodeficiencies with a simi-lar disease phenotype (Notarangelo et al. 1992, Callard et al. 1993).

Immune function in HIGMl patients

Investigation of the immune function in HIGMl has shown an inability to switchfrom IgM/IgD secretion to other isotypes (Geha et al. 1979, Levitt et al. 1983).Also on circulating B cells, surface immunoglobulin expression is restricted toIgM and IgD. Upon In vivo antigenic stimulation, the primary antibody response(mainly of the IgM isotype) develops normally; however, boosting results in poorsecondary responses, with no evidence of specific IgG production (Kyong et al.1978, Nonoyama et al. 1993). Despite lymphoid hyperplasia (which distinguishesHIGMl from X-linked agammaglobulinemia), lymph nodes are characteristicallydevoid of germinal centers (Kyong et al. 1978, Notarangelo et al. 1992). Thepathogenesis of the abnormal pattern of B-cell difTerentiation has long remainedobscure. It has been shown early that peripheral blood mononuclear cells fromHIGMl patients, when stimulated with pokeweed mitogen in vitro, secrete IgMexclusively, thus mimicking the in vivo situation (Geha et al. 1979, Levitt et al.1983, Mayer et al. 1986). In the past, this abnormality has been variably attributedto defective B- or T-cell function. However, the primary role of defective T-helpercell activity was clearly established in 1986, when it was shown that B cells fromHIGMl patients may differentiate into IgG-secreting cells if co-cultured with Tlymphoblasts (T,.^^) from a patient with a Sezary-like syndrome (Mayer et al.1986). With the notable exception of defective helper activity, in vitro (mitogen-induced proliferation, response to alloatitigens) and in vivo (delayed-type hyper-sensitivity) tests of T-cell function are usually normal in HIGMl patients (Benker-rou et al. 1990), however, thymic atrophy, with few Hassal's corpuscles, has beenwell documented (Stiehm & Fudenberg 1966).

Genetics of HIGMl

Molecular genetic analysis of HIGM I was started in 1987, when the gene respon-sible for HIGMl was tentatively mapped to Xq24-27 (Mensink et al. 1987), and

X-LINKED IMMUNODEFICIENCY WITH HYPER-IgM 41

shown to be distinct from the XLA gene (Malcolm et al. 1987). The study of alarger number of pedigrees in the past few years allowed further refinement ofthe HIGMl gene location to Xq26-27 and demonstration of a close linkage tothe HPRT locus (Padayachee et al. 1992, Padayachee et al. 1993).

HELPER T CELL ACTIVITY FOR B-CELL DIFFERENTIATION

The critical role of helper T-cell activity for B-cell function became apparent ina number of different experimental systems. First, it could be shown that directphysical interaction between T cells and B cells is necessary, since B cells prolifer-ated when co-cultured with mitogen-activated T cells, but not when incubatedwith supernatants of activated T cells (Clement et al. 1984). Physical separationof T and B cells by a membrane also prevented stimulation of B cells (Owens1988). On the other hand, membranes from activated T cells could replace wholecells when testing for T-helper cell activity (Brian 1988). In attempts to identifythe interacting molecules on B and T cells, antibodies against CD4(), a moleculeconstitutively expressed on B cells, were identified as mitogenic (Clark & Ledbetter1986, Valle et al. 1989). Later, stimulation via CD40 in the presence of accessorymolecules and appropriate lymphokines was shown to induce immunoglobulinclass switch and secretion of immunoglobulins (Zhang et al. 1991, Rousset et al.1991). The critical role of CD40 was also demonstrated by inhibiting T-helperinduced B-cell proliferation using soluble CD40 molecules (Noelle et al. 1992).

IDENTIFICATION AND CHARACTERIZATION OF THE CD40 LIGAND

Cloning ofthe human CD40 ligand (CD40L)

We originally established a collection of T-cell activation genes, i.e. genes newlyexpressed or strongly upregulated after T-cell activation. To this end a lambdagtlO cDNA library was generated from human peripheral blood T cells stimulatedwith phorbol myristate acetate (PMA) and Ca*^-ionophore A23I87. Severalsteps of differential screening followed by elimination of redundancy led to theestablishment of a collection of 100 distinct cDNA clones representing T-cellactivation genes (Mages et al., unpublished). This collection of cDNAs wasfurther analyzed by a multigene analysis method, allowing a simultaneous analysisof expression for all ofthe encoded genes (Graf et al., unpublished). One ofthecDNA clones turned out to represent a gene expressed only when using bothPMA and Ca^^-ionophore A23187 for T-cell stimulation ("two-signal gene") andthus resembled a small group of lymphokine genes with identical activationrequirements (for review see (Mages et al. 1993)). Further multigene analysisexperiments determined that the gene was expressed in activated T cells but notin many other tissues examined. Sequencing of a full length cDNA clone revealedsimilarity to tumor necrosis factor-a (TNFa) and iymphotoxin (LT), therefore

42 KROCZEK ET AL.

the encoded molecule was designated TRAP (TNF-Related Activation Protein,(Graf et a!. 1992)). The structural similarity of TRAP to TNF/LT suggested thatTRAP could interact with one of the TNF or LT receptors or a related molecule.During the ongoing characterization of the TRAP protein the sequence of aligand for the murine CD40 molecule, a member of the TNF/LT - receptorfamily, was published (Armitage et al. 1992). Sequence comparison between thismurine CD40 ligand (CD40L) and TRAP showed 82.8% identity at the nucleicacid level (coding region) and 77.4% at the amino acid level (Graf et al. 1992),strongly suggesting that TRAP was the human homologue of the murine CD40L.The binding of soluble CD40 to TRAP expressed on the cell surface of a transfec-ted murine myeloma formally proved that TRAP was the human ligand for CD40(Graf etal. 1992).

TRAP/CD40L mRNA can be detected in T cells within 30 minutes of stimula-tion in the presence of cycloheximide, defining TRAP as an immediate-early gene.TRAP/CD40L mRNA expression is transient, with a peak between 4 and 14hours (depending on the mode of activation), after 24 hours the signal is usuallyno longer detectable. The mRNA of approximately 2.3 kb encodes a type-IItransmembrane protein with a predicted intracellular a mi no-terminal tail of 22amino acid residues, a transmembrane region extending from position 23 toposition 46, and an extracellular carboxy-terminal domain of 215 amino acids(Graf et al. 1992). The use of a second potential translation initiation site atnucieotide position 117 would generate a protein without an intracytoplastnictail but with an intact hydrophobic region serving as a signal peptide with apredicted cleavage site between Arg at position 43 and Val at position 44 (Grafet al. 1992). It is therefore conceivable that the TRAP protein is also secreted, asis the case with TNFa (Vassalli 1992). Again, in analogy to TNF and LT, boththe membrane-bound and the soluble TRAP/CD40L molecules probably exist astrimers, as recent computer models suggest (Peitsch & Jongeneel 1993). Essentiallyidentical data on the structure and expression of TRAP/CD40L were obtainedindependently by two other groups (Hollenbaugh et al. 1992, Spriggs et al. 1992).

Induction and detection of CD40L on the T-cell surface

An important step for the identification of TRAP/CD40L function was the earlydevelopment of a soluble CD40 (sCD40) molecule by fusing the extracellulardomain of human CD40 to the constant region of human IgGl (Fanslow et al.1992). The use of this chimeric reagent proved extremely valuable for the analysisof HIGMl, since staining of activated T cells with this reagent allowed directtesting of the capacity of expressed TRAP/CD40L molecules to bind CD40(compare Fig. 6). Later, similar chimeric molecules were also developed by others(Noelle et al. 1992, Lane et al. 1992). To generate a polyclonal anti-TRAP serumfor studies of HIGMl patients, we used a fusion protein containing amino acids


38-183 to immunize rabbits (Korthauer et al. 1993). This anti-TRAP serumturned out to be very useful for detecting mutated TRAP/CD40L molecules onthe T-cell surface which could no longer bind sCD40 (compare Fig. 6).

To generate monoclonal antibodies (mAb) to TRAP/CD40L, we functionallyexpressed TRAP on the surface of a murine myeloma (Graf et al. 1992) and usedthis transfectant for immunization of mice (Graf et al., unpublished). We thusobtained mAb TRAPl (IgGl) and mAb TRAP2 (IgGl), and used these reagentsto determine the induction requirements for TRAP/CD40L on the surface of Tcells. Resting T cells do not express TRAP/CD40L, and the physiological induc-tion mechanism for TRAP/CD40L expression is as yet unknown, although theinvolvement of the T-cell receptor/CD3 complex seems probable (Spriggs et al.1992, Castle et al. 1993). In vitro, the most potent induction of TRAP/CD40Lcell surface expression is achieved with the combination of PMA and Ca'^-ionophore A23187, whereas PMA or Ca^'-ionophore A23187 alone are totallyineffective (Fig. 1), thus reflecting the induction requirements of a "two-signalgene" (see above). Using activated T cells we determined that TRAPl and TRAP2

log fluorescence

Figure I. Two-signal induction of TRAP/CD40L on the surface of T cells. T cells were leftunstimulated or were activated with A23187 (125 ng/ml), PMA (20 ng(ml), or PMA +A23187 for 5 h, the optimal time point for this mode of stimulation, and stained with mAbTRAPL

B

log fluorescence

Figure 2. MAb TRAP! and TRAP2 recognize TRAP/CD40L independent of the CD40binding site. T cells were stimulated by PMA + A23I87 for 5 h, stained with mAb TRAPlwithout pre-incubation (A), or after pre-incubation with sCD40 (B), and analyzed byflow cytometry. In the control staining, pre-incubation with sCD40 gave no signal abovebackground with goat anti-mouse IgG-EITC (C), tbe secondary reagent in A-B (curvesfilled in black). Identical results were obtained with mAb TRAP2 (not shown).

44 KROCZEK ET AL.

detect an epitope independent of the CD40 binding site on TRAP/CD40L (Fig.2). For comparison of the reagents. Fig. 3 shows staining of activated human Tcells with mAb TRAPl, sCD40, and anti-TRAP serum. TRAP/CD40L is e.\-pressed on CD4+ T cells (Lederman et al. 1992, Graf et al. 1992, Lane et al.1992, Spriggs et al. 1992), a small proportion of CD8^ T cells (Lane et al. 1992,Spriggs et al. 1992), and on activated basophils and mast cells (Gauchat et al.1993).

Chromosomal location of the TRAP/CD40L gene

Using a TRAP cDNA probe we isolated a 15 kb genomic clone TRAP.gen froma human placenta genomic library and verified its identity by Southern blottingand restriction analysis. Using this probe, the CD40L gene was mapped to theq26.3-27.1 region of the X-chromosome by fluorescence in situ hybridization(Graf et al. 1992 and Fig. 4). The localization of the TRAP gene was confirmedby hybridizing TRAP cDNA to a panel of somatic cell hybrids and cell linesretaining different portions of the q24-qter region of the human X chromosome(Fig. 5, Giliani et al., unpublished). Mapping of the CD40L gene close to HPRTwas consistent with segregation analysis in HIGMl families that had shown closehnkage of the HIGMl locus to HPRT (Padayachee et al. 1992, 1993).

MOLECULAR BASIS OF THE HIGMl DISEASE

Intact function of B cells in HIGMl

To exclude a malfunction of B cells in HIGM 1 definitively, we tested their capacityfor immunoglobulin synthesis. Peripheral blood lymphocytes (PBL) from HIGMlpatients, as well as PBL from age-matched controls were cultured in the presenceof pokeweed mitogen, an agent able to induce immunoglobulin synthesis byactivating B cells and T cells and allowing them to cooperate. As expected, thepatients' B cells could not secrete significant amounts of IgG, IgA or IgF.However, when the patients' B cells were triggered directly through crosslinkingof CD40 in the presence of Staphylococcus aureus Cowan I and appropriate

a-TRAPserum

Figure 3. Comparison of staining of PMATRAPl, sCD40, and ami TRAP serum.

log fluorescence

A23187 activated T cells (5 h) with mAb


232425 -26.1-26.2-!26.327:2^127.3/128 ^

Figure 4. Mapping of the TRAP/CD40L gene by fluorescence in siiu hybridization. TRAP.-gen probe (Graf et al. 1992) was biotinylated and hybridized lo metaphase chromosomesprepared from peripheral blood lymphocytes of a male donor and detected with avidin-Texas Red. The replication R-banding pattern was obtained by incorporation of bromod-eoxyuridine (BrdU) during the first half of the S-phase and by detection of incorporatedBrdU with a fluorescein-coupled anti-BrdU mAb. One pair of signals was observed on theX-chromosome in the region Xq26.3-q27.l (arrow), the inset shows the same chromosomecounterstained with 4.6-diamidino-2-phenylindoie-dihydrochloride (DAPl). The ideogramfor the X-chromosome shows an R-banding pattern. The bar on the right represents thehybridization site of the TRAP gene.

lymphokines, IgG, IgA and IgE were produce normally (Korthauer et al. 1993).These data established the unimpaired intrinsic capacity of B cells in HIGMl toundergo immunoglobuiin class switch in vitro and to produce immunoglobuhnof al! isotypes (Korthauer et al. 1993). Furthermore, these functional resultsclearly pointed to an ineffective T-cell help for B cells, and were thus in agreementwith earlier data (Mayer et al. 1986).

Defective expression of CD40L on the T-cell .surface in HIGM}

At the beginning of our studies, several lines of evidence strongly suggested thatthe TRAP/CD40L molecule on the surface of activated T cells is defective in

46 KROCZEK ET AL.

5'CD40L cDNA

Figure 5. Hybridization of TRAP-g5 cDNA clone (Graf et al. 1992) to a panel of somaticcell hybrids and cell lines retaining different portions of the Xq24-qter region of the humanX chromosome ((Maestrini et al. 1990); cell lines GM10663, GM10664, and GM11099 arefrom the NIGMS Human Genetic Mutant Cell Repository). This region had been dividedinto four segments, based on the presence of DNA sequences recognized by DNA probesDXS42, DXSIO, HPRT, and DXS115 (black bars). Hybridization of the TRAP/CD40LcDNA probe to the panel showed that the TRAP/CD40L gene co-localizes with the HPRTgene.

HIGM 1. Mapping of the TRAP gene by our group to Xq26.3-27.1 (Graf et al.1992), the same region of the X-chromosome to which HIGMl has been locatedpreviously (see above), provided the ftrst firm lead. Second, the phenotype of thedisease (low or absent IgG, IgA and IgF), indicative of a failure to switch fromIgM to the other immunoglobuhn isotypes, was compatible with the crucial roleof CD40 on B cells in Ig class switching (see above). Most importantly, thecloning of the human CD40L molecule (Graf et al. 1992, Spriggs et al. 1992,Hollenbaugh et al. 1992) and the development of sCD40 reagents (Fanslow et al.1992, Noelle et al. 1992, Lane et al. 1992) permitted the molecular analysis ofHIGMl.

Originally, we were able to analyze 3 affected males from unrelated families,in which segregation analysis was consistent with HIGMl gene location at the

X-LrNKED IMMUNODEFICIENCY WITH HYPER-IgM 47

q26 region of the X-chromosome (Padayachee et al. 1993). Cell surface analysisdemonstrated that TRAP/CD40L expression in HIGMl patients is defective,since activated T cells from all 3 patients failed to bind sCD40 (Korthauer et al.1993 and Fig. 6). The use of anti-TRAP serum provided the additional infor-mation, that (non-functional) TRAP/CD40L is expressed on activated T cells insome, but not all patients (Korthauer et al. 1993 and Fig. 6).

Mutations in the CD40L gene

To identify the molecular basis for the failure of the patients' T cells to bindsCD40, TRAP mRNA was investigated. Northern analysis performed with avail-able material indicated normal mRNA expression, thus excluding gross abnor-malities of gene transcription or RNA splicing in these patients. Subsequently,RNA from activated T cells of all 3 patients was reverse transcribed and theentire TRAP coding region cloned and sequenced. Point mutations were observedin all 3 patients (Korthauer et al. 1993), leading to protein truncation (patient 8,

sCD40

control

patient S

patient 9

patient 1

log fluorescence

Figure 6. Activated T cells from HIGMl patients fail to hind CD40. PBL were activatedwith PMA + A23187 for 5 h and stained with a biotinylated sCD40 chimeric molecule(Hermann et al, 1993). After washing, cells were reacted with extravidin-FITC and analyzedby flow cytometry. To control for specificity of the signal, PBL were stained in parallelwith biotinylated IL4R-Ig (Hermann et al. 1993). Activated PBL were also stained withTRAP-specific antiserum, washed, and reacted with a FITC-conjugated goat anti-rabbitf(ab')2 reagent.

48 KROCZEK ET AL.

4kb 2kb 2kb

1822

patients16

Figure 7. Location of protein mutations in HIGMl. Shown is the structure of TRAP/CD40L, including the regions coding for the intraeytoplasmic (IC), transmembrane (TM),and extracellular (EC) domains of the protein. The intron/exon boundaries and the size ofthe introns, as present in the genomic configuration, are also indicated.

Fig. 7, and Table I), a drastic amino acid exchange (patient 9), and to anintroduction of a positive charge into the transmembrane region of TRAP/CD40L (patient 1), which totally abrogated cell surface expression of TRAP/CD40L (compare Fig. 6). Mutations in the TRAP/CD40L gene in patients withHIGMl were also simultaneously analyzed by several other groups (Allen et al.1993a, Aruffo et al. 1993, DiSanto et al. 1993a, Fuleihan et al. 1993). Fig. 7 and

TABLE IMutations in the TRAP/CD40L gene causing HIGMl

Patient

12345

6789

101112131415161718

Met,6deletion 97-!

mutation

-* Arg115

deletion 116-136AlaiBSer|,BGIU|29deletion 137deletion 137Trp,4oTrpucTrpi4oLeU|;5Tyr,,,itisert 195Seri57ThrjiiGlu^,Gly;:,Alaj35

-^ Glu-» Arg^ Gly-^ frameshift-^ frameshift-f stop^ Gly-* stop-> Pro^ His-» frameshift- T y r-* Asp-» stop^ Val- Pro

substitionsplice site mutationsplice site mutationsubstitutionsubstitution

splice site mutationsplice site mutationsubstitutionsubstitutionsubstitutionsubstitutionsubstitutioninsertionsubstitutionsubstitutionsubstitutionsubstitutionsubstitution

Reference

Korthauer et al. 1993Ramesh et al. 1993DiSanto et al. 1993aDiSanto et al. 1993aArufTo et al. 1993

DiSanto et al. 1993aDiSanto et at. 1993aKorthauer et ai. 1993Korthauer et al. 1993Villa et al., unpublishedAllen et al. 1993aGraf et al., unpublishedVilla et al., unpublishedRamesh et al. 1993Allen et al. 1993aVilla et al. 1993Allen et al. 1993aAruffo et al. 1993


Table I summarize the location and type of mutations known to cause HIGMl.This summary also contains unpublished data from our laboratories (patients 10,12, and 13).

With the exception of the Met^^-Arg substitution that affects the transmem-brane domain of the protein, all other mutations are clustered in the extracellularpart of the molecule (and mostly within the TNF homology domain, correspond-ing to amino acid residues 123-261), and result in a failure of TRAP/CD40L tobind CD40. When analyzed at the cDNA level, different mutations have beenreported, including single base substitutions and deletions (Korthauer et al. 1993,DiSanto et al. 1993a, Aruffo et al. 1993, Allen et al. 1993a, Fuleihan et al. 1993,Ramesh et al. 1993, Villa et al. 1993). More recently, the first example of HIGM Idue to insertion in the CD40L cDNA (a CT dinucleotide insertion at aa residue195, patient 13 in Fig. 7) has been identified (Villa et al., unpublished). In acollaborative study with G. de Saint Basile, we have recently clarified the genomicorganization of the CD40L gene (discussed later); this has allowed us to establishthat deletions in patients 3, 6, and 7 are caused by splice-site mutations (Villa etal. 1994).

Interestingly, we have identified 3 unrelated patients (corresponding to individ-uals 8, 9, and 10 in Fig. 7) who carry different mutations in the same codon(Trp,4(,), resulting in a stop signal in 2 cases (patients 8 and 10, Korthauer et al.1993, Villa et al., unpublished) and in a non-conservative amino acid substitution(Trp-Gly) in the other (patient 9, Korthauer et al. 1993). This may suggest thatTrpi4Q is a hot spot for mutations in HIGMl.

Phenotype-genotype correlation in HIGM}

The identification of 3 patients carrying an identical mutation in the TRAP/CD40L gene (stop codon at aa residue 140; patients 8 and 10 in Fig. 7 and anaffected cousin UT of patient 8, Villa et al.. unpublished) that leads to theexpression of a truncated protein on the cell surface (compare Fig. 6), hasprompted us to attempt a genotype-phenotype correlation analysis. Interestingly,elevated IgM serum levels were present only in patient 10, while the other 2patients had low to normal IgM concentrations. Furthermore, while patientsUT and 10 suffered from profound neutropenia, subject 8 has anemia andthrombocytopenia. Thus, the only consistent phenotypic feature shared by theseaffected individuals are the low IgG sertim levels associated with increased suscep-tibility to infections.

The observation that mutations of the CD40L gene may result in panhypogam-maglobulinemia, with elevated, normal, or even low IgM serum concentrations,has important immunological and clinical implications. Based on the fact thatpatients with HIGMl often present with elevated IgM serum levels, it is apparentthat IgM synthesis and secretion can proceed in the absence of functional CD40L-

50 KROCZEK ET AL.

CD40 interaction. In HIGMl patients with elevated IgM, levels of IgM oftenfluctuate, and increases often coincide with infections. As a whole, these dataindicate that elevated IgM serum levels are not an obligatory consequence of theCD40L gene mutation, but most probably reflect the response to recurrentantigenic stimulation.

The variability of serum IgM profile in HIGMl patients has important impli-cations also on the correct identification of affected individuals. According tothe latest WHO classification of primary immunodeficiencies (WHO ScientificGroup, 1992), HIGMl patients have elevated serum IgM (and often IgD), andlow IgG and igA levels. The observation from our and other groups that hypo-gammaglobulinemic male patients with mutations of the CD40L gene may havelow to normal serum IgM levels, suggests that the identification criteria proposedby WHO may be inappropriate, leading to an underestimation of the diseaseprevalence. Indeed, a subgroup of patients with CD40L gene mutations wouldbe currently classified as affected with common variable immunodeficiency (CVI).Based on the identification of the molecular pathogenesis of HIGMl and on thefirst genotype-phenotype correlation analyses, we propose that the term "X-linked immunodeficiency with hyper-IgM" should be changed into "X-linkedimmunodeficiency due to CD40 ligand defect". From the practical point of view,any male patients who present at an early age (within the first year of life) withlow serum IgG and IgA (independently of IgM levels) and normal proportion ofcirculating B cells, should be evaluated for functional CD40 ligand expression.

EARLY DIAGNOSIS AND CARRIER DETECTION IN HIGMl

Ontogeny of CD40L gene expression

Recognition of HIGMl as a severe disorder implies that efforts should be madeto develop tests for prenatal diagnosis and for early, pre-symptomatic identifi-cation of affected males. The development of chimeric sCD40 constructs offersthe theoretical possibility to perform early, and even prenatal diagnosis of HIGMlthrough immunophenotyping. However, we have recently learned that this possi-bility is hampered by the physiological immaturity of the immune system in thenewborn.

We investigated the capability of activated cord blood T cells to express surfaceCD40L molecules upon in vitro activation. As shown in Fig. 8, even with potentstimuli (PMA -h ionomycin), no significant expression of CD40L could beachieved on cord blood mononuclear cells (CBMC) after 16 hours of stimulation,the optimal time point for adult lymphocytes. Occasionally, CD40L was moder-ately expressed by CBMC after 48-72 hours of stimulation (Brugnoni et al.,submitted). We have also shown that inefficient expression of CD40L by CBMCreflects functional immaturity of T cells due to lack of in vivo antigenic stimula-tion, since in vitro priming with PHA -|- IL-2, followed by re-stimulation, was


16h 48h 72h

i-a<

Q' 10'

o

Figure 8. Poor expression of CD40L by activated cord blood mononuclear cells. Adultperipheral blood mononuclear cells (aPBMC) and cord blood mononuclear cells (CBMC)were activated with PMA + ionomycin for various times, and stained for TRAP/CD40Lexpression with anti-TRAP serum.

able to induce normal expression of CD40L on activated CBMC (Brugnoni etal., submitted). Preliminary experiments have confirmed that, in vivo, antigenicstimulation is able to change the genetic program of T lymphocytes after birth,since CD40L can be expressed by activated T cells from 2-month-old infants(data not shown). Taken together, our data indicate that the failure to expressCD40L on T cells is a major factor contributing to the well known inability ofthe neonate to produce significant levels of immunoglobulins other than IgM(Hayward & Lawton 1977). At the same time these observations show the limi-tations for prenatal diagnosis of HIGMl by immunophenotyping. Although onecould attempt to use the priming-and-restimulation protocol to analyze CD40Lexpression, this is somehow cumbersome and difficult to standardize. On theother hand, molecular genetics offers simple and reliable methods for prenataldiagnosis and carrier identification (see below).

Structure of the CD40L gene

We have recently elucidated the genomic organization of the human CD40L gene(Villa et al. 1994). Hybridization of the TRAP.g5 cDNA clone (that contains thewhole coding region of the human CD40L cDNA (Graf et al. 1992)) to EcoRIdigests of DNA from normal individuals revealed two bands of 2.5 and 10 kb.Therefore, the whole transcriptiona! unit of the CD40L gene is contained in arather short stretch of genomic DNA.

52 KROCZEK ET AL.

Analysis of the intron/exon organization was accomplished by PCR amplifi-cation, using a series of oligonucleotide primers. In addition, we hypothesizedthat the 63 bp (nucleotides 368-430) deletion in patient 3 of Fig. 7 (DiSanto etal. 1993a) and the 58 bp (nucleotides 310-367) deletion in patient 2 (Ramesh etal. 1993) could reflect exon skipping, and designed primers accordingly; thisassumption proved correct. We succeeded in amplifying the whole human CD40Lgene, and the intron/exon boundaries were defined and sequenced. The overallorganization of the gene is shown in Fig. 9. The gene is composed of five exonsand four intervening introns, for a total length that is very much in keepingwith the length deduced by Southern-blot analysis, introns interrupt the cDNAsequence at codons 52, 96,115, and 136. The first exon contains the intracytoplas-mic tail, the transmembrane region, and the ftrst 6 amino acid residues of theextracellular domain, while the rest of the extracellular domain is contained inexons 2 to 5. Because expression of the CD40L gene has been reported to berestricted to activated T lymphocytes, exact knowledge of the gene organizationis instrumental for molecular analysis of suspected HIGMl patients in whichtissues other than lymphocytes are available, and may prove useful for prenataldiagnosis in families in which the mutation has been previously determined (seebelow).

GENOMIC ORGANIZATION OF THE TRAP / OD40L GENE

5'UTR 3'UTR

Eigwe 9. Organization of genomic DNA coding for the transcriptional unit of ihe TRAP/CD40L gene. Top: the exons (solid bars), the intervening introns (with the relative sizes),and the 5' and 3' untranslated regions (empty bars) are shown. Bottom: contribution ofthe exons to the protein organization. Intracytoplasmic (IC), transmembrane (TM), andextracellular (EC) domains, are shown.


Carrier detection in HIGMl

For other X-linked immunodeficiencies, molecular genetics offers the possibilityto perform carrier detection through two distinct methods: linkage analysis (usingpolymorphic DNA probes closely associated to disease loci) and evaluation of thepattern of X-chromosome inactivation (demonstrating preferential inactivation ofthe mutated X-chromosome in selected cell lineages from carrier females (Fearonet al. 1987, Conley & Puck 1988, Lau & Levinsky 1988, de Saint Basiie et al.1989, Guioli et al. 1989)). These possibilities have been also considered forHIGMl. Both B and T lymphocytes from some obligate carriers of HIGMldisplay a random pattern of X-chromosome inactivation (Hendriks et al. 1990,Goodship et al. 1991); thus, these females have two populations of T cells, onlyone of which can express functional CD40L molecules (DiSanto et al. 1993a).However, while this is true for some obligate carriers of HIGMl (and thus wouldpennit carrrier identification), results of staining with sCD40 proved variable forother obligate carriers of HIGMl: in some cases, expression was normal, whilein others it was very low (Caliard et al. 1993). On the other hand, carrier detectionin families with clear X-linked inheritance may be accomplished by segregationanalysis using either the polymorphic CA microsatellite repeat at the 3'-untrans-lated region of the CD40L gene (DiSanto et al. 1993a, Allen et al. 1993b), or thevariable number of tandem repeats (AGAT tetranucleotide repeat) of the HPRTgene, closely associated to the CD40L gene (Padayachee et al. 1992, Padayacheeet al. 1993). These highly polymorphic markers may also be used for prenataldiagnosis of HIGMl on chorionic villi DNA (DiSanto et al. 1993b).

In addition, knowledge of the CD40L gene structure may allow prenataldiagnosis by direct mutation analysis in families in which the CD40L genemutation has been previously defined. In these cases, PCR amplification ofchorionic villi genomic DNA, coupled to sequencing, may reveal or excludeHIGMl in a fetus. We have employed this approach for the first time for prenataldiagnosis in a pregnant carrier of HiGMl, belonging to a family in which aTrp|4(,-amber mutation in the CD40L cDNA had been previously identified. Asshown in Fig. 10, prenatal diagnosis was performed at 10 weeks of gestation,and a non-affected fetus was identified.

UNRESOLVED ISSUES AND PERSPECTIVES

Although molecular characterization of mutations in the CD40L gene explainsthe inability of HIGMl patients to switch from IgM to IgG production both invitro and in vivo, a number of issues are unresolved. For example, the mechanismsleading to thrombocytopenia, hemolytic anemia, and neutropenia in HIGMlpatients remain unknown and cannot be solely explained by autoimmune mechan-isms. Another intriguing clinical aspect of HIGMl is the increased susceptibility

54 KROCZEK ET AL.

A B

ACGT ACGT ACGT

Figure 10. Prenatal diagnosis of HIGMl. A female, belonging to the family of TG (patient8 in Fig. 7) was identified as a heterozygous (lanes A and B) carrier of a C-T ambermutation at codon 140. The male fetus was diagnosed as healthy by demonstration of anormal genomic sequence (lane C) on chorionic villi DNA. One of three sequenced clonesfrom the fetal DNA is shown.

to Pneumocystis carinii and Cryptosporidium infections, which is usually onlyobserved in the context of T-cell immunodeficiencies. However, in vitro testsfailed to detect any abnormality of T-cell function in HIGM1. It has been recentlydemonstrated that CD40L may also be co-mitogenic for T cells (Armitage et al.1993). While the exact molecular mechanisms of such stimulatory activity arestill unclear, it is possible that defective TRAP/CD40L expression in HIGMlpatients may in this way affect T-cell function.

Finally, an important issue to be considered is somatic cell gene therapy forHIGMl., since the in vivo response to immunoglobulin replacement therapy hasproven less effective than for XLA patients (Notarangelo et al. 1992), and bonemarrow transplantation is a real option only if HLA-identicat siblings are avail-able. However, somatic cell therapy poses a number of questions. First, optimalcellular targets have to be considered, given the fact that CD40L is only expressedon CD4^ T cells, basophils and mast cells. Furthermore, the regulation of thetransfered gene is a very important issue, since under physiological conditions


TRAP/CD40L is only transiently expressed - overexpression of the gene wouldprobably lead to hypergamtnaglobulinemia and to autoimmune phenomena.Therefore, characterization of the TRAP/CD40L gene protnoter atid the designof gene transfer systems utilizing the autologous promoter will probably berequired for successful gene therapy strategies.

SUMMARY

X-linked immunodeficiency with hyper-IgM (HIGMl) is a rare disorder, char-acterized by recurrent infections associated with very low or absent IgG and IgA,and normal to increased IgM serum levels. The disease has been earlier mappedto the q26-27 region of the X-ehromosome. We have identified a novel moleculeexpressed on the surface of activated T cells, which was designated TRAP (Tumornecrosis factor Related Activation Protein), and could demonstrate that TRAPis a ligand for the CD40 receptor expressed on B cells. Our mapping of the TRAPgene to the Xq26.3-27.l region suggested a causal relationship to HIGMl.Further work revealed that various mutations of the TRAP/CD40 ligand(CD40L) gene may lead to a defective expression of the TRAP/CD40L moleculeon the T-cell surface in HIGMl patients. A combination of structural andfunctional analyses finally demonstrated that the failure of TRAP/CD40L on Tcells to interact with CD40 on B cells is responsible for the inefficient T-cell helpfor B cells observed in HIGMl. The observations made in HIGMl allowed usto conclude that TRAP/CD40L is not required for IgM synthesis. In contrast,functional expression of TRAP is a prerequisite for effective immunogiubulinisotype switching and subsequent production of IgG, IgA and IgE by B cells invivo. The interaction of TRAP/CD40L with CD40 thus provides a very criticallink between the cellular and the humoral part of the immune system. Theknowledge of TRAP/CD40L cDNA sequence, the availability of various reagentsfor the testing of expression and function of TRAP/CD40L, and our recentelucidation of the exon-intron structure of the TRAP/CD40L gene now provideall necessary tools for early diagnosis of affected patients and the detection offemale carriers of HIGMl. The available information will also provide a basisfor future attempts at gene therapy in this disease.

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