www.elsevier.com/locate/yclim

Clinical Immunology 1

ICOS deficiency in patients with common variable immunodeficiency

Ulrich Salzera, Andrea Maul-Pavicica, Charlotte Cunningham-Rundlesb, Simon Urschelc,

Bernd H. Belohradskyc, Jiri Litzmand, Are Holme, Jose Luis Francof, Alessandro Plebanig,

Lennart Hammarstromh, Andrea Skrabli, Wolfgang Schwingeri, Bodo Grimbachera,*

aDivision of Rheumatology and Clinical Immunology, University of Freiburg, 79106 Freiburg, GermanybPediatrics and Immunobiology, Mount Sinai School of Medicine, New York, 10029, United States

cDivision of Infectious Diseases and Immunology, University Childrens Hospital, University of Munich, GermanydDepartment of Clinical Immunology and Allergology, St. Anne University Hospital, Masaryk University, Brno, Czech Republic

eResearch Institute for Internal Medicine, National Hospital, Oslo, NorwayfGrupo de Immunodeficiencias Primarias, Departamento de Microbiologıa y Parasitologıa, Facultad de Medicina,

Universidad de Antioquia, Medellin, ColombiagClinica Pediatrica, Universita di Brescia and Istituto Medicina Molecolare bAngelo NocivelliQ, Spedali Civili, Brescia, Italy

hDivision of Clinical Immunology, IMPI, Karolinska Institute at Huddinge Hospital, Stockholm, SwedeniDivision of Pediatric Hemato-Oncology, Department of Pediatrics, University of Graz, Austria

Received 18 April 2004; accepted 6 July 2004

Available online 17 September 2004

Abstract

Common variable immunodeficiency (CVID) is the most frequent clinically significant primary antibody deficiency in man, predisposing

to recurrent bacterial infections. Recently, we showed that the homozygous loss of the inducible costimulator (ICOS) on activated T cells may

result in an adult onset form of CVID with autosomal recessive inheritance (AR-CVID).

We screened 181 sporadic CVID patients and 13 CVID patients from nine families with AR-CVID for mutations in ICOS by genomic

DNA sequencing. In the AR-CVID families, the genomic integrity of the ligand for ICOS (ICOS-L) was also evaluated.

In two of the nine AR-CVID families, we identified five individuals with ICOS deficiency, carrying the identical large genomic deletion

of ICOS as previously described. In the remaining seven AR-CVID families, we subsequently sequenced the coding region of the ICOS

ligand but found no mutations.

The incidence of ICOS deficiency among patients with CVID is less than 5%. Worldwide, there are now a total of nine patients diagnosed

with ICOS deficiency most likely due to a common founder. ICOS-L deficiency could not be identified in families with AR-CVID.

D 2004 Elsevier Inc. All rights reserved.

Keywords: CVID; ICOS; Immunodeficiency

Introduction

The diagnosis of common variable immunodeficiency

(CVID) is based on markedly reduced serum levels for IgG

1521-6616/$ - see front matter D 2004 Elsevier Inc. All rights reserved.

doi:10.1016/j.clim.2004.07.002

* Corresponding author. Division of Rheumatology and Clinical

Immunology, University Hospital Freiburg, University of Freiburg,

Hugstetterstr. 55, 79106 Freiburg, Germany. Fax: +49 761 270 3531.

E-mail address: grimbacher@medizin.ukl.uni-freiburg.de

(B. Grimbacher).

and IgA or IgM, an impaired ability to specific antibody

production after vaccination or exposure, and exclusion of

secondary causes for antibody deficiency [1]. Although it is a

rare disease with an estimated prevalence of 1 in 25,000 in the

Western population, it is the second most frequent primary

immunodeficiency (PID) after selective IgA deficiency

(IgAD) and the most frequent PID requiring medical

attention. Most cases of CVID are sporadic, however, about

10% are familial with a predominance of autosomal dominant

over autosomal recessive inheritance [2,3]. CVID and IgAD

13 (2004) 234–240

Table 2

Clinical features of CVID patients (n = 194)

Sex Female 106 (55%)

Male 88 (45%)

Age b10 years 11 (6%)

10–30 years 41 (21%)

31–50 years 96 (49%)

N50 years 39 (20%)

Unknown 7 (4%)

Age of diagnosis

of CVID

b10 years 34 (17%)

10–30 years 85 (44%)

31–50 years 52 (27%)

N50 years 11 (6%)

Unknown 12 (6%)

Clinical presentation Recurrent sinusitis 123 (63%)

Recurrent bronchitis 130 (67%)

Recurrent pneumonia 98 (50%)

Bronchiectasis 52 (27%)

Recurrent gastrointestinal

infections

36 (18%)

Autoimmune phenomenaa 42 (21%)

splenomegaly 74 (38%)

Granuloma formation 14 (7%)

Malignancyb 10 (5%)

a Including idiopathic thrombocytopenic purpura, autoimmune hemolytic

anemia, pernicious anemia, rheumatoid arthritis, psoriasis, vitiligo, diabetes

mellitus, and systemic lupus erythematosus.b Including thymoma, Hodgkin lymphoma, non-Hodgkin lymphoma,

glioma, acute lymphatic leucemia, and cutaneous T cell lymphoma.

U. Salzer et al. / Clinical Immunology 113 (2004) 234–240 235

may occur in the same family, which suggests a possible

common genetic basis for these two disease entities [4].

The age of onset of CVID has two major peaks: the first

between 5 and 10 years of age, the second in early

adulthood, ages 20–30. The phenotype of CVID is

characterized by the sequelae of hypogammaglobulinemia

with recurrent upper respiratory infections by encapsulated

bacteria. In addition to these infections, the phenotype of

CVID varies and may be accompanied by autoimmune

phenomena, splenomegaly, granulomas, and the develop-

ment of certain types of cancer [1,5].

The inducible costimulator (ICOS) belongs to the family

of costimulatory T cell molecules such as CD28 and CTLA-

4 [6]. All three genes were mapped to chromosome 2q33.

ICOS spans over 25 kb and is divided into five exons and

four introns [7,8]. Fifty SNPs have been reported for ICOS:

16 lie within the promoter region, 13 are intronic, 1 is within

exon 5, 1 SNP lies within an exon–intron boundary, and the

remaining 19 SNPs are in the 3V UTR [8–10].

ICOS is only expressed on activated T cells and

coinduces the secretion of IL-4, IL-5, IL-6, GM-CSF,

TNF-a, and IFN-g but is pivotal for the superinduction of

IL-10 [11]. Highest expression of ICOS is found within the

T cell zones of secondary lymphoid organs and in the

apical light zones of germinal centers [6,11]. This

expression pattern and the cytokines induced by ICOS

point to an important role of ICOS:ICOS-L interaction in

mediating T–B cell cooperation and promoting the

terminal differentiation of B cells into memory cells and

plasma cells. This is further supported by findings in the

ICOS and ICOS-L knockout mice, both showing a defect

in germinal center formation and impaired humoral

immune responses, especially after secondary immuniza-

tions [12–14].

ICOS-L is the unique ligand for ICOS and is

constitutively expressed on both lymphoid and nonlym-

phoid tissues. ICOS-L can be up-regulated by inflamma-

tory stimuli such as TNF-a, IFN-g, and lipopolysaccharide

(LPS) [15]. ICOS-L shares 20% homology with CD80 and

Table 1

Amplification and sequencing primers for ICOS and ICOS-L

Forward primer (5VY3V)

ICOS Exon 1 TGC CTT TCA TAT AGC CAT TG

Exon 2 GCA ACA GAG ATG ACT TTA TGC ATT GA

Exon 3 AAG CCC TTT CTA TAA ACC ACA TT

Exon 4 TGG GGGATA TTC TTT TTG GTC

Exon 5 GTG TAT GAA AGG CAA TGG AGA GG

ICOS-L Exon 1 GCG GGA GCG CAG TTA GAG C

Exon 2 CAC GGG CAC GCC TGA TGT TCT

Exon 3 GGA CCT CAC CAT GAA ATG TC

Exon 4 CCA TGT CGG GGC ACA ATG

Exon 5 CCT GGC TGT GGT TGG GGT TAT C

Exon 6 GGT GTT TAG GGG GCT GCT GAG AGC

Exon 7 GGC CGA CCG CAG AAA CGC ACT T

CD86 (the ligands of CD28 and CTLA4). The gene

encoding ICOS-L is located on chromosome 21q22.3 and

consists of seven exons [16]. ICOS-L is expressed as two

splice variants designated as hGL50 and B7H2, with

hGL50 showing an expression pattern restricted to

lymphoid organs [17,18]. ICOS-L expression on naRve B

cells is down regulated via B cell receptor and ICOS

engagement or IL-4 signaling, but it can be sustained by

stimulation via CD40 [19].

We recently identified homozygous deletions in ICOS in

4 of 32 patients with CVID [20]. Therefore, the aim of this

study was to determine the incidence of ICOS deficiency

Reverse primer (5VY3V) Annealing

temperature (8C)

AGT CAT TTT GCC TTT CAT CTT T 55

CTG CAT CTA AGT GAA CTC CAA 58

TGT CTC CCT GTT GGT CAA A 55

TCT AGA ATT AGG CCT TGG AGA TGT T 58

AGA ATG CTG GCC CAT TAA AGA TGA T 62

CTC ACA GGT TCA CCC AGG GG 58

GTG CTG CGG TGC TTC GTC CTT ATT 63

GCT AAC TAA CTC AAG AGG CAA 63

GCG GTG GTT CTC ACT GTG 63

CTT GGC AGT GTC AGG AAT G 58

ACA GGC CGG CCG TGT TTT TAG C 58

GGA ACA GCC GAG GGA CAT TG 63

Table 3

Genotype frequencies of SNPs at the ICOS-locus

SNPa Genotype frequency, n/(%)

p1203 CVID T/T 5 (3) C/T 28 (17) C/C 128 (80) n = 161

HDb T/T 0 (0) C/T 15 (14) C/C 95 (86) n = 110

T-repeat CVID T[13] 130 (91) T[14] 13 (9) T[15] 0 (0) n = 145

HDb T[13] 99 (93) T[14] 5 (5) T[15] 2 (2) n = 106

p3990 CVID T/T 32 (20) G/T 63 (41) G/G 62 (39) n = 157

HDb T/T 18 (16) G/T 55 (50) G/G 37 (34) n = 110

P4031 CVID C/C 15 (10) A/C 34 (22) A/A 106 (68) n = 155

HDb C/C 3 (3) A/C 35 (32) A/A 72 (65) n = 110

a Single nucleotide polymorphism.b HD = healthy donor, refers to Ref. [10].

U. Salzer et al. / Clinical Immunology 113 (2004) 234–240236

(OMIM #607594) among patients with CVID. In addition,

we screened autosomal recessive CVID (AR-CVID) families

for disease causing mutations in the ICOS-L gene.

Patients and methods

Patients

Patients were diagnosed with CVID according to the

ESID criteria (www.esid.org). One hundred and twenty-

three patients were recruited from central Europe, 16

patients were from Scandinavia, 48 patients were from the

United States, and 7 patients were from Colombia.

Informed written consent was obtained from each indivi-

dual before participation under the internal ethics review

board-approved clinical study protocol (#239/99). Ten

milliliters of heparinized blood was obtained from each

participant of the study for isolation of genomic DNA

using DNA Blood isolation kit reagents (Puregene DNA

isolation kit; Gentra Systems, Minneapolis, USA).

Fig. 1. Pedigrees of newly identified patients with ICOS deficiency; circles, female; squares, male; slashed rhombuses, deceased; filled symbols, affected; open

symbols, unaffected. Individuals II.1 and II.2 died due to complications at birth as premature neonates. No autopsies were performed. Long-range PCR analysis

of ICOS. Genomic DNA from members of family C and D was amplified with primers covering ICOS exons 2 and 3. From DNA of a healthy donor, a 3-kb

PCR fragment could be amplified, in the heterozygote parents (family D) both a 3-kb and a shortened 1.2-kb fragment was present, and in all affected

individuals only a 1.2-kb fragment could be detected, indicating a genomic deletion.

Sequencing of ICOS and ICOS-L

The coding sequence of the five exons and the adjacent

intron–exon boundaries of ICOS was amplified from

genomic DNA using primer pairs and conditions as

indicated in Table 1. Long-range PCR from genomic

DNA was performed using Takara LA Taq (Cambrex

Bioscience Rockland Inc., Rockland, USA) with the

following primers: LRfw (5VY3V) TGG GGC TTT ATC

TTT ATT ATC AGG, LRrv (5VY3V) TGG GCG CTATCT

CAT TCT CT. The coding sequence of seven exons and

adjacent intron–exon boundaries of ICOS-L was amplified

from genomic DNA using the primer pairs and conditions as

indicated in Table 1.

PCR products were sequenced with the PCR amplifica-

tion primers. After gel electrophoresis on an ABI Prismk377 DNA Sequencer (PE Applied Biosystems, Foster City,

USA), the data were analyzed with aid of the DNA

Sequencing Analysis software, version 3.4 (PE Applied

Biosystems) and Sequenchertk version 3.4.1 (Gene Codes

Corporation, Ann Arbor, USA).

Table 4

T and B cell numbers and IgG, IgA, and IgM levels in ICOS deficiency

Family Individual Age of onset CD3+ T cells (cells/Al) CD19+ B cells (cells/Al) IgG (g/l) IgA (g/l) IgM (g/l)

Normal range

Age N16 years 700–2100 100–500 7–16 0.7–4.0 0.4–2.3

Age 3–5 years 900–4500 200–2100 5–13 0.4–1.8 0.4–1.8

C II.3 20 629 127 1.01 b0.06 0.38

II.4 18 816 28 0.57 b0.06 1.8

D II.1 15 2683 184 2.55 b0.25 0.26

II.3 4 2727 686 2.13 0.58 0.57

II.4 3 2923 882 0.54 0.2 0.43

Pathologic values are printed bold.

U. Salzer et al. / Clinical Immunology 113 (2004) 234–240 237

Genotyping of the ICOS locus

Patients showing a genomic deletion of ICOS were

genotyped at the ICOS locus using the polymorphic markers

D2S115 and D2S2289. Genotyping PCR was performed at

the recommended conditions. PCR results were analyzed on

an ABI 377 sequencer (PE Applied Biosystems) with the

Collection and Analysis software packages (PE Applied

Biosystems). Allele sizes were determined with the help of

GENOTYPER (PE Applied Biosystems).

Results

A cohort of 194 CVID patients was collected for

analysis of mutations in ICOS. All patients had a typical

clinical history of recurrent respiratory infections and were

diagnosed with CVID according to the ESID criteria

(www.esid.org). A summary of their clinical presentation is

given in Table 2. An early onset of the disease (b10 years)

was observed in 34 patients. One hundred and eighty-one

patients were sporadic cases with a negative family history

for CVID, and 13 patients originated from nine families

with AR-CVID. The parents were consanguineous in five

of these nine families.

In the 181 sporadic CVID patients, the sequence of the

five exons and the adjacent exon–intron boundaries of

ICOS revealed no mutations. Since the expression of ICOS

may be influenced by genetic polymorphisms outside the

coding region, we evaluated the single nucleotide poly-

morphisms (SNP) p1203, p2412, p3990, p4031 (names

refer to position in GenBank accession number AF488347)

and one T repeat of the ICOS gene (NCBI SNP id:

rs5837889–90). However, the frequency estimation of these

five SNPs showed no significant differences in CVID

patients when compared to a healthy control population

[10] (see Table 3).

By contrast, exons 2 and 3 could not be amplified from

the DNA of five patients from two of the nine families with

familial CVID. These findings suggested a homozygous

deletion. Therefore, we performed a long-range PCR that

spans 3 kb of genomic DNA amplifying both exons 2 and 3

of ICOS (Fig. 1). DNA from healthy controls yielded a 3-kb

DNA fragment. In contrast, all affected individuals in the

two families were homozygous for a shortened DNA

fragment of 1.2 kb. The PCR result in parent I.1 and I.2

of family D displays a clear heterozygote state with both

DNA fragments present (Fig. 1). The 1.2-kb DNA fragment

in the five affected individuals was subjected to DNA

sequencing (Fig. 2A). In all five newly diagnosed patients,

we identified the same 1815 bp deletion as has been

reported in families A and B of the original description of

ICOS deficiency [20] (Fig. 2B, shaded sequence). The

homozygous loss of 1815 bp genomic DNA results in an

mRNA frameshift with a premature stop codon leading to a

truncated ICOS protein, which putatively consists of the

signal peptide and nine dnonsenseT amino acids. The

absence of ICOS expression on activated T cells was

verified for individuals II.3 and II.4 of family C by FACS

analysis (data not shown). As all patients with ICOS

deficiency carry the same large deletion, a common founder

for all four families was anticipated. To prove this, we

examined the polymorphic marker D2S2289 that is adjacent

to the ICOS locus and two SNPs (NCBI SNP id: rs4675378

and rs4270326), flanking the deleted region. The five newly

diagnosed patients as well as the four originally published

cases [20] were homozygous for the same 203 bp allele at

D2S2289 and had identical SNPs (data not shown). Thus, a

common founder for the mutations in all four families with

ICOS deficiency is very likely.

The eight patients from the remaining seven AR-CVID

families had a normal ICOS sequence. Since we evaluated

ICOS in 194 CVID patients, we can now calculate the

incidence of ICOS deficiency in this cohort to four out of

190 unrelated families or nine out of 194 CVID patients

(approximately 5%).

Because the ICOS-L knockout mice display the very

same phenotype as the ICOS knockout mice [14], ICOS-L

was a plausible disease causing candidate gene in the

remaining seven AR-CVID families. Therefore, ICOS-L

was sequenced in these patients. In two patients from two

consanguineous families, a previously nondescribed poly-

morphism was detected in exon 3 of ICOS-L. Individual

AR6 was heterozygous and AR4 was homozygous for a G to

A transition at position 490 of the ICOS-L mRNA ref.

#NM_015259 (see Fig. 2C). The alteration results in an

amino acid change from Valine to Isoleucine at position 128

(Val128Ile) in a nonconserved part of the protein. However,

Fig. 2. (A) Sequencing results of long-range PCR products of affected individuals in families C and D and a healthy donor (HD). The PCR fragments shown in

Fig. 1 were subjected to DNA sequencing. The site of the deletion is indicated by an arrow. The starting and ending points of the deletion are located within

highly homologous regions of DNA, so that the wt and mutated sequences differ only in two single bases (indicated by asterisks) in the segment shown. (B

Deletion of 1815 bp in ICOS. Sequencing of the shortened PCR product of 1.2 kb revealed a 1815 bp deletion in ICOS from all five newly identified ICOS

deficient patients, including parts of intron 1, exon 2, intron 2, exon 3, and parts of intron 3. The shaded area designates the deleted part of ICOS. (C) Single

nucleotide polymorphism in exon 3 of ICOS-L. Exon 3 of ICOS-L was amplified by PCR and subjected to DNA sequencing. A previously undescribed

polymorphism resulting in a heterozygous (AR6) or homozygous (AR4) G to A transition is shown.

U. Salzer et al. / Clinical Immunology 113 (2004) 234–240238

)

-

U. Salzer et al. / Clinical Immunology 113 (2004) 234–240 239

we could detect this polymorphism also in control individ-

uals at a similar frequency for both heterozygotes and

homozygotes.

Therefore, we concluded that neither ICOS nor ICOS-L

is the disease causing gene in those remaining seven CVID

families.

Discussion

The CVID phenotype appears to origin from genetic

conditions resulting in the cardinal symptoms of primary

antibody deficiency. In the past, patients with other mono-

genic primary immunodeficiencies such as X-linked Agam-

maglobulinemia (XLA), X-linked lymphoproliferative

Syndrome (XLP), or hyper-IgM-syndrome (HIgM) have

been misdiagnosed as CVID since these disorders can

mimic the phenotype of CVID [21–23].

ICOS deficiency represents the first single gene defect

identified to solely cause the CVID phenotype [20]. Thus,

we screened a cohort of 194 CVID patients for mutations of

ICOS to determine how frequent a defect in ICOS causes

CVID.

The originally described four ICOS deficiency patients

were homozygous for a large genomic deletion in ICOS

[20]. The patients came from two CVID families with an

autosomal recessive trait. Heterozygote parents and siblings

had no clinical signs of immunodeficiency. The immuno-

logic laboratory findings in the heterozygote carriers were

also normal, despite a slightly weaker surface staining for

ICOS on their activated T cells [20]. We therefore concluded

that both alleles of ICOS had to be affected by mutations to

cause the phenotype of CVID. In a previous study [24],

ICOS was evaluated in 47 unrelated CVID patients by

SSCP analysis and no mutations were found. However,

SSCP has a sensitivity of about 80% and thus possible

mutations in this cohort may have been missed.

In a group of 181 sporadic CVID patients, we could not

detect any mutations in the coding regions of ICOS. The

frequency of a known polymorphism (p4031 AYC) located

in the located in the 3VUTR two bp after the stop codon was

determined to be comparable to that in control populations

[10]. Lee et al. [25] found that there was no difference in the

binding affinity to B7RP-1 between the different allelic

variants of this polymorphism. However, we cannot exclude

alterations or deficiency in ICOS expression due to mutations

in the promoter region of ICOS or putative regulatory intronic

regions in the CVID patients we analyzed since protein

expression of ICOS on activated T cells could only be

assessed by flow cytometry in 28 patients.

Among nine CVID families most likely affected by an

autosomal recessive trait, we identified two new unrelated

families with five individuals carrying the identical large

genomic deletion in ICOS as previously published [20].

Further genetic analysis of all nine ICOS deficiency patients

revealed that they share identical homozygous haplotypes in

the ICOS locus, indicating that the mutation was passed on

to them deriving from a common founder. This is further

supported by the fact that three of the four families with

ICOS deficiency originate from the same village, which is

linked to the origin of the fourth family by the long river of

Danube. The Danube was a frequently used route of

emigration in the 18th century. The observation that the

deleted region is flanked by highly homologous stretches of

DNA including a 13-bp identical segment suggests that the

deletion resulted from an aberrant recombination event.

Family C showed a childhood onset of the disease, which

was not observed in the other three families. The flow

cytometry analysis of T cells and their proliferative response

to mitogens gave similar results as in the originally

described patients [20]. The B cell compartment showed a

reduction in memory B cell subsets (Table 4), which

confirms the results obtained in the previously described

ICOS-deficient patients, a phenotype also found in about

three quarters of CVID patients.

After exclusion of mutations in ICOS, we subsequently

evaluated the ICOS-L in the remaining seven AR-CVID

families. In two patients, we could identify a previously

undescribed polymorphism located in exon 3 of ICOS-L

giving rise to an amino acid substitution from Valine to

Isoleucine. However, as both amino acids are aliphatic and

the exchange is located within a nonconserved part of the

protein [16], the structure or function of ICOS-L are

unlikely to be impaired, which is further supported by the

observation of the same polymorphism in healthy controls at

a similar frequency.

We suggest mutations of ICOS be sought in patients with

an recessive pattern of inheritance of CVID. While we did

not identify this mutation in the sporadic cases investigated

here, additional cases arising from the same or other ICOS

mutations may be identified.

Acknowledgments

We thank Dfrte Thiel, Judith Deimel, and Cristina

Wfllner for excellent technical assistance and the patients’

physicians Dr. Mezger and Prof. Dr. Vaith. This work was

supported by the Deutsche Forschungsgemeinschaft (DFG)

grant GR 1617/3 and SFB 620/project C2 to B.G and by

grants from the National Institutes of Health, AI-467320,

AI-48693, and contract N01-AI-30070, NIH-NIAID-DAIT

03-22 to C.C.

References

[1] C. Cunningham-Rundles, C. Bodian, Common variable immunode-

ficiency: clinical and immunological features of 248 patients, Clin.

Immunol. 92 (1999) 34–48.

[2] R.F. Ashman, F.M. Schaffer, J.D. Kemp, W.M. Yokoyama, Z.B. Zhu,

M.D. Cooper, J.E. Volanakis, Genetic and immunologic analysis of a

family containing five patients with common variable immune

U. Salzer et al. / Clinical Immunology 113 (2004) 234–240240

deficiency or selective IgA deficiency, J. Clin. Immunol. 12 (1992)

406–414.

[3] I. Vorechovsky, M. Cullen, M. Carrington, L. Hammarstrom, A.D.

Webster, Fine mapping of IGAD1 in IgA deficiency and common

variable immunodeficiency: identification and characterization of

haplotypes shared by affected members of 101 multiple-case families,

J. Immunol. 164 (2000) 4408–4416.

[4] P.D. Burrows, M.D. Cooper, IgA deficiency, Adv. Immunol. 65

(1997) 245–276.

[5] L. Mellemkjaer, L. Hammarstrom, V. Andersen, J. Yuen, C.

Heilmann, T. Barington, J. Bjorkander, J.H. Olsen, Cancer risk

among patients with IgA deficiency or common variable immunode-

ficiency and their relatives: a combined Danish and Swedish study,

Clin. Exp. Immunol. 130 (2002) 495–500.

[6] A. Hutloff, A.M. Dittrich, K.C. Beier, B. Eljaschewitsch, R. Kraft, I.

Anagnostopoulos, R.A. Kroczek, ICOS is an inducible T-cell co-

stimulator, structurally and functionally related to CD28, Nature 397

(1999) 263–266.

[7] A.J. Coyle, S. Lehar, C. Lloyd, J. Tian, T. Delaney, S. Manning, T.

Nguyen, T. Burwell, H. Schneider, J.A. Gonzalo, M. Gosselin, L.R.

Owen, C.E. Rudd, J.C. Gutierrez-Ramos, The CD28-related molecule

ICOS is required for effective T cell-dependent immune responses,

Immunity 13 (2000) 95–105.

[8] K. Ihara, S. Ahmed, F. Nakao, N. Kinukawa, R. Kuromaru, N.

Matsuura, I. Iwata, S. Nagafuchi, H. Kohno, K. Miyako, T. Hara,

Association studies of CTLA-4, CD28, and ICOS gene polymor-

phisms with type 1 diabetes in the Japanese population, Immunoge-

netics 53 (2001) 447–454.

[9] K.E. Haimila, J.A. Partanen, P.M. Holopainen, Genetic polymorphism

of the human ICOS gene, Immunogenetics 53 (2002) 1028–1032.

[10] A.D. Haaning Andersen, M. Lange, S.T. Lillevang, Allelic variation

of the inducible costimulator (ICOS) gene: detection of polymor-

phisms, analysis of the promoter region, and extended haplotype

estimation, Tissue Antigens 61 (2003) 276–285.

[11] K.C. Beier, A. Hutloff, A.M. Dittrich, C. Heuck, A. Rauch, K.

Buchner, B. Ludewig, H.D. Ochs, H.W. Mages, R.A. Kroczek,

Induction, binding specificity and function of human ICOS, Eur. J.

Immunol. 30 (2000) 3707–3717.

[12] A. Tafuri, A. Shahinian, F. Bladt, S.K. Yoshinaga, M. Jordana, A.

Wakeham, L.M. Boucher, D. Bouchard, V.S. Chan, G. Duncan, B.

Odermatt, A. Ho, A. Itie, T. Horan, J.S. Whoriskey, T. Pawson, J.M.

Penninger, P.S. Ohashi, T.W. Mak, ICOS is essential for effective

T-helper-cell responses, Nature 409 (2001) 105–109.

[13] C. Dong, U.A. Temann, R.A. Flavell, Cutting edge: critical role of

inducible costimulator in germinal center reactions, J. Immunol. 166

(2001) 3659–3662.

[14] S.C. Wong, E. Oh, C.H. Ng, K.P. Lam, Impaired germinal center

formation and recall T-cell-dependent immune responses in mice

lacking the costimulatory ligand B7-H2, Blood 102 (2003)

1381–1388.

[15] M.M. Swallow, J.J. Wallin, W.C. Sha, B7h, a novel costimulatory

homolog of B7.1 and B7.2, is induced by TNFalpha, Immunity 11

(1999) 423–432.

[16] V. Ling, P.W. Wu, H.F. Finnerty, K.M. Bean, V. Spaulding, L.A.

Fouser, J.P. Leonard, S.E. Hunter, R. Zollner, J.L. Thomas, J.S.

Miyashiro, K.A. Jacobs, M. Collins, Cutting edge: identification of

GL50, a novel B7-like protein that functionally binds to ICOS

receptor, J. Immunol. 164 (2000) 1653–1657.

[17] A. Aicher, M. Hayden-Ledbetter, W.A. Brady, A. Pezzutto, G.

Richter, D. Magaletti, S. Buckwalter, J.A. Ledbetter, E.A. Clark,

Characterization of human inducible costimulator ligand expression

and function, J. Immunol. 164 (2000) 4689–4696.

[18] V. Ling, P.W. Wu, J.S. Miyashiro, S. Marusic, H.F. Finnerty, M.

Collins, Differential expression of inducible costimulator-ligand splice

variants: lymphoid regulation of mouse GL50-B and human GL50

molecules, J. Immunol. 166 (2001) 7300–7308.

[19] L. Liang, E.M. Porter, W.C. Sha, Constitutive expression of the B7h

ligand for inducible costimulator on naive B cells is extinguished after

activation by distinct B cell receptor and interleukin 4 receptor-

mediated pathways and can be rescued by CD40 signaling, J. Exp.

Med. 196 (2002) 97–108.

[20] B. Grimbacher, A. Hutloff, M. Schlesier, E. Glocker, K. Warnatz, R.

Drager, H. Eibel, B. Fischer, A.A. Schaffer, H.W. Mages, R.A.

Kroczek, H.H. Peter, Homozygous loss of ICOS is associated with

adult-onset common variable immunodeficiency, Nat. Immunol. 4

(2003) 261–268.

[21] A. Soresina, V. Lougaris, S. Giliani, F. Cardinale, L. Armenio, M.

Cattalini, L.D. Notarangelo, A. Plebani, Mutations of the X-linked

lymphoproliferative disease gene SH2D1A mimicking common

variable immunodeficiency, Eur. J. Pediatr. 161 (2002) 656–659.

[22] H. Kanegane, S. Tsukada, T. Iwata, T. Futatani, K. Nomura, J.

Yamamoto, T. Yoshida, K. Agematsu, A. Komiyama, T. Miyawaki,

Detection of Bruton’s tyrosine kinase mutations in hypogammaglo-

bulinaemic males registered as common variable immunodeficiency

(CVID) in the Japanese Immunodeficiency Registry, Clin. Exp.

Immunol. 120 (2000) 512–517.

[23] L. Mouthon, P. Cohen, C. Larroche, M.H. Andre, I. Royer, P.

Casassus, L. Guillevin, Common variable immunodeficiency: one or

multiple illnesses? 3 clinical cases, Ann. Med. Interne 150 (1999)

275–282.

[24] J. Kralovicova, L. Hammarstroem, A. Plebani, D.B. Webster, I.

Vorechovsky, Fine scale mapping at IgAD1 and genome-wide linkage

analysis implicate HLA-DQ/DR as a major susceptibility locus in

selective IgA deficiency and common variable immunodeficiency, J.

Immunol. 170 (2003) 2765–2775.

[25] W.I. Lee, Q. Zhu, E. Gambine, Y. Jin, A.A. Welcher, H.D. Ochs,

Inducible CO-stimulator molecule, a candidate gene for defective

isotype switching, is normal in patients with hyper-IgM syndrome of

unknown molecular diagnosis, J. Allergy Clin. Immunol. 112 (2003)

958–964.