doi:10.1182/blood-2007-01-069781Prepublished online March 23, 2007;   

 Montserrat Hoyos and David GallardoGonzalez, David Serrano, Ildefonso Espigado, Carlos Solano, Josep M. Ribera, Josep M. Pujal,

MarcosEncuentra, Jose B. Nieto, Javier de la Rubia, Alvaro Urbano-Ispizua, Salut Brunet, Arturo Iriondo, Arianne Perez-Garcia, Rafael De la Camara, Jose Roman-Gomez, Antonio Jimenez-Velasco, Maite transplantation from HLA-identical sibling donorsCTLA-4 polymorphisms and clinical outcome after allogeneic stem cell

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CTLA-4 POLYMORPHISMS AND CLINICAL OUTCOME AFTER

ALLOGENEIC STEM CELL TRANSPLANTATION FROM HLA-IDENTICAL

SIBLING DONORS

Arianne Pérez-García, Rafael De la Cámara, Jose Román-Gómez, Antonio Jiménez-

Velasco, Maite Encuentra, Jose B. Nieto, Javier de la Rubia, Alvaro Urbano-Ispizúa,

Salut Brunet, Arturo Iriondo, Marcos González, David Serrano, Ildefonso Espigado,

Carlos Solano, Josep M. Ribera, Josep M. Pujal, Montserrat Hoyos, and David

Gallardo.

From the GvHD/Immunotherapy Committee of the Spanish Group of Hematopoietic

Stem Cell Transplantation (GETH: Grupo Español de Trasplante Hemopoyético).

Affiliations note: This study has been performed at the Alloreactivity Unit, Laboratori

de Recerca Translacional and Clinical Hematology Department from the Institut Català

d’Oncologia - IDIBELL, Hospital Duran i Reynals, L’Hospitalet, Barcelona, Spain

Grant support note: Supported by grant FIS PI050939 from the Fondo de

Investigaciones Sanitarias, Instituto de Salud Carlos III, Ministerio de Sanidad y

Consumo.

Reprints: David Gallardo. Clinical Haematology Department, Institut Català

d'Oncologia, Hospital Duran i Reynals, Av. Gran Vía s/n, km 2.7, 08907 L'Hospitalet

de Llobregat, Barcelona, Spain; e-mail: 25739dgg@comb.es

Left running head: PÉREZ-GARCÍA et al.

Right running head: CTLA-4 POLYMORPHISMS AND allo-HSCT OUTCOME

Section designation: TRANSPLANTATION

Blood First Edition Paper, prepublished online March 23, 2007; DOI 10.1182/blood-2007-01-069781

Copyright © 2007 American Society of Hematology

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Abstract

CTLA-4 is an inhibitory molecule that downregulates T-cell activation. Although

polymorphisms at CTLA-4 have been correlated with autoimmune diseases their

association with clinical outcome after allogeneic hematopoietic stem-cell

transplantation (allo-HSCT) has yet to be explored. Five CTLA-4 single-nucleotide

polymorphisms were genotyped on 536 HLA-identical sibling donors of allo-HSCT.

Genotypes were tested for an association with patients’ post-transplant outcome. The

effect of the polymorphisms on CTLA-4 mRNA and protein production were

determined in 60 healthy controls. We observed a reduction in the mRNA expression of

the soluble CTLA-4 isoform in the presence of a G allele at CT60 and +49. Patients

receiving stem cells from a donor with at least one G allele in position CT60 had worse

overall survival (56.2% vs. 69.8% at 5 years; p: 0.001; HR: 3.80; 95% CI: 1.75-8.22),

due to a higher risk of relapse (p: 0.049; HR: 1.71; 95% CI: 1.00-2.93). Acute graft-

versus-host disease (aGVHD) was more frequent in patients receiving CT60 AA stem

cells (P: 0.033; HR: 1.54; 95% CI: 1.03-2.29). This is the first study to report an

association between polymorphisms at CTLA-4 and clinical outcome post allo-HSCT.

CT60 genotype influences relapse and aGVHD, probably due to its action on CTLA-4

alternative splicing.

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Introduction

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) has been widely carried

out as a curative therapy for several haematological malignancies and non-malignant

disorders such as severe aplastic anaemia, thalassaemia or immune deficiencies. Disease

relapse and graft-versus-host disease (GVHD) are the major complications post-allo-

HSCT, being the occurrence of GVHD the main cause of morbidity and mortality1,2. A

variety of genetic and non-genetic factors affect transplant success by favouring or

hindering alloimmune responses. Polymorphisms in minor histocompatibility antigens,

cytokine-coding genes and genes involved in drug metabolism, amongst others, affect

the incidence of GVHD3-7.

Donor T lymphocytes play a critical role in alloimmune recognition and their ability to

detect non-self antigens can lead to GVHD or contribute to relapse prevention through

recognition and subsequent elimination of minimal residual disease8. Transplantation

outcome may be influenced not only by the ability of T-cells to recognize antigens but

also by their capacity to become activated or inactivated.

In the immune recognition process two signals are required for T-lymphocyte expansion

and differentiation: the T-cell receptor (TCR) binding to the HLA molecule-peptide

complex and an antigen-independent costimulatory signal provided by the B7 (CD80

and CD86)/CD28 interaction. The cytotoxic T-lymphocyte antigen 4 (CTLA-4) is a

homologous molecule of CD28 that is a competitive antagonist for B7. CTLA-4 has

greater affinity and avidity for B7 than does CD28, and its translocation to the cell

surface after T-cell activation results in B7 sequestration and transduction of a negative

signal, responsible for T-cell inactivation9-12.

The CTLA-4 gene is translated into two proteic isoforms: a full-length protein (flCTLA-

4) and a soluble counterpart (sCTLA-4), which lacks exon 3 (responsible for coding the

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transmembrane domain) due to alternative splicing. flCTLA-4 downregulates T-cell

responses by inducing cell-cycle arrest and blocking cytokine production13,14. It may

also be involved in maintaining peripheral tolerance to self-antigens15. Moreover, there

is evidence that CTLA-4 is crucial in both solid organ transplants and hematopoietic

stem cell transplantation. Many studies have examined the effect of CTLA-4-Ig and a

number of drugs that induce tolerance and prevent allograft rejection have been

developed16,17. In contrast, little is known about sCTLA-4 function. Although sCTLA-4

was thought to be expressed by non-stimulated human T cells, no evidence of this

soluble isoform has been found in the serum of healthy volunteers18,19.

The human CTLA-4 gene is located on 2q33, in a susceptibility region for autoimmune

disease. Certain polymorphisms in the CTLA-4 gene correlate with different

autoimmune disorders and with some malignancies20-26. The thymine-to-cytosine

nucleotide transitions at positions –1722 and –318, and the adenine-to-guanine changes

at –1661, +49 and CT60 have been widely investigated. Many studies have identified

cytosine and adenine alleles as protective, whereas thymine and guanine are considered

risk-associated or susceptibility alleles for several autoimmune diseases.

However, there are no studies regarding the effect of CTLA-4 polymorphisms on

alloimmune recognition after allo-HSCT.

Here we attempt to determine whether CTLA-4 gene polymorphisms –1722, -1661, -

318, +49 and CT60 influenced the clinical outcome of patients receiving an allogeneic

stem cell transplantation from an HLA-identical sibling donor. We also aimed to

elucidate the involvement of the five polymorphisms in the transcription and protein

expression levels of sCTLA-4 and flCTLA-4.

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Patients, materials and methods

Subjects

DNA isolated from peripheral blood from 536 healthy hematopoietic stem cell donors

was analyzed for the presence of polymorphisms in the CTLA-4 gene. These samples

were collected between years 1993 to 2006, from 19 Spanish transplantation centres.

The study was performed on DNA samples from donors whose HLA-identical siblings

underwent allogeneic HSCT without T cell depletion after receiving myeloablative-

conditioning regimens for the treatment of several hematological malignancies. All

patient-donor pairs meeting the selection criteria were included in the study unless there

was no donor’s DNA availability. Patient characteristics are summarized in Table 1.

This study was approved by the ethical committee from the Hospital Universitari de

Bellvitge (L’Hospitalet, Barcelona, Spain) and met with the recommendations of the

Helsinki declaration.

In order to evaluate whether the presence of CTLA-4 polymorphisms affected the

CTLA-4 mRNA and protein expression levels, 10 ml of whole blood from sixty healthy

blood donors was collected from the Blood Bank of the Hospital Universitari de

Bellvitge (BST: Banc de Sang i Teixits), Barcelona, Spain. All donors gave their written

informed consent.

CTLA-4 genotyping

DNA was obtained from 200µL of whole blood using the QIAampDNA Blood Mini

Kit (Qiagen GmbH, Hilden, Germany).

PCR−RFLP was used to genotype five bi−allelic polymorphisms of CTLA-4: -1722TC,

-1661AG, -318CT, +49AG and CT60. The primers and restriction enzymes (New

England Biolabs, Beverly, MA, USA) used were: -1722 (BbvI) and –1661 (MseI)

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forward 5′-CTA AGA GCA TCC GCT TGC ACC T-3′, reverse 5′-TTG GTG TGA

TGC ACA GAA GCC TTT T-3′; -318 (MseI) forward 5′-AAA TGA ATT GGA CTG

GAT GGT-3′, reverse 5′-TTA CGA GAA AGG AAG CCG TG-3′; +49(BbvI) forward

5′-GTC AAG GGA CCA TTA GAA G-3′; CT60 (NcoI) forward 5′-CAC CAC TAT

TTG GGA TAT ACC-3′, reverse 5′-AGC TCT ATA TTT CAG GAA GGC-3′. PCR

reactions were performed with 100 ng DNA, as previously described27,28, and 3µL of

the amplified products were digested for 1 h at 37°C with 1U of the corresponding

restriction enzyme in a total reaction volume of 10µL. The digestion products were

resolved in 2.5% agarose gels stained with ethidium bromide.

mRNA isolation and reverse transcription (RT)

Eight millilitres of whole blood was centrifuged in a Ficoll−Hypaque density gradient

and the peripheral blood mononuclear cell (PBMC) layers were collected and pelleted.

Total RNA was isolated using the Ultraspec™ RNA Isolation System (Biotecx

Laboratories, Houston, TX, USA). Five-thousand nanograms of purified RNA was

retro−transcripted using the First Strand cDNA Synthesis Kit for RT-PCR (AMV)

(Roche Applied Science, Penzberg, Germany), according to the manufacturer’s

protocol. The RT reaction consisted in incubation for 1 h at 37ºC and a reverse

transcriptase-denaturing step at 95ºC for 5 min.

Quantification of sCTLA-4, flCTLA-4 and beta-2-microglobulin mRNA expression

Specific primers for sCTLA-4, flCTLA-4 and beta-2-microglobulin mRNA were used

to amplify fragments of 93bp, 79bp and 89bp, respectively20. PCR products were

purified with the JETquick PCR Product Purification Spin Kit (Genomed GmbH,

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Löhne, Germany) and then subcloned into a pCR4-TOPO vector (Invitrogen,

Carisbad, CA, USA). The constructs were verified by direct sequencing.

A standard curve ranging from 10 to 107 copies of each construct was established to

quantify sCTLA-4, flCTLA-4 and beta-2-microglobulin mRNA copies. Beta-2-

microglobulin was used as housekeeping control transcript.

The Quantitative Real Time PCR (QRT-PCR) reaction consisted of 1µL of cDNA,

0.2µL of each primer (10mM), 0.4µL of the corresponding probe (previously

described20) (4mM), 5µL of Premix Ex Taq™ (Perfect Real Time) (Takara Bio, Otsu,

Shiga, Japan) and distilled water up to a final volume of 10µL. Amplification was

performed in a LightCycler 2.0 Instrument. A reference to the standard curve was

included in each run and all samples were replicated once. The average of the replicates

was used to determine absolute levels of sCTLA-4, flCTLA-4 and beta-2-microglobulin

mRNA copies of each sample. Comparison between samples was performed after

standardization with beta-2-microglobulin and also the sCTLA-4/flCTLA-4 mRNA

ratio was calculated.

Flow cytometric analysis

PBMCs from healthy controls were stained with mAbs conjugated with APC, PE, PE-

Cy7, FITC and PerCP fluorochromes (Becton-Dickinson, San Jose, CA, USA) against

surface CD3, CD8, CD19, CD25 and CD152. Appropriate isotype controls were

included for each sample. A total of 100.000 events were acquired for each sample

using a FacsCanto flow cytometer (Becton Dickinson, San Jose, CA, USA). The results

were expressed as the proportion of CD3+, CD3+/CD8+, CD3+/CD25+ and CD19+ cells

expressing CD152 antigen, depending on the CTLA-4 genotype. The samples were also

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analyzed after 24 h of stimulation with 50 ng/ml phytohemagglutinin (PHA) and 1µg/ml

PMA (phorbol myristate acetate).

Enzyme-linked immunosorbent assay (ELISA)

Ten microlitres of sera was used to quantify sCTLA-4 with a commercially available

kit (MedSystems Diagnostics, GmbH, Vienna, Austria). sCTLA-4 levels were also

determined after 24 h of PHA and PMA stimulation. A standard curve ranging from

0.156-10 ng/ml was established following the manufacturer’s protocol. All samples and

standards were analyzed in duplicate and the average between duplicates was

calculated. Fifty microlitres of diluted biotin conjugate was added to all wells. After 2 h

of incubation, 100µl of Streptavidin-horseradish peroxidase was added and incubated

for 1 h. Tetramethyl-benzidine (100µl) was added and incubated for 15 min, followed

by the addition of the stop solution. The optical density of each well was determined

immediately at 450 nm. Absolute levels of sCTLA-4 were extrapolated from the

standard curve.

Statistical analysis

The sample size consisted of 536 patients, providing 99% power for comparison of

overall survival between CT60 AA and CT60 AG/GG genotypes. The statistical power

calculation was based on a two-sided significance level of 0.05, assuming 850 days

median survival time on control patients and a ratio CT60 AG/GG : CT60 AA of 1:3

according to the genotypes distribution. The accrual time during which patients were

recruited was 1825 days.

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Concerning acute GVHD and relapse, the sample size was of 515 and 534 evaluable

patients, respectively, which provided a statistical power of 99% to detect differences

between both genotypes.

The chi-squared test was used to estimate linkage disequilibrium among pairs of alleles,

while haplotype frequencies were determined using the Estimating Haplotype-

frequencies (EH) program from Rockefeller University

(ftp://linkage.rockefeller.edu/software/eh/). CTLA-4 allele frequencies, genotypes and

haplotypes were also formulated by direct counting. Univariate analyses were

performed to evaluate the association between the presence of polymorphisms at –1722,

-1661, -318, +49 and CT60 of CTLA-4 and overall survival (OS), acute GVHD

(aGVHD), and relapse. Two study groups were established for each polymorphism

regarding the presence (in either homozygosis or heterozygosis) or absence of the

susceptibility allele, except for –1722. In this case, due to the low frequency of the

protective allele in homozygosis, heterozygotes were compared to homozygous subjects

for the risk allele.

The Kaplan-Meier method was applied for the analysis of OS. Curves were compared

using the Breslow Test, which favours the detection of differences in the early events.

Statistical incidence estimates were used to determine the cumulative incidence of

aGVHD and relapse in the presence of polymorphisms. Death without signs of aGVHD

was considered a competing risk in the analysis of aGVHD incidence. The competing

risk for relapse was death in complete remission. Acute GVHD was diagnosed and

graded according to standard criteria29. Only grades II and higher of aGVHD were

considered for the analysis of aGVHD incidence.

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Multivariate Cox regression models using a backward stepwise procedure with the

likelihood ratio criterion (inclusion/exclusion criteria: P≤0.05/P>0.10, respectively)

were applied to analyze the combined effects of CTLA-4 polymorphisms and other

factors on OS, relapse and aGVHD. All variables in the univariate analysis with a P

value at or below 0.2 were included in the multivariate analysis.

In the analysis of mRNA expression levels of flCTLA-4 and sCTLA-4 according to the

three different genotypes of the five polymorphisms, univariate and multiple regression

analyses relating CTLA4 expression to the G allele numbers were performed. P values

were two-sided and those lower than 0.05 were considered statistically significant.

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Results

Allele, genotype and haplotype frequencies in the Spanish population

Allele and overall genotype frequencies at the five CTLA-4 loci for the 536 allo-HSCT

donors are listed in Table 2. Genotypes corresponding to the five CTLA-4

polymorphisms did not significantly deviate from the Hardy-Weinberg equilibrium.

Previous studies have described a region of approximately 100 kb between CD28 and

the 5’ part of ICOS (the CTLA-4 flanking genes) as being a strong linkage

disequilibrium (LD) zone20. We analyzed the segregation pattern of –1722, -1661, -318,

+49 and CT60 single nucleotide polymorphisms on 780 chromosomes and identified six

major haplotypes (Table 3).

CTLA-4 polymorphisms influence clinical outcome post allo-HSCT

The presence of susceptibility alleles at the –1722, –1661 and –318 promoter positions

of CTLA-4 did not show significant correlations with overall survival, acute GVHD

incidence, or disease relapse (Table 4).

Concerning the polymorphism +49 at exon 1, higher overall survival at 5 years was

observed for patients receiving stem cells from an AA homozygous donor (+49 AA:

67.7% vs. +49 AG/GG: 55.3%; P: 0.019). In addition, the donor’s CT60 genotype

showed a significant association with a reduction in overall survival at 5 years (CT60

AA: 69.8% vs. CT60 AG/GG: 56.2) (Figure 1A). Multivariate analysis confirmed that

the CT60 AG/GG genotype was an independent risk factor for worse overall survival

(P: 0.001; HR: 3.80; 95% CI: 1.75-8.22), whereas the polymorphism at position +49

was not (Table 5).

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Disease relapse was higher in the CT60 AG/GG group (32% vs. 27% at 5 years) (Figure

1B). Multivariate analysis demonstrated that CT60 was an independent risk factor for

relapse (P: 0.049; HR: 1.71; 95% CI: 1.00-2.93) (Table 5).

Given that the relapse pattern differs depending on the diagnosis, a highly homogeneous

cohort was selected for further analysis of the effect of CTLA-4 polymorphisms on

disease relapse. Three hundred and twenty-one patients diagnosed with AML, ALL and

MDS (the main indications for allo-HSCT) were included in this analysis. In this cohort,

the CT60 G allele was associated with increased relapse rates (CT60 AA: 24% vs. CT60

AG/GG: 36% at 5 years) (Figure 2). The hazard ratio for relapse in the presence of a G

allele at CT60 was 3.31 (P: 0.006, 95% CI: 1.41-7.76), leading to worse overall survival

(CT60 AA: 71% vs. CT60 AG/GG: 48.6%, P: 0.005). Disease relapse was not affected

by polymorphisms at –1722, -1661, –318 or +49.

Risk of grades II-IV of acute GVHD was also increased by the CT60 genotype when

considering all 536 patients. Interestingly, for GVHD the susceptibility alleles were not

those previously defined as such for survival and relapse in this study or for other

autoimmune diseases. The A allele at CT60 was associated with an increased risk of

developing aGVHD while the G allele was protective. Forty-six percent of patients with

the CT60 AA genotype developed aGVHD, but the disease incidence decreased to 37%

in the presence of a G allele (Figure 1C). Multivariate analysis detected that the donor’s

CT60 AA genotype was an independent risk factor for developing grades II-IV aGVHD

(P: 0.033; HR: 1.54; 95% CI: 1.03-2.29) (Table 5). The incidence of severe aGVHD

grades (III-IV) was not significantly affected by CT60 (AA: 21.3% vs. AG/GG: 15.8%,

P: 0.335). The single-nucleotide polymorphism (SNP) +49 showed a trend towards

association with grades II-IV aGVHD although statistical significance was not reached

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(AA: 43% vs. AG/GG: 34%, P: 0.07). Severe grades of the disease were not influenced

by +49.

CTLA-4 haplotypes

The presence of linkage disequilibrium among the five CTLA-4 polymorphic sites

results in the non-random distribution of the alleles. We identified six major blocks of

inheritance or haplotypes for the CTLA-4 gene, with frequencies higher than the 6% in

our population (Table 3). There is linkage disequilibrium between CT60 and +49 SNPs,

with only three haplotypes observed at reasonable frequencies. In the first, CT60A is

linked to +49A (the A-A haplotype), the second involves CT60G and +49G (the G-G

haplotype), and in the third, CT60G is linked to +49A (the G-A haplotype). Among the

six resulting genotypes, overall survival at five years was higher for patients receiving

an A-A homozygous graft (74.1%) than for those heterozygous for it (A-A/G-A and A-

A/G-G) (58.8%), as well as for those without A-A (G-A/G-A, G-A/G-G and G-G/G-G)

(55.2%; P: 0.034). Interestingly, patients transplanted from donors with genotypes other

than CT60AA/+49AA, regardless of whether they presented one copy of the protective

haplotype or not, had similar survival rates at five years, suggesting that the presence of

a G allele at CT60 and/or at +49 was sufficient to worsen the transplantation outcome.

Given these observations, we examined whether there were differences between having

just one polymorphism with the risk allele or presenting the G allele at both CT60 and

+49. To this end, patients were stratified into three groups according to the absence of

risk alleles at CT60 and +49 (protective genotype: CT60AA/+49AA), the presence of a

risk allele at CT60 only (intermediate risk genotypes: CT60AG/+49AA and

CT60GG/+49AA), and the presence of, at least, a risk allele in both polymorphisms

(high risk genotypes: CT60AG/+49AG, CT60GG/+49AG and CT60GG/+49GG).

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Patients receiving grafts carrying the protective CTLA-4 CT60AA/+49AA genotype had

a 74.1% overall survival rate compared to 61.9% among those with the intermediate risk

genotypes. The presence of the susceptibility alleles in both positions resulted in a

dramatic decrease of OS to 54.4% (Figure 3). Multivariate analysis confirmed that the

CTLA-4 protective genotype was an independent protective factor for overall survival

(P: 0.001; HR: 0.22; 95% CI: 0.08-0.55). After accounting for the significant protective

effect on OS of the CTLA-4 protective genotype compared to all other genotypes

combined, there was no further significant variation of OS among CT60/+49 genotype

combinations.

Relapse was also evaluated according to the CT60/+49 combination. We found that

relapse was higher in patients transplanted from donors with the intermediate-risk and

high-risk genotypes. The CT60AA/+49AA combination was identified in the

multivariate analysis as protective for relapse (P: 0.05; HR: 0.39; 95% CI, 0.15-1.00).

Univariate analysis for grades II-IV of aGVHD and with respect to the CT60/+49

genotype was also statistically significant. The genetic combination considered as

protective for overall survival and relapse was, in this case, the one that conferred

higher incidence of aGVHD (49%). In contrast, the presence of a G allele resulted in a

reduction of GVHD incidence, to 39.1% when it was present at one locus and to 33%

when both CT60 and +49 displayed at least a G allele (P: 0.035).

Dominant effect of the CT60 polymorphism

The results previously reported indicate that the presence of a risk allele at both CT60

and +49 conferred worse overall survival than when this risk factor was only present at

CT60, suggesting that the polymorphism at +49 also influences outcome. However,

when a multivariate analysis adjusted for CT60 was performed, +49 was not identified

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as an independent risk factor for overall survival (P: 0.613), whereas the involvement of

CT60 remained significant. When the analysis included both the CT60 genotype and the

CT60/+49 haplotype the CT60/+49 combination was no longer significant. The same

was observed in the multivariate analysis for aGVHD and relapse. These results support

the hypothesis that CT60 itself has a dominant effect on outcome, and the results

observed for the CT60/+49 haplotype can thus be attributed to the linkage

disequilibrium between these two loci.

Expression of sCTLA-4 and flCTLA-4

QRT-PCR was performed to quantify the sCTLA-4 and flCTLA-4. Full-length CTLA-4

transcription levels were not affected by the presence of polymorphisms at either –1722,

-1661, -318, +49 or CT60. These observations were confirmed by flow cytometry. No

differences in surface protein expression according to the polymorphisms were observed

on CD3+, CD3+CD25+, CD3+CD8+ or CD19+ cell subsets,

No differences in the percentage of neither CD19+CD152+ cells nor T cell subsets

(CD3+CD8+CD25+, CD3+CD4+CD25+, CD3+CD8+CD25+CD152+,

CD3+CD4+CD25+CD152+ cells) were observed in basis of the CTLA-4

polymorphisms, even after PHA and PMA stimulation.

It has previously been suggested that CT60 may determine the efficiency of the

alternative splicing of CTLA-4, which is responsible for the production of the soluble

isoform of the protein20. We observed that depending on the CT60 polymorphism the

amount of sCTLA-4 mRNA was reduced in a G-allele dose-dependent manner. The

mean quantity of sCTLA-4 transcript in individuals carrying the CT60AA genotype was

24.7% higher than in heterozygous subjects and 38.5% higher than in homozygotes for

the G allele (P: 0.005) (Figure 4). We also observed a significant decrease in sCTLA-4

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when the +49 genetic combinations were compared (P: 0.009). The presence of

polymorphic alleles at –318, -1661 and –1722 did not influence the production of

sCTLA-4 mRNA.

We also measured sCTLA-4 production according to CT60/+49 genetic combinations.

The sCTLA-4 mRNA levels observed for individuals with the protective genotypes

were considered as a reference. The expression of the soluble isoform was 31% lower in

the intermediate-risk group and 34.5% lower in the high-risk group (P: 0.018). When

regressing sCTLA-4 on the G allele numbers for both CT60 and +49 in a multiple

regression model, CT60 remained statistically significant (P: 0.005) but +49 did not (P:

0.156). These results agree with our previous observations on the CT60/+49 genotypes,

supporting the hypothesis that the polymorphism at CT60 is responsible for the effect

observed in allogeneic stem cell transplantation outcome.

We also performed ELISA assay on the sixty controls and no sCTLA-4 was detected,

regardless of the CT60 genotype.

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Discussion

The results of this study highlight the importance of the regulatory molecule CTLA-4 in

the outcome of patients who receive allogeneic stem cell transplantation from an HLA-

identical sibling for the treatment of several haematological malignancies.

The effect of some CTLA-4 polymorphisms on the onset and evolution of several

autoimmune diseases, such as lupus erythematosus, type-1 diabetes and Grave’s

disease, has been widely investigated20-28. However, to our knowledge there are no

references in the literature about the role of CTLA-4 variants in alloimmunity. This

study is therefore the first to show that single-nucleotide polymorphisms at CTLA-4 are

predictors of allogeneic transplant outcome.

The most widely studied polymorphisms are the CT60 and +49 adenine-to-guanine

transitions. Traditionally, the A allele has been identified as protective whereas the G

allele is considered to be associated with greater susceptibility to autoimmune disease.

However, the results reported by different groups have often been controversial25,30-32.

The CTLA-4 exon 1, where the +49 polymorphism is located, encodes the leader peptide

of the protein, which is responsible for CTLA-4 trafficking to the endoplasmic

reticulum. The reason why the G allele at +49 confers susceptibility to autoimmune

disease remains unknown, although a number of hypotheses have been proposed33.

However, our findings do not support the hypothesis that the +49 polymorphism is

involved on transplant outcome. In contrast, we observed that CT60 had a dominant

effect on transplantation outcome. CT60 maps at the 3’ untranslated region of CTLA-4

and it could play a role in the alternative splicing by which sCTLA-4 is generated.

Based on a small number of subjects, Ueda et al. suggested that the G allele of CT60

produced less sCTLA-4 mRNA than did the A allele. Given these observations the

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authors concluded that sCTLA-4 expression is the functional basis for the observed

genetic association between autoimmune diseases and the CTLA-4 gene. If this

conclusion was correct, one would expect patients with autoimmune disease to have

lower levels of sCTLA-4 than healthy controls. However, although sCTLA-4 was first

thought to be produced by non-stimulated T-cells and its expression downregulated

after T-cell activation18,34, no evidence of this soluble isoform has been found in sera of

healthy controls19. The results obtained from our ELISA assays are also at odds with the

data reported by Magistrelly et al. In contrast, many studies have reported increased

protein levels of sCTLA-4 in patients with autoimmune disease, suggesting that this

isoform of the protein is responsible for pathogenesis and reflects T-cell activation35,36.

Our results from the sCTLA-4 mRNA analysis of the sixty healthy blood donors also

identified the A allele at CT60 as being responsible for a greater production of sCTLA-

4, and we found that patients receiving grafts with CT60 AA genotypes had more

alloimmune recognition processes, as indicated by the higher incidence of aGVHD

observed in this group.

We identified the A allele at CT60 as an independent risk factor for developing

moderate to severe grades of aGVHD, whereas its G counterpart conferred protection

against disease relapse. Our findings on alloimmunity, along with other results

previously reported on autoimmunity, suggest that sCTLA-4 blocks the B7-flCTLA-4

interaction, thereby enhancing T-cell reactivity by preventing the transduction of

inhibitory signals that lead to lymphocyte inactivation. Despite the highest incidence of

aGVHD in the CT60 AA group, no reduction in overall survival was observed

suggesting an enhanced graft-versus-leukaemia (GVL) effect, which protected these

patients from relapse.

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In addition, the low sCTLA-4 production associated with the G allele may explain why

patients with G alleles at CT60 had increased relapse rates and, therefore, higher

mortality. We hypothesize that in the absence of significant levels of sCTLA-4

competing with flCTLA-4 for the B7 ligand, T cells are more likely to be inactivated

and less capable of detecting neoplastic antigens, thus allowing residual tumoral cells to

escape from lymphocytic control.

In conclusion, the donor’s CT60 AA genotype has been identified as a less tolerogenic

genotype associated with a higher incidence of moderate to severe grades of aGVHD

and better transplant outcome through an increased GVL effect.

The clinical interest of these findings resides in the identification of patients diagnosed

primarily with acute leukaemia and MDS syndromes and at high risk of relapse after

allo-HSCT based on their donor’s CTLA-4 genotype at CT60. These patients could

potentially benefit from a rapid withdrawal of immunosuppressive therapy.

Thorough knowledge of the CTLA-4 molecule and its role in the allo-HSCT setting is

necessary for the better understanding of the alloreactive responses, as is the case for

other non-HLA molecules such as cytokines and minor histocompatibility antigens.

This information could improve the pre-transplant risk assessment process and serve as

a reference for treatment planning.

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Acknowledgements

The authors thank Mrs. Sònia Ramon for her technical assistance. We are also grateful

to all members of the Laboratori de Recerca Translacional (ICO Duran i Reynals) for

their useful comments and collaboration in the experimental process. We also thank Dr.

Antonio Agudo for his useful comments.

Authorship

A. Pérez-García and D. Gallardo designed the research, analyzed data and wrote the

manuscript. M. Encuentra provided statistical support. A. Pérez-García performed

research helped by M.Hoyos and J.M. Pujal. R. De la Cámara, J. Román-Gómez, A.

Jiménez-Velasco, J.B. Nieto, J. de la Rubia, A. Urbano-Ispizúa, S. Brunet, A. Iriondo,

M. González, D. Serrano, I. Espigado, C. Solano and J.M. Ribera provided donors’

samples and clinical data.

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Figure 1. Overall survival (A), cumulative incidence of relapse (B) and II-IV aGVHD

(C) according to the donor’s CTLA-4 genotype at CT60 among allogeneic transplant

recipients.

A

B

C

0,0

0,1

0,2

0,3

0,4

0,5

0 50 100 150

CT60 AA: 46%

CT60 AG/GG: 37%

P: 0.033; HR: 1.54; 95%CI: 1.03-2.29

200

II-IV

aG

VH

D

0,0

0,1

0,2

0,3

0,4

0,5

0 600 1200 1800

CT60 AA: 27%

CT60 AG/GG: 32%

P: 0.049; HR: 1.71; 95%CI: 1.00-2.93

Rel

apse

180012006000

Days post allo-HSCT

1,0

0,8

0,6

0,4

0,2

0,0

Ove

rall

Su

rviv

al

CT60 AA: 69%

CT60 AG/GG: 56.2%

P: 0.001; HR: 3.80; 95%CI: 1.75-8.22

Days post allo-HSCT

Days post allo-HSCT

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Figure 2. Association of CT60 genotypes and relapse in a homogeneous cohort

consisting of patients who received an allo-HSCT for the treatment of AML, ALL and

MDS.

0.

00.

10.

20.

30.

40.

5

Rel

apse

0,0

0,1

0,2

0,3

0,4

0,5

Days post allo-HSCT

0 600 1200 1800

CT60 AA: 24%

CT60 AG/GG: 36%

p: 0.006; HR: 3.31; 95%CI: 1.41-7.76

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Figure 3. Overall survival of patients depending on the donor’s CT60/+49 genotype

combinations.

180012006000

Days post allo-HSCT

1,0

0,8

0,6

0,4

0,2

0,0

Ove

rall

Sur

viva

l

Protective genotype: 74.1%

Intermediate risk genotype: 61.9%

High risk genotype: 54.4%

P: 0.002; HR: 0.26; 95%CI: 0.11-0.61

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Figure 4. Normalized values of sCTLA-4 mRNA according to the CTLA-4 genotype at

CT60. Median values +/- standard deviation for each genotype are represented within

the box. P value is obtained by regression analysis test relating sCTLA4 to the G allele

number (0, 1 or 2) for CT60.

GGAGAA

CT60 genotype

100

80

60

40

20

0

sCT

LA

4 m

RN

A(n

orm

aliz

ed v

alu

es)

P: 0.005

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Table 1. Clinical characteristics of the patients included in the study (n=536)

Advanced disease was considered for patients with acute leukaemia beyond first complete remission, chronic myeloid leukaemia beyond first chronic phase, and progressive disease for patients with myelodysplastic syndrome, multiple myeloma or lymphoma. Sensitized donor corresponds to donors with previous pregnancies or transfusions. PB indicates peripheral blood; BM, bone marrow; TBI, total body irradiation; CSA, cyclosporin A; MTX, methotrexate.

Age [median (range)] 34 (0-59) Sex (male: female) 311: 225 Diagnosis 124 Acute lymphoblastic leukaemia 163 Acute myelogenous leukaemia 158 Chronic myeloid leukaemia 11 Multiple myeloma 35 Non-Hodgkin’s lymphoma 2 Hodgkin’s disease 34 Myelodysplastic syndrome 9 Other Disease stage (early: advanced) 326: 175 Sex mismatch n (%) 132 (24.8) Sensitized donor n (%) 96 (20.1) Source of stem cells (PB: BM) 270: 266 Conditioning regimens 206 Cyclophosphamide + TBI 308 Busulfan + cyclophosphamide 20 Other GVHD prophylaxis 29 CSA 485 CSA + MTX 2 CSA + MTX + corticosteroids 20 CSA + corticosteroids

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Table 2. Allele and genotype frequencies at the CTLA-4 loci (n=536)

Values in parenthesis indicate percentages.

Polymorphism n (%) -1722 TT 372 (83.8) TC 70 (15.8) CC 2 (0.4) T allele 814 (91.7) C allele 74 (8.3) -1661 AA 400 (75.8) AG 128 (24.2) GG 0 (0) A allele 928 (87.9) G allele 128 (12.1) -318 TT 5 (1.1) TC 82 (18.1) CC 366 (80.8) T allele 92 (10.2) C allele 814 (89.8) +49 AA 208 (46.6) AG 189 (42.4) GG 49 (11.0) A allele 605 (67.8) G allele 287 (32.2) CT60 AA 153 (24.1) AG 248 (47.0) GG 127 (24.1) A allele 554 (52.5) G allele 502 (47.5)

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Table 3. CTLA-4 most frequent haplotypes

Table 4. Univariate analysis for CTLA-4 polymorphisms and HSCT outcome

Overall survival aGVHD (II-IV) Relapse P value HR 95% CI P value HR 95% CI P value HR 95% CI -1722 CC/CT 0.454 1.16 0.78-1.73 0.112 0.74 0.52-1.07 0.523 1.16 0.73-1.85 -1661 AG/GG 0.538 1.11 0.79-1.55 0.679 1.07 0.76-1.50 0.268 1.26 0.84-1.88 -318 CT/TT 0.393 0.83 0.55-1.26 0.882 0.97 0.65-1.44 0.854 0.96 0.61-1.50 +49 AG/GG 0.019 1.47 1.06-2.02 0.072 0.75 0.55-1.02 0.276 1.22 0.85-1.75 CT60 AG/GG 0.016 1.57 1.09-2.26 0.108 0.77 0.56-1.06 0.099 1.41 0.93-2.12

-1722 -1661 -318 +49 CT60 n chromosomes (%) T A C A A 344 (44.0) T A C G G 166 (21.3) T G T A G 62 (8.0) T G C A G 59 (7.6) C A C G G 53 (6.8) T A C A G 49 (6.3)

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Table 5. Multivariate analysis for transplantation outcome.

Patient/donor CMV+ indicates positive IgG cytomegalovirus serology. Stem cell source was stratified as peripheral blood versus bone marrow. Diagnosis was stratified in acute leukaemia/MDS versus other. Conditioning regimen was stratified in total body irradiation containing regimens versus chemotherapy alone.

Variables P value HR (95% CI) Overall Survival Early disease <0.001 0.33 (0.22-0.50) CT60 AG/GG 0.001 3.80 (1.75-8.22) +49 AG/GG 0.564 0.88 (0.56-1.37) Donor CMV+ 0.468 1.21 (0.72-2.04) Sensitized donor 0.538 1.18 (0.70-1.98) Stem cell source 0.124 1.40 (0.91-2.14) Diagnosis 0.741 1.08 (0.69-1.68) Age <30 years 0.113 1.42 (0.92-2.18) aGVHD II-IV Stem cell source 0.041 1.49 (1.01-2.20) CT60 AG/GG 0.033 0.62 (0.40-0.96) +49 AG/GG 0.587 0.87 (0.54-1.41) Donor CMV+ 0.276 1.28 (0.81-2.03) Sex mismatch 0.543 1.15 (0.73-1.79) 1722 CC/CT 0.055 1.63 (0.99-2.67) Relapse Age <30 years 0.019 0.61 (0.41-0.92) Early disease <0.001 0.37 (0.25-0.56) Patient CMV+ 0.008 2.08 (1.20-3.57) CT60 AG/GG 0.049 1.71 (1.00-2.93) Donor CMV+ 0.158 1.48 (0.86-2.53) Conditioning regimen 0.958 0.99 (0.64-1.52) Diagnosis 0.643 1.10 (0.73-1.68)

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