© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Clin Transplant 2015: 29: 612–618 DOI: 10.1111/ctr.12563
Clinical Transplantation
The influence of CTLA-4 single nucleotide polymorphisms on acute kidney allograft rejection in Turkish patients Ruhi C gin O, S€ uleymanlar G, Ersoy FF. The ß , Sallakcßi N, Ye influence of CTLA-4 single nucleotide polymorphisms on acute kidney allograft rejection in Turkish patients. Abstract: Cytotoxic T-lymphocyte antigen-4 (CTLA-4) is a cell surface protein, which down-regulates the immune response at CTLA-4/CD28/ B7 pathway. We aimed to investigate the influence of the 318C/T, +49A/G, 1661A/G and CT60A/G, and CTLA-4 gene polymorphisms on acute rejection of kidney allograft in Turkish patients. The study design was a case–control study that consists of three groups: Group 1 (n = 34) represented the kidney transplant (Ktx) recipients who experienced acute rejection, Group 2 (n = 47) was randomly assigned Ktx recipients without acute rejection, and Group 3 (n = 50) consisting of healthy volunteers to evaluate the normal genomic distribution. The polymerase chain reaction–restriction fragment length polymorphism technique was used to determine the polymorphisms. Genotype and allele frequencies among three groups denoted similar distributions for +49A/ G, 1661A/G, and CT60A/G. Conversely, 318C/T genotype was three times more frequent in the acute rejection group than in the non-rejection group (OR = 3.45; 95%CI = 1.18–10.1, p = 0.015) and two times more frequent than the healthy control group (OR = 2.45; 95% CI = 0.98 – 6.11, p = 0.047). Additionally, having a T allele at 318 position was significantly associated with acute rejection (0.147 vs. 0.043, OR = 3.45; 95% CI = 1.13–10.56, p = 0.02). 318C/T gene polymorphism and T allelic variant were found to be associated with increased acute rejection risk in Turkish kidney allograft recipients.
Cytotoxic T lymphocyte antigen 4 (CTLA-4) is a cell surface protein that plays a major role in the CTLA-4/CD28/B7 co-stimulatory pathway. This pathway represents the second signal for T-cell activation and sustains the rejection process, after the interaction of the T cell receptor with the antigens that are presented by the antigen-presenting cells (1, 2). CD28 is continuously expressed on the surface of T lymphocytes, and its interaction with B7 (CD80/CD86) yields the expansion of the immune response by the increased production of cytokines, cytokine receptors, and activation of T-cells (3). In contrast, the expression of the CTLA-4 is relatively low and increases rapidly after the initiation of the immune response. The CTLA-4 expression on activated T cells induces the suppression of the immune system (4). The interaction of CTLA-4 with CD80/CD86 downregulates the immune response via the inhibition of T-cell receptor signaling, blocking IL-2 production
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lar Ruhi, Nilgu € n Sallakc C ß ag ß i, € € leymanlar Olcay Yegin, Gultekin Su and F. Fevzi Ersoy Transplantation Center and Health Sciences Research Center, Akdeniz University Medical School, Antalya, Turkey
Key words: 318 C/T – acute rejection – cytotoxic T-lymphocyte antigen-4 – kidney transplantation – single nucleotide polymorphism lar Ruhi, Corresponding author: Dr. C ß ag Department of Nephrology, Haydarpasßa Numune Training and Research Hospital, € u €dar/I_ Tıbbiye Street, No: 40, 34668, Usk stanbul, Turkey. Tel: 90 (216) 542 32 32; fax: 90 (216) 336 05 65; e-mail: [email protected] Conflict of interest: None. Accepted for publication 9 May 2015
(5) inhibition of transcription factors (NFkB, NFAT, AP-1) (6), and increasing the CD4+ CD25+ (T-reg) lymphocyte population (7). The alternative splicing of CTLA-4 generates two different variants: one of them is the fulllength isoform (flCTLA-4) encoded by exons 1–4 and the second one is the soluble isoform (sCTLA-4) which lacks the exon 3, the transmembrane domain (8). The sCTLA-4 can be found in the human plasma, binds to CD80/CD86 ligands and is thought to be potentializing the immune response by the competitive inhibition of flCTLA-4 for binding its ligands (9). The sCTLA-4 levels have found to be elevated in various autoimmune diseases and are considered as a new potential biochemical marker for the diagnosis of autoimmune disorders (10). As CTLA-4 plays a key role in the regulation of immune response, single nucleotide polymorphism (SNP) of this gene may be responsible for the
Polymorphisms of CTLA-4 and allograft rejection reduced function and insufficient down-regulation of the immune response that leads to autoimmune disorders or allograft rejection. CTLA-4 gene SNPs have found to be associated with several autoimmune diseases such as type 1 diabetes mellitus (11), SLE (12), rheumatoid arthritis (13), and Grave’s disease (14). Regarding the CTLA-4 SNPs studies on solid organ transplantation, consequences differ significantly from each other probably due to distinct study designs, patient inclusion criteria and possible ethnical discrepancies. Therefore, we designed this study to investigate the influence of the +49A/G, 318C/T, CT60A/G and 1661A/G, and CTLA-4 SNPs on acute kidney allograft rejection in Turkish recipients.
Materials and methods Study design
The study was conducted in Akdeniz University Transplantation Center from January 2006 to December 2009. The study design was a case–control study that consists of three groups; 131 individuals were enrolled (87 male, 44 female); and all of them were adults (18–65 yr/old). Group 1 (n = 34) consisted of the kidney transplantation (Ktx) recipients who had at least one acute rejection episode during the six months of follow-up, Group 2 (n = 47) was randomly assigned Ktx recipients without acute rejection episode, and Group 3 (n = 50) were healthy volunteers who were selected from the same geographic region to evaluate the normal genomic distribution. The inclusion criteria for Ktx recipients were as follows: living donorrelated Ktx, negative lymphocyte cross-match test (LCM) prior to transplantation and having a biopsy-proven acute cellular allograft rejection for the rejectors’ group. Complement dependent cytotoxicity (CDC) was used for LCM test. Exclusion criteria for the study were having a history of autoimmune and/or inflammatory diseases, second Ktx, transplantation from a deceased donor and peak panel reactive antibody (PRA) greater than 15%. Acute rejectors with a history of non-adherence to the prescribed immunosuppressant were also excluded from the study cohort. The local ethics committee approved the investigation protocol, and written informed consents were obtained from all participants at the time of enrollment. Immunosuppressive protocols
All Ktx recipients received 1000 mg intravenous methylprednisolone post-operatively for three con-
secutive days with 50% dose reduction each day. The steroid dosage was prednisone 20 mg/d in the first month and gradually tapered to 5 mg/d through the sixth month. Anti-thymocyte globulin or monoclonal antibodies (dacluzimab or basiliximab) were used for induction of immunosuppression according to the recipient’s immunologic risk. The maintenance immunosuppressive protocol was triple therapy with prednisone, mycophenolate and calcineurin inhibitors or m-TOR inhibitors. The dose of mycophenolate was 1000 mg twice/d for mycophenolate mofetil and 720 mg twice/d for mycophenolic acid. The target through the blood concentration of the tacrolimus was 7–10 ng/mL for the first six months and 5–7 ng/mL after then. The cyclosporine dose was adjusted according to the peak concentrations of the samples collected two h post-dose (C2), and the target range was 1000–1200 ng/mL at first month, 700–1000 ng/mL for three months, and 600–900 ng/mL after the first six months. The m-TOR inhibitors were combined with steroids and calcineurin inhibitors postoperatively for three months subsequently continued with prednisone and mycophenolate. The target blood level for m-TOR inhibitors was 5–8 ng/ mL. All of the Ktx recipients were in the target range of their prescribed immunosuppressive medications during the follow-up period. In case of acute rejection, m-TOR inhibitors were withdrawn and altered to calcineurin inhibitors. The acute rejection episode was initially treated with 1000 mg methylprednisolone pulses for three consecutive days. In case of steroid resistance, the treatment was continued with antithymocyte globulin (ATG, 2–3 mg/kg/d). The ATG dosage was adjusted according to CD3 levels. Genotyping
For genotyping process, 2 mL venous blood samples were taken to EDTA-containing test tubes and stored at 80°C until the assay day. The polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) technique was applied to determine the polymorphisms. Genomic DNA was extracted by the automatic DNA isolation equipment kit (Qiagen DNA Isolation Kit, Hilden, Germany) with the magnetic bead assay to prevent contamination. DNA thermal cycler (Corbett Research) was used for the PCR amplification. Specific primer pairs were used to amplify the CTLA-4 gene to search for CT60A/G, 318C/ T, +49A/G, and 1661A/G polymorphisms (Table 1). Following the PCR, to confirm the size of amplification product, 5 lL PCR product and proper-sized DNA markers were loaded to 2%
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agarose gel. After the confirmation, specific restriction enzymes were used for cutting the product molecules. Samples were electrophoresed on 3% agarose gel (Bio-Rad, Hercules, CA, USA) for 1.5 h at 150V and stained with ethidium bromide solution (10 mg/mL) and, the gels were evaluated for specific base pairs for each polymorphism under ultraviolet light (Table 2). Fig. 1 is representing an example view of 318 region. Statistics
The results were analyzed with an SPSS 20.0 (Armonk, NY, USA) for Windows statistical package. Continuous variables were expressed as the mean ( SD) and categorical variables as a proportion (%). Comparisons among the groups were performed using Student’s t-test or ANOVA for continuous variables and the Mann–Whitney Utest or the chi-square for the categorical variables. Genotype and allele frequencies were counted directly. The chi-square test (with bootstrap method) was used for the comparison of the distribution of genotype frequencies among the groups tested with Hardy–Weinberg equilibrium. p value < 0.05 (two-sided) was considered statistically significant. Results
The study consisted of 131 individuals (81 kidney transplant recipients, 50 healthy controls). The mean ages were similar among three groups (37.7 13.4, 34.5 10.4 and 34.2 6 yr, respectively, p = 0.28). In terms of gender distribution, groups 1 and 2 had male predominance (80% vs. 74.5% male), while in Group 3, the distribution was identical (51% male, p = 0.006). There were no statistically significant differences between Group 1 and Group 2 subjects regarding the etiology of Table 1. Specific primer sequences for CTLA-4 SNPs (for all primers, annealing temperature was 55°C, SNP, single nucleotide polymorphism; F, forward; R, reverse)
SNP
Size of DNA marker (Base pairs)
CT60
216 bp
318
247 bp
+49
152 bp
1661
486 bp
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Primer sequences F 50 CAC CAC TAT TTG GGA TAT ACC G-30 R 50 AGG TCT ATA TTT CAG GAA GGC-30 F 50 AAT GAA TTG GAC TGG ATG G-30 R 50 TTA CGA GAA AGG AAG CCG TG-30 F 50 AAG GCT CAG CTG AAC CTG G-30 R 50 CTG CTG AAA CAA ATG AAA CCC-30 F 50 CAC CAC TAT TTG GGA TAT ACC G-30 R 50 AGG TCT ATA TTT CAG GAA GGC-30
Table 2. The size of the DNA markers for the confirmation of amplification, specific restriction enzymes and base pairs for each evaluated SNP (Roche, Indianapolis, IN, USA)
SNP
Size of DNA marker (Base pairs)
Restriction enzyme
CT60
216 bp
NcoI (Roche)
318
247 bp
MseI (Tru9I) (Roche)
+49
152 bp
BstEII (Roche)
1661
486 bp
MseI (Tru9I) (Roche)
Base pairs for alleles G allele 196 bp A allele 216 bp C allele 226 bp T allele 130 bp and 96 bp A allele 131 bp G allele 152 bp A allele 154 bp and 332 bp G allele 486 bp
kidney failure, dialysis modalities, total match/mismatch numbers, and immunosuppressive protocols. Patient clinical characteristics were presented in Table 3. In Group 1, acute rejection occurred with an average of 12.4 23 d (2–110 d), 80% of rejections were within the first ten d of transplantation. The multivariate analysis of acute rejection risk revealed only significant association with 318 C/T SNP (Table 4). The average follow-up times for groups 1 and 2 were 27.5 13 vs. 25.8 12.3 months, respectively. During this period, no patient or graft loss was observed in the Ktx recipients. CTLA-4 polymorphisms
CTLA-4 gene polymorphisms were evaluated for genotype and allele distribution differences among three patient groups, and the only significant difference was found at -318 promoter region. There were no significant differences for +49 A/G, 1661 A/G, CT60 A/G, their homozygote variants, and allelic distributions among the three groups (Table 5). The evaluation of 318 promoter region denoted that 318 C/T genotype was three times more frequent in the rejection group than in the non-rejection group and this relation was statistically significant (29% vs. 9% respectively, OR = 3.45; 95% CI = 1.18 to 10.1, p = 0.015). The comparison of the rejection group with the healthy control group also revealed statistically significant twofold frequent 318 C/T genotype (29% vs. 12% respectively, (OR = 2. 45; 95% CI = 0.98 to 6.11, p = 0. 047). Additionally, having a T allele in the 318 region was significantly associated with acute rejection (G1 vs. G2, 0.147 vs. 0.043 respectively, OR = 3.45; 95% CI = 1.13
Polymorphisms of CTLA-4 and allograft rejection
Fig. 1. The view of 318 region under ultraviolet light (Acute Rejection group).
Table 3. Comparison of clinical characteristics among Group 1 and Group 2 patients Group 1 (AR), n = 34 Age (years) 37.7 13.4 Gender (male %) 27–80% ESRD etiology Hypertensive 12% nephropathy Chronic 12% glomerulonephritis Urological 30% disorders/ PKD Diabetic 12% nephropathy Other etiologies 34% Dialysis modalities Preemptive 12% transplantation Hemodialysis 73% Peritoneal dialysis 15% Duration of dialysis 25.8 33.3 (months) Total mismatches 2.94 1.2 Total matches 2.94 1.0 A matches 1.06 0.60 B matches 0.85 0.61 DR matches 1.03 0.57 Induction immunosuppressive Anti-thymocyte 3% globulin (ATG) Basiliximab 61% Dacluzimab 21% None 15% Maintenance immunosuppressive Calcineurin inhibitors 45% M-TOR inhibitors 55%
Group 2 (NAR), n = 47
p-value
34.5 10.4 35–74.5%
0.23 0.60
19%
0.83
19% 17% 12% 33% 15%
0.27
64% 21% 30.9 43.4
0.57
0.51 0.37 0.91 0.15 0.64
2.77 3.17 1.04 1.04 1.09 17%
1.1 1.1 0.65 0.55 0.50
0.29
53% 13% 17% 59% 41%
0.21
AR, acute rejection; NAR, no-acute rejection; PKD, polycystic kidney disease; m-TOR, mammalian target of rapamycin.
to 10.56, p = 0.02) and was nearly statistically significant when compared G1 vs. G3 (0.147 vs. 0.060 respectively, OR = 2.45; 95% CI = 0.93 to 6.42, p = 0.06).
Table 4. The multivariate analysis of the acute rejection risk among the kidney transplant recipients Dependent variables
Mean square
OR (Odds ratio)
Sig. (p-value)
Age Gender Induction immunosuppressive Maintenance immunosuppressive Total mismatch Total match A Match B Match DR Match SNP49 SNPCT60 SNP1661 SNP318
206.180 0.048 0.256
1.484 0.263 0.281
0.227 0.610 0.598
9.179
1.166
0.283
0.606 1.035 0.005 0.709 0.061 0.384 0.920 0.115 0.862
0.434 0.813 0.013 2.140 0.214 0.894 1.652 0.474 6.352
0.512 0.370 0.910 0.147 0.645 0.347 0.202 0.493 0.014
Discussion
In the present study, we investigated the influence of four SNPs on acute rejection of kidney allograft and only found a significant association of 318 C/T gene polymorphism in the promoter region. 318C/T genotype and having a T allele in this position were significantly detected frequently at the Ktx recipients who had acute rejection than the non-rejectors and the healthy individuals. The impact of 318 promoter region SNP on allograft rejection is not clearly identified. Though several studies did not show any relationship (15–17), one of the former studies suggested that the presence of the T allele in 318 position might be preventive, but the relationship was weak and statistically insignificant (18). Conversely, several studies stated that 318C/T genotype and the T allelic variant were associated with increased acute rejection risk. For example, De Revuer et al. (19) reported an association between 318C/T and allograft rejec-
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Ruhi et al. Table 5. Comparison of the CTLA-4 SNPs genotype and allele frequencies among the groups Group 1 (AR), n = 34 318 C/T C/C 24 (71%) C/T 10 (29%) T/T 0 C 0.853 T 0.147 +49 A/G A/A 13 (38%) A/G 17 (50%) G/G 4 (12%) A 0.630 G 0.370 CT60 A/G A/A 7 (21%) A/G 17 (50%) G/G 10 (29%) A 0.455 G 0.545 1661 A/G A/A 22 (65%) A/G 12 (35%) G/G 0 A 0.823 G 0.177
Group 2 (NAR), n = 47
Group 3 (Healthy), n = 50
43 (91%) 4 (9%) 0 0.957 0.043
44 (88%) 6 (12%) 0 0.940 0.060
23 (50%) 20 (42%) 4 (8%) 0.700 0.300
24 (48%) 22 (44%) 4 (8%) 0.700 0.300
17 (36%) 19 (40%) 11 (24%) 0.560 0.440
17 (34%) 24 (48%) 9 (18%) 0.580 0.420
35 (75%) 11 (23%) 1 (2%) 0.860 0.140
34 (68%) 16 (32%) 0 0.840 0.160
G1 vs. G2, p value
G1 vs. G3, p value
p = 0.015
p = 0.047
p = 0.020
p = 0.060
p = 0.33
p = 0.34
p = 0.19
p = 0.12
p = 0.38
p = 0.75
tion in liver transplantation; while the relationship was statistically significant in univariate analysis, they concluded that it might possibly be inadequate to be effective per se. Similarly, Dimitrenko et al. found that 318C/T SNP was 2.5 times more frequent in the Ktx recipients who had acute rejection; however, the difference did not reach the statistical significance (20). In a recent study, 318 T/ T genotype was found to be four times frequent in the Chinese Ktx recipients who experienced acute rejection than the non-rejectors (8.9% vs. 1.6%); however, the significance disappeared after Bonferroni correction, and they concluded that susceptibility to acute rejection may not be the same among different ethnicities (21). These studies are consistent with our results that 318C/T gene polymorphism and T allelic variant may be considered to create some predisposition for acute allograft rejection, although available evidence is not sufficient to point the polymorphic state as a single causative factor. As we evaluate the 318 promoter region at the transcriptional and functional level, having a T allele instead of C allele in this position has shown to be linked to increased expression and arrangement of both two isoforms of CTLA-4 (flCTLA-4 and sCTLA-4) (22, 23). The sCTLA-4 has binding
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capacity to CD80/CD86. The elevated levels of sCTLA-4 have been found to be related to various autoimmune disorders, including rheumatoid arthritis (24), Crohn’s disease (25), and celiac disease (26), and were considered as a potential new biomarker for the diagnoses of autoimmune diseases (10). Additionally, in a recent study, it was shown to be associated with inflammatory markers such as IL-6, TNF-alpha, IFN-gamma, and IL-8 (27). Therefore, sCTLA-4 may be functionally different from flCTLA4 and may promote the activation of the immune system instead of its inhibition. Several studies revealed that the effect of sCTLA-4 on T-cell population is dose- and time dependent. During the inactive state of the immune system, the expression of sCTLA-4 may be low and it inhibits the effects of CD28 by binding to CD80/ CD86. Nevertheless, when the immune system is activated, sCTLA-4 probably represents a rapid increase and inhibits the effects of flCTLA-4 by competing for the same binding site (28). Considering these data, the relationship between acute rejection and 318C/T polymorphism can be explained theoretically. Any increase in the CTLA-4 expression due to 318 T allelic variant extremely increases sCTLA-4 expression and the competition with flCTLA4 to bind to the CD80/CD86 ligands. Consequently, sCTLA-4 may block the effects of flCTLA-4 and it inhibits the down-regulation of T-lymphocyte population. The influence of other two genetic polymorphisms CT60A/G and 1661A/G have not been widely investigated in Ktx. Although Muro et al. (29) found that CT60 G allelic variant might be associated with an increased risk of rejection in patients with liver transplantation, we did not find any differences between allelic variants at Ktx. Additionally, similar to a recent study, we did not find any relationship between 1661 A/G SNP and acute rejection (21). The other area of interest, +49A/G, is found in exon-1, gene polymorphisms in this site may change the cell surface expression of CTLA-4 and have shown to be related to autoimmune disorders (30, 31). In our study, we found + 49A/G genotype and G allelic variant to be unrelated to acute rejection risk, in harmony with a recent meta-analysis that revealed no relations in most studies (32). The exceptions are the findings of Gendzekhadze et al. (16), +49A/A genotype significantly prevents acute rejection in Ktx, and conversely De Reuver et al. (19), +49G allelic variant prevents in liver transplantation. Considering our findings and the results of the studies mentioned above, we might suggest that the role of gene polymorphism in the +49 site in the pathogenesis of acute rejection of
Polymorphisms of CTLA-4 and allograft rejection kidney allografts might be still unclear and probably its effect per se may not be enough to cause acute rejection in kidney allografts. Our results have shown inconsistence with various studies, especially for 318C/T gene polymorphism, the explanation may include both ethnical differences and different study designs. In our study, we aimed to expose the net effects of CTLA4 SNPs on acute kidney allograft rejection. For this purpose, only living-related Ktx recipients were included, to eliminate potential risk factors for increased rejection risk at cadaveric transplantation such as number of mismatches and cold ischemia time. As supporting our opinion, Gendhankze et al. (16) concluded a similar association about 49A/A in only living-related Ktx recipients. As we considered that most studies included only cadaveric Ktx recipients, our distinct results might be explained. It may be hypothesized that the immunologic risk factors for cadaveric transplantation are more crucial than the genetic polymorphisms and the SNPs are not sufficient to be a single cause of acute rejection. As a conclusion, 318C/T gene polymorphism and T allelic variant were significantly associated with increased acute rejection risk in Turkish kidney allograft recipients. The effects of T allelic variant on the 318 position still need to be investigated in a proper designed large-scale solid organ transplantation studies that include detailed transcriptional and functional-level investigations.
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Acknowledgements This study was supported by the Akdeniz University Research Fund. Akdeniz University, Antalya, Turkey.
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Clin Transplant 2015: 29: 612–618 DOI: 10.1111/ctr.12563
Clinical Transplantation
The influence of CTLA-4 single nucleotide polymorphisms on acute kidney allograft rejection in Turkish patients Ruhi C gin O, S€ uleymanlar G, Ersoy FF. The ß , Sallakcßi N, Ye influence of CTLA-4 single nucleotide polymorphisms on acute kidney allograft rejection in Turkish patients. Abstract: Cytotoxic T-lymphocyte antigen-4 (CTLA-4) is a cell surface protein, which down-regulates the immune response at CTLA-4/CD28/ B7 pathway. We aimed to investigate the influence of the 318C/T, +49A/G, 1661A/G and CT60A/G, and CTLA-4 gene polymorphisms on acute rejection of kidney allograft in Turkish patients. The study design was a case–control study that consists of three groups: Group 1 (n = 34) represented the kidney transplant (Ktx) recipients who experienced acute rejection, Group 2 (n = 47) was randomly assigned Ktx recipients without acute rejection, and Group 3 (n = 50) consisting of healthy volunteers to evaluate the normal genomic distribution. The polymerase chain reaction–restriction fragment length polymorphism technique was used to determine the polymorphisms. Genotype and allele frequencies among three groups denoted similar distributions for +49A/ G, 1661A/G, and CT60A/G. Conversely, 318C/T genotype was three times more frequent in the acute rejection group than in the non-rejection group (OR = 3.45; 95%CI = 1.18–10.1, p = 0.015) and two times more frequent than the healthy control group (OR = 2.45; 95% CI = 0.98 – 6.11, p = 0.047). Additionally, having a T allele at 318 position was significantly associated with acute rejection (0.147 vs. 0.043, OR = 3.45; 95% CI = 1.13–10.56, p = 0.02). 318C/T gene polymorphism and T allelic variant were found to be associated with increased acute rejection risk in Turkish kidney allograft recipients.
Cytotoxic T lymphocyte antigen 4 (CTLA-4) is a cell surface protein that plays a major role in the CTLA-4/CD28/B7 co-stimulatory pathway. This pathway represents the second signal for T-cell activation and sustains the rejection process, after the interaction of the T cell receptor with the antigens that are presented by the antigen-presenting cells (1, 2). CD28 is continuously expressed on the surface of T lymphocytes, and its interaction with B7 (CD80/CD86) yields the expansion of the immune response by the increased production of cytokines, cytokine receptors, and activation of T-cells (3). In contrast, the expression of the CTLA-4 is relatively low and increases rapidly after the initiation of the immune response. The CTLA-4 expression on activated T cells induces the suppression of the immune system (4). The interaction of CTLA-4 with CD80/CD86 downregulates the immune response via the inhibition of T-cell receptor signaling, blocking IL-2 production
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lar Ruhi, Nilgu € n Sallakc C ß ag ß i, € € leymanlar Olcay Yegin, Gultekin Su and F. Fevzi Ersoy Transplantation Center and Health Sciences Research Center, Akdeniz University Medical School, Antalya, Turkey
Key words: 318 C/T – acute rejection – cytotoxic T-lymphocyte antigen-4 – kidney transplantation – single nucleotide polymorphism lar Ruhi, Corresponding author: Dr. C ß ag Department of Nephrology, Haydarpasßa Numune Training and Research Hospital, € u €dar/I_ Tıbbiye Street, No: 40, 34668, Usk stanbul, Turkey. Tel: 90 (216) 542 32 32; fax: 90 (216) 336 05 65; e-mail: [email protected] Conflict of interest: None. Accepted for publication 9 May 2015
(5) inhibition of transcription factors (NFkB, NFAT, AP-1) (6), and increasing the CD4+ CD25+ (T-reg) lymphocyte population (7). The alternative splicing of CTLA-4 generates two different variants: one of them is the fulllength isoform (flCTLA-4) encoded by exons 1–4 and the second one is the soluble isoform (sCTLA-4) which lacks the exon 3, the transmembrane domain (8). The sCTLA-4 can be found in the human plasma, binds to CD80/CD86 ligands and is thought to be potentializing the immune response by the competitive inhibition of flCTLA-4 for binding its ligands (9). The sCTLA-4 levels have found to be elevated in various autoimmune diseases and are considered as a new potential biochemical marker for the diagnosis of autoimmune disorders (10). As CTLA-4 plays a key role in the regulation of immune response, single nucleotide polymorphism (SNP) of this gene may be responsible for the
Polymorphisms of CTLA-4 and allograft rejection reduced function and insufficient down-regulation of the immune response that leads to autoimmune disorders or allograft rejection. CTLA-4 gene SNPs have found to be associated with several autoimmune diseases such as type 1 diabetes mellitus (11), SLE (12), rheumatoid arthritis (13), and Grave’s disease (14). Regarding the CTLA-4 SNPs studies on solid organ transplantation, consequences differ significantly from each other probably due to distinct study designs, patient inclusion criteria and possible ethnical discrepancies. Therefore, we designed this study to investigate the influence of the +49A/G, 318C/T, CT60A/G and 1661A/G, and CTLA-4 SNPs on acute kidney allograft rejection in Turkish recipients.
Materials and methods Study design
The study was conducted in Akdeniz University Transplantation Center from January 2006 to December 2009. The study design was a case–control study that consists of three groups; 131 individuals were enrolled (87 male, 44 female); and all of them were adults (18–65 yr/old). Group 1 (n = 34) consisted of the kidney transplantation (Ktx) recipients who had at least one acute rejection episode during the six months of follow-up, Group 2 (n = 47) was randomly assigned Ktx recipients without acute rejection episode, and Group 3 (n = 50) were healthy volunteers who were selected from the same geographic region to evaluate the normal genomic distribution. The inclusion criteria for Ktx recipients were as follows: living donorrelated Ktx, negative lymphocyte cross-match test (LCM) prior to transplantation and having a biopsy-proven acute cellular allograft rejection for the rejectors’ group. Complement dependent cytotoxicity (CDC) was used for LCM test. Exclusion criteria for the study were having a history of autoimmune and/or inflammatory diseases, second Ktx, transplantation from a deceased donor and peak panel reactive antibody (PRA) greater than 15%. Acute rejectors with a history of non-adherence to the prescribed immunosuppressant were also excluded from the study cohort. The local ethics committee approved the investigation protocol, and written informed consents were obtained from all participants at the time of enrollment. Immunosuppressive protocols
All Ktx recipients received 1000 mg intravenous methylprednisolone post-operatively for three con-
secutive days with 50% dose reduction each day. The steroid dosage was prednisone 20 mg/d in the first month and gradually tapered to 5 mg/d through the sixth month. Anti-thymocyte globulin or monoclonal antibodies (dacluzimab or basiliximab) were used for induction of immunosuppression according to the recipient’s immunologic risk. The maintenance immunosuppressive protocol was triple therapy with prednisone, mycophenolate and calcineurin inhibitors or m-TOR inhibitors. The dose of mycophenolate was 1000 mg twice/d for mycophenolate mofetil and 720 mg twice/d for mycophenolic acid. The target through the blood concentration of the tacrolimus was 7–10 ng/mL for the first six months and 5–7 ng/mL after then. The cyclosporine dose was adjusted according to the peak concentrations of the samples collected two h post-dose (C2), and the target range was 1000–1200 ng/mL at first month, 700–1000 ng/mL for three months, and 600–900 ng/mL after the first six months. The m-TOR inhibitors were combined with steroids and calcineurin inhibitors postoperatively for three months subsequently continued with prednisone and mycophenolate. The target blood level for m-TOR inhibitors was 5–8 ng/ mL. All of the Ktx recipients were in the target range of their prescribed immunosuppressive medications during the follow-up period. In case of acute rejection, m-TOR inhibitors were withdrawn and altered to calcineurin inhibitors. The acute rejection episode was initially treated with 1000 mg methylprednisolone pulses for three consecutive days. In case of steroid resistance, the treatment was continued with antithymocyte globulin (ATG, 2–3 mg/kg/d). The ATG dosage was adjusted according to CD3 levels. Genotyping
For genotyping process, 2 mL venous blood samples were taken to EDTA-containing test tubes and stored at 80°C until the assay day. The polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) technique was applied to determine the polymorphisms. Genomic DNA was extracted by the automatic DNA isolation equipment kit (Qiagen DNA Isolation Kit, Hilden, Germany) with the magnetic bead assay to prevent contamination. DNA thermal cycler (Corbett Research) was used for the PCR amplification. Specific primer pairs were used to amplify the CTLA-4 gene to search for CT60A/G, 318C/ T, +49A/G, and 1661A/G polymorphisms (Table 1). Following the PCR, to confirm the size of amplification product, 5 lL PCR product and proper-sized DNA markers were loaded to 2%
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agarose gel. After the confirmation, specific restriction enzymes were used for cutting the product molecules. Samples were electrophoresed on 3% agarose gel (Bio-Rad, Hercules, CA, USA) for 1.5 h at 150V and stained with ethidium bromide solution (10 mg/mL) and, the gels were evaluated for specific base pairs for each polymorphism under ultraviolet light (Table 2). Fig. 1 is representing an example view of 318 region. Statistics
The results were analyzed with an SPSS 20.0 (Armonk, NY, USA) for Windows statistical package. Continuous variables were expressed as the mean ( SD) and categorical variables as a proportion (%). Comparisons among the groups were performed using Student’s t-test or ANOVA for continuous variables and the Mann–Whitney Utest or the chi-square for the categorical variables. Genotype and allele frequencies were counted directly. The chi-square test (with bootstrap method) was used for the comparison of the distribution of genotype frequencies among the groups tested with Hardy–Weinberg equilibrium. p value < 0.05 (two-sided) was considered statistically significant. Results
The study consisted of 131 individuals (81 kidney transplant recipients, 50 healthy controls). The mean ages were similar among three groups (37.7 13.4, 34.5 10.4 and 34.2 6 yr, respectively, p = 0.28). In terms of gender distribution, groups 1 and 2 had male predominance (80% vs. 74.5% male), while in Group 3, the distribution was identical (51% male, p = 0.006). There were no statistically significant differences between Group 1 and Group 2 subjects regarding the etiology of Table 1. Specific primer sequences for CTLA-4 SNPs (for all primers, annealing temperature was 55°C, SNP, single nucleotide polymorphism; F, forward; R, reverse)
SNP
Size of DNA marker (Base pairs)
CT60
216 bp
318
247 bp
+49
152 bp
1661
486 bp
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Primer sequences F 50 CAC CAC TAT TTG GGA TAT ACC G-30 R 50 AGG TCT ATA TTT CAG GAA GGC-30 F 50 AAT GAA TTG GAC TGG ATG G-30 R 50 TTA CGA GAA AGG AAG CCG TG-30 F 50 AAG GCT CAG CTG AAC CTG G-30 R 50 CTG CTG AAA CAA ATG AAA CCC-30 F 50 CAC CAC TAT TTG GGA TAT ACC G-30 R 50 AGG TCT ATA TTT CAG GAA GGC-30
Table 2. The size of the DNA markers for the confirmation of amplification, specific restriction enzymes and base pairs for each evaluated SNP (Roche, Indianapolis, IN, USA)
SNP
Size of DNA marker (Base pairs)
Restriction enzyme
CT60
216 bp
NcoI (Roche)
318
247 bp
MseI (Tru9I) (Roche)
+49
152 bp
BstEII (Roche)
1661
486 bp
MseI (Tru9I) (Roche)
Base pairs for alleles G allele 196 bp A allele 216 bp C allele 226 bp T allele 130 bp and 96 bp A allele 131 bp G allele 152 bp A allele 154 bp and 332 bp G allele 486 bp
kidney failure, dialysis modalities, total match/mismatch numbers, and immunosuppressive protocols. Patient clinical characteristics were presented in Table 3. In Group 1, acute rejection occurred with an average of 12.4 23 d (2–110 d), 80% of rejections were within the first ten d of transplantation. The multivariate analysis of acute rejection risk revealed only significant association with 318 C/T SNP (Table 4). The average follow-up times for groups 1 and 2 were 27.5 13 vs. 25.8 12.3 months, respectively. During this period, no patient or graft loss was observed in the Ktx recipients. CTLA-4 polymorphisms
CTLA-4 gene polymorphisms were evaluated for genotype and allele distribution differences among three patient groups, and the only significant difference was found at -318 promoter region. There were no significant differences for +49 A/G, 1661 A/G, CT60 A/G, their homozygote variants, and allelic distributions among the three groups (Table 5). The evaluation of 318 promoter region denoted that 318 C/T genotype was three times more frequent in the rejection group than in the non-rejection group and this relation was statistically significant (29% vs. 9% respectively, OR = 3.45; 95% CI = 1.18 to 10.1, p = 0.015). The comparison of the rejection group with the healthy control group also revealed statistically significant twofold frequent 318 C/T genotype (29% vs. 12% respectively, (OR = 2. 45; 95% CI = 0.98 to 6.11, p = 0. 047). Additionally, having a T allele in the 318 region was significantly associated with acute rejection (G1 vs. G2, 0.147 vs. 0.043 respectively, OR = 3.45; 95% CI = 1.13
Polymorphisms of CTLA-4 and allograft rejection
Fig. 1. The view of 318 region under ultraviolet light (Acute Rejection group).
Table 3. Comparison of clinical characteristics among Group 1 and Group 2 patients Group 1 (AR), n = 34 Age (years) 37.7 13.4 Gender (male %) 27–80% ESRD etiology Hypertensive 12% nephropathy Chronic 12% glomerulonephritis Urological 30% disorders/ PKD Diabetic 12% nephropathy Other etiologies 34% Dialysis modalities Preemptive 12% transplantation Hemodialysis 73% Peritoneal dialysis 15% Duration of dialysis 25.8 33.3 (months) Total mismatches 2.94 1.2 Total matches 2.94 1.0 A matches 1.06 0.60 B matches 0.85 0.61 DR matches 1.03 0.57 Induction immunosuppressive Anti-thymocyte 3% globulin (ATG) Basiliximab 61% Dacluzimab 21% None 15% Maintenance immunosuppressive Calcineurin inhibitors 45% M-TOR inhibitors 55%
Group 2 (NAR), n = 47
p-value
34.5 10.4 35–74.5%
0.23 0.60
19%
0.83
19% 17% 12% 33% 15%
0.27
64% 21% 30.9 43.4
0.57
0.51 0.37 0.91 0.15 0.64
2.77 3.17 1.04 1.04 1.09 17%
1.1 1.1 0.65 0.55 0.50
0.29
53% 13% 17% 59% 41%
0.21
AR, acute rejection; NAR, no-acute rejection; PKD, polycystic kidney disease; m-TOR, mammalian target of rapamycin.
to 10.56, p = 0.02) and was nearly statistically significant when compared G1 vs. G3 (0.147 vs. 0.060 respectively, OR = 2.45; 95% CI = 0.93 to 6.42, p = 0.06).
Table 4. The multivariate analysis of the acute rejection risk among the kidney transplant recipients Dependent variables
Mean square
OR (Odds ratio)
Sig. (p-value)
Age Gender Induction immunosuppressive Maintenance immunosuppressive Total mismatch Total match A Match B Match DR Match SNP49 SNPCT60 SNP1661 SNP318
206.180 0.048 0.256
1.484 0.263 0.281
0.227 0.610 0.598
9.179
1.166
0.283
0.606 1.035 0.005 0.709 0.061 0.384 0.920 0.115 0.862
0.434 0.813 0.013 2.140 0.214 0.894 1.652 0.474 6.352
0.512 0.370 0.910 0.147 0.645 0.347 0.202 0.493 0.014
Discussion
In the present study, we investigated the influence of four SNPs on acute rejection of kidney allograft and only found a significant association of 318 C/T gene polymorphism in the promoter region. 318C/T genotype and having a T allele in this position were significantly detected frequently at the Ktx recipients who had acute rejection than the non-rejectors and the healthy individuals. The impact of 318 promoter region SNP on allograft rejection is not clearly identified. Though several studies did not show any relationship (15–17), one of the former studies suggested that the presence of the T allele in 318 position might be preventive, but the relationship was weak and statistically insignificant (18). Conversely, several studies stated that 318C/T genotype and the T allelic variant were associated with increased acute rejection risk. For example, De Revuer et al. (19) reported an association between 318C/T and allograft rejec-
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Ruhi et al. Table 5. Comparison of the CTLA-4 SNPs genotype and allele frequencies among the groups Group 1 (AR), n = 34 318 C/T C/C 24 (71%) C/T 10 (29%) T/T 0 C 0.853 T 0.147 +49 A/G A/A 13 (38%) A/G 17 (50%) G/G 4 (12%) A 0.630 G 0.370 CT60 A/G A/A 7 (21%) A/G 17 (50%) G/G 10 (29%) A 0.455 G 0.545 1661 A/G A/A 22 (65%) A/G 12 (35%) G/G 0 A 0.823 G 0.177
Group 2 (NAR), n = 47
Group 3 (Healthy), n = 50
43 (91%) 4 (9%) 0 0.957 0.043
44 (88%) 6 (12%) 0 0.940 0.060
23 (50%) 20 (42%) 4 (8%) 0.700 0.300
24 (48%) 22 (44%) 4 (8%) 0.700 0.300
17 (36%) 19 (40%) 11 (24%) 0.560 0.440
17 (34%) 24 (48%) 9 (18%) 0.580 0.420
35 (75%) 11 (23%) 1 (2%) 0.860 0.140
34 (68%) 16 (32%) 0 0.840 0.160
G1 vs. G2, p value
G1 vs. G3, p value
p = 0.015
p = 0.047
p = 0.020
p = 0.060
p = 0.33
p = 0.34
p = 0.19
p = 0.12
p = 0.38
p = 0.75
tion in liver transplantation; while the relationship was statistically significant in univariate analysis, they concluded that it might possibly be inadequate to be effective per se. Similarly, Dimitrenko et al. found that 318C/T SNP was 2.5 times more frequent in the Ktx recipients who had acute rejection; however, the difference did not reach the statistical significance (20). In a recent study, 318 T/ T genotype was found to be four times frequent in the Chinese Ktx recipients who experienced acute rejection than the non-rejectors (8.9% vs. 1.6%); however, the significance disappeared after Bonferroni correction, and they concluded that susceptibility to acute rejection may not be the same among different ethnicities (21). These studies are consistent with our results that 318C/T gene polymorphism and T allelic variant may be considered to create some predisposition for acute allograft rejection, although available evidence is not sufficient to point the polymorphic state as a single causative factor. As we evaluate the 318 promoter region at the transcriptional and functional level, having a T allele instead of C allele in this position has shown to be linked to increased expression and arrangement of both two isoforms of CTLA-4 (flCTLA-4 and sCTLA-4) (22, 23). The sCTLA-4 has binding
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capacity to CD80/CD86. The elevated levels of sCTLA-4 have been found to be related to various autoimmune disorders, including rheumatoid arthritis (24), Crohn’s disease (25), and celiac disease (26), and were considered as a potential new biomarker for the diagnoses of autoimmune diseases (10). Additionally, in a recent study, it was shown to be associated with inflammatory markers such as IL-6, TNF-alpha, IFN-gamma, and IL-8 (27). Therefore, sCTLA-4 may be functionally different from flCTLA4 and may promote the activation of the immune system instead of its inhibition. Several studies revealed that the effect of sCTLA-4 on T-cell population is dose- and time dependent. During the inactive state of the immune system, the expression of sCTLA-4 may be low and it inhibits the effects of CD28 by binding to CD80/ CD86. Nevertheless, when the immune system is activated, sCTLA-4 probably represents a rapid increase and inhibits the effects of flCTLA-4 by competing for the same binding site (28). Considering these data, the relationship between acute rejection and 318C/T polymorphism can be explained theoretically. Any increase in the CTLA-4 expression due to 318 T allelic variant extremely increases sCTLA-4 expression and the competition with flCTLA4 to bind to the CD80/CD86 ligands. Consequently, sCTLA-4 may block the effects of flCTLA-4 and it inhibits the down-regulation of T-lymphocyte population. The influence of other two genetic polymorphisms CT60A/G and 1661A/G have not been widely investigated in Ktx. Although Muro et al. (29) found that CT60 G allelic variant might be associated with an increased risk of rejection in patients with liver transplantation, we did not find any differences between allelic variants at Ktx. Additionally, similar to a recent study, we did not find any relationship between 1661 A/G SNP and acute rejection (21). The other area of interest, +49A/G, is found in exon-1, gene polymorphisms in this site may change the cell surface expression of CTLA-4 and have shown to be related to autoimmune disorders (30, 31). In our study, we found + 49A/G genotype and G allelic variant to be unrelated to acute rejection risk, in harmony with a recent meta-analysis that revealed no relations in most studies (32). The exceptions are the findings of Gendzekhadze et al. (16), +49A/A genotype significantly prevents acute rejection in Ktx, and conversely De Reuver et al. (19), +49G allelic variant prevents in liver transplantation. Considering our findings and the results of the studies mentioned above, we might suggest that the role of gene polymorphism in the +49 site in the pathogenesis of acute rejection of
Polymorphisms of CTLA-4 and allograft rejection kidney allografts might be still unclear and probably its effect per se may not be enough to cause acute rejection in kidney allografts. Our results have shown inconsistence with various studies, especially for 318C/T gene polymorphism, the explanation may include both ethnical differences and different study designs. In our study, we aimed to expose the net effects of CTLA4 SNPs on acute kidney allograft rejection. For this purpose, only living-related Ktx recipients were included, to eliminate potential risk factors for increased rejection risk at cadaveric transplantation such as number of mismatches and cold ischemia time. As supporting our opinion, Gendhankze et al. (16) concluded a similar association about 49A/A in only living-related Ktx recipients. As we considered that most studies included only cadaveric Ktx recipients, our distinct results might be explained. It may be hypothesized that the immunologic risk factors for cadaveric transplantation are more crucial than the genetic polymorphisms and the SNPs are not sufficient to be a single cause of acute rejection. As a conclusion, 318C/T gene polymorphism and T allelic variant were significantly associated with increased acute rejection risk in Turkish kidney allograft recipients. The effects of T allelic variant on the 318 position still need to be investigated in a proper designed large-scale solid organ transplantation studies that include detailed transcriptional and functional-level investigations.
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Acknowledgements This study was supported by the Akdeniz University Research Fund. Akdeniz University, Antalya, Turkey.
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