Objectives: Costimulatory gene polymorphisms have been proposed to affect the hepatitis B virus pathogenesis by affecting regulation of immune responses. The association between costimulatory molecule gene polymorphisms including CTLA4, PD.1, ICOS, and CD28 with hepatitis B virus infection has been evaluated in hematopoietic stem cell transplant patients.

Materials and Methods: In a cross-sectional study, single-nucleotide polymorphisms in the loci of the costimulatory molecules were analyzed in 3 study groups. Hepatitis B virus infection was evaluated in plasma samples of each allogeneic and autologous hematopoietic stem cell transplant patients by a third generation HBsAg enzyme-linked immunosorbent assay kit according to the manufacturer’s instructions.

Results: Hepatitis B virus infection was found in 19 of 72 allogeneic (26.3%) and 26 of 59 autologous patients (44.1%). The T allele of CTLA4-318 and CC genotype of CD28+17 polymorphisms are significantly more frequent in hepatitis B virus-infected allogeneic hematopoietic stem cell transplant patients. The CC genotype of CD28 +17 was seen more frequently in hepatitis B virus-infected allogeneic hematopoietic transplant patients. The C allele of the PD.1.9 was seen more frequently in hepatitis B virus-infected allogeneic hematopoietic patients experiencing graft-versus-host disease. Also, the frequency of CT genotype and T allele of the PD.1.9 was significantly increased in hepatitis B virus-infected allogeneic hematopoietic stem cell transplant patients experiencing low-grade graft-versus-host disease.

Conclusions: Associations of CTLA4 -318 and CD28 +17 with hepatitis B virus infection in allogeneic hematopoietic stem cell transplant patients was reported, also it was determined that PD.1.9 genotypes and hepatitis B virus infection in allogeneic hematopoietic stem cell transplant patients experiencing low-grade graftversus- host disease was associated. However, better evaluations of the relations between costimulatory gene polymorphisms with hepatitis B virus infection in hematopoietic stem cell transplant patients requires further investigations.

Key words : Hepatitis B virus; Stem cell transplantation, Graft-versus-host disease, PDCD1, CTLA4

Introduction

Potent immune response is essential for the complete struggle with hepatitis B virus (HBV) infection and widespread disease spectrum. Inefficiency of the host immune responses has been associated with the chronic and complicated HBV infection.^1,2 Immuno-logic genetic factors have played a role in altering the immune response and particularly, the outcome of HBV infection.^1,3 Therefore, understanding single nucleotide polymorphisms (SNPs) of several immunoregulatory genes (ie, costimulatory molecules) may be crucial for developing effective strategies against HBV.

Cytotoxic T-lymphocyte antigen-4 (CTLA4, also known as CD152) encoded by a gene in chromosome 2q33, plays a pivotal role in the negative regulation of T-cell proliferation, activates and indirectly controls effector T cells by its constitutive expression on T-regulatory cells leading to preservation of T-cell homeostasis.^4-6 To date, more than 10 SNPs have been identified in the CTLA-4 gene region.⁷ Single nucleotide polymorphisms in the promoter region can affect the binding affinity of transcriptional factor involved in regulating gene expression.⁸

Cytotoxic T-lymphocyte antigen-4 gene polymorphisms, which may affect the function of CTLA4 in regulating the immune response, has been proposed to affect the susceptibility and chronicity of the disease in patients with HBV infection; however, the results are still controversial.^9-12,2 Similar to CTLA4, CD28 also binds to B7 family of receptors (CD80 and CD86), but it provides a positive costimulatory signal for T-cell proliferative response after being expressed on T cells.^13,14

Several polymorphisms in the CD28 gene may affect the intensity of T-cell–mediated immunity, resulting in various autoimmune diseases, and the occurrence of post transplant allograft rejection.^15,16 Moreover, the CD28 gene polymorphism has been shown to affect the inhibitory/activating functions of CD28.¹⁷

The programmed cell death-1 (PDCD-1 or PD.1) gene is another costimulatory molecule located in chromosome 2q37.3, encoding a 55 kDa glycoprotein member of CD28 immunoglobulin superfamily receptors.^18,19 Programmed cell death-1 is an inhibitory immunoreceptor expressed on different hematopoietic cell types including activated T cells, B cells, and myeloid cells, plays a crucial role in the down-regulation of immune responses.^3,20,21 Studies have shown a strong association between HBV viremia and higher expression of PD.1 on T cells. Anti-HBV therapy-induced suppression of HBV replication results in a significant reduction of PD.1 transcription and PD.1 expression on the T-cell surface; thus, reducing its negative effect on T-cell activation and function.^22-25 Also, PD.1 gene polymorphisms have recently been demonstrated to be associated with the susceptibility and disease progression of chronic HBV infection.¹

Inducible costimulator (ICOS) is a T-cell positive regulator that can regulate helper T-cell differentiation.²⁶ Its functions also are involved in B-cell development and antibody secretion.²⁷ Previous studies indicate that the ICOS gene polymorphism is related to autoimmune diseases.²⁶ Additionally, blockade of ICOS stimulation could prolong cardiac and liver allograft. Hematopoietic stem cell transplant (HSCT) has been studied only in a mouse model, and the results are inconclusive.^26,30,31 Therefore, in this study, the possible association between costimulatory molecule gene polymorp­hisms including CTLA4, PD.1, ICOS, and CD28 with HBV infection was evaluated in HSCT patients.

Materials and Methods

Patients and samples
All of the patients received HSCT at the Bone Marrow Transplant Unit of Namazee Hospital affiliated to the Shiraz University of Medical Sciences, between 2008 and 2012 were enrolled in this cross-sectional study. The studied patients were subgrouped to 72 allogeneic and 59 autologous HSCT recipients. The EDTA-treated blood samples were collected from each allogeneic and autologous transplant patients after HSCT.

Acute graft-versus-host disease (GVHD) has been graded according to the classic Glucksberg-Seattle criteria and the International Bone Marrow Transplant Registry.^32-34 Signs and symptoms were identified by an expert European hematology team for blood and marrow transplant criteria. This study was approved by Research Ethics Committee of our institute. Written, informed consent was obtained from all of the patients, and all of the protocols conformed with the ethical guidelines of the 1975 Helsinki Declaration.

Conditioning chemotherapy regimen included busulfan 16 mg/kg, and cyclophosphamide 120 to 200 mg/kg in leukemia patients (acute myelogenous leukemia, acute lymphogenous leukemia, and chronic myelogenous leukemia), and cyclophosphamide 60 to 120 mg/kg +ATG 90 kg/mg for severe aplastic anemia and Fanconi’s anemia. Graft-versus-host disease prophylaxis consisted of cyclosporine and methotrexate. Prophylactic antibiotic, antifungal, and antiviral drugs were prescribed for all patients. All blood products were irradiated with gamma rays to prevent posttransfusion GVHD. Human leukocyte antigen typing is routine in our center.

DNA extraction
Genomic DNA was extracted from each Buffy coat of EDTA-treated blood samples by (DNP kit, CinnaGen,Tehran, Iran) according to the manu-facturer’s instruction.

Polymerase chain reaction restriction fragment length polymorphism protocols
The SNPs of costimulatory molecule genes including the CTLA4 gene,^35,36 the PD.1 gene,^37,38 the ICOS gene,³⁹ and the CD28 gene⁴⁰ in chromosome 2q33-37, were evaluated by polymerase chain reaction restriction fragment length polymorphism methods. After PCR, products were digested by restriction enzyme, and the amplified products were monitored by agarose gel electrophoresis and ethidium bromide staining.

Hepatitis B virus enzyme-linked immunosorbent assay
The presence of hepatitis B virus infection (HBsAg) was evaluated in plasma samples of each allogeneic and autologous HSCT patients by a third generation of enzyme-linked immunosorbent assay (ELISA) kits (Dia.Pro - Diagnostic Bioprobes; Milan, Italy) according to manufacturer’s instruction.

Statistical analyses
Allele and genotype frequencies were calculated in both allogeneic and autologous HSCT patients by direct gene counting. Statistical analyses were performed with SPSS software (SPSS: An IBM Company, version 15.0, IBM Corporation, Armonk, NY, USA). The frequencies of alleles/genotypes and the relation between costimulatory molecule SNPs with HBV infection were analyzed in both groups of HSCT patients using the chi-square test, and the Fisher exact test. The odds ratios and 95% confidence intervals (CIs) for relative risks (RRs) were calculated. A 2-tailed test with P < .05 was considered statistically significant. Arlequin software version 3.1 was used to estimate Hardy-Weinberg equilibrium.

Results

Patients’ profiles
62.5% of the allogeneic transplant patients were male, and 37.5% were female (age range, 7-52 y). All of allogeneic patients who received a graft from related HLA-matched donors and were subgrouped to experienced GVHD and did not experience (non-GVHD) HSCT patients. Based on the laboratory and clinical indices, the GVHD was graded as I-IV.³² The mean age of GVHD and non-GVHD HSCT patients was 24.66 ± 10.06 and 22.25 ± 10.8 years. Male-to-female ratio (M/F) was 2.2 (20/9) in the GVHD group, and 1.3 in the non-GVHD group (25/18). 71.2% of autologous transplant patients were male, and 28.8% were female (age range, 14-55 y). The M/F ratio was 2.47 in autologous HSCT patients (42/17).

The most frequent age range in allogeneic and autologous HSCT patients was 20 to 30 years old (23 patients; 32.9%) and 30 to 50 years old (31 patients; 63.3%). Also, the most frequent blood group in both allogeneic and autologous HSCT patients was O+ (52.9% and 38.8%).

Excepting CD28, CTLA4 +49 A/G and -1722, other genotypes were in agreement with Hardy-Weinberg equilibrium in both groups of patients. The Cochran-Armitage test for trend was used to check the association of genotypes with acute rejection whenever the Hardy-Weinberg equilibrium did not meet. The significance of this allele and genotype, however, did not survive the Bonferroni correction, which suggests the striking of the threshold of P value from conventional .05 to .017.

Acute GVHD was found in 29 of 72 HSCT recipients (41.3%); low and severe grades of GVHD were found in 9 of 29 (31.03%) and 20 of 29 of HSCT patients (68.96%) respectively. Also, 43 of 72 patients (59.7%) did not show GVHD symptoms.

Costimulatory molecule gene polymorphisms and hepatitis B virus infection
Hepatitis B virus infection was detected in 19 of 72 allogeneic (26.3%) and 26 of 59 autologous HSCT patients (44.1%). The TT genotype of PD-1.9 7625 T/C was not found in HBV-infected autologous HSCT patients. The T allele of the CTLA4 -318 T/C genotype was significantly more frequent in HBV-infected allogeneic HSCT patients compared with autologous transplant patients (P = .01; OR 169.20, 95%CI: 26.58-1453.85; study power=100%) (Table 1). The TT genotype and T allele of CD28 +17 C/T were significantly less frequent in HBV infected allogeneic HSCT patients (P = .01; OR 3.83,95% CI: 1.13-13.32; study power=69%; P = .001; OR 3.71, 95% CI: 1.53-9.02; study power=86%). Also, the CC genotype of  CD28 +17 C/T was significantly more frequent in HBV-infected allogeneic HSCT patients (P = .02; OR 0.23, 95% CI: 0.05-1.02, study power=50%) (Table 2). The frequency of the TT genotype and the T alleles of CD28 +17 C/T were significantly less frequent in HBV-infected allogeneic HSCT patients without GVHD (P = .004; OR 9.72, 95% CI: 1.41-84.59, study power=90%; P = .0001; OR 8.14,95% CI: 2.27-30.37 study power=95%). Also, the T alleles of CD28 +17 C/T were significantly more frequent in HBV-infected allogeneic HSCT patients without GVHD compared with patients experiencing GVHD (P = .05; OR 0.27, 95% CI: 0.06-1.26, study power = 53%) (Table 3).

The frequency of C allele of the PD.1.9 was significantly more frequent in HBV infected allogeneic HSCT patients experiencing GVHD (P = .04, OR 3.56, 95% CI: 0.87-14.96, study power=50%) (Table 4).

The CC genotype and the C allele of the PD-1.9 7625 T/C and the A allele of the CTLA4 - 1661 A/G were seen significantly less frequently in HBV-infected allogeneic HSCT patients experiencing low-grade GVHD (P = .04; OR 0.001, 95% CI: 0.001-5.38, study power=29%; P = .05; OR 0.001, 95% CI: 0.001-5.15, study power=21%; P = .04; OR 11.0, 95% CI: 0.58-424.27, study power=21%). Also, the CT genotype, PD.1.9 was seen significantly more frequently in HBV-infected allogeneic HSCT patients experiencing low-grade GVHD (P = .04 study power=29%).

Additionally, after classifying the allogeneic HSCTs according to their sex, the T allele and the TT genotype of CD28 were seen more frequently in HBV-negative male patients (P = .004; study power=74%; P = .009; study power=78%). Also, the G allele and the GG genotype of CTLA4 -1661 were seen more frequently in HBV-positive female patients (P = .05; study power=46%; P = .05; study power=38%) (Table 2).

Costimulatory molecule gene polymorphisms and risk factors
Significant associations were found between acute myelogenous leukemia (as an underlying disease) 1720 T/C, CTLA4 1661-A/G, and CD28 17-T/C gene polymorphisms (P = .01; P = .02; and P = .02). A significant association was found between age and ICOS-1720-T/C gene polymorphism in the allogeneic HSCT patients experiencing GVHD (P = .05). Also, a significant association was found between O+ blood group with the PD.1.3 7146 A/G gene polymorphism in allogeneic HSCT patients without GVHD (P = .01).

Discussion

Acute graft-versus-hostdisease of the liver, veno-occlusive disease, and viral infections can cause hepatitis with varying degree of severityafter HSCT. Impairing the immune response in HSCT patients can affect the clinical and serologic outcome of HBV infection, as the severity of the disease depends mainly on the immune system’s ability to attack infected hepatocytes.⁴¹ The diversity in a HBV clinical course was largely related to the host immunologic genetic variety especially costimulatory molecules which have roles in immune responses.^10,11,9,42 Therefore according to, the importance of costimulatory molecules, in this study their genetic variations was evaluated in allogeneic and autologous HSCT patients with and without HBV.

The CTLA-4 preserves T-cell homeostasis and induces Fas-independent apoptosis of activated T cells elicited.¹² Several reports have shown that CTLA-4 gene polymorphisms may be associated with the susceptibility and chronicity of the disease in HBV-infected patients; however, the results are controversial.^9-12 Gu and associates reported that the A allele instead of CTLA4 49A/G (rs231775) polymorphisms enhances the inhibitory effect on T-cell activation, and the A/G variant may decrease the HBV clearance capability of T cells; thus, increasing HBV-related HCC susceptibility.¹²

Duan and associates evaluated the CTLA-4 49A/G and 318 T/C polymorphisms in 172 chronic HBV-infected patients. The AA genotype and A allele of the CTLA-4 49A/G, and the genotype CC of the CTLA-4 -318 C/T polymorphisms was seen more frequently in chronic HBV-infected patients.² Thio and associates have shown a significant association of the CTLA-4 49A/G with clearance of HBV infection.⁹ Alizadeh et al and Schott et al have shown a significant association between CTLA-4 -318 C/T with a susceptibility to chronic HBV infection.^10,11 Jiang and associates showed that the GG genotype of the CTLA-4 49A/G in Chinese liver transplant patients is related to a reduced risk of the HBV recurrence.⁴³ Han and associates have shown a relation between the GG genotype of the CTLA-4 49A/G, with the lower TNF-α and IFN-γ levels in patients with chronic HBV infection.⁴⁴ Similar to earlier reports, a significant association was found between the higher frequency of the T allele of the CTLA-4 318 T/C polymorphisms with HBV infection in allogeneic HSCT patients compared with autologous transplant patients.

CD28 is bound to the B7 ligand on antigen-presenting cells. The CD28 molecule is expressed on T cells and induces T-cell responses.¹⁴ The CD28 gene polymorphisms may influence signals for T-cell proliferation.^14,15

As we know, there has been no study on the relations between CD28 and HBV in HSCT patients, so we cannot compare our results with others. Few studies have focused on the clinical importance of the CD28 gene polymorphisms. Marron and associates showed a weak association between the CD28 gene polymorphisms and/or the D2S72 genetic marker with insulin-dependent diabetes mellitus.⁴⁵

In this study, the TT genotype and the T allele of CD28 +17 C/T were significantly less frequent in HBV-infected allogeneic HSCT patients. Also, the C allele and the CC genotype of the CD28 gene were significantly more frequent in HBV-infected allogeneic HSCT patients without GVHD compared with patients with GVHD compared with patients with GVHD. The main duty of the CD28+17 T/C is still unknown; however, it is known that the above polymorphism is located near the splice acceptor site; therefore, it may influence the mRNA splicing efficiency and consequently change CD28 molecule expression. Further studies should investigate its functional effects accurately.

The PD-1:PD-L pathway is involved in T-cell function and progression of established chronic infections.⁴⁶ Up-regulation of PD.1 on HBV-specific T-CD8+ cells influences dysfunction of T cells in chronic HBV-infected patients with high viremia.^22,47 Based on the importance of PD.1 in anti-HBV specific T-cell response, the PD.1 gene polymorphisms may affect expression or function of PD.1.^1,21 However, we showed that the frequency of the C allele of the PD.1.9 was significantly greater in HBV-infected allogeneic HSCT patients with GVHD. Also, the PD.1.9 7625 CT genotype and the T allele were seen significantly more frequently in HBV-infected allogeneic HSCT patients with low-grade GVHD.

Additionally, previous reports have shown that in different diseases, this polymorphism may play different role. For example, it is a risk factor for ankylosing spondylitis in a Korean population.⁴⁴ The PD.1.6 GG genotype and the G allele were less abundantly seen in HBV patients than they were in controls.¹ There are studies about the relation of the PD.1 polymorphism and HBV, but there have been no studies regarding the relation between PD.1.9 and HBV in HSCT patients. PD.1.9 in exon5 is a nonsynonymous polymorphism (Val215Ala), located in the intracellular domain of PD.1, that also might affect signal transduction; however, side chains of valine and alanine appear to be biochemically equivalent.²² Further studies are needed to determine whether this amino acid change alters the protein structure and affects its function. Altering expression and function in PD.1 conferred by the PD.1 polymorphism may affect T-cell activation and antiviral response via PD.1: PD-L pathway in chronic infection.¹

Ye and associates showed that an accurate balance between positive and negative costimulatory regulation, such as PD-1/PD-L pathway, may contribute to the outcome of HBV-related disease.²⁴ Zhang and associates analyzed the association between the PD.1-specific memory T-CD8+ cells and developing acute HBV infection. The authors also showed that PD.1 was significantly up-regulated efficiently with attenuated HBV-specific T-CD8+ cell effector’s function.¹ Zhang and associates also have shown that in patients with PD-1-606, the AA genotype had lower TNF-α and IFN-γ levels. Hepatitis B virus infection patients with PD.1 +8669 GG genotype confer higher level of TNF-α as well as this genotype have lower frequency HBV-infected patient.³ Kong and associates and Meng and associates showed that the PD.1.1 at-531G/A and-8669 G/A (previously called “PD.1.6”) at -8738G/A polymorphisms are common among Chinese HBV-infected patients.^48,49 In another study, Zhang and associates indicated that the G allele and the GG genotype of the PD.1.1 606G/A polymorphisms had no association with chronic HBV infection.¹ However, the G allele and GG genotype PD.1.6 8669 G/A SNPs were significantly different between chronic HBV patients and controls. These findings suggest that the PD.1 gene may play a role in chronic HBV infection in the Chinese population.¹

In conclusion, the finding of significantly higher frequency of the C allele and the CT genotype of the PD.1.9 7625 in HBV-infected individuals, and those with GVHD experienced allogeneic HSCT patients; present the inducible role of this PD.1.9 polymorphism in promoting and introducing of post-HSCT outcomes, especially GVHD. However, further studies are recommended to confirm these associations in larger populations of HBV-infected HSCT patients with GVHD.

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