Objectives: Human leukocyte antigen-G is an immuno-modulatory factor that affects acute allograft rejection and autoimmune diseases such as type 1 diabetes mellitus. In this study, possible associations between human leukocyte antigen-G 14-bp insertion/deletion polymorphism and acute pancreas rejection were investigated.

Materials and Methods: Human leukocyte antigen-G genotyping was assessed in 102 Iranian pancreas transplant recipients (including 41 with acute rejection and 61 with nonacute rejection). Results were compared with 100 individuals in a normal control group.

Results: No significant differences in genotype frequencies of human leukocyte antigen-G 14-bp insertion/deletion were observed in recipients who had acute rejection episodes. On the other hand, the insertion/insertion genotype was a risk factor for susceptibility to type 1 diabetes mellitus (odds ratio = 3.82, 95% confidence interval, 1.37- 11.22; P = .005).

Conclusions: Our results provided evidence revealing that the human leukocyte antigen-G insertion/insertion genotype might be involved in the pathogenesis of type 1 diabetes mellitus.

Key words : Acute rejection, Autoimmune disease, Human leukocyte antigen G, Pancreas transplantation

Introduction

Human leukocyte antigen-G (HLA-G) is considered to be involved in down-regulation of the immune response to avoid allograft rejection in organ transplant patients or protects the fetus as a semi-allograft from rejection by the mother’s immune system.^1,2 Expression of HLA-G may lead to a decrease in immune response against the allograft, consequently reducing allograft rejection. Human leukocyte antigen-G impairs the cytotoxic activity of natural killer (NK) and CD8 T cells and the maturity of dendritic cells (DC).^3,4 Expression of HLA-G correlates with improved kidney,⁵ heart,⁶ liver, and lung³ allograft acceptance as well as improved acceptance of combined liver-kidney and kidney-pancreas transplant.⁷ Heart transplant patients with expression of HLA-G in myocardial cells and plasma have been shown to experience fewer acute rejection episodes, as well as no chronic allograft rejection, compared with HLA-G-negative patients.⁶ In kidney transplant patients, expression of HLA-G was associated with low-level production of alloantibody, immune tolerance, and better graft acceptance. Alloantibodies have an important role in pathogenesis of both acute and chronic rejection in recipients.⁵

The effect of HLA-G to increase allograft survival has been shown in in vivo and in vitro studies. In vivo immunization of HLA-G improves graft survival. In addition, interaction of tetrameric com-plexes of HLA-G with paired immunoglobulin-like inhibitory receptor (the receptor of HLA-G in mice) impairs murine DC maturation. Indeed, suppression of T cells resulted in better graft survival when HLA-G tetramer-coated beads were injected into mice before skin transplant.⁸

During pregnancy, the trophoblast cells produce HLA-G to restrict the immune response against semi-allogeneic fetal tissues. Human leukocyte antigen-G stimulates CD4+ T cells to produce interleukin 4 (IL-4), which has a protective effect on pregnancy outcome.^2,9 In addition, HLA-G impairs B- and T-cell proliferation and cytotoxic activity of NK cells and also inhibits NK-mediated lysis.10 Indeed, HLA-G is associated with maternal-fetal immunotolerance through its immunoregulatory roles by interaction with various immune cells.¹¹

Moreover, HLA-G is correlated with autoimmune diseases such as type 1 diabetes mellitus (DMT1) in which the destruction of pancreatic β cells occurs during immune response by activated T cells.^12,13 A high level of soluble HLA-G molecules was also observed in individuals with impaired glucose metabolism.¹

The role of HLA-G was also emphasized in control of other autoimmune and inflammatory diseases such as Crohn’s disease, pemphigus, celiac disease, systemic lupus erythematosus (SLE), rheumatoid arthritis, juvenile idiopathic arthritis, and asthma.²

Human leukocyte antigen-G is a nonclassical HLA class I that contains 8 exons and 7 introns and is located on the short arm of chromosome 6 (6p21.2-21.3).^14,15 Alternative splicing of the primary transcript of HLA-G leads to formation of 7 isoforms, including 4 membrane-bound isoforms (HLA-G1 to HLA-G4) and 3 soluble isoforms (HLA-G5 to HLA-G7), both of which can be involved in the allogeneic response.^16,17 Several polymorphisms in the 5´ upstream regulatory region and the 3´ untranslated regions (3´-UTR) regulate HLA-G production at the level of transcription and posttranscription.16 The HLA-G 14-bp insertion/deletion polymorphism in 3´ UTR can affect mRNA stability.¹⁸ Presence of 14 bp (5´-ATTTGTTCATGCCT-3´) in the 3´-UTR promotes alternative splicing that results in 92-bp deletion of exon 8 in mature HLA-G mRNA; consequently, a more stable transcript is generated.^19,20 In addition, the presence of the 14-bp fragment is related to production of low expression levels of HLA-G mRNA.3 Human leukocyte antigen-G has been shown to be expressed in different conditions in various cells such as the pancreas, intestine, thymic epithelium, and peripheral blood monocyte cells.¹⁶

To the best of our knowledge, this is the first study investigating the possible association of the genetic polymorphism of HLA-G 14-bp insertion/deletion in acute rejection in pancreas transplant recipients. Because all transplant patients had DMT1 as the underlying disease of pancreas transplant, the risk of HLA-G 14-bp insertion/deletion in DMT1 was also assessed (for the first time) in Iranian DMT1 patients.

Materials and Methods

Patients and controls
Data were collected on 102 pancreas transplant recipients admitted to Namazi Hospital (affiliated with Shiraz University of Medical Sciences, Shiraz, Iran) from April 2006 to June 2014. The ethics committee of Shiraz University of Medical Sciences approved all protocols in accordance with the ethical guidelines of the 1975 Helsinki Declaration. Informed consent was provided by all participants.

Immunosuppressive drugs given to patients included tacrolimus, mycophenolate mofetil, and prednisolone. The intake of tacrolimus was started orally at 1.0 mg/kg/day. Mycophenolate mofetil was given orally at 2.0 g/day, twice per day. Prednisolone 20 mg was given intravenously and tapered to zero within 3 months.

Pancreas transplant recipients were followed for 6 months after transplant to monitor and record any episodes of acute rejection during this period. The diagnosis of rejection was based on paraclinical findings, manifested as an increase in serum amylase and lipase levels, and further confirmed by tissue biopsies based on Banff criteria.²¹ Our control group comprised 100 healthy volunteers from the Iranian Blood Transfusion Organization in Shiraz, Iran.

Patients were divided into 2 groups: acute rejection group (AR group) and nonacute rejection group (NAR group). All pancreas transplant recipients had DMT1 as the underlying disease. Demographic and laboratory data were collected from patient medical records.

Genotyping
DNA extraction was performed from peripheral blood mononuclear cells using the DNP kit DNG plus DNA extraction kit (Sinagen, Tehran, Iran). The concentration of DNA was investigated by measuring the optimal density at 260 nm.

Typing of HLA-G was performed by specific primers for exon 8 of the HLA-G gene, which was amplified at 224 bp and 210 bp of DNA in insertion and deletion genotypes, respectively, using polymerase chain reaction (PCR). The sequence of forward and reverse primers was 5´-GTG ATG GGC TGT TTA AAG TGT CAC C-3´ and 5´-GGA AGG AAT GCA GTT CAG CAT GA-3´, respectively. Reaction of PCR was mixed in the final volume (20 μL), which included 2.5 μL of 10× PCR buffer, 0.75 μL of 50 mM MgCl2, 0.75 μL of 10 nM dNTP, 0.5 mL of each primer (10 μM), 0.5 μL of 5 U/μL Taq, 9.5 μL of water, and 5 mL of template DNA. The PCR was performed by initial denaturation at 95°C for 5 minutes, followed by 30 cycles at 92°C for 10 seconds, 64°C for 1 minute, 72°C for 2 minutes, and a final extension of 72°C for 10 minutes. The PCR products were observed by 3% gel electrophoresis.

Statistical analyses
Allele differences and genotype frequencies between patients and the healthy control group and between the AR and NAR groups were analyzed by 2-tailed Fisher exact test, using SPSS version 22 (SPSS: An IBM Company, IBM Corporation, Armonk, NY, USA). Odds ratio (OR) and 95% confidence intervals (95% CI) were calculated to determine the magnitude of the effect. Furthermore, differences shown with regard to age, sex, and blood group type of patients were calculated with t test and Fisher exact test. P < .05 was considered statistically significant.

Results

Clinical characteristics of patients
The transplant recipient group consisted of 63 male (61.8%) and 39 female (38.2%) patients, with mean age of 32.96 ± 9.2 years (range, 14-56 y). Although we observed a significant difference in age (P = .003) between the transplant and control group (which including 61 male [61%] and 39 female [39%] participants with mean age of 37.59 ± 12.6 y and range of 20-68 y), no significant difference in age was shown between the AR and NAR group (P = .58). The mean age of the AR group (n =41) was 32.34 ± 8.8 years (range, 19-55 y), and the mean age of the NAR group (n= 61) was 38.33 ± 9.50 years (range, 14-56 y) (Table 1).

We observed no significant distribution of male versus female participants between the transplant and control groups (P = 1.0) or between the AR and NAR groups (P = .49). The AR group consisted of 27 male (65.9%) and 14 female (34.1%) patients, and the NAR group consisted of 36 male (59%) and 25 female (41%) patients (Table 1).

The most frequent blood group was type O; this finding was not significant between the transplant and the control groups (P = .6) or between the AR and NAR groups (P = .51) (Table 1).

HLA-G 14-bp insertion/deletion genotype frequency
The frequencies of HLA-G 14-bp insertion/deletion genotypes and alleles were compared between pancreas transplant recipients versus healthy control participants. Because all transplant recipients had DMT1 as the underlying disease, the association between HLA-G 14-bp insertion/deletion genotypes and DMT1 was also investigated (Table 2). Differences in frequency of homozygote genotype (14-bp insertion/14-bp insertion) were significant between the pancreas recipients and the control group and was shown to be associated with DMT1 (OR = 3.82, 95% CI, 1.37-11.22; P = .005) (Table 2). Moreover, the heterozygote genotype (14-bp insertion/14-bp deletion) showed a significant protective effect (OR = 0.46, 95% CI, 0.25-0.84; P = .007) (Table 2).

For investigation of association of HLA-G 14-bp insertion/deletion genotypes and alleles and susceptibility to acute rejection, transplant patients were divided into 2 groups (AR and NAR). No significant association was observed between HLA 14-bp insertion/deletion genotypes and alleles with acute rejection (Table 3).

Discussion

The effect of HLA-G on alloimmune response modulation was confirmed by the interaction of HLA-G with specific receptors such as the killer immunoglobulin-like receptor KIR2DL4/CD158d and the leukocyte immunoglobulin-like receptors LILRB1/IL-2/CD85j and LILRB2/IL-4/CD85d. Although KIR2DL4 is only presented on NK cells, IL-2 and IL-4 are expressed by various cells: IL-2 is expressed by monocytes, NK cells, DCs, and T and B cells, and IL-4 is expressed by myeloid cells such as DCs, monocytes, neutrophils, and macrophages. The presence of HLA-G receptors on a variety of cells induces different immune functions. It has been demonstrated that HLA-G leads to impairment in DC maturation and in CD8 cell and NK cell cytotoxic activity and inhibits CD4+ T-cell alloproliferation.^3,22 Interaction of HLA-G with IL-2/IL-4 expressed by T cells and antigen-presenting cells induces dif-ferentiation of T cells into suppressor cells (CD3+CD4^low and CD3+CD8^low), which are related to the immune tolerance and consequently graft acceptance. In fact, it was demonstrated that expression of HLA-G leads to down-regulation of CD8 and CD4 expression at both the transcription and posttranslation levels of T cells. In addition, HLA-G mediates the down-regulation of CD4 and CD8 production, which can lead to increased plasma levels of soluble CD4 and CD8 molecules in liver and combined liver-kidney transplant patients. Soluble CD4 and CD8 co-receptors are defined as potent inhibitors of T-cell activation compared with membrane-anchored co-receptors. The presence of CD3+CD4^low and CD3+CD8^low T cells has been shown to increase the production of IL-4, IL-10, and IL-13 and to decrease the production of interferon-γ levels through the increased representation of CD45RA cells. Associations among HLA-G, IL-10, and CD3+CD4^low and CD3+CD8^low suppressor T cells have also been confirmed. Therefore, HLA-G is associated with immunologic markers included in immunotolerance.²³

The 14-bp deletion genotype was associated with high expression levels of soluble HLA-G, which impaired the alloimmune responses and is associated with better graft function.²⁰ The expression of HLA-G has been detected in various organ transplant recipients with no evidence of acute rejection, including liver,²⁴ kidney,⁵ heart,^6,25 lung,²⁶ and combined liver-kidney transplant.²⁷ This is the first study in which the association of HLA-G 14-bp insertion/deletion polymorphism and acute rejection of pancreas transplant was assessed in Iranian pancreas transplant recipients. Our results showed that there were no significant associations between any genotypes of HLA-G 14-bp insertion/deletion and acute rejection. We previously investigated the association between HLA-G 14-bp insertion/deletion polymorphism and acute rejection in kidney and liver transplant. Similarly, the HLA-G 14-bp insertion/deletion genotypes were not correlated with acute rejection in both kidney and liver transplant.^28,29 However, other studies have indicated an association between HLA-G insertion/deletion polymorphism and acute rejection.^30-32 In heart transplant, the 14-bp deletion/deletion genotype has been shown to be associated with high levels of HLA-G production, resulting in low acute rejection rate.²⁵ The HLA-G 14-bp insertion/insertion genotype and insertion alleles were associated with nearly 4-fold and 2-fold increased risk of acute rejection in kidney transplant recipients, respectively; in addition, the HLA-G 14-bp insertion/insertion genotype was correlated with lower survival in kidney transplant patients. The HLA-G 14-bp deletion allele was defined as a protective allele30; however, a risk of acute rejection was noted for the 14-bp insertion allele in kidney transplant patients in other studies.^31,32

With respect to the role of HLA-G on auto-immune diseases, low levels of soluble HLA-G and reduction of HLA-G expression in monocytes were detected in patients with rheumatoid arthritis and multiple sclerosis. Interferon therapy (interferon-β), which is usually used for patients with multiple sclerosis, has been shown to help increase HLA-G levels in monocytes.³³

In patients with rheumatoid arthritis, allele and genotype frequencies of HLA-G 14-bp insertion/-deletion were not different from frequencies shown in control patients. In addition, different genotypes were not associated with disease severity and clinical manifestations.³⁴ Conflicting results in SLE patients have been reported. There was an increased frequency of 14-bp insertion/14-bp insertion genotype and 14-bp insertion allele in Italian SLE patients,35 but no asso-ciation was observed among Chinese SLE patients.³⁶

Regarding HLA-G in the pancreas, it was confirmed that pancreatic endocrine cells have low expression levels of HLA-G.³⁷ Unusual recycling of HLA-G from the Golgi to the endoplasmic reticulum leads to prolonged lifespan of intracellular HLA-G compared with other HLA molecules and prepares the interaction of HLA-G with multiple low-affinity peptides, defined as autoantigens; therefore, it is possible that HLA-G may contribute to lack of presence of self-antigens on the cell surface and islet immunologic ignorance. On the other hand, HLA-G has been detected in the secretory granules, which are composed of many autoantigens. During insulin secretion, HLA-G could be increased on the cell surface through exocytosis. Because T-cell activation relies on the density of the autoantigen/HLA complex on the cell surface, it was suggested that local clustering of HLA-G during exocytosis may prevent deleterious T-cell activation.^33,37

Based on tolerogenic function of HLA-G, this is the first study that investigated the possible association between HLA-G 14-bp insertion/deletion polymorphism and susceptibility to DMT1 as cause for pancreas transplant in Iranian patients. Our results indicated 3.82-fold susceptibility with homozygote genotypes (HLA-G 14-bp insertion/insertion) among DMT1 patients and indicated that the heterozygote genotype (14-bp insertion/deletion) had a protective effect. The presence of the 14-bp deletion allele may also have a protective effect. The correlation between risk of DMT1 and the HLA-G 14-bp deletion/deletion genotype and deletion allele has been previously defined as a risk factor for DMT1.¹² An association between HLA-G 14-bp insertion/deletion and onset of DMT1 was assessed, with a correlation noted between HLA-G 14-bp deletion/deletion genotypes and early-age onset of DMT1. In addition, the HLA-G 14-bp insertion allele was associated with later-age onset of DMT1, suggesting the important role of HLA-G in pathogenesis of DMT1.¹³

Conclusions

This is the first study to assess the correlation between HLA-G 14-bp insertion/deletion polymorphism and acute pancreas rejection and DMT1 in an Iranian population. The HLA-G 14-bp insertion/deletion polymorphism did not show an association with acute rejection, but the homozygote genotype of HLA-G 14-bp (insertion/insertion) was shown to be a risk factor for DMT1. Rejection is a complex process caused by interactions between both cellular and humeral axes of the immune system. However, further studies on the exact role of HLA-G genetic variations in DMT1 development are required. Furthermore, design of future HLA-G-based therapies, such as up-regulators of HLA-G expression, may induce a tolerogenic environment that is useful in management protocols of solid-organ transplant or DMT1.

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