Objectives: HLA-E is located on the nonclassical major histocompatibility complex class I and acts as the ligand for natural killer cells. Consequently, it has a main role in the regulation of innate immune responses by involving cell identification by natural killer cells. Differences in expression levels among HLA-E alleles have been suggested to affect transplant outcomes. In this study, we evaluated the effects of different HLA-E genotypes on allogeneic hema­topoietic stem cell transplant in southern Iran.

Materials and Methods: We investigated 200 patients (donors and recipients) who underwent allogeneic hematopoietic stem-cell transplant and 100 normal participants (control group) in a case-control study. Detection of HLA-E polymorphisms was performed using a sequence-specific primer polymerase chain reaction method.

Results: Statistical analyses indicated that genotypes in the transplant group were not distributed in accordance with Hardy-Weinberg equilibrium (χ2 = 76.56; P < .001), whereas genotypes in the control group were distributed in accordance with Hardy-Weinberg equilibrium (χ2 = 0.39; P = .53). No significant differences were observed in cumulative incidence of acute (P = .76; hazard ratio = 0.80; 95% confidence interval, 0.19-3.31) and chronic (P = .75, hazard ratio = 0.048; 95% confidence interval, 0.00) graft-versus-host disease in recipients harboring HLA-E*0103 allele compared with those homozygous for the HLA-E*0101 allele. The HLA-E*0103 allele showed a trend toward lower cumulative incidence of relapse compared with the homozygous HLA-E*0101 genotype (8% vs 21.5%; P = .37; hazard ratio = 2.50; 95% confidence interval, 0.32-19.20).

Conclusions: Genotypes of the HLA-E molecule did not correlate with acute and chronic graft-versus-host disease in hematopoietic stem cell transplant recipients except for the HLA-E*0101*/*0103 genotype, which was protective in survival of our study patients.

Key words : Graft-versus-host disease, Human leukocyte antigen E, Natural killer cells

Introduction

Hematopoietic stem cell transplant (HSCT) is currently recognized as an important and definitive treatment for blood malignancies and plays an increasing role in the treatment of hemoglobino­pathies and bone marrow defects.¹ Stem cells used in this type of transplant procedure have the ability to produce different types of blood cells in patients undergoing severe chemotherapy and even in patients with myeloablative bone marrow. Hematopoietic stem cells are extracted from 3 sources: bone marrow, peripheral blood, and umbilical cord blood. In autologous transplant procedures, hematopoietic stem cells are the own stem cells of the patient; however, in allogeneic transplant, another person’s stem cells are used.² One of the more important problems of allogeneic transplant is graft-versus-host disease (GVHD). In this disease, the white blood cells (WBCs) of a donor (transplanted WBCs) identify the patient’s host cells as the alien cells and attack them.

Graft-versus-host disease is generally treatable by steroids or other immunosuppressive drugs. Many studies are underway to find a way to prevent GVHD occurrence.^3,4 It is well shown that HLA plays an important role in GVHD.5 HLA is the most polymorphic gene in the human genome and is located in chromosome 6p21. Antigen presentation is the main role of HLA.6 The greater similarity in the components of HLA between donor and host equals a lesser risk of rejection of transplant and also GVHD.⁵

HLA molecules are divided into HLA class I and HLA class II. HLA class I is classified into 2 major groups: the classical (Ia) and nonclassical (Ib) groups. HLA-A, HLA-B, and HLA-C are members of classical HLA class I, whereas HLA-E, HLA-F, HLA-G, MICA, and MICB are members of nonclassical HLA class I and produce molecules of the same name.⁷ Classical HLA I molecules are more polymorphic than nonclassical HLA class I, and they are present in most tissues. Nonclassical HLA class I molecules have limited expression. Moreover, the HLA-E gene has the most expression among nonclassical HLA class I genes in tissues.⁸

HLA-E has the lowest polymorphism among all HLA class I members. Only 8 alleles of HLA-E have been identified in different populations, which produce 3 proteins. However, only 2 alleles (HLA-E*0101 and HLA-E*0103) have significant prevalence among populations (about 50%). Different com­binations of these 2 alleles lead to creation of 3 different genotypes, including HLA-E*0101/*0101, HLA-E*0103/*0103, and HLA-E*0101/*0103.^9,10 These 2 alleles have only 1 amino acid difference at position 107 on the A2 domain of the heavy chain; an arginine at position 107 (HLA-ER) defines HLA-E*0101, whereas a glycine at this position (HLA-EG) defines HLA-E*0103.^11,12 The functional differences between the HLA-E alleles are related to the relative peptide affinity and cell surface expression. The HLA-E*0103 allele is expressed at a higher level than HLA-E*0101 and has a higher affinity to peptides and therefore higher surface stability.¹³

The HLA-E polymorphism has been associated with infection, cancer, recurrent spontaneous abortion, autoimmune diseases, and transplant outcomes.^14-17

In this study, we investigated the effects of different genotypes of the HLA-E molecule on the prevalence of GVHD in allogeneic HSCT recipients in southern Iran.

Materials and Methods

Study participants
This case-control study included 200 samples from 100 patients who were recipients of HSCT and 100 individuals who were donors (patient group [cases]) and 100 healthy people (healthy control group). The 100 recipients received transplants from their HLA-identical siblings at the Bone Marrow Transplantation Unit, Namazi Hospital (affiliated with the Shiraz University of Medical Sciences). This project was started in 2014, and patient follow-up ended in 2019. The patients were followed for about 24 months. The relapse of primary disease and incidence of GVHD (acute and chronic) were recorded. This study was confirmed by the ethical committee of Shiraz University of Medical Sciences, and informed consent was obtained from all participants.

HLA typing was done using the serologic method (Bio-Rad, Hercules, CA, USA) for HLA class I typing and sequence-specific oligonucleotide probe method (Inno-Lipa HLADRB1 plus) for HLA class II typing. HLA-E genotyping was performed using a sequence-specific primer polymerase chain reaction (PCR) method.

Conditioning regimens and prophylaxis for graft-versus-host disease
Conditioning regimens included intravenous busulphan (12.8 mg/kg/day for 4 days; day -8 to day -5) and intravenous cyclophosphamide (120 mg/kg for 3 days; day -4 to day -2).

Prophylaxis for GVHD included intravenous cyclosporine 3 mg/kg from day -1, which was changed to oral form (3 mg/kg) once the patient could swallow for 6 months posttransplant.

HLA-E typing
DNA was extracted from peripheral blood of recipients and donor by using a DNP kit (SinaClon, Co., Tehran, Iran). A sequence-specific primer PCR typing system was used for detection of HLA-E polymorphisms. We carried out PCR reactions in 25 μL containing 100 ng DNA, 10 pM of each forward 5′-ATACCCGCGGAGGAAGCGCCT-3′ and reverse 5′-TCCCAGATTCACCCCAAG-3′ primers, 1× PCR buffer, 200 μL dNTP mix, 1.5 mM MgCl2, and 2.5 U smarTaq DNA polymerase (SinaClon, Co.). Polymerase chain reaction cycling was done in an Eppendorf gradient machine as initial denaturation for 2 minutes at 95°C, which was followed by 35 cycles at 94°C for 45 seconds, 61°C for 1 minute, 72°C for 1 minute, and finally 72°C for 5 minutes. The resulting 280-base pair fragment was visualized by electro­phoresis on 1.5% agarose gel.

Statistical analyses
Numerical data are expressed as means ± standard deviation. Hardy-Weinberg equilibrium was tested using Pearson chi-square (χ2) test. Chi-square test (Fisher exact test) was used to examine associations between qualitative variables. Survival analysis was done using the Kaplan-Meier method. Comparisons between survival curves were done using log-rank tests. Estimation of the hazard ratio (HR) was done using Cox regression model. Hazard ratio with 95% confidence interval (95% CI) was used for risk assessment. Survival was used to test the proportional hazards assumption. The assumption (proportionality) of the Cox proportional hazard models was met. Relapse incidence was estimated by the cumulative incidence method with death, without relapse, and treated as a competing risk. Transplant-related mortality was calculated using cumulative incidence with relapse and treated as a competing risk. P < .05 was considered statistically significant.

Results

Patients characteristics
The patient group included 67 male and 33 female patients. Median age was 33.54 years (range, 7-63 y). Diagnoses included 51 patients (51%) with acute myeloid leukemia, 30 patients (30%) with acute lymphoblastic leukemia, and 19 patients (19%) with other malignant disorders. Furthermore, 34% exhibited relapse, 77% exhibited remission, and 44% had acute GVHD after transplant. Patient characteristics are summarized in Table 1.

Frequency of HLA-E genotypes and alleles
Analyses of HLA-E alleles in the patient group indicated high frequency (94%) of HLA-E*0101/*0103 genotype, whereas both homozygote genotypes presented at low frequency (3% each). The frequency for the HLA-E*0101 and the HLA-E*0103 alleles was 50%. Further statistical analyses (Table 2) clearly showed that distribution in the patient group was not in Hardy-Weinberg equilibrium (χ2 = 76.56; P < .001).

In the control group, analyses of HLA-E genotypes revealed that the HLA-E*0101/*0101 was detected in 16%, HLA-E*0101/*0103 in 52%, and HLA-E*0103/*0103 in 32%. The allele frequency was 42% for the HLA-E*0101 allele and 58% for the HLA-E*0103 allele. The distribution of genotypes was in Hardy-Weinberg equilibrium (χ2 = 0.39; P = .53).

Association of HLA-E genotype with clinical outcome
HLA-E genotype was not significantly associated with patient characteristics (Table 1). There was no significant difference in cumulative incidence of acute GVHD at 100 days between recipients harboring the HLA-E*0103 allele and those homozygous for the HLA-E*0101 allele (P = .76; HR = 0.80; 95% CI, 0.19-3.31) (Figure 1). Similarly, there was no significant difference in cumulative incidence of severe/chronic GVHD at 2 years between recipients with the HLA-E*0103 allele and those homozygous for the HLA-E*0101 allele (P = .75; HR = 0.048; 95% CI, 0.00) (Table 3 and Figure 2).

The median duration of follow-up was 48 months (range, 0.5-65 mo). The cumulative incidence of relapse at 2 years was 9.5%. Those with the HLA-E*0103 allele showed a trend toward lower cumulative incidence of relapse at 2 years versus that shown in those with the homozygous HLA-E*0101 genotype (8% vs 21.5%; P = .37, HR = 2.50, 95% CI, 0.32-19.20) (Figure 3).

Discussion

Human HSCT is a highly complex process due to complications in HLA molecules and their interactions, which can result in graft rejection, relapse, and GVHD.^7,18-21 The HLA-E molecule provides a ligand for receptors on cytotoxic T lymphocytes and natural killer (NK) cells. Therefore, the HLA-E molecule participates in both innate and adaptive immunity. Moreover, HLA-E alleles have different levels of expression, which are suggested to have different effects on transplant outcomes.^13,22-25

Our study found comparable frequencies for the HLA-E*0101 and HLA-E*0103 alleles, a finding in line with other studies in White, African American, and Hispanic populations. However, in Japanese and Chinese populations, the frequency of HLA-E*0103 has been shown to be higher than the frequency of the HLA-E*0101 allele. Furthermore, in our study group, the HLA-E*0101/*0103 genotype was more frequent than the HLA-E*0101/*0101 and HLA-E*0103/*0103 genotypes in donors and patients, an observation also consistent with those previously reported by other groups.^18,26,27

In our study, there was no significant difference in the cumulative incidence of extensive relapse in recipients harboring the HLA-E*0103 allele versus those homozygous for HLA-E*0101 (P = 0.37). In contrast to the study from Mossallam and colleagues,²⁸ GVHD and disease stage did not affect risk of relapse after allogeneic transplant. Our results revealed that those with HLA-E*0103/*0103 genotype had lower survival, with an average survival time of 94 days versus survival time of 648 days for those with the HLA-E*0101/*0103 genotype.

Some researchers have clearly shown that incidence of relapse after HSCT is because of evasion of malignant cells from graft-versus-leukemia effect, which is considerably significant for elimination of residual malignant cells before or after HSCT.^7,8,18,21 Furthermore, the HLA-E molecule has a consequential function in the modulation of NK cell interactions, which is highly important for elimination of residual malignant cells. In addition, evidence has shown that patients with the HLA-E*0103 allele have a stronger graft-versus-leukemia effect.^29-32

The prevalence of different HLA-E genotypes
is not related to occurrence of acute GVHD and transplant-related mortality, Furthermore, the prevalence of acute GVHD in those with the HLA-E*0101/*0103 genotype was higher than in those with the other genotypes. Moreover, our results offer no association between HLA-E genotype and increased overall survival following HLA-E genoidentical allogeneic HSCT. Together, these considerations suggest no proof for the role of polymorphisms of HLA-E in allogeneic HSCT and its effects.

We could not find any association between progressive occurrence of acute GVHD and HLA-E polymorphism, which is in agreement with Hosseini and associates,³³ who reported on unrelated donor HLA-matched transplant. Moreover, we could not find any connection between cumulative incidence of chronic GVHD and HLA-E, although Hosseini and associates⁷ reported reduced risk of chronic GVHD in those with the HLA-E*0103/*0103 genotype.

Tamouza and colleagues¹⁷ showed an elevation in bacterial infection in transplant patients with HLA-E*0103 homozygous unrelated donors, which may be because of ineffectual exposure of bacterial peptides by this HLA-E molecule. Nevertheless, we could not observe a similar relationship in our population due to low incidence of bacterial infection in our transplant patients. In our study, only 8 patients had urinary tract bacterial infections and only 10 had cytomegalovirus infections after HSCT, with no association with HLA-E genotypes. Other researchers have reported a protective effect of the HLA-E*0103 allele on acute GVHD in related and unrelated transplants patients.^18,34,35 Furthermore, Tamouza and associates18 declared that com­petitiveness between HLA-E*0103 and classical class I HLA molecules for presentation of mHAg to T cells and reduced tissue damage mediated by NK cells may be involved in protection from acute GVHD.

Conclusions

A clear understanding of the association between HLA-E polymorphisms and alleviation of risk of relapse requires further investigations in larger cohorts. Investigations into the role of HLA-E polymorphisms in graft-versus-leukemia disease will help researchers to better understand T-cell and NK cell alloreactivity and their role in immunotherapy after autologous HSCT.

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