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Protective Effect of HLA-E*0101/*0103 Genotype in Survival of Patients After Allogeneic Hematopoietic Stem Cell Transplant

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


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