HLA-E Alleles Research Articles

The HLA‐ER/HLA‐ERGenotype Affects the Natural Course of Hepatitis C Virus (HCV) Infection and Is Associated with HLA‐E–Restricted Recognition of an HCV‐Derived Peptide by Interferon‐γ–Secreting Human CD8+T Cells

Recently, we showed chronic hepatitis C to be associated with increased expression of HLA-E and identified peptide hepatitis C virus (HCV) core amino acids 35-44 as a ligand for HLA-E that stabilizes HLA-E expression, favoring inhibition of natural killer cell cytotoxicity. Here we describe HLA-E-restricted recognition of peptide HCV core amino acids 35-44 by CD8(+) T cells. Frequency of HLA-E-restricted responses was significantly higher in patients homozygous for the HLA-E(R) allele (60% vs 38%; P = .038). Moreover, we found that the HLA-E(R) allelic variant confers protection against chronic infection with HCV genotypes 2 and 3. Taken together, our data indicate an important immunomodulating function of HLA-E in hepatitis C.

Clinical Significance of HLA-E*0103 Homozygosity on Survival After Allogeneic Hematopoietic Stem-Cell Transplantation

Hematopoietic stem-cell transplantation is a well-established treatment in various hematologic malignancies, but the outcome depends on disease relapse, infections, and the development and severity of acute and chronic graft-versus-host disease. Some evidence has revealed an important role for the nonclassical major histocompatibility complex class I molecules in transplantation, most notably human leukocyte antigen (HLA)-E. This study evaluates the impact of HLA-E alleles on transplantation outcome after HLA-matched allogeneic HSCT. We genotyped DNA for HLA-E polymorphism from 83 recipients and their respective donors by real-time polymerase chain reaction after melting curve analysis and compared the results with clinical outcome. HLA-E*0103 homozygous patients showed a higher probability of overall survival (P=0.003) and disease-free survival (P=0.001) in a univariate model. Cox regression analysis confirmed HLA-E*0103, 0103 (P=0.006; relative risk 1.12; 95% confidence interval 0.31-1.94) and early stage of disease (P=0.005; relative risk 1.16; 95% confidence interval 0.45-1.86) as independent factors improving overall survival. Moreover, homozygosity for HLA-E*0103 was associated with a significant decreased incidence of transplant-related mortality (P=0.01). We found an association between HLA-E*0103 homozygosity and the significant reduction of transplant-related mortality in related and unrelated HSCT. The risk of posttransplant complications was significantly reduced when the donor possesses the HLA-E*0103, 0103 genotype, and this was translated in a better overall survival.

Influence of Hsp70 and HLA-E on the killing of leukemic blasts by cytokine/Hsp70 peptide-activated human natural killer (NK) cells

This study compared the effects of the human 70-kDa stress protein (Hsp70) peptide, TKDNNLLGRFELSG (TKD), proinflammatory cytokines, or a combination of both on the repertoire of receptors expressed by human natural killer (NK) cells and their capacity to kill human CX colon carcinoma cells, K562 erythroleukemic cells, and leukemic blasts from two patients with acute myelogenous leukemia. Low-dose interleukin (IL) 2/IL-15 and TKD increase the expression density of activatory (NKG2D, NKp30, NKp44, NKp46, CD94/NKG2C) and inhibitory (CD94/NKG2A) receptors on NK cells. Concomitantly, IL-2/TKD treatment enhances the cytotoxicity of NK cells (as reflected by their secretion of granzyme B) against Hsp70 membrane-positive and human leukocyte antigen (HLA)-E membrane-negative (Hsp70(+)/HLA-E(-)) CX(+) and K562 cells. However, it had no effect on the responsiveness to Hsp70(-)/HLA-E(-) CX(-) cells over that induced by IL-2 alone. The cytotoxicity of IL-2/TKD-activated, purified NK cells and peripheral blood mononuclear cells against Hsp70(+)/HLA-E(+) leukemic blasts was weaker than that against Hsp70(+)/HLA-E(-) K562 cells. Hsp70-blocking and HLA-E transfection experiments confirmed membrane-bound Hsp70 as being a recognition/activatory ligand for NK cells, as cytotoxicity was reduced by the presence of the anti-Hsp70 monoclonal antibody cmHsp70.2 and by inhibiting Hsp70 synthesis using short interference ribonucleic acid. HLA-E was confirmed as an inhibitory ligand, as the extent of NK cell-mediated lysis of K562 cell populations that had been transfected with HLA-E(R) or HLA-E(G) alleles was dependent on the proportion of HLA-E-expressing cells. These findings indicate that Hsp70 (as an activatory molecule) and HLA-E (as an inhibitory ligand) expression influence the susceptibility of leukemic cells to the cytolytic activities of cytokine/TKD-activated NK cells.

Genetic fixity in the human major histocompatibility complex and block size diversity in the class I region including HLA-E

BackgroundThe definition of human MHC class I haplotypes through association of HLA-A, HLA-Cw and HLA-B has been used to analyze ethnicity, population migrations and disease association.ResultsHere, we present HLA-E allele haplotype association and population linkage disequilibrium (LD) analysis within the ~1.3 Mb bounded by HLA-B/Cw and HLA-A to increase the resolution of identified class I haplotypes. Through local breakdown of LD, we inferred ancestral recombination points both upstream and downstream of HLA-E contributing to alternative block structures within previously identified haplotypes. Through single nucleotide polymorphism (SNP) analysis of the MHC region, we also confirmed the essential genetic fixity, previously inferred by MHC allele analysis, of three conserved extended haplotypes (CEHs), and we demonstrated that commercially-available SNP analysis can be used in the MHC to help define CEHs and CEH fragments.ConclusionWe conclude that to generate high-resolution maps for relating MHC haplotypes to disease susceptibility, both SNP and MHC allele analysis must be conducted as complementary techniques.

HLA‐E polymorphism in Amerindians from Mexico (Mazatecans), Colombia (Wayu) and Chile (Mapuches): evolution of MHC‐E gene

Human leukocyte antigen (HLA)-E is a nonclassical class I (Ib) gene with a restricted polymorphism. Only eight DNA alleles and three proteins of this gene have been described and their frequencies analyzed in Caucasian, Oriental, Asian Indian, and Negroid populations. In the present study, HLA-E polymorphism has been analyzed in six Amerindian and Mestizo populations from North and South America and compared with previously described populations. HLA-E*0101 is the most frequent allele found in all populations except in Afrocolombian and Wayu Amerindians, in which blood group analyses show a high admixture with Caucasian and African populations. Mazatecan and Mapuche (two Amerindian groups from North and South America, respectively) presented similar HLA-E frequencies, whereas Wayu Indians are more similar to the Afrocolombian population. The Mexican and Colombian Mestizo show similar allele frequencies to Amerindians with high frequencies of HLA-E*0101 and HLA-E*010302 alleles. Also, frequencies in Negroids and Asian Indians present a similar distribution of HLA-E alleles. These data are in agreement with worldwide restricted polymorphism of HLA-E because no new allele was detected in the six populations studied. The allelic frequencies show differences among Caucasian, Oriental, Mestizo and Indian populations. Ape major histocompatibility complex-E allelism is also very restricted: common chimpanzee (one allele); bonobo (two alleles); gorilla (two alleles); orangutan (one allele); rhesus monkey (eight alleles); cynomolgus monkey (two alleles); and green monkey (two alleles).

HLA‐E*0101 and HLA‐G*010101 reduce the risk of Behcet’s disease

The nonclassical human leukocyte antigen (HLA)-E and -G molecules have previously been shown to inhibit natural killer- and cytotoxic T-lymphocyte-mediated cell lysis and have also been shown to prevent the proliferation of CD4 T cells and secrete cytokines that appear to be important in the modulation of the Behcet's disease (BD) immune systems. Polymorphisms in the HLA-E and HLA-G genes have been associated with differential expression and function. Thus, we conducted an analysis of the HLA-E and HLA-G alleles using Amplification Refractory Mutation System-polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphism techniques in a study comprising 312 patients with BD and 486 controls. The HLA-E*0101 and HLA-G*010101 alleles were associated with a reduced risk of BD (P = 0.0002, odds ratio (OR) = 0.7 and P = 0.002, OR = 0.7, respectively). By way of contrast, the variants HLA-E*010302, HLA-G*010102, G*0105N alleles and 3741_3754ins14bp were all associated with an increased risk of BD (P < 0.0001, OR = 1.6; P = 0.002, OR = 1.8; P = 0.024, OR = 2.0 and P = 0.003, OR = 1.4, respectively). Individuals carrying both the HLA-E*0101 and the HLA-G*010101 alleles evidenced significantly lower frequency in the patients than in the controls (35.6% vs 49.6%; P < 0.0001, OR = 0.6). These results indicate that variant HLA-E and HLA-G molecules appear to function independently and synergistically, increasing the risk of BD, and may result in an imbalance of lymphocytic functions, which may culminate in the development of BD.

Homozygous Status for HLA-E*0103 Confers Protection from Acute Graft-Versus-Host Disease and Transplant-Related Mortality in HLA-Matched Sibling Hematopoietic Stem Cell Transplantation

The posttransplant period following hematopoietic stem cell transplantation (HSCT) is potentially high risk for developing survival-compromising complications, many of which are known to be under the control of immunogenetic factors. Given the dual role of human leukocyte antigen (HLA)-E molecules in innate and adaptive immune processes, we analyzed the impact of HLA-E polymorphism in genoidentical HSCT setting. We analyzed 187 HLA-genoidentical sibling pairs for HLA-E polymorphism. To explore its potential association with the incidence of acute and chronic graft versus host disease (aGVHD, cGVHD), severe infections, risk for transplant-related mortality (TRM), and overall survival, HLA-E locus was genotyped by a polymerase chain-reaction-sequence-specific primer (PCR-SSP) strategy. Multivariate analysis, taking into account the patient-, donor- and transplant-related factors, showed that the incidence of aGVHD and TRM at day 180 were low when the genotype was HLA-E*0103/E*0103, either in the donor or in the recipient, the pairs being identical for HLA-E alleles (hazard ratio [HR]=0.71, P=0.009; and HR=0.42, P=0.04, respectively). We also found a trend towards association between E*0103 homozygosity and improved survival (P=0.05). There was no association between HLA-E polymorphism and incidence of severe infections. These data suggest that the homozygous state for HLA-E*0103 allele behaves as a protective genetic factor against aGVHD and TRM and likely contributes to improved survival in HLA-genoidentical bone marrow transplantation.

HLA‐E and immunobiology of pregnancy

Recently, it has been suggested that non-classical antigens such as human leukocyte antigen (HLA)-G and HLA-E may interact with KIR receptors of NK cells which results into downregulation of immune response and helps in the maintenance of pregnancy. In the present study, we have investigated HLA-E polymorphism in normal fertile women and recurrent spontaneous aborters to assess the effect of HLA-E alleles on the success of pregnancy. Allele E*0101 was found to be significantly higher in patients with recurrent spontaneous abortion (chi(2) = 4.097 and P = 0.0430). Differential expression, peptide affinity, and stability of E*0101 may be one of the reasons.

HLA‐E polymorphism in patients with nasopharyngeal carcinoma

Nasopharyngeal carcinoma (NPC) has been known to be associated with HLA class I region. The aim of this study was to investigate the association between HLA-E and genetic susceptibility to nasopharyngeal carcinogenesis by comparing the frequencies of HLA-E alleles in 100 Thai NPC patients and 100 healthy controls. HLA-E typing was performed by means of polymerase chain reaction-sequence-specific oligonucleotide probe method. The frequency of the HLA-E*0103 allele and HLA-E*0103, 0103 genotype, but not others, was increased in NPC patients, compared to controls. This observation suggests a possible role for HLA-E in NPC development, possibly via natural killer cell or cytotoxic lymphocyte function.

Pig-tailed macaques (Macaca nemestrina) possess six MHC-E families that are conserved among macaque species: implication for their binding to natural killer receptor variants.

MHC loci encode highly polymorphic molecules involved in the presentation of self and non-self peptides to cells of the adaptive and innate immune systems. Although variable, MHC-E genes are well conserved among primates and provide signals to natural killer cells. In this study, we sequenced and analyzed MHC-E alleles of pig-tailed macaque (Macaca nemestrina), a nonhuman primate used for HIV pathogenesis and vaccine studies. Among a group of seven macaques, the characterization of eight Mane-E alleles revealed an increased number of polymorphic sites compared with human HLA-E alleles. Phylogenetic analyses of MHC-E alleles from pig-tailed macaque, rhesus monkey (Macaca mulatta) and cynomolgus macaque (Macaca fascicularis) demonstrated that the three macaque species shared six families of macaque MHC-E alleles and indicated that these families existed in the common ancestor 5.5 million years ago. Polymorphic Mane-E sites were not concentrated within the peptide-binding pockets, but were distributed throughout the entire ORF. The peptide-binding domain of Mane-E is similar to its human analogue, and peptide substrates theoretically capable of binding to Mane-E molecules were found in the leader sequence of classical Mane-A and -B molecules. Additionally, the polymorphic amino acids located in the alpha(1) and alpha(2) domains of Mane-E molecules have side chains expected to be oriented toward solvent and away from the peptide-binding groove, suggesting that some of them (positions 19, 73, 79 and 145) might be available for interaction with polymorphic receptors of natural killer cells.

HLA-E Allelic Variants: CORRELATING DIFFERENTIAL EXPRESSION, PEPTIDE AFFINITIES, CRYSTAL STRUCTURES, AND THERMAL STABILITIES

Previous studies of HLA-E allelic polymorphism have indicated that balancing selection may be acting to maintain two major alleles in most populations, indicating that a functional difference may exist between the alleles. The alleles differ at only one amino acid position, where an arginine at position 107 in HLA-E*0101 (E(R)) is replaced by a glycine in HLA-E*0103 (E(G)). To investigate possible functional differences, we have undertaken a study of the physical and biochemical properties of these two proteins. By comparing expression levels, we found that whereas steady-state protein levels were similar, the two alleles did in fact differ with respect to cell surface levels. To help explain this difference, we undertook studies of the relative differences in peptide affinity, complex stability, and three-dimensional structure between the alleles. The crystal structures for HLA-E(G) complexed with two distinct peptides were determined, and both were compared with the HLA-E(R) structure. No significant differences in the structure of HLA-E were induced as a result of binding different peptides or by the allelic substitution at position 107. However, there were clear differences in the relative affinity for peptide of each heavy chain, which correlated with and may be explained by differences between their thermal stabilities. These differences were completely consistent with the relative levels of the HLA-E alleles on the cell surface and may indeed correlate with functional differences. This in turn may help explain the apparent balancing selection acting on this locus.

Analysis of HLA-E expression in human tumors

Downregulation of MHC class Ia molecule expression is a widespread mechanism used by tumor cells to escape antitumor T-cell-mediated immune responses. However, it is not known why NK cells cannot lyse these MHC class-Ia-deficient tumor targets. Tumors must select additional routes of escape from NK cells. An attractive hypothesis is that the aberrant expression of nonclassical HLA class Ia molecules in tumors provides the required inhibitory signal to NK cells, rendering tumor cells resistant to NK lysis. To analyze the possible role of HLA-E molecules in providing tumor cells with an NK escape mechanism, we studied the cell surface expression of this HLA class Ib molecule in a variety of tumor cell lines with well-defined HLA class Ia alterations. Tests were done with the monoclonal antibody 3D12 recognizing cell surface HLA-E molecules. Our results indicate that HLA-E was mainly detected in leukemia-derived cell lines. In addition, HLA-E was detected in tumor cell lines of different origin. This expression was related with the availability of free beta(2)-microglobulin (beta(2)m) in the cytoplasm of tumor cells. Expression was detected in tumor cell lines showing an imbalance in heavy chain/beta(2)m expression, particularly in tumor cell lines with alterations in the expression of heavy-chain genes. Several lines of evidence favor these conclusions: (1) In the FM55 and NW145 melanoma tumor systems, the reduction in HLA class Ia expression paralleled the increased cell surface detection of HLA-E. (2) A cervical tumor (808) and a melanoma cell line (R22.2) expressing a single HLA-A1 allele also expressed HLA-E. (3) The addition of human beta(2)m to tumor cell lines that expressed the HLA-E(G) allele increased HLA-E cell surface expression. (4) There was no HLA-E cell surface expression in tumor cell lines with total loss of HLA class Ia expression, including cell lines with low transcription of HLA class I heavy chains or with beta(2)m mutations. Our findings suggest that the biological consequences of these cumulative genetic and molecular changes in tumor cells lead to the appearance of HLA-E in a limited number of tumor cell lines with peculiar phenotypic and genotypic characteristics, namely: HLA-class Ia downregulation, free beta(2)m and HLA-E(G) genotype. The aberrant HLA-E expression might be of particular biological relevance in those HLA tumor phenotypes that express a single HLA-A allele when NK inhibition is markedly reduced due to the downregulation of HLA-B and -C alleles.

Nomenclature for factors of the HLA system, 2002.

This chapter provides the nomenclature for factors of the HLA system, 2002. A number of previously described class I and II gene fragments within the HLA region are named in this system. Official designations are given to these gene fragments. The names LMP2 and LMP7 used previously for the two proteasome genes in the HLA class II region have been renamed by the Human Genome Nomenclature committee (HGNC) as PSMB9 and PSMB8, respectively. This system introduces the additional digit for synonymous variation and all allele names that are currently five digits or above are renamed accordingly. The use of an optional “N’” or “L” suffix to an allele name to indicate either “Null” or “Low” expression was introduced in previous Nomenclature Reports. Three new suffixes are introduced in this system. An “S” to denote an allele specifying a protein that is expressed as a soluble “Secreted” molecule but is not present on the cell surface; a “C” to indicate an allele product that is present in the “Cytoplasm” but not at the cell surface; an “A” to indicate “Aberrant” expression where there is some doubt as to whether a protein is expressed or not. There is evidence of differential splicing of HLA-G that leads to the production of both membrane-bound and soluble forms of the same allele. The IMGT/HLA Sequence Database contains sequences for all HLA alleles officially recognized by the WHO Nomenclature Committee for Factors of the HLA System and, provides users with online tools and facilities for their retrieval and analysis.

Implications of HLA-E allele expression and different HLA-E ligand diversity for the regulation of NK cells

The interaction of HLA-E with CD94/NKG2A is dependant on the binding of HLA class I signal sequence derived peptides to HLA-E. In the caucasoid population two HLA-E alleles are observed at equal frequencies. Here we study the functional differences between the two HLA-E molecules with regard to cell surface expression, peptide binding, and potential to inhibit lytic activity of a CD94/NKG2A + NK cell line. In contrast to the HLA-E R allele, the HLA-E G allele shows considerable cell surface expression even in the absence of endogenous HLA class I signal sequence derived HLA-E ligands. Eighteen HLA-E allele/HLA-E ligand combinations were analyzed. No correlation between cell surface expression of HLA-E and NK cell inhibition was observed. The peptides present in the signal sequences of HLA-B15, -Cw0402, and -Cw7 bound to both HLA-E alleles but did not lead to an inhibition of NK cell lysis. In our experimental system the peptides A2 and G were not effective with regard to NK cell inhibition when bound to the HLA-E R allele. These results may be of functional significance particularly in the placenta where the only HLA-E ligands are derived from HLA-G and -C.

HLA-G and HLA-E polymorphisms in an indigenous African population

Studies of HLA-G and HLA-E polymorphisms in different populations from many industrialized countries have uniformly reported little sequence variation within these genes. To date, the polymorphism of these genes has not been characterized in populations from developing countries where more sequence variation would be anticipated due to greater exposure to microbial pathogens. In order to address this issue, we have recently investigated the polymorphism of these MHC genes in an indigenous African population (Shona ethnic group). HLA-G and HLA-E alleles were determined by PCR-restriction fragment length polymorphism (PCR-RFLP) and amplification refractory mutation system (ARMS) analysis in 108 unrelated women recruited from maternity clinics and hospitals in Harare (Zimbabwe). The genotyping analysis identified six different HLA-G alleles and three HLA-E alleles in this population. Of interest, the null-allele HLA-G∗0105N was found with 11.1% of frequency, which is higher than in other populations tested so far. We observed the presence of 15 distinct HLA-G genotypes and 6 HLA-E genotypes. These data indicate that this African population contains low levels of allelic polymorphism similar to ethnic groups from industrialized countries. This is the first report describing HLA-G and HLA-E polymorphisms in an indigenous African population.

HLA-E polymorphism in patients with recurrent spontaneous abortion.

The aim of this study was to compare the frequencies of five HLA-E alleles in 82 women with recurrent spontaneous abortions with that of 150 random Danish controls. PCR sequence-specific oligonucleotide typing was carried out to detect polymorphism in exons 2 and 3 of the HLA-E gene. In informative samples sequencing of these two exons was also undertaken to confirm the presence of the HLA-E*01031 allele. HLA-E*0101, HLA-E*01032 and HLA-E*01031 were detected with frequencies of 56.7%, 33.6% and 9.6% in controls and 58,5%, 32.9% and 8.5% in patients with recurrent abortion, respectively. No HLA-E*0102 and E*0104 alleles could be detected. Since the HLA-E allele distribution was similar in women with recurrent spontaneous abortion and controls, it is suggested that maternal HLA-E polymorphism per se does not play any role in the pathogenesis of this disorder of pregnancy.

Description of a New HLA-E (E∗01031) Allele and Its Frequency in the Spanish Population

An HLA-E polymorphism study by oligotyping and DNA sequencing was carried out in the Spanish population. As a result, a new HLA-E allele (E∗01031) initially assigned by polymerase chain reaction oligotyping as E∗0104 was found. This allele presents a synonymous change at codon 77 (AAT → AAC; Asn) when compared with the E∗01032 allele. This position is located in the α-helix (α1-domain) and is involved in the peptide binding region of the hypothetical HLA-E molecule. Among 60 Spanish individuals, HLA-E∗0101 presents the highest phenotype frequency, followed in decreasing order by E∗01032, E∗01031 (new allele), and E∗0102. Also, new partial intron 1 and complete intron 2 sequences from E∗0101, E∗01031, and E∗01032 are described; the sequences are identical among the three forms. However, the intron 2 sequence of the E∗0102 allele bears a two-base deletion not found in apes.

Population genetic studies of HLA-E: Evidence for Selection

HLA-E is a nonclassical, class I gene (Ib) of unknown function. This study was initiated to determine the amount and nature of the variation in the class Ib gene HLA-E in diverse ethnic groups. A single basepair substitution (A → G at 382, exon 3) resulting in a change from an arginine (R) to a glycine (G) at codon 107 was found. A glycine was present at position 107 in individuals from four ape species, suggesting that EG107 is the older of the two alleles. The two human alleles were present in all samples studied. The alleles were in linkage disequilibrium with HLA-A (W = 0.58), HLA-B (W =0.59) and HLA-C (W = 0.55) in the Hutterites. The frequencies of the two HLA-E alleles were more equal than expectations based on neutrality in inbred and outbred Caucasian samples (Watterson's F = 0.506, p = 0.02 and F = 0.512, p = 0.047, respectively) and nearly significant in African-American and Hispanic samples ( F =0.513, p = 0.063 and F = 0.508, p = 0.053). These data suggest that this polymorphism arose before the expansion of Homo sapiens and has been maintained in diverse populations by stabilizing selection.

P367 - On the diversity of HLA-E: Identification of a novel HLA-E allele

P367 - On the diversity of HLA-E: Identification of a novel HLA-E allele

Polymorphism at the HLA-E locus predates most HLA-A and -B polymorphism

The extensive polymorphism of the classic class I antigens has been well described. In contrast, the nonclassic HLA antigens are distinguished by their low polymorphism. We examine here the HLA polymorphism of the HLA-E locus by examining the DNA sequence of cDNA from nine ethnically diverse individuals. From this analysis, we show that there is no polymorphism in the regions including exon 1 and from exon 4 to exon 8, the 3′ untranslated exon. In exons 2 and 3, there are two base substitutions, one of which is at a replacement site and the other silent. The replacement substitution changes an arginine to a glycine at position 107, defining two alleles at the HLA-E locus. Using the PCR on exon 3 from genomic DNA and hybridization with oligonucleotide probes, we have examined 90 HLA-typed individuals to determine the relative frequency of the two alleles in the population and their association with the classical antigens. This analysis showed that these two alleles were present at nearly equal frequencies in the population. Surprisingly, both alleles were found in an essentially random association with all but one HLA-A and -B haplotype. The single exception was to the A1-B8 haplotype, which appeared to be linked to only one of the two alleles. One implication of this random association is that these HLA-E alleles may have existed before most of the HLA-A and B polymorphism. Thus, selection has maintained the HLA-E locus essentially unaltered during a time when considerable polymorphism was being selected for at the HLA-A and -B loci. This finding may also have important consequences in an unrelated bone marrow transplant, where it is predicted that 37% of HLA-A and -B matched donors are mismatched at the HLA-E locus.