Resolving unknown nucleotides in the IPD-IMGT/HLA database by extended and full-length sequencing of HLA class I and II alleles.

In the past, identification of HLA alleles was limited to sequencing the region of the gene coding for the peptide binding groove, resulting in a lack of sequence information in the HLA database, challenging HLA allele assignment software programs. We investigated full-length sequences of 19 HLA class I and 7 HLA class II alleles, and we extended another 47 HLA class I alleles with sequences of 5' and 3' UTR regions that were all not yet available in the IPD-IMGT/HLA database. We resolved 8638 unknown nucleotides in the coding sequence of HLA class I and 2139 of HLA class II. Furthermore, with full-length sequencing of the 26 alleles, more than 90 kb of sequence information was added to the non-coding sequences, whereas extension of the 47 alleles resulted in the addition of 5.5 kb unknown nucleotides to the 5' UTR and > 31.7 kb to the 3' UTR region. With this information, some interesting features were observed, like possible recombination events and lineage evolutionary origins. The continuing increase in the availability of full-length sequences in the HLA database will enable the identification of the evolutionary origin and will help the community to improve the alignment and assignment accuracy of HLA alleles.

Specific amino acid patterns define split specificities of HLA-B15 antigens enabling conversion from DNA-based typing to serological equivalents.

The HLA-B15 typing by serological approaches defined the serological subgroups (or splits) B62, B63, B75, B76, B77 and B70 (B71 and B72). The scarcity of sera with specific anti-HLA antibodies makes the serological typing method difficult to discriminate a high variety of HLA antigens, especially between the B15 antigen subgroups. Advancements in DNA-based technologies have led to a switch from serological typing to high-resolution DNA typing methods. DNA sequencing techniques assign B15 specificity to all alleles in the HLA-B*15 allele group, without distinction of the serological split equivalents. However, the presence of antibodies in the patient defined as split B15 antigens urges the identification of HLA-B*15 allele subtypes of the donor, since the presence of donor-specific antibodies is an important contraindication for organ transplantation. Although the HLA dictionary comprises information regarding the serological subtypes of HLA alleles, there are currently 394 B15 antigens out of 516 in the IPD-IMGT/HLA database (3.38.0) without any assigned serological subtype. In this regard, we aimed to identify specific amino acid patterns for each B*15 serological split, in order to facilitate the assignment of B*15 alleles to serological equivalents after high-resolution molecular typing. As a result, serological specificities of 372/394 not yet assigned alleles could be predicted based on amino acid motifs. Furthermore, two new serological types were identified and added, B62-Bw4 and B71-Bw4.

Human leukocyte antigen typing using buccal swabs as accurate and non-invasive substitute for venipuncture in children at risk for celiac disease.

BACKGROUND AND AIM: Human leukocyte antigen (HLA) typing is an important step in the diagnostic algorithm for celiac disease (CD) and is also used for screening purposes. Collection of blood is invasive and accompanied with emotional impact especially in children. Genetic technological progress now enables HLA typing from buccal cell samples. This study evaluated the reliability and feasibility of HLA typing for CD-associated HLA polymorphisms using buccal swabs as routine test in high-risk individuals. METHODS: Blood and buccal swabs of 77 children and adolescents with high risk for CD were prospectively collected in this cohort study. Buccal swab collection was performed either by the investigator at the outpatient clinic or by the patient or its parents at home. To evaluate the possibility of self-administration, three families performed the test at home. DNA was extracted using an adapted QIAamp method. Quantity, quality, and purity of DNA were recorded. HLA-DRB1, HLA-DQA1, and HLA-DQB1 typing was examined on buccal cell-derived and blood-derived DNA at low and, if necessary, high resolution level, using sequence-specific oligonucleotide and sequence-based typing, respectively. RESULTS: DNA isolation using buccal swabs yielded a good quality and sufficient quantity of DNA to perform HLA-DQ typing in all individuals. HLA typing results on buccal cell-derived DNA were identical to typing on blood-derived DNA, also for the self-administered samples. CONCLUSION: Introduction of the buccal swab test for HLA typing of CD risk in routine diagnostics can omit the current venipuncture and enables self-administration at home. Therefore, the buccal swab test is beneficial for individuals with a clinical suspicion for CD, as well as for screening purposes in high-risk populations.

Analysis of FCGR3A-p.176Val variants in women with recurrent pregnancy loss and the association with CD16a expression and anti-HLA antibody status.

Natural Killer (NK) cells have been implicated in recurrent pregnancy loss (RPL). The p.Val176Phe (or Val158Phe) Single Nucleotide Polymorphism (SNP) in the FCGR3A gene encoding the FcγRIIIA or CD16a receptor has been associated with an enhanced affinity for IgG and stronger NK-mediated antibody-dependent cellular cytotoxicity. We hypothesized that the presence of at least one p.176Val variant associates with RPL and increased CD16a expression and alloantibodies e.g., against paternal human leukocyte antigen (HLA). In 50 women with RPL, we studied frequencies of the p.Val176Phe FCGR3A polymorphisms. Additionally, CD16a expression and anti-HLA antibody status were analyzed by flowcytometry and Luminex Single Antigens. In woman with RPL, frequencies were: 20% (VV), 42% (VF) and 38% (FF). This was comparable to frequencies from the European population in the NCBI SNP database and in an independent Dutch cohort of healthy women. NK cells from RPL women with a VV (22,575 [18731-24607]) and VF (24,294 [20157-26637]) polymorphism showed a higher expression of the CD16a receptor than NK cells from RPL women with FF (17,367 [13257-19730]). No difference in frequencies of the FCGR3A-p.176 SNP were detected when comparing women with or without class I and class II anti-HLA antibodies. Our study does not provide strong evidence for an association between the p.Val176Phe FCGR3A SNP and RPL.

Polymorphic differences within HLA-C alleles contribute to alternatively spliced transcripts lacking exon 5.

The HLA genes are amongst the most polymorphic in the human genome. Alternative splicing could add an extra layer of complexity, but has not been studied extensively. Here, we applied an RNA based approach to study the influence of allele polymorphism on alternative splicing of HLA-C in peripheral blood. RNA was isolated from these peripheral cells, converted into cDNA and amplified specifically for 12 common HLA-C allele groups. Through subsequent sequencing of HLA-C, we observed alternative splicing variants of HLA-C*04 and *16 that resulted in exon 5 skipping and were co-expressed with the mature transcript. Investigation of intron 4 sequences of HLA-C*04 and *16 compared with other HLA-C alleles demonstrated no effect on predicted splice sites and branch point. To further investigate if the unique polymorphic positions in exon 5 of HLA-C*04 or *16 may facilitate alternative splicing by acting on splicing regulatory elements (SRE), in-silico splicing analysis was performed. While the HLA-C*04 specific SNP in exon 5 had no effect on predicted exonic SRE, the HLA-C*16 specific exon 5 SNP did alter exonic SRE. Our findings provide experimental and theoretical support for the concept that polymorphisms within the HLA-C alleles influence the alternative splicing of HLA-C.

HLA Class I Molecules as Immune Checkpoints for NK Cell Alloreactivity and Anti-Viral Immunity in Kidney Transplantation.

Natural killer (NK) cells are innate lymphocytes that can kill diseased- or virally-infected cells, mediate antibody dependent cytotoxicity and produce type I immune-associated cytokines upon activation. NK cells also contribute to the allo-immune response upon kidney transplantation either by promoting allograft rejection through lysis of cells of the transplanted organ or by promoting alloreactive T cells. In addition, they protect against viral infections upon transplantation which may be especially relevant in patients receiving high dose immune suppression. NK cell activation is tightly regulated through the integrated balance of signaling via inhibitory- and activating receptors. HLA class I molecules are critical regulators of NK cell activation through the interaction with inhibitory- as well as activating NK cell receptors, hence, HLA molecules act as critical immune checkpoints for NK cells. In the current review, we evaluate how NK cell alloreactivity and anti-viral immunity are regulated by NK cell receptors belonging to the KIR family and interacting with classical HLA class I molecules, or by NKG2A/C and LILRB1/KIR2DL4 engaging non-classical HLA-E or -G. In addition, we provide an overview of the methods to determine genetic variation in these receptors and their HLA ligands.

Long-Read Nanopore Sequencing Validated for Human Leukocyte Antigen Class I Typing in Routine Diagnostics.

Matching of human leukocyte antigen (HLA) gene polymorphisms by high-resolution DNA sequence analysis is the gold standard for determining compatibility between patient and donor for hematopoietic stem cell transplantation. Single-molecule sequencing (PacBio or MinION) is a newest (third) generation sequencing approach. MinION is a nanopore sequencing platform, which provides long targeted DNA sequences. The long reads provide unambiguous phasing, but the initial high error profile prevented its use in high-impact applications, such as HLA typing for HLA matching of donor and recipient in the transplantation setting. Ongoing developments on instrumentation and basecalling software have improved the per-base accuracy of 1D2 nanopore reads tremendously. In the current study, two validation panels of samples covering 70 of the 71 known HLA class I allele groups were used to compare third field sequences obtained by MinION, with Sanger sequence-based typing showing a 100% concordance between both data sets. In addition, the first validation panel was used to set the acceptance criteria for the use of MinION in a routine setting. The acceptance criteria were subsequently confirmed with the second validation panel. In summary, the present study describes validation and implementation of nanopore sequencing HLA class I typing method and illustrates that nanopore sequencing technology has advanced to a point where it can be used in routine diagnostics with high accuracy.

Insights into the polymorphism in HLA-DRA and its evolutionary relationship with HLA haplotypes.

HLA-DRA encodes the alpha chain of the HLA-DR protein, one of the classical HLA class II molecules. Reported polymorphism within HLA-DRA is currently limited compared with other HLA genes, as only a single polymorphism encodes an amino acid difference in the translated protein. Since this SNP (rs7192, HLA00662.1:g.4276G>T p.Val217Leu) lies within exon 4, in the region encoding the cytoplasmic tail, the resulting protein is effectively monomorphic. For this reason, in-depth studies on HLA-DRA and its function have been limited. However, analysis of sequences from the 1000 Genomes Project and preliminary data from our lab reveals unrepresented polymorphism within HLA-DRA, suggesting a more complex role within the MHC than previously assumed. This study focuses on elucidating the extent of HLA-DRA polymorphism, and extending our understanding of the gene's role in HLA-DR~HLA-DQ haplotypes. Ninety-eight samples were sequenced for full-length HLA-DRA, and from this analysis, we identified 20 novel SNP positions in the intronic sequences within the 5711 bp region represented in IPD-IMGT/HLA. This polymorphism gives rise to at least 22 novel HLA-DRA alleles, and the patterns of intronic and 3' UTR polymorphism correspond to HLA-DRA~HLA-DRB345~HLA-DRB1~HLA-DQB1 haplotypes. The current understanding of the organization of the genes within the HLA-DR region assumes a single lineage for the HLA-DRA gene, as opposed to multiple gene lineages, such as in HLA-DRB. This study suggests that the intron and 3' UTR polymorphism of HLA-DRA indicates different lineages, and represents the HLA-DRA~HLA-DRB345~HLA-DRB1~HLA-DQB1 haplotypes.

Clinical relevance of pretransplant donor-directed antibodies detected by single antigen beads in highly sensitized renal transplant patients.

BACKGROUND: Highly sensitized (HS) patients (>85% panel-reactive antibodies) have a lower chance of receiving a donor kidney. Within Eurotransplant the Acceptable Mismatch (AM) program was developed to increase the chances of HS patients to receive a crossmatch-negative donor kidney. The standard crossmatch in the AM program is based on complement-dependent cytotoxicity. METHODS: In this study we wanted to determine the clinical relevance of human leukocyte antigen donor-directed antibodies (DDA) detected by the single antigen (SA) bead technique, in the pretransplant sera of HS patients transplanted in our center through the Eurotransplant AM program. RESULTS: From 34 AM patients, 27 were transplanted with 1 to 5 mismatches and 7 received a 0-mismatched graft. From the mismatched patients, retrospectively, 13 proved to possess pretransplant DDA by SA whereas 14 did not. No antibodies were found in the 0-mismatched group. Comparison of the DDA+ and DDA- patients in the human leukocyte antigen-mismatched donor/recipient combinations revealed a trend to an earlier and higher number of rejection episodes in DDA+ patients (P=0.08). No detrimental effect of DDA on graft survival was observed. CONCLUSIONS: This single-center study showed that in the AM program DDA detected by SA, and not by less-sensitive methods, may be related to acute rejection episodes but is not detrimental to long-term graft outcome. These findings question the increasing use of more-sensitive screening techniques for the allocation of organs.

A comprehensive overview of FCGR3A gene variability by full-length gene sequencing including the identification of V158F polymorphism.

The FCGR3A gene encodes for the receptor important for antibody-dependent natural killer cell-mediated cytotoxicity. FCGR3A gene polymorphisms could affect the success of monoclonal antibody therapy. Although polymorphisms, such as the FcγRIIIA-V158F and -48L/R/H, have been studied extensively, an overview of other polymorphisms within this gene is lacking. To provide an overview of FCGR3A polymorphisms, we analysed the 1000 Genomes project database and found a total of 234 polymorphisms within the FCGR3A gene, of which 69%, 16%, and 15% occur in the intron, UTR, and exon regions respectively. Additionally, only 16% of all polymorphisms had a minor allele frequency (MAF) > 0.01. To facilitate (full-length) analysis of FCGR3A gene polymorphism, we developed a FCGR3A gene-specific amplification and sequencing protocol for Sanger sequencing and MinION (Nanopore Technologies). First, we used the Sanger sequencing protocol to study the presence of the V158F polymorphism in 76 individuals resulting in frequencies of 38% homozygous T/T, 7% homozygous G/G and 55% heterozygous. Next, we performed a pilot with both Sanger sequencing and MinION based sequencing of 14 DNA samples which showed a good concordance between Sanger- and MinION sequencing. Additionally, we detected 13 SNPs listed in the 1000 Genome Project, from which 11 had MAF > 0.01, and 10 SNPs were not listed in 1000 Genome Project. In summary, we demonstrated that FCGR3A gene is more polymorphic than previously described. As most novel polymorphisms are located in non-coding regions, their functional relevance needs to be studied in future functional studies.

Full-length extension of HLA allele sequences by HLA allele-specific hemizygous Sanger sequencing (SSBT).

The gold standard for typing at the allele level of the highly polymorphic Human Leucocyte Antigen (HLA) gene system is sequence based typing. Since sequencing strategies have mainly focused on identification of the peptide binding groove, full-length sequence information is lacking for >90% of the HLA alleles. One of the goals of the 17th IHIWS workshop is to establish full-length sequences for as many HLA alleles as possible. In our component "Extension of HLA sequences by full-length HLA allele-specific hemizygous Sanger sequencing" we have used full-length hemizygous Sanger Sequence Based Typing to achieve this goal. We selected samples of which full length sequences were not available in the IPD-IMGT/HLA database. In total we have generated the full-length sequences of 48 HLA-A, 45 -B and 31 -C alleles. For HLA-A extended alleles, 39/48 showed no intron differences compared to the first allele of the corresponding allele group, for HLA-B this was 26/45 and for HLA-C 20/31. Comparing the intron sequences to other alleles of the same allele group revealed that in 5/48 HLA-A, 16/45 HLA-B and 8/31 HLA-C alleles the intron sequence was identical to another allele of the same allele group. In the remaining 10 cases, the sequence either showed polymorphism at a conserved nucleotide or was the result of a gene conversion event. Elucidation of the full-length sequence gives insight in the polymorphic content of the alleles and facilitates the identification of its evolutionary origin.

Relevance of posttransplant flow cytometric T- and B-cell crossmatches in tacrolimus-treated renal transplant patients.

BACKGROUND: De novo development of anti-human leukocyte antigen (HLA) antibodies after transplantation is associated with increased rejection and decreased graft survival. In this study, the effect of posttransplant HLA antibodies on clinical outcome was evaluated in patients treated with tacrolimus by means of flow cytometric crossmatches (FCXm). METHODS: T- and B-cell FCXm were performed retrospectively on posttransplant sera of patients who received a graft between 1997 and 1999. Ninety-four kidney-only recipients were tested and all FCXm positive sera were investigated for the presence of HLA class I and II antibodies by Flow panel reactive antibodies. RESULTS: From 94 patients with a negative pretransplant complement-dependent cytotoxicity crossmatch, seven (7%) showed a positive pretransplant FCXm. After transplantation the FCXm became positive in five patients (6%). The predictive value of a positive FCXm after transplantation, and the log-transformed relative change in fluorescence ratio between pretransplant and posttransplant serum, were not significant to rejection within six months, nor to graft survival censored for death. CONCLUSIONS: The presence of HLA antibodies before rejection or graft failure could only be shown in a minority of patients; most antibodies were detected after graft failure, especially after transplantectomy. Monitoring through antibody testing after transplantation on the basis of our results has no added value with tacrolimus-based immunosuppression.

Sequence-based typing of the complete coding sequence of DQA1 and phenotype frequencies in the Dutch Caucasian population.

Typing of DQA1 by sequencing has been a challenge because of a 3-nucleotide deletion in exon 2 in half of the alleles. Furthermore, 19 of the 28 alleles cannot be identified on basis of exon 2 alone, but need additional exon information. With the sequencing strategy presented here the complete exons 1-4 are sequenced heterozygously, enabling identification of all DQA1 alleles by sequence-based typing (SBT). Exons 1-4 were amplified and sequenced separately, the combined sequences were used for automated allele assignment. The method was validated by typing 21 individuals with all possible different allele group combinations. In addition 26 quality control samples were correctly typed by this method. To determine the phenotype frequencies 155 unrelated Dutch Caucasian individuals were DQA1 typed. In total 15 known and two new DQA1 alleles were identified. DQA1*0103 and *0505 were the most frequent alleles with phenotype frequencies of 30% and 29%, respectively. The SBT method presented here is an improvement compared to already existing protocols in that the complete exon sequence is obtained for all coding exons, using identical polymerase chain reaction conditions. Furthermore, all exons are sequenced heterozygously, facilitating allele assignment and reducing the number of amplification reactions.

Uncommon HLA alleles identified by hemizygous ultra-high Sanger sequencing: haplotype associations and reconsideration of their assignment in the Common and Well-Documented catalogue.

Although the number of HLA alleles still increases, many of them have been reported being uncommon. This is partly due to lack of full length gene sequencing, especially for those alleles belonging to an allele ambiguity in which the first discovered allele has been assigned as the most frequent one. As members of the working group on Common and Well Documented (CWD) alleles and since we implemented full length group-specific sequencing as standard method routinely, we have investigated the presence of presumably rare alleles in our collection of HLA typing data. We identified 50 alleles, that were not previously encountered as Common or Well Documented. Sixteen of them should be added to the CWD catalogue, since we encountered them in 5 or more unrelated individuals. Another 11 could be added, based upon our results and the data present in the IMGT database and the rare allele section of the allele frequencies database. Furthermore, tight associations were observed between several different alleles even at the level of synonymous and non-coding sequences. In addition, in several cases the uncommon allele was found to be more frequent than its common counterpart.

Severe pulmonary sarcoidosis is strongly associated with the haplotype HLA-DQB1*0602-DRB1*150101.

Sarcoidosis is a multiorgan granulomatous disease of unknown etiology. Several lines of evidence suggest a genetic predisposition and associations have been demonstrated with HLA antigens. HLA-DQB1 has been proposed as one of the candidate genes. To investigate the relationship between DQB1 and sarcoidosis at the allele level, we typed 149 Dutch Caucasian sarcoidosis patients for DQB1 by sequence-based typing as the ultimate technique to identify all DQB1 alleles. Phenotype frequencies were compared with controls. Both groups were also typed for HLA-A, -B, and -DRB1 at the low-resolution level. To decide on the possible linkage with DR, all DRB1*15-positive patients were subsequently sequence-based typed. Results showed a statistically significant increase of DQB1*0602 in sarcoidosis patients. The increase was also proven for DRB1*150101. Because of the high linkage disequilibrium between DRB1*1501 and DQB1*0602 in Caucasians, it could not be decided which one was the primary association. The increase was most pronounced in patients with severe pulmonary sarcoidosis indicated by radiographic stages II-IV. Although not statistically significant, DRB1*03 and DQB1*0201 were increased in radiographic stage I compared with II-IV. This study provides evidence that the combination DQB1*0602/DRB1*150101 is a strong positive marker for severe pulmonary sarcoidosis.

Sequence-based typing of the HLA-A10/A19 group and confirmation of a pseudogene coamplified with A*3401.

The strategy for sequencing human leukocyte antigen (HLA)-A was based on separate amplification of exons 2 and 3, followed by forward and reverse heterozygous sequencing of the alleles. Validation of the method was obtained by sequencing 11 individuals carrying alleles from all different HLA-A allele groups, except *43. All alleles could be correctly identified except A*3401. Unexpected polymorphic positions were identified in exon 3, even in individuals homozygous for A*3401. In addition, the pseudogene HLA-COQ or HLA-DEL linked to A*3401 was coamplified and sequenced. The problem was solved by using different amplification primers for exon 3 with mismatches for the two pseudogenes. A total of 252 unrelated individuals with at least one allele belonging to the A10 or A19 group were typed for HLA-A by this strategy. Ten different alleles were identified in the A10 group and 14 in the A19 group. As second allele a further 30 different subtypes from all different groups were sequenced. In 21 individuals, sequencing exon 1 was necessary to distinguish A*7401 from A*7402. The sequencing strategy, with separate amplification of the exons, has proven to be a robust method, resulting in reliable and efficient high-resolution HLA-A typing.

Elucidation of exon 1, 4, and 5 sequences of 39 infrequent HLA-B alleles.

More than 590 human leukocyte antigen (HLA)-B alleles have been identified by sequence analysis. Although the polymorphic exon 2 and 3 sequences of all HLA-B alleles are described, the sequences of the other exons of a number of infrequent B-alleles are unknown. In this study, the exon 1, 4, and 5 sequences of 39 different HLA-B alleles were elucidated by allele-specific sequencing. Overall, these exon sequences showed identity with the majority of the known sequences from the corresponding allele groups, except for four alleles B*4010, B*4415, B*4416, and B*5606. The exon 1 sequence of B*4010 had nucleotide differences with all B*40 alleles, but was identical to the B*54, *55, *56, and *59 allele groups. B*4416 differed from B*440201 at position 988, which was previously considered a conserved position. B*4415 showed exon 1, 4, and 5 sequences deviating from the other B*44 alleles, but identical to B*4501. The exon 1 and 4 sequences of B*5606 differed from other B*56 alleles, but were in complete agreement with B*7801. The deviating exon sequences of B*4415 and B*5606 confirmed the evolutionary origin of these alleles suggested by the sequences of exons 2 and 3. The polymorphism observed in exons 1, 4, and 5 merely reflects the lineage-specificity of HLA-B.

No HLA-A gene detectable on one of the haplotypes in a Caucasian family.

An unusual haplotype was detected in a family of a caucasian transplant patient. Human leukocyte antigen (HLA) analysis of the family demonstrated the absence of HLA-A on one of the haplotypes present in two family members. One was serologically typed A24, the other A2. Because they had one haplotype in common, the HLA-A allele of the shared haplotype was supposed to be a null allele. Different molecular typing methods identified only one allele in both individuals. The results suggest a deletion of the complete HLA-A gene or a major part of it. For confirmation, microsatellite analysis of the HLA-A region was performed with six microsatellite markers. Both family members were heterozygous for all markers, and a deletion of HLA-A could not be proven. Fluorescent in situ hybridization (FISH) was performed with cosmid and PAC probes encompassing the HLA-A gene. Both probes demonstrated an identical normal distribution pattern for diploid results. The absence of any serologic and molecular reaction with the results of the microsatellite and FISH analysis make a deletion of a narrow region, encompassing the HLA-A gene, the most plausible explanation.

Association of HLA DQB1 0602 in sarcoidosis patients with small fiber neuropathy.

BACKGROUND AND AIM: Sarcoidosis has been reported to be associated with the HLA genes, in particular DQB1. METHODS: High resolution DQB 1 of 103 sarcoidosis patients was obtained by sequence-based typing; low resolution HLA-A/B/DRB 1 typing was performed by serological and molecular methods. Small fiber neuropathy (SFN) was established by thermal threshold testing. RESULTS: Sixty-seven patients suffered from SFN (SFN+), in 36 patients SFN was absent (SFN-). Comparing HLA DQB 1 typings of SFN+ patients, SFN- patients and control individuals revealed a significant increase of the allele DQB 1 0602 in SFN+ patients compared to controls. CONCLUSION: This association might be correlated with a severe course of the disease.

HLA-E regulates NKG2C+ natural killer cell function through presentation of a restricted peptide repertoire.

UNLABELLED: NK cells interact with the HLA-E molecule via the inhibitory receptor NKG2A and the activating receptor NKG2C. Hence, HLA-E can have a dual role in the immune response. In the present study, we aim to investigate the functional consequences of HLA-E for NKG2A and NKG2C expressing NK cell subsets by using a panel of HLA-E binding peptides derived from CMV, Hsp60 and HLA class I. PBMC derived from healthy subjects were used as targets for isolated NK cells and NK cell activation was examined by analysis of the expression of the degranulation marker CD107a. Peptide induced HLA-E expression inhibited degranulation of NKG2A+ NK cell subsets with almost all peptides, whereas NKG2A- NKG2C+ NK cell responses were enhanced only after incubation with four peptides; 1.3-fold with CMV(I), A80 and B13 and 3.2-fold with HLA-G derived peptide. In addition, the HLA-E:G peptide complex triggered NKG2C receptor internalization, as evidenced by reduction in the percentage of NKG2C+ NK cells when incubated with the peptide, which could be restored by addition of Bafilomycin. IN CONCLUSION: in contrast to NKG2A, NKG2C is regulated by HLA-E only when HLA-E is in complex with a restricted peptide repertoire, especially in combination with the HLA-G leader peptide.