High-Allelic Variability in HLA-C Mrna Expression: Association with HLA-Extended Haplotypes

ORIGINAL ARTICLE High-allelic variability in HLA-C mRNA expression: association with HLA-extended haplotypes

F Bettens1,2, L Brunet1,2 and J-M Tiercy1

Human leukocyte antigen (HLA)-C is a clinically relevant transplantation antigen in unrelated hematopoietic stem cell and cord blood transplantation. Furthermore, HLA-C antigens, as ligands for killer immunoglobulin-like receptors expressed on natural killer cells, have a central role in HIV control. Several studies have reported significant correlations between HLA-C mRNA and cell surface expression with polymorphisms in the 50- and 30-regions of the HLA-C locus. We determined HLA-C mRNA in blood donors by using locus as well as allele-specific real-time–PCR and focused the analysis on HLA-extended haplotypes. High inter-individual variability of mRNA expression was disclosed. A lower inter-individual variability for C*07:01 but a higher variability for C*06:02, C*04:01 and C*03:04 alleles were detected. The previously reported associations between HLA-C cell surface expression and À 32 kb/ À 35 kb single nucleotide polymorphisms were not confirmed. Related and unrelated individuals sharing the same two A-B-C-DRB1 or B-C haplotypes show strikingly similar levels of HLA-C mRNA expression in each of the different haplotypic combinations tested. Altogether, our results suggest that HLA-C expression levels best correlate with the extended HLA haplotype rather than with the allotype or with polymorphisms in the 50-region of the HLA-C locus.

Genes and Immunity (2014) 15, 176–181; doi:10.1038/gene.2014.1; published online 6 February 2014 Keywords: HLA-C mRNA expression; HLA-A,B,C,DRB1 haplotypes; alloreactivity; autoimmunity; stem cell transplantation

INTRODUCTION CD4 þ -T-cell counts and HLA-C mRNA levels in individuals of Hematopoietic stem cell transplantation (HSCT) is a recognized European ancestry.16,17 Thomas et al.18 reported that HLA-C alleles treatment for a number of hematological malignancies and in linkage disequilibrium with the À 35C SNP had a higher cell disorders of the immune system. In the western countries, surface expression, as determined by DT9 antibody staining. hematopoietic stem cells (HSCs) from highly matched unrelated However, Corrah et al.19 reported that the differences in donors are increasingly used.1 Matching criteria in most transplant expression levels could be accounted by the low expression of centers include allelic compatibility for the human leukocyte HLA-Cw7 antigen only, which is strongly linked to À 35T. antigen (HLA)-A,B,C,DRB1,DQB1 loci, a so-called 10/10 match.2,3 In HLA-C*07/*07- and À 35TT-positive individuals showed a the absence of a compatible donor for HSCT, a donor with a single fivefold lower cell surface expression of HLA-C compared with allele mismatch is often the best option. Among such 9/10 C*07-negative À 35CC individuals. The protective À 35C geno- matched donors, HLA-C incompatibilities account for a large type does not associate with higher HLA-C expression they fraction of the HLA class I/II disparities,4–7 and their clinical argued, but rather the nonprotective À 35T allele was associated relevance has been documented in several studies.2,3,6–9 The with the much lower expression of a single allele, C*07.19 Further impact of HLA-C disparities on T-cell alloreactivity could be insight in the regulation of HLA-C expression was provided by influenced by the expression level of HLA-C antigens at the cell Kulkarni et al.20 who showed that a polymorphism in the surface. Low-expression HLA-C alleles could thus possibly 30-untranslated region (UTR) controls the binding of micro-RNA represent good candidates for more permissive mismatches. (miR)-148, with lower expression of HLA-C alleles that bind HLA-C surface expression is controlled at the transcriptional, miR-148 and higher expression of C alleles that do not bind translational and post-translational levels. HLA-C mRNA has a miR-148. Interestingly, this miR-148 binding site was present in the higher turn-over rate which could account for the lower HLA-C cell common ancestor of all the extant HLA-C alleles.21 A correlation of surface expression compared with HLA-A or -B antigens.10 In the À 35-kb SNP and the 30-UTR polymorphism and HLA-C surface addition, owing to a selective preference in the peptide repertoire, expression as determined by DT9 binding has also been reported HLA-C heavy chains associate with b2-microglobulin less on a small cohort of healthy blood donors.22 efficiently than HLA-A and -B antigens.11–14 A higher HLA-C expression might allow a greater recognition by A first genome-wide association study screening of HIV-infected the KIR2DL receptors during natural killer (NK) cell maturation, patients led to the discovery of a single nucleotide polymorphism leading to more effective natural killer responses against (SNP) 35 kb upstream of the HLA-C transcription start site infected cells,18 although HLA-C expression was recently ( À 35C/T) that was associated with the viral load set point. reported to have no effect on natural killer cell function in The À 35C allele correlated with lower viral set point,15 higher healthy individuals.22 Higher HLA-C expression is also expected to

1National Reference Laboratory for Histocompatibility, Transplantation Immunology Unit, Department of Genetics and Laboratory Medicine, University Hospital, Geneva, Switzerland. Correspondence: Dr J-M Tiercy, National Reference Laboratory for Histocompatibility, Transplantation Immunology Unit, Department of Internal Medicine and Department of Genetics and Laboratory Medicine, Geneva University Hospitals and University of Geneva, 4 rue Gabrielle-Perret-Gentil, 1211 Geneva, Switzerland. E-mail: [email protected] 2These authors contributed equally to this study. Received 8 October 2013; revised 6 December 2013; accepted 13 December 2013; published online 6 February 2014 Haplotype-linked HLA-C mRNA expression F Bettens et al 177 allow a more efficient presentation of viral peptides to cytotoxic HLA-C*04:01 (n ¼ 4) and C*07:02 (n ¼ 5) homozygous blood donors T lymphocytes.23 (open circles in Figure 2) showed a homogenous distribution. Haplotype-specific variations in transcripts as determined by Considering a mean HLA-C expression value of 11.5 (horizontal microarray hybridization have been reported for a number of dotted line in Figure 2), several alleles such as C*01:02, C*02:02, major histocompatibility complex genes, including the classical C*03:03, C*07:02 or C*16:01 found in heterozygous individuals HLA class I or II loci, upon analysis of the transcriptome of three were associated with a lower expression, whereas other alleles HLA homozygous cell lines COX, QBL and PGF.24 Haplotype- such as C*03:04, C*07:04 or C*15:02 were linked to a higher specific differences in gene expression was reported for 96 genes mean expression level. For example, as compared with the across the major histocompatibility complex. In particular, HLA-C C*03:04-positive individuals, mean expression levels were signifi- expression was higher in QBL (C*05:01) compared with COX cantly (Mann–Whitney test) lower for C*01:02-(P ¼ 0.05), C*02:02- (C*07:01) and PFG (C*07:02). Alternative splicing was also (P ¼ 0.04), C*07:02-(P ¼ 0.02) and C*16:01-positive individuals haplotype-dependant and 526 exons showed haplotypic (P ¼ 0.02). differences, including HLA-DPB2, DQB2, HLA-C or HLA-G.24 An Interestingly, three samples from the same family with the analysis of HLA-C mRNA expression in skin samples from healthy B*49:01-C*07:01 haplotype and three different C allotypes on the donors and psoriatic patients revealed a large heterogeneity in second haplotype (C*03:04, C*04:01 and C*15:02) showed a both C*06:02-positive and C*06:02-negative patients.25 strikingly high HLA-C mRNA expression level with a mean As a C/T dimorphism at À 35 kb of HLA-C locus has been expression value of 15.4. As these belonged to the same family reported to be predictive of HLA-C expression,18 we aimed to and originated from nonEuropean ancestry they were not reproduce this result in order to use this SNP as a surrogate included in Figure 2. marker for HLA-C expression in the HSCT setting. We furthermore Because of the coamplification of two different alleles in tested the hypothesis that HLA-C expression could be, at least heterozygous samples, we sought to investigate the impact of the partially, regulated in a HLA haplotype-dependant manner. We second allele. We therefore grouped the C*07:01- (Figure 3 left report here a large heterogeneity in HLA-C allotypes mRNA panel) and C*04:01-positive (Figure 3 right panel) individuals expression, and show correlations with HLA-A-B-DRB1 haplotypes according to the same heterozygous combinations of C allotypes. but not with the À 35C/T dimorphism. Whereas C*07:01/03:04 and C*07:01/04:01 individuals exhibited heterogenous mRNA expression levels, C*07:01/01:02 had significantly lower (P ¼ 0.03) but more homogenous expression levels RESULTS (Figure 3), suggesting that both C*07:01 and C*01:02 alleles are Correlation with À 35 kb/ À 32 kb SNPs associated with lower rates of expression. When comparing HLA-C mRNA levels were quantified by reverse transciptase C*04:01/03:04, C*04:01/07:01 and C*04:01/07:02 individuals, we (RT)–PCR in 130 donors using locus-specific primers. Of these again found a significant difference in HLA-C expression between 130 donors, 107 were typed in parallel for the two SNPs at À 35 kb C*04:01/03:04 and C*04:01/07:01 donors (P ¼ 0.05), as well as and À 32 kb, respectively. As shown in (Figure 1), HLA-C mRNA C*04:01/07:02 donors (P ¼ 0.02). analysis did not reveal any significant association between the expression levels and the -35CC, CT and TT genotypes. Furthermore, when analyzing the C*07-35TT homozygous donors Allele-specific HLA-C mRNA expression separately, neither any difference was disclosed nor any correla- To unambiguously demonstrate variability within individual tion was revealed with the À 32-kb genotypes (data not shown). alleles, we set up an allele-specific RT–PCR assay for the four HLA-C allotypes represented in heterozygous blood donors with Total HLA-C mRNA expression the largest number of samples. As shown in Figure 4a, C*07:01 mRNA expression was low and slightly more homogenous HLA-C mRNA levels, as determined by locus-specific RT–PCR, are represented in Figure 2 for each allotype in 130 HLA-C allele-typed individuals. This study group (heterozygosity rate ¼ 87.7%) 18 showed, as expected, a large representation of the most frequent alleles C*04:01, C*05:01, C*06:02, C*07:01 and C*07:02. In the whole 16 study group, a large variability in mRNA levels was disclosed. 14

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n=49 01:02 02:02 03:02 03:03 03:04 04:01 05:01 06:02 07:01 07:02 07:04 07:18 08:02 12:02 12:03 14:02 15:02 15:04 16:01 16:02 n=49 HLA-C alleles HLA-C expression 5 5 Figure 2. Total HLA-C mRNA expression of 130 individuals plotted according to HLA-C alleles. Mean expression of individual alleles is indicated by a solid bar and mean expression of all alleles is 0 0 CC CT TT CX TT represented by the dotted line (11.5). When removing the 26 patients from the analysis, the distribution pattern of the 104 Figure 1. Lack of correlation between À 35 kb SNP and total HLA-C healthy individuals was very similar with a mean expression of 11.43 mRNA expression as determined in 107 individuals. Distribution (not shown). Note that each heterozygous individual is plotted twice of the donors (a) according to CC, CT and TT genotypes and (once for each C allele). Open circles correspond to homozygous (b) according to C/x and TT genotypes. Numbers of individuals per individuals. All homozygous C*04:01 and 07:02 individuals are B35 genotype are indicated. and B7 homozygous, respectively.

& 2014 Macmillan Publishers Limited Genes and Immunity (2014) 176 – 181 Haplotype-linked HLA-C mRNA expression F Bettens et al 178 compared with the expression patterns detected with the C*03:04 expression (haplotype B*15:01-C*03:04), whereas for other coamplification of the two C alleles (referred to as total HLA-C samples, high HLA-C total mRNA expression was linked to high expression). Interestingly, 22/27 HLA-C*07:01-positive individuals expression of both alleles. had the B*08:01 allele and 18/27 had the A1-B8-DR3 or A1-B8 or B8-DR3 haplotype. In contrast, the C*06:02-, C*04:01- and C*03:04- specific mRNA expression were more heterogenous (Figures 4b–d, Haplotype-linked HLA-C mRNA expression respectively). Pairwise comparisons between total and specific Despite the high HLA-A,B,DRB1 haplotypic diversity of the expression, as represented in lower panels of Figure 4, showed individuals tested, we investigated possible correlation of HLA- poor correlation coefficients (R2 ¼ 0.3 for C*07:01, R2 ¼ 0.6 for C*04:01-specific mRNA expression with different haplotypes. C*06:02, R2 ¼ 0.8 for C*04:01 and R2 ¼ 0.4 for C*03:04), indicating Interestingly, among the group of 26 C*04:01 heterozygous that total HLA-C mRNA is the sum of two alleles with possibly very individuals with normalized expression varying from 8.00–10.97 different expression levels. Extreme examples are found in (Figure 4c), a few haplotypes exhibited more homogenous C*04:01 Figure 4d where the individual (C*03:04/16:01) with the lowest mRNA expression levels. For example, four A*11:01-B*35:01- total HLA-C mRNA expression has a moderate C*03:04 expression C*04:01 haplotypes associated with nonDRB1*11:01 alleles were (haplotype B*40:01-C*03:04) and the individual (C*03:04/04:01) expressed at a relatively high level (10.36–10.65) and three with the highest total HLA-C mRNA expression has a very low A*03:01-B*35:01-C*04:01-DRB1*01:01 haplotypes had also a narrow range of expression (9.36–10.28). Two HLA-C*04:01 homozygous donors with the A*01:01-B*35:02-C*04:01-DRB1*13:02/A*01:01- HLA-C*07:01 HLA-C*04:01 B*35:01-C*04:01-DRB1*01:01 haplotypes expressed very similar C*04:01 mRNA levels (8.83–9.41) (data not shown). p=0.03 p=0.02 As variation within individuals sharing the very same HLA-C 15 p=0.03 15 p=0.05 genotypes, and variations of allele-specific expression levels were observed, we hypothesized that HLA-C mRNA expression could be 13 13 at least partly associated with the HLA haplotypes. As shown in Figure 5, unrelated and related individuals sharing the same two 11 11 HLA-A,B,C,DRB1 haplotypes (8/8 matched individuals) did show low inter-individual variability. This was particularly striking for the 9 9 C*07:02/07:02 (groups 3 and 4), C*02:02/07:02 (group 5) and C*05:01/12:03 (group 10) individuals. Blood donors with the same HLA-C expression 7 7 two HLA-A-B-C-DRB1 haplotypes tended to have less variable inter-individual mRNA expression levels as compared with all the 5 5 individuals positive for either one of the two HLA-C alleles 03:04 04:01 01:02 03:04 07:01 07:02 (Figure 2). For example, an s.d. of 1.43 was measured in all C*16:01 Figure 3. Total HLA-C mRNA expression of the HLA-C*07:01- (left individuals (Figure 2), the s.d. was 0.58 only for the C*12:03/16:01 panel) and C*04:01-positive (right panel) individuals from Figure 2 heterozygotes (Figure 5, group 7). Similarly, the s.d. of B*07:02- grouped according to the second allele indicated on the x-axis. C*07:02 homozygotes (groups 3 and 4) was 0.35, but 1.02 in all Significant differences (Mann–Whitney test) in expression levels are C*07:02 individuals. Three blood donors (group 11) with the same indicated. B*08:01/40:01-C*07:01/03:04 haplotypic combinations showed also

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5 5 5 5 normalized expression levels Total HLA-C HLA-C*07:01 Total HLA-C HLA-C*06:02 Total HLA-C HLA-C*04:01 Total HLA-C HLA-C*03:04 Figure 4. Upper panels: inter-individual locus (total HLA-C mRNA) versus allele-specific HLA-C mRNA expression in C*07:01 (a), C*06:02 (b), C*04:01 (c) and C*03:04 (d) donors. For each HLA-C allele, the distribution of the most frequent B–C haplotypes is as follows: HLA-C*07:01: B*08:01 (n ¼ 22/27), B*49:01 (n ¼ 3/27) HLA-C*06:02: B*13:02 (n ¼ 7/20), B*57:01 (n ¼ 6/20), B*37:01 (n ¼ 4/20) HLA-C*04:01: B*35:01 (n ¼ 10/26), B*35:02 (n ¼ 6/26), B*44:03 (n ¼ 4/26) HLA-C*03:04: B*40:01 (n ¼ 12/15) Lower panels: paired sample measurements of total and specific mRNA expression of single individuals as indicated by connecting lines. Note that total and specific HLA-C RT–PCRs were done at the same time in order to avoid possible variations linked to cDNA preparations.

Genes and Immunity (2014) 176 – 181 & 2014 Macmillan Publishers Limited Haplotype-linked HLA-C mRNA expression F Bettens et al 179 18 group HLA-A* HLA-B* HLA-C* HLA-DRB1*

16 1 01:01/32:01 08:01/27:05 01:02/07:01 03:01/03:01 2 01:01/29:01 08:01/55:01 01:02/07:01 03:01/13:01 14 3 11:01/11:01 07:02/07:02 07:02/07:02 11:01/15:01 12 4 02:01/24:02 07:02/07:02 07:02/07:02 14:01/15:01 5 23:01/24:02 07:02/44:03 02:02/07:02 01:01/13:02 10 6 01:01/01:01 35:01/35:02 04:01/04:01 01:01/13:02 7 11:01/29:02 35:03/44:03 12:03/16:01 04:04/04:05

HLA-C expression 8 8 01:01/02:01 13:02/44:02 06:02/07:04 12:01/16:01 6 9 01:01/33:01 08:01/51:01 07:01/15:02 03:01/11:01 10 02:01/24:02 39:01/44:02 05:01/12:03 11:04/16:01 4 11 MM 08:01/40:01 03:04/07:01 MM 1234567891011 haplotype groups Figure 5. Total HLA-C mRNA expression of 10 groups of HLA-A-B-C-DRB1 (8/8)-matched individuals and one group of HLA-B-C-matched individuals allocated according to their same haplotypes. Groups 1–7 include unrelated individuals and groups 8–11 include related individuals. HLA typing for the different groups is indicated in the figure. MM: mismatched. grouped HLA-C expression levels, although HLA-A and -DRB1 were 4 related) with the same two HLA-A-B-C-DRB1 or HLA-B-C different. The fact that blood from individuals sharing identical haplotypes (Figure 5) revealed a rather narrow variation in the haplotypes have been received, extracted and tested at different HLA-C mRNA expression levels. times excludes an experimental artefact. Two studies have reported gradients of HLA-C expression on peripheral blood lymphocytes as determined by DT9 antibody binding in European-American18 and in African-American DISCUSSION individuals.23 A correlation with the À 35-kb SNP was reported The crucial role of HLA-C molecules as ligands of killer in the first study.18 Subsequently, Kulkarni et al.20 showed that immunoglobulin-like receptors on natural killer cells has led to a variation in the 30-UTR-263 miR-148 binding site correlated best growing interest in investigating the expression of HLA-C antigens with HLA-C expression on the basis of in vitro studies using HLA-C both at the mRNA and protein levels in relationship with HIV transfectants. However, by comparing HLA-C expression levels as infection.15,18,23 On the basis of the hypothesis that in mismatched determined by DT9 binding18,23 with the 30-UTR-263 indel allogeneic HSCT, T-cell alloreactivity could be modulated by polymorphism defining HLA-C expression,20 somewhat opposite differential expression of HLA-C target antigens, we analyzed conclusions are observed. For example, the two alleles classified HLA-C mRNA levels in a large cohort of patients and HSC donors. with the highest average DT9 binding (C*01:02, C*14:02)23 are We choose to use RT–PCR for the determination of HLA-C characterized by an intact miR-148 binding site resulting in HLA-C expression rather than cell surface expression as quantified by the downregulation.20 Similarly, C*04:01 lies among the C alleles with DT9 antibody. One reason resided in the debated issue on the a high cell surface expression, but nevertheless is associated with crossreactivity of the DT9 antibody with HLA-E,18,19 but the main the 263ins genotype linked to low expression. These apparently argument was that the allele-specific PCR allowed us to test and divergent results suggest that the control of HLA-C expression compare the expression of single HLA-C alleles in a large sample might be more complex than expected. Interestingly, a most of donors. First, we report that the analysis of mRNA expression recent study showed that differential expression levels of with generic primers allowed robust, reproducible and precise miR-148a, as determined by a polymorphism at the MIR-148A inter-individual comparisons, although its limitation resides in the locus changed the HLA-C expression levels, but only in individuals lack of discrimination of individual HLA-C alleles except in homozygous for the ins263 intact binding site.26 The presence of homozygotes. The purpose of this study was to disclose possible an additional putative miR181a binding site in the 30-UTR26,27 adds relationship between expression levels and polymorphisms in the to further diversity and may also contribute to HLA-C expression 50-region of HLA-C locus and HLA-extended haplotypes. variability. It is furthermore interesting to note that the C*02:10 Although most tested individuals are heterozygous and high allele, frequent in some African populations, was found to inter-individual variability is observed, certain allotypes showed be associated with both the 30-UTR 263ins (low-expression) higher mean expression levels (Figure 2). Particularly striking are and 263del (high-expression) alleles.27 Altogether, the lack of the C*15:02-positive individuals that are associated with a higher correlation between the À 35-kb SNP and HLA-C expression mean expression compared with C*01:02-(P ¼ 0.01), C*02:02- reported here and in previous studies in both Caucasoid and (P ¼ 0.004) C*04:01-(P ¼ 0.03), C*07:01-(P ¼ 0.03), C*07:02- African population groups27,28 show that À 35 kb genotyping (P ¼ 0.003) and C*16:01-positive (P ¼ 0.007) donors. Similar to cannot be used as a marker for HLA-C expression. Although we previous studies18,19 we find a lower, but not significant, confirm the lower expression of C*07:02 alleles observed by mean expression level for C*07:02-positive individuals. Strikingly, others,19,23 it is not easy to compare mean HLA-C expression levels C*07:04-positive individuals exhibited a high mean expression reported in different populations with presumably completely level with all donors sharing the B*44:02 haplotype. different HLA haplotypes. We consider that the differences As a new finding reported in this study, the large heterogenity observed for some HLA-C allotypes in this and previous in HLA-C mRNA expression correlated best with HLA-B-C studies18,23 can be accounted for by the different haplotypic haplotypes. Several arguments support a HLA-B-C haplotype- associations in the study groups. specific expression of HLA-C antigens rather than a control by Our results on the basis of allele-specific RT–PCR show that a À 35 kb/ À 32 kb SNPs. First, although a large heterogeneity of given allele might not be simply classified as a high- or low- HLA-C mRNA expression was detected for most HLA-C allotypes expression allele, and that the HLA-A-B-DRB1 haplotypic associa- (Figure 2), the expression was homogenous in HLA-B, -C tion could significantly impact on the level of expression in homozygous individuals (for example B*35-C*04:01 and B*07- addition to the miR-148a regulatory pathway.20,26 As determined C*07:02 donors, legend to Figure 2). Second, 81% of HLA-C*07:01- in lymphoblastoid cell lines, transcription maps of three common positive individuals showing more homogenous allele-specific major histocompatibility complex haplotypes have shown signi- expression have B*08:01-positive haplotypes (Figure 4). Third, the ficant variations in the transcriptional activity of 46.4% genes analysis of 11 groups of heterozygous individuals (7 unrelated and in the major histocompatibility complex including HLA-C.24

& 2014 Macmillan Publishers Limited Genes and Immunity (2014) 176 – 181 Haplotype-linked HLA-C mRNA expression F Bettens et al 180 HLA-B and HLA-C genes are 88 kb apart and transcribed in the The primers used for total HLA-C expression were located in exons 5 and 7, same orientation. Considering the haplotype-specific transcript- respectively: forward primer 50-ATCGTTGCTGGCCTGGCTGTCCT-30 and ionally active regions described in lymphoblastoid cell lines,24 it is reverse primer 50-TCATCAGAGCCCTGGGCACTGTT-30.18 For the 18S amplification the forward and reverse primers 50-GTAACCCGTTGA possible that long-range regulatory elements may affect 0 0 0 transcription of the whole subregion. Haplotype-linked variation ACCCCATT-3 and 5 -CCATCCAATCGGTAGTAGCG-3 were used. HLA-C allele-specific primers have been previously described.37 The following in HLA-C expression is expected to impact on allorecognition of forward/reverse primers were used: specific target HLA-C antigen mismatches in transplantation. 50-GGACCGGGAGACACAGAAC-30,50-CGCACGGGCCGCCTCCA-30 for HLA- Although expressed at a lower level compared with HLA-A or -B C*07:01,50-CACAGACTGACCGAGTGAG-30,50-CCGCCGTGTCCGCGGCA-30 for antigens, incompatible HLA-C molecules can be efficiently HLA-C*06:02,50-CCGAGTGAACCTGCGGAAA-30,50-CCCCAGGTCGCAGCCAA- recognized by alloreactive T lymphocytes as shown in HSCT.29–31 30 HLA-C*04:01, and 50-CACAGACTGACCGAGTGAG-30,50-AGCGTCTCCTTCC- Cytotoxic T-lymphocyte precursor frequency assays have disclosed CATTCTT-30 for HLA-C*03:04.37 The specificity of the amplifications was several HLA-C-mismatched combinations which did not induce a controlled by amplification of allele-positive and -negative cDNAs and positive test.32 It could be expected that in cytotoxic T-lymphocyte sequence analysis of the respective amplicons. For each primer pair cDNA precursor frequency-negative pairs, target HLA-C antigens are dilutions curves showed the same slope (Supplementary Figure S2), indicating that allele-specific and locus-specific HLA-C amplification are encoded by a haplotype with a lower transcriptional activity. On comparable. Furthermore, control experiments where HLA-C expression of the other hand, HSC donors with a lower self-HLA-C expression in different blood samples of the same donor taken at different time points the thymus could become high responders toward incompatible and kept in sodium citrate up to 7 days allowed us to monitor HLA-C antigens because of a more limited negative selection of reproducibility and storage time of blood samples. cDNAs obtained from T cells. peripheral blood lymphocytes isolated from blood samples kept up to 3 In disease association studies, the importance of HLA-C days gave the same results and were thus included in the study expression in modulating the immune response has been (Supplementary Figure S3). As a further control, repeated total HLA-C demonstrated by the highly significant correlation between high amplifications of the same cDNA samples at different experimental dates HLA-C expression and a more efficient control of HIV infection,18,23 (n ¼ 3 for 5 cDNAs) revealed a low 0.4 100/DCt s.d. One blood sample 23 taken from a healthy volunteer donor at one-year interval showed similar but a higher risk of Crohn’s disease (CD). A direct effect of HLA-C 100/DCt values: 12.42 in 2012, 13.07 in 2013. expression level on HIV control and on CD was recently shown to be mediated by a MIR-148A polymorphism.26 We consider the possibility that the highly expressed HLA-C alleles conferring a CONFLICT OF INTEREST higher risk of CD23,26 are found on different haplotypes compared The authors declare no conflict of interest. with healthy individuals. Interestingly, a genome-wide association study analysis of a Japanese population disclosed a B-C-DRB1 33 haplotype that correlated with decreased susceptibility to CD In ACKNOWLEDGEMENTS several autoimmune diseases HLA class II loci (mainly DRB1) stand 34 We are grateful to the technicians of the National Reference Laboratory for out as the major genetic risk determinants, but class I and II Histocompatibility for their technical assistance. This work has been supported by alleles seem to contribute to susceptibility, such as HLA-C*08 in grant N1 31-0030-146306/1 from the Swiss National Science Foundation. CD, and HLA-C*05 in type I diabetes and rheumatoid arthritis.35 It is tempting to suggest that the previously reported associations of 36 HLA-A and -B alleles with type I diabetes could be accounted for REFERENCES by the strong linkage with HLA-C alleles that has an impact on 1 Passweg J, Baldomero H, Gratwohl A, Bregni M, Cesaro S, Dreger P et al. 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