Nina Lauterbach

Full-length HLA-DPB1 diversity in multiple alleles of individuals from Caucasian, Black, or Oriental origin.

Despite DP antigens have been shown to be stimulators of the mixed lymphocyte reaction, human leukocyte antigen-DPB1 is not considered in the matching criteria for hematopoietic stem cell transplantation (HSCT). The role of DPB1 matching in HSCT remains inconclusive because of contradictory findings in different studies. The concept of permissible and non-permissible mismatches might clarify these contradictory results. Although several groups have attempted to identify immunogenic epitopes in exon 2 to establish permissive and non-permissive allele groups, the direct correlation between individual exon 2 amino acids and epitopes with DPB1 immunogenicity is still not evident. We hypothesize that polymorphism within the entire molecule, including polymorphic variability in different ethnic groups, is crucial to unravel the function of DPB1 polymorphism. Using an RNA-based approach, we sequenced all frequent and available non-frequent DPB1 alleles full length from 148 samples representing 28 different DPB1 alleles from either Black, Caucasian, or Oriental origin. We identified various DPB1 alleles with, in addition to the exon 2 polymorphism, polymorphisms in exons 1, 3, 4, and 5. Based on this polymorphism outside exon 2, we defined one new allele. Two alleles with identical exon 2 polymorphism but differing outside exon 2 were identified in individuals of different ethnic groups. As T cell binding is not restricted to the polymorphic groove and polymorphism in the β2 domain of the DP molecule affects CD4 interaction, full-length polymorphism should be considered to determine immunogenicity. Eventually, this knowledge will provide new insights in the classification of DPB1 polymorphism and more importantly will add new perspectives to the concept of permissiveness in transplantation.

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.

Molecular typing of HLA-E.

Human leukocyte antigen-E (HLA-E) is a non-classical HLA class I gene that shows a limited degree of polymorphism compared to the classical HLA genes. The HLA-E molecule can bind peptides derived from the leader sequence of various HLA class I alleles and some viral homologues, including CMV. The HLA-E peptide complex can act as a ligand for the CD94/NKG2 receptors expressed on the surface of natural killer cells and T cell subsets. Differences in expression levels between the different HLA-E alleles have been reported and a role for HLA-E polymorphism in stem cell transplantation has been postulated. This chapter focuses on routine technologies for HLA-E typing: the sequence-specific primer-PCR method that uses sequence-specific primers, the PCR sequence-specific oligonucleotides Luminex method, using sequence-specific probes attached to beads and the sequencing-based typing method, where sequencing of the alleles is performed.

Peptide-induced HLA-E expression in human PBMCs is dependent on peptide sequence and the HLA-E genotype

Human Leukocyte Antigen (HLA)-E is a low-polymorphic non-classical HLA class I molecule which plays a crucial role in immune surveillance by presentation of peptides to T and natural killer (NK) cells. HLA-E polymorphism is related to HLA-E surface expression and is associated with patient outcome after stem cell transplantation. We aim to investigate the regulation of HLA-E expression level in peripheral blood mononuclear cells (PBMCs) of healthy individuals homozygous for HLA-E*01:01 or HLA-E*01:03, by using a panel of HLA-E binding peptides derived from CMV, Hsp60 and HLA class I. Basal and peptide-induced HLA-E surface expression levels were higher in PBMC from HLA-E*01:03 homozygous subjects as compared to PBMC from HLA-E*01:01 homozygous subjects. HLA-E mRNA levels were comparable between the two genotypes and remained constant after peptide stimulation. HLA-E surface expression seemed to be not only dependent on the HLA-E genotype, but also on the sequence of the peptide as evidenced by the profound difference in HLA-E upregulation with the Hsp60 and the B7 peptide. Our results showed that peptide-induced HLA-E expression is regulated at the posttranscriptional level as extracellular peptide stimulation did not influence RNA expression. This study provides new insights in the mechanism by which HLA-E expression is regulated and underlines a new role for extracellular peptides in inducing HLA-E translation, which may represent a defense mechanism against lytic viral infections and necrosis.

Effects of transmembrane region variability on cell surface expression and allorecognition of HLA-DP3

The functional relevance of polymorphisms outside the peptide binding groove of HLA molecules is poorly understood. Here we have addressed this issue by studying HLA-DP3, a common antigen relevant for functional matching algorithms of unrelated hematopoietic stem cell transplantation (HSCT) encoded by two transmembrane (TM) region variants, DPB1(*)03:01 and DPB1(*)104:01. The two HLA-DP3 variants were found at a overall allelic frequency of 10.4% in 201 volunteer stem cell donors, at a ratio of 4.2:1. No significant differences were observed in cell surface expression levels of the two variants on B lymphoblastoid cell lines (BLCL), primary B cells or monocytes. Three different alloreactive T cell lines or clones showed similar levels of activation marker CD107a and/or CD137 upregulation in response to HLA-DP3 encoded by DPB1(*)03:01 and DPB1(*)104:01, either endogenously on BLCL or after lentiveral-vector mediated transfer into the same cellular background. These data provide, for the first time, direct evidence for a limited functional role of a TM region polymorphism on expression and allorecognition of HLA-DP3 and are compatible with the notion that the two variants can be considered as a single functional entity for unrelated stem cell donor selection.

Allorecognition of HLA-DP by CD4+ T cells is affected by polymorphism in its alpha chain.

Alloreactivity to HLA-DP molecules, class II heterodimers of an oligomorphic alpha and a polymorphic beta chain, is increasingly being studied due to its relevance in clinical transplantation. We hypothesized that not only polymorphisms in the peptide binding groove encoded by exon 2 of HLA-DPB1, but also in other regions of the molecule and the alpha chain, could play a role in CD4+ T cell allorecognition. To test this possibility, we comparatively investigated CD4+ T cell allorecognition, measured by upregulation of the activation marker CD137, against HLA-DPB1*13:01, *05:01, *03:01, *17:01 or their allele counter parts DPB1*107:01, *135:01, *104:01, *131:01, with identical exon 2 sequences but polymorphism in exons 1, 3 or 4, in the context of different HLA-DPA1 (DPA1) polymorphisms (DPA1*01:03 and *02:01). No significant differences in CD4+ T cell allorecognition levels could be demonstrated for any of the beyond exon 2 DPB1 variants studied. Interestingly, however, the mean fold change in CD4+ CD137+ cells was significantly higher when the target shared at least one DPA1 allele with the allogeneic stimulator, compared to a distinct DPA1 background (1.65 vs 0.23, P<0.005). Structural homology modeling suggested specific amino acid residues in the alpha chain, in particular position 31, to impact CD4+ T cell allorecognition of HLA-DP. Our data argue against a significant role of beyond exon 2 DPB1 polymorphisms for T cell alloreactivity, but show relevance of DPA1 polymorphism in this mechanism. These new findings impact HLA matching strategies in unrelated stem cell transplantation.