Axel Seltsam

Missense mutations outside the catalytic domain of the ABO glycosyltransferase can cause weak blood group A and B phenotypes.

BACKGROUND: Only little is known about the impact of amino acid substitutions outside an enzymes active site on A and Bu2003transferase activity.

A single amino-acid polymorphism in pocket A of HLA-A*6602 alters the auxiliary anchors compared with HLA-A*6601 ligands

In this study we have sequenced peptides eluted from a truncated recombinant HLA-A*6602 molecule, and compared their features with data reported for peptides presented in the A*6601 molecule. A striking change in the amino-acid binding preferences was observed at peptide position P1, which interacts with pocketxa0A of the HLA peptide-binding region. For A*6601, aspartic acid and glutamic acid, both of which possess polar acidic side-chains, have been described as auxiliary anchors. This is in marked contrast to A*6602, where we observed serine, which has a neutral polar side-chain, as auxiliary anchor at P1. Accordingly, this shift in the physico-chemical properties of the auxiliary anchor may be best explained by the HLA amino-acid polymorphism at position 163, where arginine (hydrophilic, alkaline) in A*6601 has been replaced by glutamic acid in A*6602. This amino-acid exchange results in a shift towards higher acidity in pocket A, apparently resulting in the loss of preference for acidic auxiliary anchors, and leading to the preference for the neutral amino acid serine. The change of the auxiliary anchor residue at P1 is likely to alter the spectrum of peptides presented by A*6602 compared with A*6601, which may result in allogenicity in the case of a mismatch in allogeneic stem cell transplantation.

ABO glycosyltransferases as potential source of minor histocompatibility antigens in allogeneic peripheral blood progenitor cell transplantation

BACKGROUND: Most studies indicate that the incidence of graft‐versus‐host disease (GVHD) is not increased in ABO‐mismatched allogeneic peripheral blood progenitor cell transplantation. These studies exclusively looked at ABO phenotypes without considering the fact that different genotypes hide behind identical phenotypes that encode for different sets of glycosyltransferases, thus providing a source for minor histocompatibility antigens (mHags).

Prokaryotic versus eukaryotic recombinant Lutheran blood group protein for antibody identification.

BACKGROUND: At present, identification of antibodies against high‐frequency antigens is limited to reference laboratories having panels of rare red blood cell (RBC) specimens in stock. Antibodies against Lub are among the most frequent clinically relevant antibody specificities directed against high‐frequency antigens.

Peptide-binding motif of HLA-A*6603

The peptide motif of HLA-A*6603 was determined and compared with the available data on the peptide motifs of A*6601 and A*6602. A*6601 differs from A*6602 by two amino acids at positions 90 (Asp90Ala; outer loop) and 163 (Arg163Glu; pocket A). A*6603 differs from A*6601 and A*6602 by a single amino-acid exchange at position 70 (His70Gln; pockets A, B and C). No significant differences were found between the A*6602 and A*6603 peptide motifs suggesting that the Gln70His variation is of minor importance. However, the auxiliary anchors at position P1 of peptides bound by A*6601 (polar/acidic: Asp, Glu) and A*6602/6603 (polar/neutral: Ser) had striking differences. This finding may be best explained by the Arg163Glu substitution that results in a shift towards higher acidity in pocket A of A*6602/6603, apparently leading to the loss of preference for acidic auxiliary anchors. The similarity of A*6602 and A*6603 peptide motifs suggests low allogenicity when mismatched in stem cell transplantation. Inversely, the differences in A*6601 versus A*6602/6603 peptide motifs suggest that mismatches will have a higher allogenicity. These data will contribute to both assessing permissive mismatches in the A*66 group and weighting the impact of this individual amino-acid variation for matching and peptide binding algorithms.

NKG2A silencing in effector cells to improve effectivity of cell based therapeutics

The heterodimeric NKG2A/CD94 receptor delivers an inhibitory signal upon recognition of HLA-E molecules. In several studies it has been demonstrated that signalling via NKG2A/CD94 receptor significantly reduces T or NK cell cytotoxic activity and cytokine production. In hematopoietic stem cell transplantation, expression of NKG2A on NK and T cells has been shown to compromise the graft-versus-leukemia effect. In addition, NKG2A was shown to inhibit tumor-specific T cell responses. In this study, we developed a RNAi-based approach to permanently silence the expression of NKG2A molecules on NK and T-cells. The functional relevance of NKG2A silencing for the cytotoxic potential of genetically engineered NK and T-cells was evaluated. n nNKG2A+ cells were isolated from fresh PBMCs. Lentiviral vectors were designed to express short hairpin RNA sequences (shRNA) targeting NKG2A transcripts. The level of NKG2A suppression was measured by flow cytometry and real-time RT-PCR. The effect of NKG2A receptor silencing on the cytolytic potential of NK and T cells was evaluated in cytotoxicity assays using K562 and B-LCL cells as targets. In addition, granzyme B mRNA transcript levels were detected by real-time RT-PCR. n nThe transduction of inducible RNAi cassettes containing the sequences for shRNAs targeting NKG2A caused a reduction of protein expression by up to 80% in NK and T cells. In cytotoxicity assays, it was demonstrated that NKG2A silencing was effective to enhance NK and CD8+ T cell lysis by up to 40%. In comparison with unmodified cells, granzyme B transcript levels were upregulated by up to 12-fold in NKG2A silenced cells after target exposure. Expression of NKG2A-specific shRNA did not affect the expression of the activating markers NKp44 on NK cells and CD25 on T cells. n nOur data suggest that RNAi-mediated silencing of NKG2A in effector cells could improve the effectivity of cell-based immunotherapeutics and might be of particular interest in an autologous approach.

Haemolytic disease of fetus and newborn caused by ABO antibodies in a cisAB offspring

ABO hemolytic disease of fetus and newborn (ABO-HDFN) occurs almost exclusively in infants of blood group A or B who are born to group O mothers because IgG anti-A or -B occurs more commonly in group O than in group A or B individuals. We report a case of clinically significant ABO-HDFN where the mother was blood group O with elevated IgG anti-A and anti-B titers and delivered a child with an A2B phenotype. This unusual ABO constellation between mother and infant was based on the inheritance of a rare ABO allele encoding for a glycosyltransferase capable of synthesizing both A and B antigens. Because both anti-A and anti-B antibodies may have been involved in hemolysis in this case, it may be relevant to consider the cisAB phenomenon when monitoring ABO-incompatible pregnancies and births.