Allele-specific peptide presentation of human leukocyte antigens: implications for tumor immunotherapy.

BACKGROUND: Human leukocyte antigen (HLA) class I plays a central role in the immune defense against microbes and tumors by presenting endogenously processed peptides to T-cells. Few studies have addressed the question as to what extent variants of the same allelic HLA group differ in their peptide motif. MATERIALS AND METHODS: To answer this question for the HLA-A*66 family, which is a sub-group of the widespread serological HLA-A10 group, peptides eluted from recombinant A*6602 and A*6603 molecules were sequenced and have with data previously reported for A*6601. RESULTS: The amino acid exchange between A*6602 and A*6603 (Gln70His) has no impact on the peptide motif. By contrast, the amino acid exchange Arg163Glu that distinguishes A*6601 from A*6602/6603 results in a change of the peptides preferentially bound. CONCLUSION: Minor amino acid differences between HLA molecules may have a large impact on peptide presentation. For immunotherapy based on tumor-related peptides, it is crucial that the tumor specific peptide is presented by the patients' HLA molecules. For this reason, patients need to be typed not only at the HLA group level, but also at the allelic level.

Aberrant expression of HLA-B*3565Q is associated with a disrupted disulfide bond.

The identification of expression variants is a challenge in HLA diagnostics. We here describe the identification of the novel allele HLA-B*3565Q. The serological HLA class I type, as determined by a lymphocytotoxicity test, was A11,24; B38; Bw4; Cw-; whereas PCR-sequence-specific primers resulted in A*11,*24, B*35,*38; Cw*12, thus suggesting the presence of a nonexpressed B*35 allele. To clarify the lack of serological HLA-B35 reactivity, exons 2 and 3 were sequenced following haplotype-specific amplification. At position 564 from the beginning of the coding region (exon 3), a transversion (C-->G) was observed, which, at the amino acid level, results in a substitution from cysteine to tryptophane at position 164 of the mature polypeptide. Because this position is essential for the formation of a disulfide bond linking the cysteine residues at positions 101 and 164, which is strongly conserved in functional class I molecules of vertebrates, the disruption of this bond is very likely to be the reason for the lack of serological detectability. We later found the same novel allele in a second unrelated individual, of whom we were able to establish a lymphoblastoid cell line (B-LCL). Serological testing of this B-LCL indicated a very low aberrant expression of HLA-B*3565Q, which cannot be expected to be detected by standard serology techniques.

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.

NQO1 C609T polymorphism in distinct entities of pediatric hematologic neoplasms.

BACKGROUND AND OBJECTIVES: NAD(P)H:quinone oxidoreductase 1 (NQO1) is an enzyme that protects cells against mutagenicity from free radicals and toxic oxygen metabolites. The gene coding for NQO1 is subject to a genetic polymorphism at nucleotide position 609 (C-->T) of the human NQO1 cDNA. Heterozygous individuals (C/T) have intermediate activity and homozygotes for the variant allele (T/T) are deficient in NQO1 activity. In previous studies, genotypes conferring lower NQO1 activity have been associated with an increased risk of acute leukemia, particularly infant leukemia carrying MLL/AF4 fusion genes. In the present study, we investigated this association in our population and extended the analysis to other subgroups of pediatric hematologic neoplasms characterized by specific fusion genes. DESIGN AND METHODS: We genotyped 138 patients with childhood acute lymphoblastic leukemia (ALL) carrying distinct fusion genes (MLL/AF4=35; BCR/ABL=31; TEL/AML1=72), 71 cases of pediatric sporadic Burkitt's lymphoma and 190 healthy control individuals for the NQO1 C609T polymorphism. RESULTS: When compared to the healthy control group, only children with Burkitt's lymphoma significantly more often had NQO1 genotypes associated with lower NQO1 activity (odds ratio, 1.81; p=0.036), predominantly at a younger age (< 9 years at diagnosis: odds ratio, 3.02; p=0.003). INTERPRETATION AND CONCLUSIONS: Our results suggest that in our population the NQO1 C609T polymorphism does not confer an increased risk of the investigated entities of childhood ALL. However, there may be a modulating role for NQO1 in the pathogenesis of pediatric sporadic Burkitt's lymphoma.

Platelet P-selectin and GPIIb/IIIa expression after liver transplantation and resection.

Platelet dysfunction contributes to haemostatic defects, possibly leading to bleeding complications. We hypothesised that liver transplantation and liver resection, together with portal clamping time, might be a potential stimulus for platelet activation. Therefore, we determined the expression of platelet GPIIb/IIIa and P-selectin, representing important platelet activation markers, and the thrombopoietin (TPO) serum level after transplantation and resection. Twenty patients [ten that had undergone orthotopic liver transplantation (OLT), ten with liver resection (LRX)] were included in the study. From sequential venous blood samples, surface expression of GPIIb/IIIa and P-selectin was quantified by flow cytometry, and TPO serum levels were determined by ELISA. Baseline GPIIb/IIIa receptor expression on circulating platelets was significantly reduced in the OLT group compared to the LRX group and healthy volunteers. GPIIb/IIIa expression after activation with TRAP-6 increased significantly ( P<0.001) in the LRX group but not in the OLT group. P-selectin expression after TRAP-6 stimulation increased significantly ( P<0.001) in the LRX group, being comparable to that in healthy volunteers, whereas only a very low increase in the OLT group was found. In the OLT group, TPO serum levels were in the lower normal range and rose above the upper limit of normal values 24 h after reperfusion. These data indicate that neither liver transplantation nor liver resection influences GPIIb/IIIa and P-selectin expression on circulating platelets. There was a lack of expression in cirrhotic patients and unimpaired baseline expression and functional reserve in non-cirrhotic liver-resection patients. After liver transplantation, increasing serum TPO levels, which indicated a recovering graft function, resulted in rising peripheral platelet counts.

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 pocket A 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.

Lack of influence of the COX inhibitors metamizol and diclofenac on platelet GPIIb/IIIa and P-selectin expression in vitro.

BACKGROUND: The effect of non-steroidal anti-inflammatory drugs (NSAIDs) for reduced platelet aggregation and thromboxane A2 synthesis has been well documented. However, the influence on platelet function is not fully explained. Aim of this study was to examine the influence of the COX-1 inhibiting NSAIDs, diclofenac and metamizol on platelet activation and leukocyte-platelet complexes, in vitro. Surface expression of GPIIb/IIIa and P-selectin on platelets, and the percentage of platelet-leukocyte complexes were investigated. METHODS: Whole blood was incubated with three different concentrations of diclofenac and metamizol for 5 and 30 minutes, followed by activation with TRAP-6 and ADP. Rates of GPIIb/IIIa and P-selectin expression, and the percentage of platelet-leukocyte complexes were analyzed by a flow-cytometric assay. RESULTS: There were no significant differences in the expression of GPIIb/IIIa and P-selectin, and in the formation of platelet-leukocyte complexes after activation with ADP and TRAP-6, regarding both the time of incubation and the concentrations of diclofenac and metamizol. CONCLUSIONS: Accordingly, the inhibitory effect of diclofenac and metamizol on platelet aggregation is not related to a reduced surface expression of P-selectin and GPIIb/IIIa on platelets.

Impact of different modalities of continuous venovenous hemofiltration on sepsis-induced alterations in experimental pancreatitis.

BACKGROUND: Continuous venovenous hemofiltration (CVVH) is assumed to attenuate systemic complications in septic diseases. The impact of different treatment intensities of CVVH on immunologic and systemic alterations in experimental pancreatitis was evaluated. METHODS: Eighty-four minipigs were allocated either to an untreated control group (group 1) or to one of six treatment groups (groups 2 to 7) that underwent CVVH in different modalities: (1): "late" CVVH, started after a decline of total peripheral resistance of 30% versus "prophylactic" CVVH started immediately after the induction of pancreatitis; (2) no change of hemofilters versus a periodic change of filters every 12 hours; (3) low-volume CVVH with a filtrate turnover of 20 mL/kg body weight (BW)/h versus high-volume CVVH (100 mL/kg/h). Pancreatitis was induced by intraductal injection of sodium-taurocholate (3%, 1 mL/kg BW) and enterokinase (2 U/kg BW). We focused on the occurrence of sepsis, serum cytokines, down-regulation of major histocompatibility complex II (MHC II) and the endotoxin receptor CD14 expression, bacterial translocation/endotoxemia, and pulmonary and renal histologic alterations. RESULTS: CVVH delayed or definitively prevented the occurrence of sepsis. Pancreatitis was associated with a tremendous initial tumor necrosis factor-alpha (TNF-alpha) response prior to a return to near baseline levels in the late course of sepsis. Endotoxin hyporesponsiveness, suggested by the dissociation of decreasing TNF-alpha levels and increasing endotoxemia in end-stage sepsis, was favorably influenced by CVVH. Down-regulation of MHC II and CD14 expression was prevented in non-septic animals. CVVH-related sepsis-protection led to a significant attenuation of histological injury in lungs and kidneys. "Prophylactic" CVVH prevented histological changes more effectively than "late" CVVH. CONCLUSIONS: CVVH offers a therapeutic option for supportive treatment in severe pancreatitis. The efficiency of CVVH is associated with the duration of filter use and cumulative plasma turnover. Since CVVH may lead to sepsis-protection and long-term survival, further evaluation in controlled, clinical trials is warranted.

The nature of introns 4-7 largely reflects the lineage specificity of HLA-A alleles.

For most HLA-A alleles the phylogeny of the 3' non-coding regions has not yet been studied systematically. In this study, we have determined the sequences of introns 4-7 in 50 HLA-A variants, and have computed nucleotide substitution rates and phylogenetic relationships. The A2/A28, A9, and A10 groups were characterized by clear lineage specificity. For the A19 group, lineage specificity was weaker. A*3001 clustered together with the alleles of the A1/A3/A11/A36 serological family, but not with the A19 group alleles. Reduced lineage specificity was also observed for the alleles of the A1/A3/A11/A36groups. The 3' intron sequences of A*8001 were clearly distinct from all other alleles studied. In several cases two allelic groups shared identical intron sequences, whereby the patterns varied with the introns. A similar situation has been previously described for the 5' introns. Since recombination is the major mechanism of HLA diversification, the intronic lineage specificity corresponds to the comparatively lower recombination rate of the HLA-A 3' exons. The low level of recombination within the 3' region of HLA-A is supported by the low CpG content with a maximum of 3.0% in this region compared with up to 10.7% in the 5' region. Apart from phylogenetic studies of HLA diversity and diversification, the sequence data obtained in our study may prove valuable for the development of a haplotype-specific sequencing strategy for the HLA-A3' exons and for the explanation of recombination events in newly described HLA class I alleles.