Establishment of the reversible peptide-major histocompatibility complex (pMHC) class I Histamer technology: tool for visualization and selection of functionally active antigen-specific CD8(+) T lymphocytes.

Multimers of soluble peptide-major histocompatibilty complex (pMHC) molecules are used in both basic and clinical immunology. They allow the specific visualization and isolation of antigen-specific T cells from ex vivo samples. Adoptive transfer of antigen-specific T cells sorted by pMHC multimers is an effective strategy for treatment of patients with malignancies or infectious diseases after transplantation. We developed a new reversible pMHC multimer called 'Histamer' to enable the specific detection and isolation of antiviral T cells from peripheral blood. HLA-A*02:01/CMVpp65 (495-503) Histamer (A02/CMV Histamer) was generated by coupling 6xHis-tagged pMHC molecules onto cobalt-based magnetic beads. The specificity of the Histamer was evaluated by flow cytometry. Sorting of antiviral CD8(+) cytotoxic T lymphocytes (CTLs) was performed by magnetic cell separation, followed by the monomerization of the Histamer after addition of the competitor L-histidine. Sorted T cells were analyzed for phenotype and function. The reversible pMHC Histamer proved to be highly specific and sensitive. CMV-specific T cells of up to 99.6% purity were isolated using the Histamer technology. Rapid and complete disassembly of the T-cell surface-bound A02/CMV Histamer followed by the subsequent dissociation of the pMHC monomers from CD8(+) CTL receptors was achieved using 100 mM L-histidine. The function of CMV-specific T cells enriched by Histamer staining did not differ from CTLs induced by standard T-cell assays. This reversible T-cell staining procedure preserves the functionality of antigen-specific T cells and can be adapted to good manufacturing practice conditions. The pMHC Histamer technology offers full flexibility and fulfills all requirements to generate clinical-grade T lymphocytes.

Position 156 influences the peptide repertoire and tapasin dependency of human leukocyte antigen B*44 allotypes.

BACKGROUND: Polymorphic differences between donor and recipient human leukocyte antigen class I molecules can result in graft-versus-host disease due to distinct peptide presentation. As part of the peptide-loading complex, tapasin plays an important role in selecting peptides from the pool of potential ligands. Class I polymorphisms can significantly alter the tapasin-mediated interaction with the peptide-loading complex and although most class I allotypes are highly dependent upon tapasin, some are able to load peptides independently of tapasin. Several human leukocyte antigen B*44 allotypes differ exclusively at position 156 (B*44:02(156Asp), 44:03(156Leu), 44:28(156Arg), 44:35(156Glu)). From these alleles, only the high tapasin-dependency of human leukocyte antigen B*44:02 has been reported. DESIGN AND METHODS: We investigated the influence of position 156 polymorphisms on both the requirement of tapasin for efficient surface expression of each allotype and their peptide features. Genes encoding human leukocyte antigen B*44 variants bearing all possible substitutions at position 156 were lentivirally transduced into human leukocyte antigen class I-negative LCL 721.221 cells and the tapasin-deficient cell line LCL 721.220. RESULTS: Exclusively human leukocyte antigen B*44:28(156Arg) was expressed on the surface of tapasin-deficient cells, suggesting that the remaining B*44/156 variants are highly tapasin-dependent. Our computational analysis suggests that the tapasin-independence of human leukocyte antigen B*44:28(156Arg) is a result of stabilization of the peptide binding region and generation of a more peptide receptive state. Sequencing of peptides eluted from human leukocyte antigen B*44 molecules by liquid chromatography-electrospray ionization-mass spectrometry (LTQ-Orbitrap) demonstrated that both B*44:02 and B*44:28 share the same overall peptide motif and a certain percentage of their individual peptide repertoires in the presence and/or absence of tapasin. CONCLUSIONS: Here we report for the first time the influence of position 156 on the human leukocyte antigen/tapasin association. Additionally, the results of peptide sequencing suggest that tapasin chaperoning is needed to acquire peptides of unusual length.

Structural insight into the stereoselective inhibition of MMP-8 by enantiomeric sulfonamide phosphonates.

Potent and selective inhibitors of matrix metalloproteinases (MMPs), a family of zinc proteases that can degrade all the components of the extracellular matrix, could be useful for treatment of diseases such as cancer and arthritis. The most potent MMP inhibitors are based on hydroxamate as zinc-binding group (ZBG). alpha-Arylsulfonylamino phosphonates incorporate a particularly favorable combination of phosphonate as ZBG and arylsulfonylamino backbone so that their affinity exceptionally attains the nanomolar strength frequently observed for hydroxamate analogues. The detailed mode of binding of [1-(4'-methoxybiphenyl-4-sulfonylamino)-2-methylpropyl]phosphonate has been clarified by the crystal structures of the complexes that the R- and S-enantiomers respectively form with MMP-8. The reasons for the preferential MMP-8 inhibition by the R-phosphonate are underlined and the differences in the mode of binding of analogous alpha-arylsulfonylamino hydroxamates and carboxylates are discussed.

Heme Oxygenase-1 Inhibits HLA Class I Antibody-Dependent Endothelial Cell Activation.

Antibody-mediated rejection (AMR) is a key limiting factor for long-term graft survival in solid organ transplantation. Human leukocyte antigen (HLA) class I (HLA I) antibodies (Abs) play a major role in the pathogenesis of AMR via their interactions with HLA molecules on vascular endothelial cells (ECs). The antioxidant enzyme heme oxygenase (HO)-1 has anti-inflammatory functions in the endothelium. As complement-independent effects of HLA I Abs can activate ECs, it was the goal of the current study to investigate the role of HO-1 on activation of human ECs by HLA I Abs. In cell cultures of various primary human macro- and microvascular ECs treatment with monoclonal pan- and allele-specific HLA I Abs up-regulated the expression of inducible proinflammatory adhesion molecules and chemokines (vascular cell adhesion molecule-1 [VCAM-1], intercellular cell adhesion molecule-1 [ICAM-1], interleukin-8 [IL-8] and monocyte chemotactic protein 1 [MCP-1]). Pharmacological induction of HO-1 with cobalt-protoporphyrin IX reduced, whereas inhibition of HO-1 with either zinc-protoporphyrin IX or siRNA-mediated knockdown increased HLA I Ab-dependent up-regulation of VCAM-1. Treatment with two carbon monoxide (CO)-releasing molecules, which liberate the gaseous HO product CO, blocked HLA I Ab-dependent EC activation. Finally, in an in vitro adhesion assay exposure of ECs to HLA I Abs led to increased monocyte binding, which was counteracted by up-regulation of HO-1. In conclusion, HLA I Ab-dependent EC activation is modulated by endothelial HO-1 and targeted induction of this enzyme may be a novel therapeutic approach for the treatment of AMR in solid organ transplantation.

Development of a single-antigen magnetic bead assay (SAMBA) for the sensitive detection of HPA-1a alloantibodies using tag-engineered recombinant soluble ß3 integrin.

Timely and accurate testing for human platelet antigen 1a (HPA-1a) alloantibodies is vital for clinical diagnosis of neonatal alloimmune thrombocytopenia (NAIT). Current antigen-specific assays used for the detection of HPA-1 alloantibodies are technically very complex and cumbersome for most diagnostic laboratories. Hence, we designed and applied recombinant soluble (rs) ß3 integrins displaying HPA-1a or HPA-1b epitopes for the development of a single-antigen magnetic bead assay (SAMBA). Soluble HPA-1a and HPA-1b were produced recombinantly in human embryonic kidney 293 (HEK293) cells and differentially tagged. The recombinant soluble proteins were then immobilized onto paramagnetic beads and used for analysis of HPA-1 alloantibodies by enzyme-linked immunosorbent assay (ELISA). HPA-1a serum samples (n=7) from NAIT patients, inert sera and sera containing non-HPA-1a antibodies were used to evaluate the sensitivity and specificity of the SAMBA. Fusion of V5-His or GS-SBP-His tags to the rsß3 integrins resulted in high-yield expression. SAMBA was able to detect all HPA-1a and -1b alloantibodies recognized by monoclonal antibody-specific immobilization of platelet antigens assay (MAIPA). No cross-reactions between the sera were observed. Two out of seven of the HPA-1a alloantibody-containing sera demonstrated weak to moderate reactivity in MAIPA but strong signals in SAMBA. SAMBA provides a very reliable method for the detection of HPA-1 antibodies with high specificity and sensitivity. This simple and rapid assay can be adapted for use in any routine laboratory and can be potentially adapted for use on automated systems.

Isolation and purification of blood group antigens using immuno-affinity chromatography on short monolithic columns.

Monolithic columns have gained increasing attention as stationary phases for the separation of biomolecules and biopharmaceuticals. In the present work the performance of monolithic convective interaction media (CIM(®)) chromatography for the purification of blood group antigens was established. The proteins employed in this study are derived from blood group antigens Knops, JMH and Scianna, equipped both with a His-tag and with a V5-tag by which they can be purified. In a first step a monoclonal antibody directed against the V5-tag was immobilized on a CIM(®) Disk with epoxy chemistry. After this, the immobilized CIM(®) Disk was used in immuno-affinity chromatography to purify the three blood group antigens from cell culture supernatant. Up-scaling of the applied technology was carried out using CIM(®) Tubes. In comparison to conventional affinity chromatography, blood group antigens were also purified via His-tag using a HiTrap(®) metal-affinity column. The two purifications have been compared regarding purity, yield and purification speed. Using the monolithic support, it was possible to isolate the blood group antigens with a higher flow rate than using the conventional bed-packed column.

Mismatches outside exons 2 and 3 do not alter the peptide motif of the allele group B*44:02P.

Sequence variations outside exons 2 and 3 do not appear to affect the function of human leukocyte antigen (HLA) class I alleles. HLA-B*44:02:01:01 and -B*44:27 are considered functionally identical because they differ by a single amino acid substitution of Val > Ala at position 199, which is located in the α3 domain. To validate that HLA-B*44:02:01:01 and -B*44:27 represent functionally identical alleles that might reflect a permissive mismatch in hematopoetic stem cell transplantation (HSCT), we determined their peptide-binding features. B-lymphoblastic cells were lentivirally transduced with B*44:02 and B*44:27 constructs and soluble recombinant molecules were purified by affinity chromatography. Peptides were isolated and sequencing of single peptides was performed using liquid chromatography-electrospray ionization-tandem mass spectrometry (LTQ-Orbitrap) technology. We demonstrate that the peptide motif of B*44:02(199Val) and B*44:27(199Ala) is identical. Both variants feature E at P2 and Y, F, or W at PΩ in their ligands. Most of the identified peptides are 9 to 11 amino acids in length and approximately 20% of these ligands are shared between the alleles. Our results lead to the conclusion that B*44:02:01:01 and B*44:27 might have the same immune function, validating a theory that is now being used in deciding which donors to select in HSCT when there is no identical donor available.

N-Hydroxyurea as zinc binding group in matrix metalloproteinase inhibition: mode of binding in a complex with MMP-8.

The first crystallographic structure of an N-hydroxyurea inhibitor bound into the active site of a matrix metalloproteinase is reported. The ligand and three other analogues were prepared and studied as inhibitors of MMP-2, MMP-3, and MMP-8. The crystal structure of the complex with MMP-8 shows that the N-hydroxyurea, contrary to the analogous hydroxamate, binds the catalytic zinc ion in a monodentate rather than bidentate mode and with high out-of-plane distortion of the amide bonds.

Clinical relevance of matrix metalloproteinase-13 determined with a new highly specific and sensitive ELISA in ascitic fluid of advanced ovarian carcinoma patients.

Matrix metalloproteinases (MMPs) are involved in many physiological and pathophysiological processes, including tumor cell invasion and metastasis. For one member of this family, MMP-13 (collagenase-3), a new, highly specific ELISA with a sensitivity of 0.5 ng MMP-13/ml was established. The protein levels of MMP-13 in ascitic fluids of 30 patients with advanced ovarian cancer FIGO stage III (n = 19) and IV (n = 11) were measured with this ELISA. Using a cut-off value of 0.5 ng MMP-13/mg total protein, two patient subpopulations with short (median 16 months) and long (median 36 months) overall survival were identified. Together with other prognostic markers, determination of MMP-13 in ascitic fluid may help to identify patients at risk for early death and help to individualize adjuvant therapy.