Epitope mapping of an HLA-DR-specific monoclonal antibody produced by using human-mouse transfectant cells

A polymorphic HLA-DR mAb, TAL15.1, was produced against L cells transfected with DR alpha- and beta-chain cDNA from a cell line homozygous for HLA-DRw8. This antibody reacted with DRw8 plus all other DR types except DR3 and DRw52. DR3 and DRw52 differ uniquely from other other DR antigens at position 77 in the beta 1-domain of their beta-chains where there is asparagine instead of threonine. In Western blots the antibody reacted with DR alpha/beta-dimer but not with free alpha- or beta-chains. Two-dimensional gel analysis of a DRw11, DRw52 cell line showed that TAL15.1 immunoprecipitated the DR products. Although it also coprecipitated the DQ beta chain products, flow microfluorimetric analysis with various transfectant cell lines showed that TAL15.1 failed to bind the DQ or DP products tested. We conclude that TAL15.1 is a DR-specific polymorphic antibody whose activity correlates with a specific residue. It has already proved to be a valuable reagent for distinguishing DR3 homozygotes from DR3, DRw6 heterozygotes, which have in the past been difficult to separate.     

Molecular definition of an elusive third HLA-A9 molecule: HLA-A9.3

The HLA-A9 family has been characterized as possessing two well defined specificities; HLA-A23 and A24. Serological studies have suggested the presence of a third member of this family HLA-A9.3, however there is doubt surrounding the existence of this specificity. HLA-A23, A24, and the putative A9.3 proteins were analyzed biochemically by immunoprecipitation and isoelectric focusing. Both HLA-A24 and A9.3 have identical isoelectric points whereas A23 is different. We have sequenced cDNA encoding HLA-A23, A24, and A9.3. From the observed protein sequences, we found A9.3 to differ from A24 by two amino acid substitutions located in the ₂ helix of the class I molecule. These substitutions are expected to significantly change the shape of the peptide binding cleft.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession numbers M64740 (HLA-A ^*2402); M64741 (HLA-A ^*2403); M64742 (HLA-A ^*2301). Address correspondence and offprint requests to: P. Parham.     

Investigation of peptide involvement in T cell allorecognition using recombinant HLA class I multimers

Alloreactive T cells are involved in injurious graft rejection and graft-vs-host disease. However, they can also evoke beneficial responses to tumor Ags restricted by foreign MHC molecules. Manipulation of these alloreactivities requires information on the basis of T cell allorecognition. The vigorous T cell response to foreign MHC molecules may arise from peptide-independent recognition of polymorphic residues of foreign MHC molecules or peptide-specific recognition of novel peptides presented by foreign MHC molecules. We investigated CD8+ T cell allorecognition using recombinant HLA class I/peptide complexes. Peptide-specific allorecognition was examined using tetramers of HLA-A*0201 representing five peptides derived from ubiquitously expressed self-proteins that are known to bind endogenously to HLA-A*0201. Distinct subsets of CD8+ T cells specific for each HLA-A*0201/peptide combination were detected within four in vitro-stimulated T cell populations specific for foreign HLA-A*0201. Peptide-independent allorecognition was investigated using artificial Ag-presenting constructs (aAPCs) coated with CD54, CD80, and functional densities of a single HLA-A*0201/peptide combination for four different peptides. None of the four T cell populations specific for foreign HLA-A*0201 were stimulated by the aAPCs, whereas they did produce IFN-gamma upon stimulation with cells naturally expressing HLA-A*0201. Thus, aAPCs did not stimulate putative peptide-independent allorestricted T cells. The results show that these alloreactive populations comprise subsets of T cells, each specific for a self-peptide presented by foreign class I molecules, with no evidence of peptide-independent components.     

New HLA-A2 variants defined by monoclonal antibodies and cytotoxic T lymphocytes

Three HLA-A2 variants, A2-DW, A2-KC, and A2-Lee, were identified in three Chinese donors using a panel of monoclonal antibodies. A2-DW was negative with two of the ten HLA-A2 monoclonal antibodies tested, whereas A2-KC was negative with five of the ten and A-2 Lee was negative with one.Epstein-Barr virus-specific cytotoxic T cells generated from the A2-DW donor recognized and killed target cells prepared from the A2-KC donor, but did not recognize target cells from HLA-A2.1, –A2.2, or –A2.4 donors. In isoelectric focusing studies, A2-DW and A2-KC focus in identical positions more acidic than the other HLA-A2 antigens tested.     

Photoperiodic growth enhancement in a tropical batch spawning atherinopsid,pike silverside Chirostoma estor

The effect of photoperiod on growth and survival in early life was determined in the tropical batch spawning atherinopsid, pike silverside Chirostoma estor. The results demonstrate high sensitivity of newly hatched C. estor to photoperiod treatments up to 90 days post hatch shown by improved growth in mass (43%) under continuous illumination. This is accompanied by increased fat deposition, which suggests a critical interaction between different photoperiod‐mediated mechanisms. A thorough understanding of these mechanisms can help to optimize the development of aquaculture of C. estor and similar species.     

Genetic deletion or TWEAK blocking antibody administration reduce atherosclerosis and enhance plaque stability in mice

Clinical complications associated with atherosclerotic plaques arise from luminal obstruction due to plaque growth or destabilization leading to rupture. Tumour necrosis factor ligand superfamily member 12 (TNFSF12) also known as TNF-related weak inducer of apoptosis (TWEAK) is a proinflammatory cytokine that participates in atherosclerotic plaque development, but its role in plaque stability remains unclear. Using two different approaches, genetic deletion of TNFSF12 and treatment with a TWEAK blocking mAb in atherosclerosis-prone mice, we have analysed the effect of TWEAK inhibition on atherosclerotic plaques progression and stability. Mice lacking both TNFSF12 and Apolipoprotein E (TNFSF12^−/−ApoE^−/−) exhibited a diminished atherosclerotic burden and lesion size in their aorta. Advanced atherosclerotic plaques of TNFSF12^−/−ApoE^−/− or anti-TWEAK treated mice exhibited an increase collagen/lipid and vascular smooth muscle cell/macrophage ratios compared with TNFSF12^+/+ApoE^−/− control mice, reflecting a more stable plaque phenotype. These changes are related with two different mechanisms, reduction of the inflammatory response (chemokines expression and secretion and nuclear factor kappa B activation) and decrease of metalloproteinase activity in atherosclerotic plaques of TNFSF12^−/−ApoE^−/−. A similar phenotype was observed with anti-TWEAK mAb treatment in TNFSF12^+/+ApoE^−/− mice. Brachiocephalic arteries were also examined since they exhibit additional features akin to human atherosclerotic plaques associated with instability and rupture. Features of greater plaque stability including augmented collagen/lipid ratio, reduced macrophage content, and less presence of lateral xanthomas, buried caps, medial erosion, intraplaque haemorrhage and calcium content were present in TNFSF12^−/−ApoE^−/− or anti-TWEAK treatment in TNFSF12^+/+ApoE^−/− mice. Overall, our data indicate that anti-TWEAK treatment has the capacity to diminish proinflamatory response associated with atherosclerotic plaque progression and to alter plaque morphology towards a stable phenotype.     

BCR/ABL-specific CD8<Superscript>+</Superscript> T cells can be detected from CML patients,but are only expanded from healthy donors

The BCR/ABL p210 fusion protein has long been considered an ideal target antigen for the development of immunotherapeutic strategies in chronic myeloid leukaemia (CML) due to its central role in malignant transformation and to its unique novel amino acid sequence solely expressed in leukaemia cells. However, the feasibility to expand BCR-ABL-specific T cells remains still controversial. Using BCR/ABL peptide/MHC tetramers, significantly higher frequencies of tetramer positive cells were detected in the peripheral blood of HLA-A*0301 (mean 0.38%) and HLA-B*0801 (mean 0.28%) CML patients than in healthy donors (P = 0.0025 and 0.0026, respectively). However, following stimulation with autologous peptide-pulsed DCs, BCR/ABL-specific T cells were only expanded from some healthy donors, suggesting that CML patients may have a specific immune deficit with respect to the BCR/ABL antigen. Professor A. I. Dodi passed away recently. This paper is an original contribution from the meeting which took place 28–29 May 2008 in Nottingham, UK, celebrating the contribution of Prof. A. I. “Tony” Dodi (29 January 2008) to the EU project “Network for the identification and validation of antigens and biomarkers in cancer and their application in clinical tumour immunology (ENACT)”.