"CD19 stained purified B cells were incubated with a cocktail of HLA-A*0201 multimer (HLA-A*0201/MP58–66, HLA-A*0201/HCw1, HLA-A*0201/pp65; synthesized as previously described ) and then with 2.5 uL of single HLA-class I antigens (at room temperature for 20 min in the dark). 300 uL of PBS/BSA/EDTA was added before the acquisition. "
[Show abstract][Hide abstract] ABSTRACT: In order to characterize the reactivity of B cells against nominal antigens, a method based on the coupling of antigens onto the surface of fluorescent core polystyrene beads was developed. We first demonstrate that murine B cells with a human MOG-specific BCR are able to interact with MOG-coated beads and do not recognize beads coated with human albumin or pp65. B cells purified from human healthy volunteer blood or immunized individuals were tested for their ability to interact with various nominal antigens, including viral, vaccine, self and alloantigens, chosen for their usefulness in studying a variety of pathological processes. A substantial amount of B cells binding self-antigen MOG-coated beads can be detected in normal blood. Furthermore, greater frequencies of B cell against anti-Tetanic Toxin or anti-EBNA1 were observed in primed individuals. This method can reveal increased frequencies of anti-HLA committed B cells in patients with circulating anti-HLA antibodies compared to unsensitized patients and normal individuals. Of interest, those specific CD19 cells were preferentially identified within CD27(-)IgD(+) (i-e naïve) subset. These observations suggest that a broad range of medical situations could benefit from a tool that allows the detection, the quantification and the characterization of antigen-specific blood B cells.
PLoS ONE 12/2013; 8(12):e84273. DOI:10.1371/journal.pone.0084273 · 3.23 Impact Factor
"HLA-A0201/peptide α3-mutated monomers were generated as previously described  except that the original biotinylation sequence was replaced by the AviTag sequence (GLNDIFEAQKIEWHE) (Avidity). Recombinant proteins were produced in GMP conditions by PX'Therapeutics as inclusion bodies in E. coli, dissolved in 8 M urea, and refolded with either clinical grade Melan-AA27L peptide (ELAGIGILTV) or MELOE-136-44 peptide (TLNDECWPA) (C S Bio, Menlo Park, CA, USA). "
[Show abstract][Hide abstract] ABSTRACT: A number of trials of adoptive transfer of tumor-specific T lymphocytes have been performed in the last 20 years in metastatic melanoma, with increasingly encouraging results as the relevant melanoma antigens were identified and the purity/specificity of injected T cells improved. We have previously described a sorting method of epitope-specific T lymphocytes that uses magnetic beads coated with HLA/peptide complexes and we suggested that this method could be applied to a clinical setting. In the present work, we provide a detailed description of the whole GMP process of sorting and amplification of clinical grade T cells specific for the melanoma antigens Melan-A and MELOE-1. All the reagents used in this process including the sorting reagent were produced in GMP conditions and we document the optimization of the different steps of the process such as peptide stimulation, sorting, and amplification. The optimized procedure, validated in 3 blank runs in a clinical setting, allowed the production of at least 10
pure (>90%) Melan-A- and MELOE-1-specific T cells within 28 days starting with 100 mL of blood from metastatic melanoma patients. This GMP process is thus ready to be used in an upcoming phase I/II clinical trial on metastatic melanoma patients.
"Tetramers incorporating HLA-B*3501 were refolded in 1 l with highly diluted heavy chain added over a period of several hours or days to reduce aggregation. The HLA-B*3501 heavy chain used contained a single amino acid substitution (Alanine 245 fi Valine 245 ) in the a3 domain that results in a diminished interaction with CD8 (Salter et al, 1989, 1990) and increased specificity of tetramer staining (Bodinier et al, 2000). For HLA-A*2402, the codons for the first 12 amino acids were exchanged for synonymous codons preferentially used by Escherichia coli; these substitutions significantly increase the yield of expressed protein (Sato et al, 2002). "
[Show abstract][Hide abstract] ABSTRACT: Cytomegalovirus (CMV) causes significant morbidity and mortality in patients after haematopoietic stem cell transplantation (HSCT). Due to limitations of current antiviral therapies, alternative approaches, involving transfer of donor-derived CMV-specific CD8(+) T cells, have been considered. Levels of such cells correlating with protection against CMV infection and disease have only been reported in patients expressing HLA-A*0201 and HLA-B*0702. This is despite an increasing number of reports describing cells targeting CMV peptides presented by other human leucocyte antigens (HLAs). Considering several frequent HLA alleles, our findings suggest that HLA-A*2402/pp65 (341-349)- and HLA-B*3501/pp65 (123-131)-specific CD8+ T cells correlate with protection from CMV reactivation at significantly lower cell levels than HLA-A*0101/pp50 (245-253)- and HLAA* 0201/pp65 (495-503)-specific CD8+ T cells, both in HSCT recipients posttransplant and in healthy CMV seropositive volunteers. This may result from a differing efficiency of the responses restricted by the two sets of HLA alleles. These findings add to the knowledge of immunodominance and differences in antigen processing that are coordinated in individuals with different HLA alleles and have direct implications for therapy and monitoring in patients.
British Journal of Haematology 01/2010; 148(2):311-22. DOI:10.1111/j.1365-2141.2009.07969.x · 4.71 Impact Factor
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