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Two doping cases of homologous blood transfusion (HBT) during Tokyo 2020 Summer Olympics have shown that more controls are needed. The method of detection using flow cytometry to evaluate the expression of minor blood group antigens from red blood cells (RBCs) and identify different RBC populations is efficient but still complex to perform with multiple antigens detection. Recently, the interest of using forensic DNA analysis was also highlighted as a potential new method to detect HBT, with possibility to start from dried blood spots (DBS) instead of fresh blood. After a first phase of development, a protocol was validated for HBT detection using DNA analysis after extraction from DBS. Presence of a second DNA was clear down to 2% of donor blood in vitro. A flow cytometry protocol was also developed with preparation and analysis in 96-well plates and detection of two different antigens per well using two secondary antibodies with distinct fluorophores. The objective of the project was to evaluate the window of detection of an HBT performed in vivo with 150 mL of RBC concentrate. Blood samples obtained over 7 weeks post-transfusion were analyzed. DNA profiling from DBS was not sensitive enough to detect the presence of a second DNA even 1 day after transfusion. On the contrary, the flow cytometry protocol was very efficient and allowed identification of several double populations of RBC (expressing/non-expressing several antigens) until day 50 post-transfusion. This protocol can be fully validated for a future application to doping control samples., (© 2023 The Authors. Drug Testing and Analysis published by John Wiley & Sons Ltd.) more...

The M88.7 T cell clone recognizes an antigen presented by HLA B*1302 on the melanoma cell line M88. A cDNA encoding this antigen (NA88-A) was isolated using a library transfection approach. Analysis of the genomic gene's sequence identified it is a processed pseudogene, derived from a retrotranscript of mRNA coding for homeoprotein HPX42B. The NA88-A gene exhibits several premature stop codons, deletions, and insertions relative to the HPX42B gene. In NA88-A RNA, a short open reading frame codes for the peptide MTQGQHFLQKV from which antigenic peptides are derived; a stop codon follows the peptide's COOH-terminal Val codon. Part of the HPX42B mRNA's 3' untranslated region codes for a peptide of similar sequence (MTQGQHFSQKV). If produced, this peptide can be recognized by M88.7 T cells. However, in HPX42B mRNA, the peptide's COOH-terminal Val codon is followed by a Trp codon. As a result, expression of HPX42B mRNA does not lead to antigen production. A model is proposed for events that participated in creation of a gene coding for a melanoma antigen from a pseudogene. more...

To design an efficient procedure to expand high avidity melanoma reactive T cells and to perform immunotherapies, we compared conditions of peripheral blood lymphocyte (PBL) stimulation by Melan-A/MART-1 peptides. Avidity of induced CTLs was evaluated by measuring their lysis and cytokine secretion to peptide-pulsed transporter-associated protein-deficient cells and to melanoma cells. In side-by-side experiments, we show that melanoma cells, either allogeneic or autologous, induced the growth of high avidity Melan-A-reactive CTLs from all donors, whereas essentially low avidity T cells were induced by peptide-pulsed PBLs. We also show that at least two cytokines, interleukin-6 and interleukin-2, were required to promote the growth of high avidity CTLs. Once sorted by tetramer labeling or cloning, the specificity and reactivity to tumor cells of peptide-specific T cells induced by allogeneic melanoma cells were confirmed. We then describe a relatively simple and efficient procedure that allowed us to obtain systematically high amounts (in the range of billion) of high avidity Melan-A/ MART-1-specific T cells from the PBLs of HLA-A2 melanoma patients and healthy donors in 3 months. Because this antigen is expressed by most melanoma tumors, this procedure should be useful for checking the efficiency of adoptive immunotherapy of melanoma tumors and using functionally well-defined Melan-A/MART-1-specific CTLs in a large group of patients. more...

Several tumor antigens have been described as candidates for immunotherapy. Our study compared HLA-A2-restricted epitopes from 5 antigens commonly expressed by melanomas, i.e., Melan-A/MART-1 peptides (26-35 and 27-35), tyrosinase (368-376), gp-100 (280-288), MAGE-3 (271-279) and NA17-A (1-10), for their relative capacity to promote the development of cytotoxic and cytokine-producing specific CD8+ lymphocytes within melanoma-invaded lymph nodes. We used short-term cultured melanoma-invaded lymph node lymphocytes (MILLs) and tested responses developed by these cells to peptide-pulsed TAP-deficient T2 cells. We measured both the lytic response developed by MILLs and the fraction of these cells that secreted interferon-gamma (IFN-gamma), as deduced from intracellular cytokine labeling. Reverse transcription-polymerase chain reaction (RT-PCR) was used to analyze the expression of the 5 antigens within melanoma-invaded lymph nodes. Melan-A/MART-1, tyrosinase and gp-100 peptides were recognized by MILLs derived, respectively, from 8 of 20, 5 of 19 and 4 of 20 melanoma-invaded lymph nodes expressing these antigens. Most MILLs specific for Melan-A/MART-1 and tyrosinase exhibited both lysis and IFN-gamma responses, whereas most of those specific for gp-100 developed only lysis. Weak lysis without IFN-gamma secretion was developed against NA17-A and MAGE-3 peptides by MILLs from, respectively, 3 of 9 and 2 of 14 lymph nodes expressing these antigens. Our data show a prevalence of both cytotoxic and IFN-gamma-secreting effector T cells specific for differentiation antigens within HLA-A2 melanoma-invaded lymph nodes, which makes these antigens attractive targets for specific immunotherapy. more...

T cell activation is known to depend not only on efficient antigen recognition and subsequent signaling through TCR, but also on interactions involving multiple adhesion and accessory molecules such as CD28/B7, LFA-1/ICAM-1 and LFA-3/CD2. The present study dissects the role of LFA-3/CD2 interactions in the activation of melanoma-specific CD8+ T cell clones. To this end we analyzed the influence of LFA-3 density on melanoma cells on lysis and cytokine production (TNF, IL-2, IFN-gamma) by T cells following activation by various amounts of antigenic peptides. Our results indicate that increasing LFA-3 density on melanoma cells variably affects their lysis susceptibility, but systematically and considerably enhances cytokine production by melanoma-specific cytotoxic T lymphocyte (CTL) clones. At any stimulatory antigen density, LFA-3 increased the fraction of responding cells and/or cytokine amounts produced by individual cells, without affecting TCR down-regulation. These results show that CD2 engagement increases cytokine gene activation essentially by providing to T cells a TCR-independent co-activation signal. From a practical point of view, our data demonstrate that the level of LFA-3 expressed on tumors critically affects cytokine production by specific CTL and thus the efficiency of specific immune reactions mediated by these cells. more...