High-scale expansion of melanoma-reactive TIL by a polyclonal stimulus: predictability and relation with disease advancement.

The rationale of treating melanoma patients by infusion with tumor-infiltrating leukocytes (TIL) is to perform an adoptive therapy through injection of tumor-specific T cells. Nonetheless, methods currently used for ex vivo TIL expansion have not been evaluated for their efficacy to expand TAA-specific T cells. We have addressed this question here, using a culture method in which high TIL growth was induced by a polyclonal T cell stimulus. Intracellular cytokine assays were performed to measure the proportion of T cells responding to autologous tumor cells among the lymphocytes from lymph node biopsies (TIL) of 26 patients with stage III melanoma. The data show that TIL from 18 of these patients contained detectable amounts of tumor-specific T cells before expansion. Although they decreased somewhat in percent abundance during expansion, they were still present afterwards, ranging from 0.3 to 13.8%. Since a median number of 1.7 x 10(10) TIL was obtained from these patients (starting from 3.6 x 10(6) TIL), a total amount of tumor-reactive cytokine-secreting TIL of between 2.8 x 10(6) and 1.12 x 10(9) was obtained in each case from 18 patients. The TIL populations from 8 patients did not contain tumor-reactive T cells: neither before expansion, nor after expansion. Lack of tumor-reactive TIL only occurs for patients bearing several tumor-invaded lymph nodes (40%), but not for those having a single invaded lymph node. Therefore, high numbers of tumor-reactive T cells can be produced, through a polyclonal TIL stimulation, from most early stage III melanoma patients but from only about half of the patients with a more disseminated disease. For this last group, the possibility of getting tumor-reactive TIL can be predicted by checking the presence of these cells before expansion.

Conception and organisation of a cell therapy unit of Nantes, France.

Manufacturing of cell therapy products has to follow several requirements to obtain sanitary security and quality of the product. Thus, at its conception, the cell therapy unit (CTU) of Nantes choose to integrate a quality assurance system: - The good manufacturing practices (GMP's) are a technical reference for the Unit. They are a quality criteria necessary to guarantee the security of products in term of staff, premises, material, matter and method; - The ISO 9001 standards are a model for quality assurance in design, development, production, installation and servicing. They established a quality system; - The creation, the running and the maintenance of premises are an essential aspect of the quality system and they are described in this paper. Thus, from October 1994 to June 1998, 450 cell processing (with or without cryopreservation and storage of cells) have been realised at the CTU of Nantes, leading to 160 injections without major undesirable effect and without microbiological contamination.

[Identification of spermatozoa L-selectin and two potential pellucida ligands]. / Identification d'une L-sélectine spermatique et de 2 ligands pellucidaires potentiels.

At the molecular level, gamete interactions partly depend on zona pellucida-glycoproteins fucosylation. We show, by immunocytochemistry, the sialyl-lewisx and sialyl-lewisa oligosaccharides on human zonae pellucidae. These epitopes are potential ligands of cell adhesion molecules named selectins which are known to play a role in endothelium-leukocyte interactions. By immunofluorescence, we find the leukocyte selectin (L-selectin) on the spermatozoa head. Preincubation of spermatozoa with an anti-L-selectin monoclonal antibody produces a significant inhibition of zona pellucida tight binding, under hemizona assay conditions. In contrast, preincubation of the zonae pellucidae with anti-sialyl-lewisx or anti-sialyl-lewisa antibodies does not produce a significant inhibition of spermatozoa binding. Western blot analysis of spermatozoa-detergent extracts revealed a band at approximatively 90 kDa with the anti-L-selectin monoclonal antibody. This spermatozoa selectin could play a role in human spermatozoa-zona pellucida binding. Zona-ligands have yet to be precisely defined.

Composition of human zona pellucida as revealed by SDS-PAGE after silver staining.

The macromolecular composition of zona pellucida (ZP) isolated from human oocytes and embryos was characterized by one- and two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (1-D- and 2-D-SDS-PAGE) under reducing conditions after silver staining. ZP specimens obtained after in vitro fertilization were removed from pools of heavily fragmented embryos and inseminated oocytes that failed to fertilize. For unfertilized oocytes, two major bands with an apparent molecular weight of, respectively, 96 and 76-54 kDa were observed after 1-D-SDS-PAGE and silver staining. When ZP were isolated from fragmented embryos, the electrophoretic pattern showed a marked attenuation of the 96-kDa band. Silver-stained 2-D-SDS-PAGE analysis of ZP components from unfertilized oocytes revealed the presence of four protein trains: ZP1 (Mr = 92-80 kDa, pl = 4.9-5.9), ZP2 (Mr = 66-58 kDa, pl = 5.0-6.0), ZP3H (high) (Mr = 72-58 kDa, pl = 3.5-5.1), and ZP3L (low) (Mr = 62-54 kDa, pl = 3.5-5.1). The human ZP3 family (ZP3H and ZP3L) showed marked heterogeneity. Fertilization-associated changes were apparent in the electrophoretic pattern. ZP1 (Mr = 92-86 kDa, pl = 5.0-5.8) displayed a dramatic decrease in intensity, and a new component had migrated to a position similar to that of ZP2. This modification may have been responsible for one aspect of the zona reaction, and could have contributed to a zona block to polysperma.

A fucose-containing epitope potentially involved in gamete interaction on the human zona pellucida.

The oligosaccharide moiety of human, porcine and bovine zonae pellucidae was studied with lectins and monoclonal antibodies specific for tri- or tetra-saccharidic epitopes containing at least one terminal alpha-L-fucose. Animal eggs were collected from follicular aspirates, human eggs were collected from in-vitro fertilization and embryo transfer programmes and pooled into six groups. By direct immunofluorescence, the lectins reactivity was detected for the animal or the human zonae pools in the same way. Reactivity of Aleuria aurantia lectin demonstrated the presence of alpha-L-fucose terminal residues in the zonae from the three species studied. By indirect immunofluorescence, the 2-25 antibody reactivity was detected in every pool of human zonae whereas there was no evidence of any antibody reactivity on animal zonae. Using an anti-Lewis-b blood group antibody (2-25), we observed expression of this antigen as an intrinsic component of the human zona pellucida, independently of patients' Lewis red blood cell phenotypes. Antibody 2-25 inhibited the spermatozoa-zona binding in a hemizona assay, suggesting that this fucose-containing antigen could be part of a sperm-zona receptor.

[The bovine zona pellucida: differences in macromolecular composition between oocytes, pre-treated with A-23187 or not, and embryos]. / La zone pellucide bovine: différences de composition macromoléculaire entre ovocytes, prétraités ou non à l'A 23187, et embryons.

The SDS-PAGE method was used to determine the composition of isolated bovine zona pellucida (ZP). This egg extracellular coat appears to be characterized by 3 major glycoproteins (ZP1, ZP2, ZP3), with apparent molecular weight (MW) of 80-70 kD, 66-63 kD and 60 kD, respectively, as revealed by 1-dimensional SDS-PAGE. After 2-dimensional SDS-PAGE, the zona pellucida electrophoretic pattern indicates a fourth glycoprotein, thus called ZP4. SDS-PAGE analysis of ZP isolated from oocytes and embryos after transit through the genital tract of A23187 pretreated oocytes allowed the description of modifications in glycoproteinic composition.