This protocol was approved by the Institutional Ethics Committee and registered with the regulatory state authority in France (Nantes). Moreover, TILs were expended in a specific genetic and cellular therapy unit, Human Tissue Specimens Tumor-infiltrating lymphocytes (TILs) were provided by Pr. F, 2002. ,
, France) under good manufacturing practice (GMP) conditions. Blood from healthy donors was purchased from the New York Blood Center, Nantes
, 50%]) was purchased from Biomol. The MMP-2 enzyme was inactivated either by heating to 56 C for 45min or by addition of the MMP-2 inhibitor III at 100nM (Calbiochem) for 20 min. rhPEX was purchased from Genway. rhMMP-9 (Calbiochem) was used as a mixture of the proenzyme (50%) and the active protein (50%), p.80
, Lyophilized peptides were reconstituted in DMSO and were used either individually (2 mM) or as a pool (2 mM each). rhGM-CSF was purchased from Immunex. rhIL-12, rhIL-4, rhIL-7, and rhIL-2 were from R&D Systems. The kits used for basophil isolation and IFN-g-secreting cell enrichment
, Antibodies to human Vb chains covering 70% of the repertoire (IOTest Beta Mark; Beckman Coulter) were used according to the manufacturer's intructions. Fluorescein isothiocyanate-conjugated antibody to IL-12p35/IL-12p70 (B-T21) and IFNAR1 (EP899Y) were from Abcam. Phycoerythrine-conjugated antibodies to IL-12p40/IL-12p70 (C8.6), IL-10 (B-T10), CD203c (FR3-16A11) were from Miltenyi Biotec. Blocking antibody to HLA-DQ (SPV-L3; 20 mg/ml) was from NeoMarkers. Blocking antibodies to HLA-DP (B7/21; 20 mg/ml) or HLA-DR (L243; 20 mg/ml), phycoerythrine-conjugated antibodies to phosphorylated (Y701) STAT1 (4a), TNF-a (MAb11), IFN-g (25723.11), perforin (dG9), GranzymeB (GB11), CD40 (5C3), CD80 (L307.4), CD83 (HB15e), CD86 (IT2.2), HLA-DR (TU36), fluorescein isothiocyanate-conjugated antibodies to CD45RA (HI100) and CD45RO (UCHL1), and antibody to CD4 (RPA-T4) were purchased from BD Biosciences PharMingen. Alexa fluor 488-conjugated antibody to IL-17 (eBio64DEC17)
Stimulation and Priming Peripheral blood mononuclear cells (PBMCs) were purified from healthy donor-(HD) or cord blood (CB) donor-derived buffy coats (New York Blood Center) by Ficoll-Paque Plus (GE Healthcare) centrifugation. CD4 + /CD25 -cells were enriched (>90%) by magnetic cell sorting (Miltenyi Biotec) and primed/ stimulated for 12-15 days either with irradiated (35 Gy) autologous CD4 -cells or with autologous mature DCs in IMDM (GIBCO) supplemented with 1 mM HEPES (Life Technologies ,
involving enrichment of IFN-g-secreting cells upon short-term culture and peptide stimulation to generate MMP-2 responsive clones. Although MMP-2-specific cells could be isolated, it was realized that IFN-g secretion was marginal compared with their secretion of IL-4 and TNF-a. IFN-gsecreting cells in response to MMP-2 peptide pool were enriched by cytokine-guided magnetic cell sorting (Miltenyi Biotec) and cloned the following day by limiting dilution in the presence of irradiated allogeneic PBMCs, 1 mg/ml phytohemagglutinin-L (Sigma) and 150 UI/ml rhIL-2. Tumor infiltrating lymphocytes (TILs) were provided by, UI/ml)/penicillin (100 mg/ml) (Sigma) and 5% heat inactivated pooled human serum (PHS; Valley Biomedical) in the presence of rhIL-2 (10 UI/ml) and IL7 (5 ng/ml) (R&D Systems), 2002. ,
, 10 6 cells/10 ml/dish in complete IMDM with 5% PHS. Cells were allowed to adhere for 2 hr at 37 C. Nonadherent cells were removed. The monocyteenriched fraction was supplemented with 100 UI/ml rhGM-CSF and 300 UI/ml rhIL-4 (R&D Systems) on days 0, 2, and 4. Immature DCs were harvested on day 5 and matured using poly(I:C) at 5 mg/ml/10 6 DCs (Amersham). Secretion of IL-12p70, Dendritic Cell Preparation and Activation PBMCs were purified from healthy-(HD) or cord blood-(CB) donors and plated at 40
, 000 cells/100 ml/well), polyclonal T cell populations (100,000 cells/100 ml/well), DCs (50,000 cells/100 ml/well), and basophils (10,000 cells/100 ml/well) was determined by ELISA, Enzyme-Linked Immunosorbent Assay Activation of T cell clones
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