title: Phenotypic and functional analysis of lymphocytes infiltrating paediatric tumours, with a characterization of the tumour phenotype.
abstract: Tumour-infiltrating lymphocytes (TIL) of paediatric tumours obtained from 37 lesions of different histotype (12 osteosarcomas, 5 Wilms' tumours, 7 soft-tissue sarcomas, 5 neuroblastomas and 8 miscellaneous) were studied to establish their potential for therapy. Fresh isolated TIL were cultured for the first 2 weeks with low doses of interleukin-2 (IL-2) (20 Cetus U/ml) to select for "tumour-specific" lymphocytes potentially present in the neoplastic lesion, followed by culture with high doses of IL-2 (1000 Cetus U/ml) to achieve TIL expansion. TIL were grown with more than 10-fold expansion in only 9 cases (mean expansion: 58-fold, range 13.5-346). In 17 cases no viable cells were obtained. After 30 days of culture with IL-2 the proliferative ability of TIL declined sharply in the majority of cases and TIL became refractory to any further stimulus, including addition of IL-4, tumour necrosis factor alpha (TNF alpha) or interferon gamma, and activation with OKT3 in solid phase. In 20 out of 37 cases TIL were available for phenotypic and functional analysis. TIL after long-term culture were predominantly CD3+ but 2 cases of osteosarcoma showed a predominance of CD3+TcR gamma/delta cells. The CD4/CD8 ratio was more than 1 in 10 cases, without correlation with tumour histology, site of lesion or TIL growth. The number of CD16+ and CD25+ lymphocytes decreased progressively during culture, the latter concomitantly with a reduction of TIL growth rate. The lytic pattern of TIL against allogenic and autologous tumour (Auto-Tu) cells was variable, but specific lysis of Auto-Tu was seen in only one case (Wilms' tumour) after culture with TNF alpha and irradiated Auto-Tu cells. The immunohistochemical analysis of tumour lesions revealed a limited lymphocyte infiltrate, a low expression of histocompatibility leukocyte antigens (HLA) class I and of the adhesion molecules ICAM1, LFA3, and a significant production of transforming growth factor beta (TGF beta). These data indicate that TIL obtained from paediatric patients are difficult to expand at levels required for immunotherapy and lack a significant number of tumour-specific T lymphocytes. A low expression of immunomodulatory molecules on tumour cells or the production of suppressive factors may prevent activation and expansion of TIL in paediatric tumours.
authors.affiliation: Division of Experimental Oncology D, Istituto Nazionale Tumori, Milan, Italy.
authors.forename: L
authors.initials: L
authors.lastname: Rivoltini
authors.forename: F
authors.initials: F
authors.lastname: Arienti
authors.forename: A
authors.initials: A
authors.lastname: Orazi
authors.forename: G
authors.initials: G
authors.lastname: Cefalo
authors.forename: M
authors.initials: M
authors.lastname: Gasparini
authors.forename: C
authors.initials: C
authors.lastname: Gambacorti-Passerini
authors.forename: F
authors.initials: F
authors.lastname: Fossati-Bellani
authors.forename: G
authors.initials: G
authors.lastname: Parmiani
mesh_terms: D015703:Antigens, CD / Q000032:analysis; D000945:Antigens, Differentiation, T-Lymphocyte / Q000502:physiology; D017252:CD3 Complex; D016516:CD4-CD8 Ratio; D015815:Cell Adhesion Molecules / Q000276:immunology; D002455:Cell Division; D002648:Child; D003602:Cytotoxicity, Immunologic; D006680:HLA Antigens / Q000276:immunology; D006801:Humans; D007371:Interferon-gamma / Q000494:pharmacology; D007376:Interleukin-2 / Q000494:pharmacology; D015847:Interleukin-4 / Q000494:pharmacology; D016131:Lymphocyte Subsets / Q000276:immunology*; D016246:Lymphocytes, Tumor-Infiltrating / Q000276:immunology*; D009369:Neoplasms / Q000276:immunology*; D011948:Receptors, Antigen, T-Cell / Q000502:physiology; D016212:Transforming Growth Factor beta / Q000378:metabolism; D014407:Tumor Cells, Cultured; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D009396:Wilms Tumor / Q000276:immunology
chemical_list: D015703:Antigens, CD; D000945:Antigens, Differentiation, T-Lymphocyte; D017252:CD3 Complex; D015815:Cell Adhesion Molecules; D006680:HLA Antigens; D007376:Interleukin-2; D011948:Receptors, Antigen, T-Cell; D016212:Transforming Growth Factor beta; D014409:Tumor Necrosis Factor-alpha; D015847:Interleukin-4; D007371:Interferon-gamma