title: Functional and molecular characterization of tumour-infiltrating lymphocytes and clones thereof from a major-histocompatibility-complex-negative human tumour: neuroblastoma.
abstract: Neuroblastoma (NB) is a major-histocompatibility-complex(MHC)-negative neuroectodermal tumour that is often infiltrated with lymphocytes. A detailed characterization of NB-associated tumour-infiltrating lymphocytes (TIL) has never been carried out. Here we have investigated the immunophenotype and the cytotoxic activities of TIL from nine and seven NB patients respectively. Furthermore, the T cell receptor (TcR) variability and the patterns of cytokine gene expression of fresh versus recombinant (r) interleukin (IL)-2-cultured TIL were studied in four NB cases. The results obtained showed the following: (1) freshly isolated TIL were comprised of a mixture of CD4+ and CD8+ T cells partially expressing HLA-DR and/or CD25. The CD4/CD8 ratio ranged from 0.5 to 5 in the different cases. Upon culture of TIL with rIL-2, an increased proportion of CD56+ and CD8+ lymphocytes was consistently observed; (2) IL-2-expanded TIL lysed natural-killer(NK)sensitive and lymphokine-activated-killer(LAK)-sensitive target cell lines; (3) reverse-transcriptase/polymerase-chain-reaction (RT-PCR) experiments showed that most TcR V beta genes were expressed both in fresh and in cultured TIL, suggesting that such cell populations were polyclonal; (4) interferon gamma, IL-4, IL-5, tumour necrosis factor (TNF) alpha, IL-8, IL-10 mRNA and, to a lesser extent, IL-2 mRNA were expressed by cultured TIL, as assessed by RT-PCR; the corresponding tumour samples consistently contained TNF alpha, IL-8 and IL-10 mRNA, whereas IL-2 and IFN gamma mRNA were faintly expressed in some NB tumours and IL-4 and IL-5 mRNA were never detected. A total of 90 clones were subsequently raised from IL-2-expanded TIL from six NB patients; 87/90 clones were of T cell lineage with a CD4+ or CD8+ immunophenotype, whereas the 3 remaining clones were of NK cell origin. Upon triggering of the CD3-TcR complex, 64% CD4+ and 77% CD8+ T cell clones killed the murine P815 mastocytoma cell line. Virtually no T cell clone lysed a LAK-sensitive NB cell line whereas 15% CD4+ and 17% CD8+ clones mediated NK-like activity against the K562 cell line. Finally, the patterns of cytokine production by CD4+ clones were roughly consistent with those of a T helper (TH) 1 profile and similar to those observed in CD8+ clones.
authors.affiliation: Laboratory of Oncology, Scientific Institute G. Gaslini, Genoa, Italy.
authors.forename: P
authors.initials: P
authors.lastname: Facchetti
authors.forename: I
authors.initials: I
authors.lastname: Prigione
authors.forename: F
authors.initials: F
authors.lastname: Ghiotto
authors.forename: P
authors.initials: P
authors.lastname: Tasso
authors.forename: A
authors.initials: A
authors.lastname: Garaventa
authors.forename: V
authors.initials: V
authors.lastname: Pistoia
mesh_terms: D001483:Base Sequence; D002648:Child; D002675:Child, Preschool; D002999:Clone Cells; D016207:Cytokines / Q000096:biosynthesis / Q000235:genetics; D003602:Cytotoxicity, Immunologic; D015870:Gene Expression / Q000502:physiology; D006801:Humans; D016130:Immunophenotyping; D007223:Infant; D007376:Interleukin-2 / Q000494:pharmacology; D016246:Lymphocytes, Tumor-Infiltrating / Q000276:immunology / Q000378:metabolism / Q000502:physiology*; D008285:Major Histocompatibility Complex / Q000276:immunology*; D008969:Molecular Sequence Data; D009447:Neuroblastoma / Q000276:immunology* / Q000378:metabolism / Q000473:pathology; D011948:Receptors, Antigen, T-Cell / Q000502:physiology; D011994:Recombinant Proteins / Q000494:pharmacology
chemical_list: D016207:Cytokines; D007376:Interleukin-2; D011948:Receptors, Antigen, T-Cell; D011994:Recombinant Proteins