FUNCTIONAL AND MOLECULAR CHARACTERIZATION OF TUMOR-INFILTRATING LYMPHOCYTES AND CLONES THEREOF FROM A MAJOR-HISTOCOMPATIBILITY-COMPLEX-NEGATIVE HUMAN TUMOR - NEUROBLASTOMA

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 lymphoc ytes (TIL) has never been carried out. Here we have investigated the i mmunophenotype and the cytotoxic activities of TIL from nine and seven NE patients respectively. Furthermore, the T cell receptor (TcR) vari ability and the patterns of cytokine gene expression of fresh versus r ecombinant (r) interleukin(IL)-2-cultured TIL were studied in four NE cases. The results obtained showed the following: (1) freshly isolated TIL were comprised of a mixture of CD4(+) and CD8(+) T cells partiall y 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 observe d; (2) IL-2-expanded TIL lysed natural-killer(NK)-sensitive and lympho kine-activated-killer (LAK)-sensitive target cell lines; (3) reverse-t ranscriptase/ polymerase-chain-reaction (RT-PCR) experiments showed th at most TcR V beta genes were expressed both in fresh and in cultured TIL, suggesting that such cell populations were polyclonal; (4) interf eron gamma, IL-4, IL-5, tumour necrosis factor (TNF) alpha, IL-8, IL-1 0 mRNA and, to a lesser extent, IL-2 mRNA were expressed by cultured T IL, as assessed by RT-PCR; the corresponding tumour samples consistent ly contained TNF alpha, IL-8 and IL-10 mRNA, whereas IL-2 and IFN gamm a mRNA were faintly expressed in some NE tumors and IL-4 and IL-5 mRNA were never detected. A total of 90 clones were subsequently raised fr om IL-2-expanded TIL from six NE patients; 87/90 clones were of T cell lineage with a CD4(+) or CD8(+) immunophenotype, whereas the 3 remain ing clones were of NK cell origin. Upon triggering of the CD3-TcR comp lex, 64% CD4(+) and 77% CD8(+) T cell clones killed the murine P815 ma stocytoma cell line. Virtually no T cell clone lysed a LAK-sensitive N E cell Line, whereas 15% CD4(+) and 17% CD8(+) clones mediated NK-like activity against the K562 cell line. Finally, the patterns of cytokin e production by CD4(+) clones were roughly consistent with those of a T helper (Th)1 profile and similar to those observed in CD8(+) clones.