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.

Pediatric Cancers Are Infiltrated Predominantly by Macrophages and Contain a Paucity of Dendritic Cells: a Major Nosologic Difference with Adult Tumors

“…Clonal analysis showed that the majority of lymphocytes in neuroblastomas were CD3-positive T cells; in addition, of 90 neuroblastoma-derived clones, only three represented NK cells (11). Infiltrating T cells in pediatric cancers are infrequent, difficult to clone, and functionally ineffective (14). We are not aware of a previous study documenting the presence or absence of dendritic cells among the TALs in pediatric cancers, or whether that finding has any association with prognosis.…”

IFN-γ enhances HOS and U2OS cell lines susceptibility to γδ T cell-mediated killing through the Fas/Fas ligand pathway

“…Rivoltini et al [39] found that although there was a predominance of γδ T cells in TIL from patients with osteosarcoma, TIL obtained from osteosarcoma was difficult to expand at levels required for immunotherapy, owing to a low expression of immunomodulatory molecules on tumor cells or the production of suppressive factors in paediatric tumors. In recent years, γδ T cells derived from human peripheral blood, which can be activated by phospho-antigens and aminobisphosphonates such as Zol to achieve reliably large-scale expansion in vitro, started to emerge as a rewarding target for immunotherapeutic strategies against cancer [14].…”

Phenotypic and functional analysis of lymphocytes infiltrating osteolytic tumors: use as a possible therapeutic approach of osteosarcoma

“…Moreover, their reduced response in the tumor tissue may be due to the suppressive influence under the tumor microenvironment. In this context, TIL have been identified in numerous neoplasia, such as melanoma [5-9], various carcinomas [10-17], myeloma [18], pediatric tumors [19] and sarcomas [20-22] and the therapeutic relevance of these TIL has been also documented in both animal models and clinical trials [23-28]. Unfortunatly, up to date, very few data is available on the phenotypic and functional characterization of TIL isolated from adult bone-associated tumors.…”