Restored T-cell activation mechanisms in human tumour-infiltrating lymphocytes from melanomas and colorectal carcinomas after exposure to interleukin-2

Although the immune system has the ability to recognise and kill tumour cells through its lymphoid effectors, as shown by the findings of both in vitro and in vivo studies (Topalian and Rosenberg, 1989), it is well known that cancer patients generally have an inadequate antitumour response. Probably several mechanisms underlie the lack of efficient spontaneous immune reactions observed in cancer patients and are responsible for the poor response rates to immunotherapy (Oliver and Nouri, 1992; Sulitzeanu, 1993; Atkins et al, 1999). For many years, researchers have focused attention on the effects of tumour-induced immunosuppression on T lymphocytes infiltrating, or associated with, solid tumours (Whiteside, 1999). There is evidence from numerous investigations that tumour-infiltrating lymphocytes (TIL) isolated from a wide range of tumours are functionally impaired, as manifested by decreased proliferative responses and decreased ability to mediate cytotoxicity (Whiteside, 1992; Miescher et al, 1998). Abnormalities in signal transduction molecules associated with T-cell receptor (TCR) function have been identified as one of the main mechanisms by which the tumour microenvironment affects immune cells. In particular, reduced expression of TCR-associated ζ chain is one of the more consistent findings. Initially discovered in TIL of mice bearing colon carcinoma MCA-38 cells (Mizoguchi et al, 1992), abnormalities in ζ chain expression were then isolated in TIL of patients with various types of cancer, including colorectal carcinoma, renal cell carcinoma and metastatic melanoma (Finke et al, 1993; Nakagomi et al, 1993; Rabinowich et al, 1996). It has been shown that decreased or absent expression of the ζ chain results in signalling defects in TIL or tumour-associated lymphocytes (TAL), such as altered expression of the tyrosine kinase p56lck and a decreased ability to mobilise intracellular Ca2+ in response to activation signals (Mizoguchi et al, 1992; Finke et al, 1993; Nakagomi et al, 1993; Lai et al, 1996; Rabinowich et al, 1996). Altered ζ chain is also associated with impaired cytokine production, as shown by decreased mRNA and protein levels of interleukin 2 (IL-2) and interferon-γ (Rabinowich et al, 1996) in T cells. It has been suggested that ζ chain degradation in TIL and TAL is caused by tumour-induced activation of intracellular peptidases in T cells, and that this process may ultimately lead to a caspase-dependent apoptotic cascade in activated lymphocytes (Rabinowich et al, 1998; Whiteside, 1999). The biological significance of ζ chain degradation has recently been confirmed by the results of a retrospective study conducted in patients with oral carcinoma, where low or absent ζ chain expression in TIL was found to predict poor survival, independently of other factors (Reichert et al, 1998). Another mechanism that may be playing an important role in mediating tumour-induced immunosuppression is the Fas/FasL pathway (Whiteside and Rabinowich, 1998). Recent studies have shown that tumour cells can assume characteristics similar to those of immune-privileged tissues such as the low expression or absence of surface Fas receptor (Fas) and the expression of Fas ligand (FasL) (Hahne et al, 1996; Nagata, 1996; O'Connell et al, 2001). In addition to other mechanisms (i.e. inhibitory cytokines), tumour FasL surface expression may contribute to T-cell damage and apoptosis (Whiteside and Rabinowich, 1998). However, this hypothesis, formulated on the basis of several authors' independent observations (Whiteside, 1999; Restifo, 2000), has yet to be confirmed. The finding that tumour-induced degradation of signalling molecules can be ‘reverted’, and proliferative and cytotoxic activities of TIL restored by culture with exogenous cytokines (especially IL-2), at least in vitro, has important potential therapeutic implications (Salvadori et al, 1993; Rabinowich et al, 1996; Zier et al, 1996), although data about the in vivo effects of cytokines administration on T-cell signalling molecules of cancer patients are still scarce (Farace et al, 1994; Rabinowich et al, 1996). In the present study, we carried out a multiparameter analysis of TIL used for therapeutic reinfusion in patients with advanced melanoma and colorectal cancer who had previously undergone metastasectomy. In particular, we evaluated TCR ζ and ɛ chains, p56lck, Bcl-2 and Bax expression, FasL and Fas expression on the surface of TIL from surgically obtained tumour samples, and reassessed the same parameters, with the addition of perforin, after TIL were coincubated with IL-2. In parallel, apoptosis markers in cancer cells of surgically obtained specimens were also examined. The aims of the study were to provide an overview of the parameters involved in the mechanisms of immunosuppression in human TIL from melanomas and colorectal carcinomas cultivated for therapeutic purposes and to assess the in vitro effects of exogenous IL-2 on such parameters.