Tumor Infiltrating Lymphocytes (TIL) are easily detectable in different neoplasms, including colon, kidney and lung cancer and melanoma. In the past, the presence of an infiltrate of "inflammatory" cells has been related to a good prognosis, whereas the absence was always considered highly negative. Thus, in 1985, a number of laboratory started the study of these cells, in order to better define their functional characteristics and the potential application in experimental cancer therapy. For this, the population of TIL was compared with Tumor Associated Lymphocytes (TAL, detectable in draining lymph nodes as well as in neoplastic fluids, such as bladder washings and ascitis) and lymphocytes derived from the peripheral blood. In this report, we describe the peculiar differences detectable in the three different districts and define the fine characteristics of lymphocytes involved in cancer control.

Isolation of lymphocytes.

Different methods, including mechanical and enzymatic dissociation, differential centrifugation and positive selection with magnetic immunobeads has been used to isolate the population of lymphocyte within (or associated) to cancer masses. These methods has been extensively reported in the past by our group (1, 2).

Expansion of cancer cells in vitro and in SCID mice.

The expansion of a highly purified population of cancer cells is necessary to analyse the functional and cytolytic properties of lymphocytes derived from an infiltrating population. While in vitro expansion has been extensively described (1), an original method which employs SCID mice has been developed by our group. This in vivo technique allows the expansion of several billions of cancer cells, characterized by a very good proliferative potential in vitro (3).

Bulk expansion and clonal analysis of infiltrating lymphocytes.

Bulk expansion was used in the first experiments in order to define the proliferative capacities of the infiltrating population. For this, a large scale expansion was started as described (1, 2) and the phenotypic and functional features of these cells were accurately reported (1,2,4). More recently, a clonal approach was attempted to better define the antigenic specificities of lymphoid cells detectable in cancer masses (3, 4).

Analysis of the oligoclonality of expanded population of T lymphocytes.

The definition of poli-, oligo- and monoclonaly of a T cell population can be performed by analysing the restriction patterns of the constant regions of b-chain of T cell receptor. This approach has been used by our group in the characterization of in vitro expanded lymphocytes derived from cancer masses and extensively described (1,2,5).

Proliferative capacities

The accurate analysis of the proliferative capabilities of lymphocytes detectable near the tumor site has shown that these cells have an impaired proliferative potential. This is particularly evident in some special districts, such as lymph nodes which are heavily infiltrated by neoplastic cells. Nevertheless, TIL and TAL derived from some non-small-cell lung cancer (NSCLC) and melanoma, allow a massive expansion of effector cells, suitable for further use in protocols of adoptive immunotherapy in humans. On the other hand, lymphocytes derived from bladder washings have an extremely reduced proliferative capability. Furthermore, these lymphocytes can be expanded in vitro only in samples derived from patients treated with BCG in the course of local immunotherapy of superficial bladder cancer (6).

Phenotypic features

Phenotypic analyses of a large number of samples containing either TIL or TAL have shown that the unique population of lymphocytes present near the tumor site belongs to the T cell lineage, whereas both B and NK cells are virtually absent. In addition g/d TCR+ cells are absent (1,2,4). The relative proportion of both CD4+ and CD8+ lymphocyte is extremely variable as well as the percentage of T lymphocytes expressing activation molecules, such as HLA-DR and CD25. The accurate analysis of the phenotypic distribution of different lymphoid subsets derived from the tumor site, when compared with that of peripheral blood obtained from the same patient, allows the definition of the origin of the infiltrating population. For this, the statistical analysis of relative frequencies clearly demonstrates whether lymphoid cells derived from the tumor site were actively concentrated by some local specific mechanism of recruitment, or, on the contrary, were directly derived from blood via microlesions in vessels near the tumor site (6).

Cytolytic properties

Specific cytolytic lymphocytes, able to recognize autologous cancer cells via the CD3/TCR - HLA class I pathway are present, but extremely rare (1,2,4). On the contrary, a very well represented population of NK-like cytolytic T cells is detectable within TIL and TAL (1,2,4).

Restriction pattern analysis of the T cell receptor of expanded population of cancer-related lymphocytes

The oligoclonality of lymphocytes related to cancer has been demonstrated in many different districts. In particular, oligoclonal lymphocytes, are easily detectable in TIL derived from NSCLC (1) and melanoma (2). On the contrary, oligoclonal cells can only be isolated within the in vivo activated population of TAL derived from draining lymph nodes of NSCLC (4). Bladder washings derived lymphocytes have special features. Oligoclonal populations were expanded only in BCG treated patients, since this is the only patient subset which gave rise to proliferating T cell cultures (6).

Clonal analysis of the effector population

Clonal analysis of T cell clones derived from TIL, TAL ad peripheral blood of patient with cancer was performed in the presence of autologous cancer cells expanded in SCID mice. Several different effector mechanisms were studied (3).

A number of trials, based on these studies, started in past years in several Institution, including ours. These studies evaluated the efficacy of both the immunotherapy (7-11) and adoptive immunotherapy (12-15).

These studies evaluated first the feasibility of the approach and further described the clinical efficacy of the use of both immunotherapy and adoptive immunotherapy in advance cancer patients. In particular, adoptive immunotherapy resulted suitable to control the cancer progression in surgically resectable non-small-cell lung cancer and in metastatic lesions of the liver. In these two cases, the population of in vitro activated lymphocytes infused resulted in an efficient control of residual cancer growth (12-14) and significant reduction of tumor masses (15). More importantly, these approach demonstrated that, differently from other approaches, long lasting complete response can be obtained in treated patients, in the absence of significant toxic effects (14).

In conclusion, the analysis of the immunoresponse against cancer cells demonstrated that efficient mechanisms of cancer control can be derived from tumor infiltrating lymphocytes and tumor associated lymphocytes. These in vitro expanded cells, infused into the patient in the course of controlled clinical trials, strongly contribute to the control of cancer progression and, consequently, represent a novel and efficient clinical approach to be included in our therapeutic armamentarium.