Silvia Bruno, Claudia Semino, Gabriella Pietra, Maurizio Meta,
Francesca Cagnoni, Giuliana Cangemi and Giovanni Melioli
Laboratorio di Citometria, Servizio di Patologia Clinica,
Centro di Biotecnologie Avanzate,
Istituto Nazionale per la Ricerca sul Cancro, Genova (Italy)



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).

- Specific cytolytic lymphocytes, recognizing autologous cancer cells via the CD3/TCR - HLA class I pathway. These cells are extremely rare (in general 1/ 107) and detectable only in a small group of patients.

- NK like T cells, expressing the CD8+ phenotype. These cells recognize both autologous and allogeneic cancer cells on the basis of the reduction of HLA class I protective expression

- Th0 "helper" CD4+ cells, which drive a lymphokine cascade in order to improve the specific and non-specific cytolytic capabilities

- CD4+ T cells which recognize and kill via induction of apoptosis in autologous CD95+ cancer cells overexpressing HLA class I following Interferon treatment. This mechanism of specific apoptotic lysis has been demonstrated in Burkitt Lymphoma cells.

Adoptive Immunotherapy studies.

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.

1) Melioli G., Ratto G.B., Guastella M., Meta M., Biassoni R., Semino C., Casartelli G.L., Pasquetti W., Catrullo A and Moretta L. Isolation and in vitro expansion of lymphocytes infiltrating non-small cell lung carcinoma: functional and molecular characterization for their use in adoptive immunotherapy. Eur. J. Cancer, 30, 1, 97-102, 1994

2) Melioli G., Guastella M., Semino C., Meta M., Pietra G., Ponte M., Queirolo P., Sertoli M.R., Martini L. and Reali U.M. Proliferative, phenotypic, functional and molecular characteristics of tumor infiltrating lymphocytes, obtained from unselected patients with malignant melanomas and expanded in vitro in the presence of rIL-2. Melanoma Res., 4, 127-133, 1994

3) Semino C., Cilli M., Ratto GB., Bruno S., Barocci S., Deng W., Ceppa P., Pietra G., Cangemi G., Thuroff E., Melioli G.. Clonal analysis of lymphocytes reacting with non-small-cell lung cancer: functionally heterogeneous effectors efficiently lyse autologous cancer cells (Submitted)

4) Melioli G., Ferrari I., Casartelli G. and Ragni N. Lymphocytes isolated from the peritoneal fluid of patients with advanced ovarian carcinoma differ significantly from autologous peripheral blood lymphocytes. Gynecol. Oncol, 48, 301-307, 1993.

5) Meta M., Ponte M., Guastella M., Semino C., Pietra G., Ratto G.B. and Melioli G. Detection of oligoclonal T lymphocytes in lymph nodes draining from advanced non-small-cell lung cancer. Cancer Immunol Immunother 40, 235-240, 1995

6) Bruno S., Machi' A.M., Semino C., Meta M., Varaldo M., Curotto A., Ferlazzo G., Decensi A. and Melioli G. Phenotypic, functional and molecular analysis of lymphocytes associated with bladder cancer. Cancer Immunol Immunother, 42, 47-54, 1996

7) Rossi G., Felletti R., Balbi B., Sacco O., Cosulich E., Risso A., Melioli G. and Ravazzoni C. Symptomatic Treatment of recurrent malignant pleural effusions with into pleurally administration Corynebacterium parvum. Clinical responses is not associated with evidence of enhancement of local cellular-mediated immunity. Am Rev Resp Dis, 135, 885-890, 1987

8) Melioli G., Baldini E., Mingari M.C., De Maria A., Sertoli M.R., Badellino F., Percivale P.L., Catturich A., Bertoglio S., Civalleri D., Santi L., Moretta L. Phenotypic and functional characteristic of tumor-associated lymphocytes in patients with malignant ascites receiving intraperitoneal infusion recombinant Interleukin-2 (rIL-2). Int J Cancer: 43, 231-234 (1989).

9) Melioli G., Sertoli M.R., Bruzzone M., Nobile M.T., Rosso R., Percivale P.L., Catturich A., Badellino F., Balletto N., Civalleri D., Machi' A., Galatska A., Santi L. and Moretta L. A phase I study of recombinant Interleukin-2 intraperitoneal infusion in patients with neoplastic ascites: toxic effects and immunologic results. Am J Clin Oncol. 14, 3, 231-237, 1991

10) Melioli G., Margarino G., Scala M., Mereu P., Bertoglio S., Schenone G., Barbaresi M., Machì A.M., Santi L., Badellino F. and Moretta L. Perilymphatic injections of recombinant interleukin-2 (rIL-2) partially correct the immunologic defects in patients with advanced head and neck squamous cell carcinoma. Laryngoscope, 102, 572-578,1992

11) Ferlazzo G., Magno C., Lupo G., Rizzo M., Iemmo R., Semino C. and Melioli G. A phase I study of intravesical continuous perfusion of recombinant interleukin-2 (rIL-2) in patients with superficial bladder cancer. Am. J. Clin. Oncol. 18, 2, 100-104, 1995.

12) Ratto G.B., Melioli G., Zino P., Mereu C., Mirabelli S., Fantino G., Ponte M., Minuti P., Verna A., Noceti P., Tassara E. and Rovida S. Immunotherapy using tumor-infiltrating lymphocytes and Interleukin-2 as adjuvant treatment in stage III non-small-cell lung cancer. A pilot study. J. Thor Cardiovasc. Surg., 105, 1212-1217, 1995

13) Melioli G., Ratto G.B., Ponte M., Guastella M., Semino C., Fantino G., Tassara E., Pasquetti W., Mereu C., Merlo F., Reggiardo G., Morasso G., Santi L. and Moretta L. Treatment of Stage IIIb non small cell lung cancer with surgery followed by infusion of Tumor Infiltrating Lymphocytes and Recombinant Interleukin-2: a pilot study. J. Immunother., 19(3), 224-230, 1996

14) Ratto G.B., Zino P., Mirabelli S., Minuti P., Acquilina R., Fantino G., Spessa E., Ponte M., Bruzzi P and Melioli G. A randomized trial of adoptive immunotherapy with tumor infiltrating lymphocytes and interleukin-2 vs. standard therapy in the postoperative treatment of resected non-small-cell lung cancer. Cancer, 78/2, 244-251, 1996

15) Ferlazzo G., Scisca C., Iemmo R., Cavaliere R., Quartarone G., Adamo V. and Melioli G. Intralesional sonographically guided injections of lymphokine activated killer cells and recombinant interleukin-2 for the treatment of liver tumors: a pilot study. J. Immunother. (in press)

Financial support: this work was in part supported by grants of the Associazione Italiana per la Ricerca sul Cancro (AIRC), Consiglio Nazionale per le Ricerche (CNR) and Istituto Superiore di Sanità.

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