GASTROENTEROLOGY 1990;98:531-534
EDITORIAL
Tumor-Infiltrating Lymphocytes: New Therapy, New Hones A
cytes (TIL) and their infusion into cancer patients The contribution of immunology to clinical medicine concomitantly with IL2 (8). Following the same trend continues with increasing speed in an ever expanding set by LAK cells, TIL have also quickly moved from fashion. In the last few decades immunosuppressive the laboratory bench to the bedside, and clinical trials drugs, bone marrow transplant, monoclonal antibodusing TIL have already been reported (9,lO). ies, cytokines, and activated lymphocytes have been What are TIL? Solid tumors are known to harbor a incorporated into the treatment of allergic, autoimmune, immunodeficiency, and neoplastic diseases (1,Z). heterogeneous population of immune cells, including helper, suppressor, and cytotoxic T cells, B cells, In this group of heterogeneous medical problems, the natural killer (NK) cells, and macrophages, which are one where progress has been relatively slow is by far believed to represent a marker of the host response to the immune therapy of solid tumors. Earlier apneoplastic growth (11). Although the exact role of these proaches to cancer, such as nonspecific enhancement infiltrating mononuclear cells is not fully understood, of immune reactivity by the administration of BCG, the classical although empirical observation that an met only with very limited success. However, an abundant lymphocytic infiltration is associated with a improved understanding of the type and function of less aggressive tumor behavior has long supported the the various subpopulations of immune cells, their complex interactions, and the ample repertoire of notion that the infiltrating cells, particularly T cells, may be a contributing mechanism to the host’s imsecretory products (lymphokines, monokines, and cymune defense. In agreement with this hypothesis is tokines), have drastically changed this situation. Following a series of novel and imaginative investithe original report by Rosenberg et al. that TIL exgations conducted at the National Cancer Institute panded in IL2 are 50-100 times more potent than LAK (NCI), the use of “adoptive immunotherapy” for maligcells in destroying lung and hepatic metastases in mice nant tumors has almost overnight become a realistic with sarcoma or colon adenocarcinoma (12). therapeutic option (3). Initially, lymphokine-activated An impressive amount of data on the isolation, killer (LAK) cell activity was described in animals and characterization, and antitumor potential of TIL in a humans. In this phenomenon, mononuclear cells from variety of human cancers has accumulated rapidly. various body compartments are transformed into poHuman TIL from different cancers appear to share tent although nonspecific cytotoxic cells able to kill some common characteristics, such as a weak refresh autologous or allogeneic tumor cells after in vitro sponse to mitogens (131, a low proliferative frequency stimulation with the lymphokine interleukin 2 (IL2) as compared with peripheral blood T cells (131, and (45). With amazing speed and daring, this experimenmediation of cytotoxic activity mostly by Leul9+ tal observation was translated into practical applica(nonmajor histocompatibility complex [MHC]-retion, resulting in clinical trials where patients with stricted NK cell] cells, that are both CD3 + (pan T cell] metastatic cancer unresponsive to conventional therand CD3-(14,151. The cytolytic potential of TIL is apy received infusions of large numbers of in vitro usually low or not expressed when cells are freshly expanded autologous LAK cells and substantial doses isolated, requiring induction by IL2 (16). Upon expoof recombinant human IL2 (6,7]. Variable results were sure to this lymphokine, TIL become active killer cells, observed, ranging from lack of response to remarkable and, in addition to destroying transformed cell lines shrinking of the tumor mass, that certainly justify more and allogeneic tumors, they generally display cytotoxinvestigation into this original form of cancer treaticity against autologous tumor cells (17-20). In rement. sponse to continuous stimulation with IL2 (3 to 6 wk), Even before assessment of the LAK phenomenon the number of TIL expands dramatically, usually and its cancer therapeutic potential have been fully more than peripheral blood mononuclear cells (PBMC], explored and understood, the same group of investigaand, on a per culture basis, frequently achieve higher tors at the NC1 has now described another type of antitumor activity than PBMC (21). These observations antineoplastic effector cell and an alternate form of suggest that TIL represent a population of cytolytic adoptive immunotherapy: tumor-infiltrating lymphoprecursor cells selectively accumulated at a tumor site.
532
EDITORIAL
The apparent lack of specificity against autologous tumor cells observed in most TIL cultures is not a constant finding. In contrast to other tumors, TIL from malignant melanomas often exhibit the highest level of lysis against autologous tumor cells (22,231. In this system, CD8+ (suppressor/cytotoxic cell) T cells are the predominant population before and after expansion in IL2, and are primarily responsible for autologous antitumor activity in vitro. Pretreatment of fresh melanoma cells with monoclonal antibodies to MHC antigens inhibits TIL-specific killing, strongly supporting the immunologically restricted nature of this phenomenon (24). Thus, malignant melanomas appear to contain precursors of classical cytolytic T cells (CTL) sensitized in vivo to autologous cancer cell antigens. This extremely important finding suggests that CTL may exist in other tumors, even though their specific lytic activity may not be apparent because of a dilutional effect of other nonspecific TIL populations (25). If true, cloning and expansion of CTL could offer a highly specific and enriched arsenal of TIL for almost any form of cancer. Much remains to be done before the biology of TIL is fully understood. One of the major problems with currently used TIL immunotherapy is the heterogeneity of the IL2-expanded cells being infused. In most studies TIL are grown in high doses of IL2 (around 1,000 U/ml], resulting in heterogeneous populations of lymphocytes, composed of several subsets that vary in number and phenotype within one culture over time and from one patient to another (9,lO). If the antitumor activity is restricted to a particular TIL subset(s) for a given type of cancer, then each patient is likely to receive different numbers of the appropriate effector cells. Such disparity makes it quite difficult to interpret clinical results among patients with the same form of cancer and compare results among various types of malignancies. Furthermore, it probably contributes to the great variability observed in the response of different patients to TIL infusions. This makes the identification of those TIL subsets with the most pronounced antitumor activity an absolute priority. Another unanswered question regards the role of those TIL populations that grow in IL2 but apparently do not mediate killing of autologous tumor cells. Ongoing research in our laboratory shows that most of the lytic activity mediated by TIL from renal cell carcinoma is due to a numerically minor population of Leul9 + cells and not by CD3 + Leul9 - cells that constitute the largest population of TIL (unpublished observation). These results are in agreement with others (13,14) and raise the possibility that CD3 + LeulS-cells do not participate in the immune response against renal cancer but represent a random accumulation of peripherally derived lymphocytes. Alternatively, CD3+Leul9-cells may be part of the local immune
GASTROENTEROLOGY
Vol. 98, No. 2
response, but their function has yet to be defined. This hypothesis is supported by the observation that TIL are frequently UCHLl+ and CDw29 +, indicating cell activation associated with T cells previously primed to antigens (261. Whether these activated TIL represent memory cells induced by tumor antigens is under investigation. In addition, CD3 + Leul9-cells may still have antitumor activity even though this is not demonstrable in a routine 4-h cytotoxicity assay. Many CD3 +LeulS-TIL are cytolytic in a 18-h assay and most are able to destroy monolayers of autologous cancer cells in 72 h (unpublished observation]. Finally, recent work by Stotter et al. has demonstrated that pretreatment of tumor cells with some cytokines, such as tumor necrosis factor (Yand interferon y, greatly enhances the susceptibility of autologous cancer cells to lysis by TIL but not LAK cells (27). A substantial amount of basic work has yet to be performed to define the optimal experimental conditions allowing TIL to express their maximal cytotoxic potential. Only then will the real therapeutic value of TIL be properly appraised. Few patients with colon cancer have received adoptive immunotherapy with LAK cells, invariably with poor results (6,7), and none has been reported for TIL. A study by Trudel et al. found that LAK cells derived from the intestinal mucosa are totally devoid of cytolytic activity against autologous and allogeneic colon cancer cells (28). Therefore, the study by Yoo et al describing the functional and phenotypic characteristics of TIL from human colon carcinomas in this issue of GASTROENTEROLOGY is particularly important (29). This form of cancer is one of the most common and deadly. Depending on stage, its response to surgery, chemotherapy, and irradiation is limited or negligible. New and innovative approaches are needed, making the investigation of TIL in colon cancer challenging and exciting. According to Yoo et al, freshly isolated colon cancer TIL are weakly cytotoxic against autologous tumor cells, proliferate better than PBMC, and display greater total cytotoxic activity per culture than PBMC. The predominant proliferating population expresses the CD3 + LeulSphenotype, although CD3 + Leul9 + and CD3 - Leul9 + lymphocytes also grow in long-term culture. As far as the cytolytic activity of colon cancer TIL, this is greater against autologous than allogeneic tumor cells, but at a low level. On a per cell basis TIL are less cytotoxic than PBMC, although on a per culture basis TIL display higher killing of autologous tumor than PBMC. These results are certainly not terribly encouraging, and cast serious doubts on the value of future clinical trials with colon cancer TIL. This apparently disappointing picture, however, must be carefully considered in view of 2 peculiar aspects. The first is the special environment where
February 1990
colonic adenocarcinoma arises. The intestinal mucosa is rich in diffuse lymphoid tissue, including intraepithelial lymphocytes (IEL) and lamina propria mononuclear cells, which originate in organized lymphoid tissue (Peyer’s patches) and have unique circulatory and homing patterns. In addition, the intestinal mucosa lymphoid cells are almost devoid of LeulS+ cells, the most common type of effector TIL (30). Thus, an initial question is: Do colon cancer TIL represent a population locally derived or do they originate from other distant compartments? A recent study by Jarry et al. lends support to the first possibility (31). Based on the expression of an antigen defined by the monoclonal antibody HML-1, which recognizes 95% of human IEL and 40% of lamina propria T cells (321, most colon cancer TIL are HML-l+, as determined by immunohistochemistry. Therefore, given the phenotypic and cytotoxic characteristics of human intestinal mucosa mononuclear cells, the weak cytolytic potential of colon cancer TIL is not surprising (33). The second question is: Are there any locally derived antitumor CTL in the midst of the bulk of IL2expanded colon cancer TIL? According to a recent study, the intestinal mucosa is endowed with CTL precursors (34), and, at least theoretically, the potential for developing CTL to local tumor antigens should be present. It is far too early to hypothesize what the real value of TIL might be in the therapy of human colon cancer. Even assuming that TIL became of common clinical use in the future, it is unlikely that they alone will be the single ideal anticancer weapon. Better clinical results will probably be achieved by associating the immunotherapeutic benefits of TIL to complementary forms of treatment, such as cytokines, chemotherapy (35), administration of anti-CD3 antibodies (361, hormone-conjugated anti-CD3 antibodies (37). antibodytargeting (38), etc. Perhaps the goal of obtaining “designer lymphocytes” might be closer than we think (391, and custom-made adoptive immunotherapy might be more effective than we expect. CLAUDIO FIOCCHI. M.D. JAMES H. FINKE, Ph.D.
Department of Gastroenterology and Research Institute Cleveland Clinic Foundation Cleveland, Ohio
References Fahey JL, Sarna G, Gale RP, Seeger R. Immune intervention in disease. Ann Intern Med 1987;106:257-274. Rosenberg SA. Lotze MT, Mule’ J. New approaches to the immunotherapy of cancer using interleukin-2. Ann Intern Med 1988;108:853-864. Rosenberg SA. Lymphokine-activated killer cells: A new approach to immunotherapy of cancer. JNCI 1985;75:595-603. Mazumder A, Rosenberg SA. Successful immunotherapy of
EDITORIAL
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natural-killer-resistant established pulmonary melanoma metastases by the intravenous adoptive transfer of syngeneic lymphocytes activated in vitro by interleukin 2. J Exp Med 1984;159:495-507. 5. Grimm EA, Mazumder A, Zhang HZ, Rosenberg SA. The lymphokine-activated killer cell phenomenon: Lysis of NKresistant fresh solid tumor cells by IL2 activated autologous peripheral human blood lymphocytes. J Exp Med 1982;155:18231841. 6. Rosenberg SA, Lotze MT, Muul LM, Chang AE, Avis FP, Leitman S, Linehan WM, Robertson CN, Lee RE, Rubin JT, Seipp CA, Simpson CG, White DE. A progress report on the treatment of 157 patients with advanced cancer using lymphokine-activated killer cells and interleukin-2 or high doses of interleukin-2. N Engl J Med 1987;316:889-897, 7. West WH, Tauer KW, Yanelli JR, Marshall GD, Orr DW, Thurman GB, Oldham RK. Constant-infusion recombinant interleukin-2 in adoptive immunotherapy of advanced cancer. N Engl J Med 316;1987:898-905, 8. Rosenberg SA. The development of new immunotherapies for the treatment of cancer using interleukin-2. Ann Surg 1988;208: 121-135. 9. Rosenberg SA, Packard BS, Aebersold PM, Solomon D, Topalian SL, Toy ST, Simon P, Lotze MT, Yang JC, Seipp CA, Simpson C, Carter C, Bock S, Schwartzentruber D, Wei JP, White DE. Use of tumor-infiltrating lymphocytes and interleukin2 in the immunotherapy of patients with metastatic melanoma. N Engl J Med 1988;319:1676-1680. 10. Kradin RL, Kurnick JT Lazarus DS, Preffer FI, Dubinnet SM, Pinto CE, Gifford J, Davidson E, Grove B, Callahan RJ. Strauss HW. Tumor-infiltrating lymphocytes and interleukin-2 in treatment of advanced cancer. Lancet 1989;1:577-580. 11. Vose BM, Moore M. Human tumor-infiltrating lymphocytes: a marker of host response. Semin Hematol1985;22:27-40. 12. Rosenberg SA, Spiess P, Lafreniere R. A new approach to the adoptive immunotherapy of cancer with tumor-infiltrating lymphocytes.Science 1986;233:1318-1321. 13. Miescher S, Whiteside TL, Carrel S, von Fliedner V. Functional properties of tumor-infiltrating and blood lymphocytes in patients with solid tumors: effects of tumor cells and their supernatants on proliferative responses of lymphocytes. J Immunol 1986;136:1899-1907. 14. Whiteside TL. Heo DS, Tagaki S, Johnson JT, Iwatsuki S, Herberman RB. Cytolytic tumor effector cells in long-term cultures of human tumor-infiltrating lymphocytes in recombinant interleukin 2. Cancer Immunol Immunother 1988;26:1-10. 15. Heo DS, Whiteside TL, Kanbour A, Herberman RB. Lymphocytes infiltrating human ovarian tumors. I. Role of Leu 19 (NKHl)-positive recombinant IL-Z-activated cultures of lymphocytes infiltrating human ovarian tumors. J Immunol 1988;140: 4042-4049. 16. Miescher S, Whiteside TL, Moretta L, von Flieder V. Clonal and frequency analysis of tumor-infiltrating T lymphocytes from human solid tumors. J Immunol1987;138:4004-4011. 17. Kurnick JT, Kradin RL, Blumberg R, Schneeberger EE, Boyle LA. Functional characterization of T lymphocytes propagated from human lung carcinomas. Clin Immunol Immunopathol 1986:38:367-380. 18. Belldegrum A, Muul LM, Rosenberg SA. Interleukin 2 expanded tumor-infiltrating lymphocytes in human renal cell cancer: isolation, characterization and antitumor activity, Cancer Res 1988;48:206-214. 19. Finke JH, Tubbs R, Connelly B, Pontes E. Montie J. Tumorinfiltrating lymphocytes in patients with renal cell carcinoma, Ann NY Acad Sci 1988;532:387-394. 20. Tagaki S, Chen K, Schwarz R, Iwatsuki S, Herberman RB, Whiteside TL. Functional and phenotypic analysis of tumor-
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infiltrating lymphocytes isolated from primary and metastatic liver tumors and cultured in recombinant interleukin 2. Cancer 1989;83:102-111. 21.Heo DS, Whiteside TL, Johnson JT, Chen K, Barnes EL, Herberman RB. Long-term interleukin 2-dependent growth and cytotoxic activity of tumor-infiltrating lymphocytes from human squamous cell carcinomas of the head and neck. Cancer Res 1987;47:8353-8382. 22. Itoh K, Tilden AB, Balch CM. Interleukin 2 activation of cytotoxic T-lymphocytes infiltrating into human metastatic melanoma. Cancer Res 1988;48:3011-3017. 23. Muul LM, Spiess PJ, Director EP, Rosenberg SA. Identification of cytolytic immune responses against autologous tumor in humans bearing malignant melanoma. J Immunol1987;138:989995. 24. Itoh K, Platsoukas CD, Balch CM. Autologous tumor-specific cytotoxic T lymphocytes in the infiltrate of human metastastatic melanoma. Activation by interleukin 2 and autologous tumor cells, and involvement of the T cell receptor. J Exp Med 1988;188:1419-1441. 25. Topalian SL, Solomon D, Rosenberg SA. Tumor-specific cytolysis by lymphocytes infiltrating human melanomas. J Immunol 1989;142:3714-3725. 28. Sanders ME, Makgoba MW, Shaw S. Human naive and memory T cells: reinterpretation of helper-inducer and suppressorinducer subsets. Immunol Today 1988;9:195-199. 27. Stotter H, Wiebke EA, Tomita S, Belldegrun A, Topalian S. Rosenberg SA, Lotze MT. Cytokines alter target cell susceptibility to lysis. II. Evaluation of tumor infiltrating lymphocytes. J Immunol1989;142:1787-1773. 28. Trudel JL, Youngman KR, West GA, Fazio VW, Fiocchi C. Lymphokine-activated killer (LAK) cells from human intestinal mucosa: Cytotoxic activity against tumor cell lines and modified self but not autologous and allogeneic cancer cells. J Surg Res 1988;44:445-454, 29. Yoo Y-K, Heo DS. Hata K, Van Thiel DH, Whiteside TL. Tumorinfiltrating lymphocytes from human colon carcinomas. Functional and phenotypic characteristics after long-term culture in recombinant interleukin 2. Gastroenterology 1989;2:259-268.
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30. Fiocchi C, Youngman KR, Yen-Lieberman B, Tubbs RR. Modulation of intestinal immune reactivity by interleukin 2. Phenotypic and functional analysis of lymphokine-activated killer cells from human intestinal mucosa. Dig Dis Sci 1988;33:13051315. 31. Jarry A, Cerf-Bensussan N, Brousse N, Guy-Grand D, Muzeau F, Potet F. Same peculiar subset of HML-l+ lymphocytes present within normal intestinal epithelium is associated with tumoral epithelium of gastrointestinal carcinomas. Gut 1988;29: 1632-1638. 32. Cerf-Bensussan N, Jarry A, Brousse N, Lisowska-Grospierre B, Guy-Grand D, Griscelli C. A monoclonal antibody (HML-1) defining a novel membrane molecule present on human intestinal lymphocytes. Eur J Immunol1987;17:1279-1285. 33. James SP, Strober W. Cytotoxic lymphocytes and intestinal disease. Gastroenterology 1986;90:235-240. 34. Shanahan F, Deem R, Nayersina R, Leman B, Targan S. Human mucosal T-cell cytotoxicity. Gastroenterology 1988;94:960-967. 35. Hanna MG, Key ME. Immunotherapy of metastases enhances subsequent immunotherapy. Science 1982;217:367-369. 36. Ellenhorn JD, Hirsch R. Schreiber H, Bluestone JA. In vivo administration of anti-CD3 prevents malignant progressor tumor growth. Science 1988;242:569-571. 37. Liu MA, Nussbaum SR, Eisen HN. Hormone conjugated with antibody to CD3 mediates cytotoxic T cell lysis of human melanoma cells. Science 1988;239:395-398. 38. Greiner JW, Guadagni F, Noguchi P, Pestka S, Colcher D, Fisher PB, Schlom J. Recombinant interferon enhances antibodytargeting of carcinoma lesions in vivo. Science 1987;235:895898. 39. Culliton BJ. Fighting cancer with designer cells. Science 1989244: 1430-1433.
Address requests for reprints to Claudio Fiocchi. M.D., Research Institute, Cleveland Clinic, 9500 Euclid Avenue, Cleveland, Ohio 44195. 0 1990 by the American Gastroenterological Association
EDITORIAL
Tumor-Infiltrating Lymphocytes: New Therapy, New Hones A
cytes (TIL) and their infusion into cancer patients The contribution of immunology to clinical medicine concomitantly with IL2 (8). Following the same trend continues with increasing speed in an ever expanding set by LAK cells, TIL have also quickly moved from fashion. In the last few decades immunosuppressive the laboratory bench to the bedside, and clinical trials drugs, bone marrow transplant, monoclonal antibodusing TIL have already been reported (9,lO). ies, cytokines, and activated lymphocytes have been What are TIL? Solid tumors are known to harbor a incorporated into the treatment of allergic, autoimmune, immunodeficiency, and neoplastic diseases (1,Z). heterogeneous population of immune cells, including helper, suppressor, and cytotoxic T cells, B cells, In this group of heterogeneous medical problems, the natural killer (NK) cells, and macrophages, which are one where progress has been relatively slow is by far believed to represent a marker of the host response to the immune therapy of solid tumors. Earlier apneoplastic growth (11). Although the exact role of these proaches to cancer, such as nonspecific enhancement infiltrating mononuclear cells is not fully understood, of immune reactivity by the administration of BCG, the classical although empirical observation that an met only with very limited success. However, an abundant lymphocytic infiltration is associated with a improved understanding of the type and function of less aggressive tumor behavior has long supported the the various subpopulations of immune cells, their complex interactions, and the ample repertoire of notion that the infiltrating cells, particularly T cells, may be a contributing mechanism to the host’s imsecretory products (lymphokines, monokines, and cymune defense. In agreement with this hypothesis is tokines), have drastically changed this situation. Following a series of novel and imaginative investithe original report by Rosenberg et al. that TIL exgations conducted at the National Cancer Institute panded in IL2 are 50-100 times more potent than LAK (NCI), the use of “adoptive immunotherapy” for maligcells in destroying lung and hepatic metastases in mice nant tumors has almost overnight become a realistic with sarcoma or colon adenocarcinoma (12). therapeutic option (3). Initially, lymphokine-activated An impressive amount of data on the isolation, killer (LAK) cell activity was described in animals and characterization, and antitumor potential of TIL in a humans. In this phenomenon, mononuclear cells from variety of human cancers has accumulated rapidly. various body compartments are transformed into poHuman TIL from different cancers appear to share tent although nonspecific cytotoxic cells able to kill some common characteristics, such as a weak refresh autologous or allogeneic tumor cells after in vitro sponse to mitogens (131, a low proliferative frequency stimulation with the lymphokine interleukin 2 (IL2) as compared with peripheral blood T cells (131, and (45). With amazing speed and daring, this experimenmediation of cytotoxic activity mostly by Leul9+ tal observation was translated into practical applica(nonmajor histocompatibility complex [MHC]-retion, resulting in clinical trials where patients with stricted NK cell] cells, that are both CD3 + (pan T cell] metastatic cancer unresponsive to conventional therand CD3-(14,151. The cytolytic potential of TIL is apy received infusions of large numbers of in vitro usually low or not expressed when cells are freshly expanded autologous LAK cells and substantial doses isolated, requiring induction by IL2 (16). Upon expoof recombinant human IL2 (6,7]. Variable results were sure to this lymphokine, TIL become active killer cells, observed, ranging from lack of response to remarkable and, in addition to destroying transformed cell lines shrinking of the tumor mass, that certainly justify more and allogeneic tumors, they generally display cytotoxinvestigation into this original form of cancer treaticity against autologous tumor cells (17-20). In rement. sponse to continuous stimulation with IL2 (3 to 6 wk), Even before assessment of the LAK phenomenon the number of TIL expands dramatically, usually and its cancer therapeutic potential have been fully more than peripheral blood mononuclear cells (PBMC], explored and understood, the same group of investigaand, on a per culture basis, frequently achieve higher tors at the NC1 has now described another type of antitumor activity than PBMC (21). These observations antineoplastic effector cell and an alternate form of suggest that TIL represent a population of cytolytic adoptive immunotherapy: tumor-infiltrating lymphoprecursor cells selectively accumulated at a tumor site.
532
EDITORIAL
The apparent lack of specificity against autologous tumor cells observed in most TIL cultures is not a constant finding. In contrast to other tumors, TIL from malignant melanomas often exhibit the highest level of lysis against autologous tumor cells (22,231. In this system, CD8+ (suppressor/cytotoxic cell) T cells are the predominant population before and after expansion in IL2, and are primarily responsible for autologous antitumor activity in vitro. Pretreatment of fresh melanoma cells with monoclonal antibodies to MHC antigens inhibits TIL-specific killing, strongly supporting the immunologically restricted nature of this phenomenon (24). Thus, malignant melanomas appear to contain precursors of classical cytolytic T cells (CTL) sensitized in vivo to autologous cancer cell antigens. This extremely important finding suggests that CTL may exist in other tumors, even though their specific lytic activity may not be apparent because of a dilutional effect of other nonspecific TIL populations (25). If true, cloning and expansion of CTL could offer a highly specific and enriched arsenal of TIL for almost any form of cancer. Much remains to be done before the biology of TIL is fully understood. One of the major problems with currently used TIL immunotherapy is the heterogeneity of the IL2-expanded cells being infused. In most studies TIL are grown in high doses of IL2 (around 1,000 U/ml], resulting in heterogeneous populations of lymphocytes, composed of several subsets that vary in number and phenotype within one culture over time and from one patient to another (9,lO). If the antitumor activity is restricted to a particular TIL subset(s) for a given type of cancer, then each patient is likely to receive different numbers of the appropriate effector cells. Such disparity makes it quite difficult to interpret clinical results among patients with the same form of cancer and compare results among various types of malignancies. Furthermore, it probably contributes to the great variability observed in the response of different patients to TIL infusions. This makes the identification of those TIL subsets with the most pronounced antitumor activity an absolute priority. Another unanswered question regards the role of those TIL populations that grow in IL2 but apparently do not mediate killing of autologous tumor cells. Ongoing research in our laboratory shows that most of the lytic activity mediated by TIL from renal cell carcinoma is due to a numerically minor population of Leul9 + cells and not by CD3 + Leul9 - cells that constitute the largest population of TIL (unpublished observation). These results are in agreement with others (13,14) and raise the possibility that CD3 + LeulS-cells do not participate in the immune response against renal cancer but represent a random accumulation of peripherally derived lymphocytes. Alternatively, CD3+Leul9-cells may be part of the local immune
GASTROENTEROLOGY
Vol. 98, No. 2
response, but their function has yet to be defined. This hypothesis is supported by the observation that TIL are frequently UCHLl+ and CDw29 +, indicating cell activation associated with T cells previously primed to antigens (261. Whether these activated TIL represent memory cells induced by tumor antigens is under investigation. In addition, CD3 + Leul9-cells may still have antitumor activity even though this is not demonstrable in a routine 4-h cytotoxicity assay. Many CD3 +LeulS-TIL are cytolytic in a 18-h assay and most are able to destroy monolayers of autologous cancer cells in 72 h (unpublished observation]. Finally, recent work by Stotter et al. has demonstrated that pretreatment of tumor cells with some cytokines, such as tumor necrosis factor (Yand interferon y, greatly enhances the susceptibility of autologous cancer cells to lysis by TIL but not LAK cells (27). A substantial amount of basic work has yet to be performed to define the optimal experimental conditions allowing TIL to express their maximal cytotoxic potential. Only then will the real therapeutic value of TIL be properly appraised. Few patients with colon cancer have received adoptive immunotherapy with LAK cells, invariably with poor results (6,7), and none has been reported for TIL. A study by Trudel et al. found that LAK cells derived from the intestinal mucosa are totally devoid of cytolytic activity against autologous and allogeneic colon cancer cells (28). Therefore, the study by Yoo et al describing the functional and phenotypic characteristics of TIL from human colon carcinomas in this issue of GASTROENTEROLOGY is particularly important (29). This form of cancer is one of the most common and deadly. Depending on stage, its response to surgery, chemotherapy, and irradiation is limited or negligible. New and innovative approaches are needed, making the investigation of TIL in colon cancer challenging and exciting. According to Yoo et al, freshly isolated colon cancer TIL are weakly cytotoxic against autologous tumor cells, proliferate better than PBMC, and display greater total cytotoxic activity per culture than PBMC. The predominant proliferating population expresses the CD3 + LeulSphenotype, although CD3 + Leul9 + and CD3 - Leul9 + lymphocytes also grow in long-term culture. As far as the cytolytic activity of colon cancer TIL, this is greater against autologous than allogeneic tumor cells, but at a low level. On a per cell basis TIL are less cytotoxic than PBMC, although on a per culture basis TIL display higher killing of autologous tumor than PBMC. These results are certainly not terribly encouraging, and cast serious doubts on the value of future clinical trials with colon cancer TIL. This apparently disappointing picture, however, must be carefully considered in view of 2 peculiar aspects. The first is the special environment where
February 1990
colonic adenocarcinoma arises. The intestinal mucosa is rich in diffuse lymphoid tissue, including intraepithelial lymphocytes (IEL) and lamina propria mononuclear cells, which originate in organized lymphoid tissue (Peyer’s patches) and have unique circulatory and homing patterns. In addition, the intestinal mucosa lymphoid cells are almost devoid of LeulS+ cells, the most common type of effector TIL (30). Thus, an initial question is: Do colon cancer TIL represent a population locally derived or do they originate from other distant compartments? A recent study by Jarry et al. lends support to the first possibility (31). Based on the expression of an antigen defined by the monoclonal antibody HML-1, which recognizes 95% of human IEL and 40% of lamina propria T cells (321, most colon cancer TIL are HML-l+, as determined by immunohistochemistry. Therefore, given the phenotypic and cytotoxic characteristics of human intestinal mucosa mononuclear cells, the weak cytolytic potential of colon cancer TIL is not surprising (33). The second question is: Are there any locally derived antitumor CTL in the midst of the bulk of IL2expanded colon cancer TIL? According to a recent study, the intestinal mucosa is endowed with CTL precursors (34), and, at least theoretically, the potential for developing CTL to local tumor antigens should be present. It is far too early to hypothesize what the real value of TIL might be in the therapy of human colon cancer. Even assuming that TIL became of common clinical use in the future, it is unlikely that they alone will be the single ideal anticancer weapon. Better clinical results will probably be achieved by associating the immunotherapeutic benefits of TIL to complementary forms of treatment, such as cytokines, chemotherapy (35), administration of anti-CD3 antibodies (361, hormone-conjugated anti-CD3 antibodies (37). antibodytargeting (38), etc. Perhaps the goal of obtaining “designer lymphocytes” might be closer than we think (391, and custom-made adoptive immunotherapy might be more effective than we expect. CLAUDIO FIOCCHI. M.D. JAMES H. FINKE, Ph.D.
Department of Gastroenterology and Research Institute Cleveland Clinic Foundation Cleveland, Ohio
References Fahey JL, Sarna G, Gale RP, Seeger R. Immune intervention in disease. Ann Intern Med 1987;106:257-274. Rosenberg SA. Lotze MT, Mule’ J. New approaches to the immunotherapy of cancer using interleukin-2. Ann Intern Med 1988;108:853-864. Rosenberg SA. Lymphokine-activated killer cells: A new approach to immunotherapy of cancer. JNCI 1985;75:595-603. Mazumder A, Rosenberg SA. Successful immunotherapy of
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natural-killer-resistant established pulmonary melanoma metastases by the intravenous adoptive transfer of syngeneic lymphocytes activated in vitro by interleukin 2. J Exp Med 1984;159:495-507. 5. Grimm EA, Mazumder A, Zhang HZ, Rosenberg SA. The lymphokine-activated killer cell phenomenon: Lysis of NKresistant fresh solid tumor cells by IL2 activated autologous peripheral human blood lymphocytes. J Exp Med 1982;155:18231841. 6. Rosenberg SA, Lotze MT, Muul LM, Chang AE, Avis FP, Leitman S, Linehan WM, Robertson CN, Lee RE, Rubin JT, Seipp CA, Simpson CG, White DE. A progress report on the treatment of 157 patients with advanced cancer using lymphokine-activated killer cells and interleukin-2 or high doses of interleukin-2. N Engl J Med 1987;316:889-897, 7. West WH, Tauer KW, Yanelli JR, Marshall GD, Orr DW, Thurman GB, Oldham RK. Constant-infusion recombinant interleukin-2 in adoptive immunotherapy of advanced cancer. N Engl J Med 316;1987:898-905, 8. Rosenberg SA. The development of new immunotherapies for the treatment of cancer using interleukin-2. Ann Surg 1988;208: 121-135. 9. Rosenberg SA, Packard BS, Aebersold PM, Solomon D, Topalian SL, Toy ST, Simon P, Lotze MT, Yang JC, Seipp CA, Simpson C, Carter C, Bock S, Schwartzentruber D, Wei JP, White DE. Use of tumor-infiltrating lymphocytes and interleukin2 in the immunotherapy of patients with metastatic melanoma. N Engl J Med 1988;319:1676-1680. 10. Kradin RL, Kurnick JT Lazarus DS, Preffer FI, Dubinnet SM, Pinto CE, Gifford J, Davidson E, Grove B, Callahan RJ. Strauss HW. Tumor-infiltrating lymphocytes and interleukin-2 in treatment of advanced cancer. Lancet 1989;1:577-580. 11. Vose BM, Moore M. Human tumor-infiltrating lymphocytes: a marker of host response. Semin Hematol1985;22:27-40. 12. Rosenberg SA, Spiess P, Lafreniere R. A new approach to the adoptive immunotherapy of cancer with tumor-infiltrating lymphocytes.Science 1986;233:1318-1321. 13. Miescher S, Whiteside TL, Carrel S, von Fliedner V. Functional properties of tumor-infiltrating and blood lymphocytes in patients with solid tumors: effects of tumor cells and their supernatants on proliferative responses of lymphocytes. J Immunol 1986;136:1899-1907. 14. Whiteside TL. Heo DS, Tagaki S, Johnson JT, Iwatsuki S, Herberman RB. Cytolytic tumor effector cells in long-term cultures of human tumor-infiltrating lymphocytes in recombinant interleukin 2. Cancer Immunol Immunother 1988;26:1-10. 15. Heo DS, Whiteside TL, Kanbour A, Herberman RB. Lymphocytes infiltrating human ovarian tumors. I. Role of Leu 19 (NKHl)-positive recombinant IL-Z-activated cultures of lymphocytes infiltrating human ovarian tumors. J Immunol 1988;140: 4042-4049. 16. Miescher S, Whiteside TL, Moretta L, von Flieder V. Clonal and frequency analysis of tumor-infiltrating T lymphocytes from human solid tumors. J Immunol1987;138:4004-4011. 17. Kurnick JT, Kradin RL, Blumberg R, Schneeberger EE, Boyle LA. Functional characterization of T lymphocytes propagated from human lung carcinomas. Clin Immunol Immunopathol 1986:38:367-380. 18. Belldegrum A, Muul LM, Rosenberg SA. Interleukin 2 expanded tumor-infiltrating lymphocytes in human renal cell cancer: isolation, characterization and antitumor activity, Cancer Res 1988;48:206-214. 19. Finke JH, Tubbs R, Connelly B, Pontes E. Montie J. Tumorinfiltrating lymphocytes in patients with renal cell carcinoma, Ann NY Acad Sci 1988;532:387-394. 20. Tagaki S, Chen K, Schwarz R, Iwatsuki S, Herberman RB, Whiteside TL. Functional and phenotypic analysis of tumor-
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infiltrating lymphocytes isolated from primary and metastatic liver tumors and cultured in recombinant interleukin 2. Cancer 1989;83:102-111. 21.Heo DS, Whiteside TL, Johnson JT, Chen K, Barnes EL, Herberman RB. Long-term interleukin 2-dependent growth and cytotoxic activity of tumor-infiltrating lymphocytes from human squamous cell carcinomas of the head and neck. Cancer Res 1987;47:8353-8382. 22. Itoh K, Tilden AB, Balch CM. Interleukin 2 activation of cytotoxic T-lymphocytes infiltrating into human metastatic melanoma. Cancer Res 1988;48:3011-3017. 23. Muul LM, Spiess PJ, Director EP, Rosenberg SA. Identification of cytolytic immune responses against autologous tumor in humans bearing malignant melanoma. J Immunol1987;138:989995. 24. Itoh K, Platsoukas CD, Balch CM. Autologous tumor-specific cytotoxic T lymphocytes in the infiltrate of human metastastatic melanoma. Activation by interleukin 2 and autologous tumor cells, and involvement of the T cell receptor. J Exp Med 1988;188:1419-1441. 25. Topalian SL, Solomon D, Rosenberg SA. Tumor-specific cytolysis by lymphocytes infiltrating human melanomas. J Immunol 1989;142:3714-3725. 28. Sanders ME, Makgoba MW, Shaw S. Human naive and memory T cells: reinterpretation of helper-inducer and suppressorinducer subsets. Immunol Today 1988;9:195-199. 27. Stotter H, Wiebke EA, Tomita S, Belldegrun A, Topalian S. Rosenberg SA, Lotze MT. Cytokines alter target cell susceptibility to lysis. II. Evaluation of tumor infiltrating lymphocytes. J Immunol1989;142:1787-1773. 28. Trudel JL, Youngman KR, West GA, Fazio VW, Fiocchi C. Lymphokine-activated killer (LAK) cells from human intestinal mucosa: Cytotoxic activity against tumor cell lines and modified self but not autologous and allogeneic cancer cells. J Surg Res 1988;44:445-454, 29. Yoo Y-K, Heo DS. Hata K, Van Thiel DH, Whiteside TL. Tumorinfiltrating lymphocytes from human colon carcinomas. Functional and phenotypic characteristics after long-term culture in recombinant interleukin 2. Gastroenterology 1989;2:259-268.
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30. Fiocchi C, Youngman KR, Yen-Lieberman B, Tubbs RR. Modulation of intestinal immune reactivity by interleukin 2. Phenotypic and functional analysis of lymphokine-activated killer cells from human intestinal mucosa. Dig Dis Sci 1988;33:13051315. 31. Jarry A, Cerf-Bensussan N, Brousse N, Guy-Grand D, Muzeau F, Potet F. Same peculiar subset of HML-l+ lymphocytes present within normal intestinal epithelium is associated with tumoral epithelium of gastrointestinal carcinomas. Gut 1988;29: 1632-1638. 32. Cerf-Bensussan N, Jarry A, Brousse N, Lisowska-Grospierre B, Guy-Grand D, Griscelli C. A monoclonal antibody (HML-1) defining a novel membrane molecule present on human intestinal lymphocytes. Eur J Immunol1987;17:1279-1285. 33. James SP, Strober W. Cytotoxic lymphocytes and intestinal disease. Gastroenterology 1986;90:235-240. 34. Shanahan F, Deem R, Nayersina R, Leman B, Targan S. Human mucosal T-cell cytotoxicity. Gastroenterology 1988;94:960-967. 35. Hanna MG, Key ME. Immunotherapy of metastases enhances subsequent immunotherapy. Science 1982;217:367-369. 36. Ellenhorn JD, Hirsch R. Schreiber H, Bluestone JA. In vivo administration of anti-CD3 prevents malignant progressor tumor growth. Science 1988;242:569-571. 37. Liu MA, Nussbaum SR, Eisen HN. Hormone conjugated with antibody to CD3 mediates cytotoxic T cell lysis of human melanoma cells. Science 1988;239:395-398. 38. Greiner JW, Guadagni F, Noguchi P, Pestka S, Colcher D, Fisher PB, Schlom J. Recombinant interferon enhances antibodytargeting of carcinoma lesions in vivo. Science 1987;235:895898. 39. Culliton BJ. Fighting cancer with designer cells. Science 1989244: 1430-1433.
Address requests for reprints to Claudio Fiocchi. M.D., Research Institute, Cleveland Clinic, 9500 Euclid Avenue, Cleveland, Ohio 44195. 0 1990 by the American Gastroenterological Association
