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Culture of tumour-infiltrating lymphocytes from melanoma and colon carcinoma: Removal of tumour cells does not affect tumour-specificity

  • Original Article
  • Tumour-Infiltrating Lymphocytes, Colon Carcinoma, Melanoma, CD3 MAb, CD28 MAb, Phenotype
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Cancer Immunology, Immunotherapy Aims and scope Submit manuscript

Abstract

The therapeutic potential of adoptive therapy using tumour-infiltrating lymphocytes (TIL) has been demonstrated in a number of clinical trials. However, freshly isolated tumour-infiltrating lymphocytes (TIL) are often impaired in their proliferative and cytotoxic responses, which limits their use in immunotherapy. Several hypotheses with regard to the poor effector function of TIL have been postulated, including the production of immunosuppressive factors by tumour cells. In a previous paper we reported the efficient expansion of immunoreactive TIL from a variety of solid tumours by stimulation with a combination of monoclonal antibodies (mAbs) against CD3 and CD28. In the present study we analysed whether this protocol would be improved by the removal of tumour cells at the start of the culture. We tested a highly immunogenic tumour, melanoma, and a poorly immunogenic tumour, colon carcinoma. Removal of tumour cells highly improved anti-CD3/CD28 stimulated expansion of TIL from colon carcinoma, resulting in a significantly higher percentage of potentially tumour-specific CD8-positive T-cells and a reduced CD4/CD8 ratio compared to expansion in the presence of tumour cells. In contrast, expansion and CD4/CD8 ratio of melanoma-derived TIL was not significantly influenced by the removal of autologous tumour cells. CD3/CD28-stimulated melanoma TIL cultured in the absence of tumour cells showed specific lysis of autologous tumour cells comparable to melanoma TIL cultured in high-dose IL2. However, no cytotoxicity could be detected in colon TIL irrespective of the culture conditions used. On the other hand, 3/8 colon carcinoma TIL cultures and 9/12 melanoma-derived TIL cultures showed IFNγ secretion upon stimulation with autologous tumour cells. We conclude that stimulation of TIL with a combination of mAbs to CD3 and CD28 in the absence of tumour cells induces efficient expansion of potentially tumour-specific cells from a highly and a poorly immunogenic tumour. Removal of tumour cells does not have a negative influence on the generation of tumour-specific T cells, while cell yield improves. Therefore, for large-scale cultures this protocol can efficiently induce the outgrowth of tumour-specific TIL, at the same time providing a useful source of autologous tumour cells that can be stored and used to direct or test antitumour specificity.

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References

  1. Aebersold P, Hyatt C, Johnson S, Hines K, Korcak L, Sanders M, Lotze M, Topalian S, Yang J, Rosenberg SA (1991) Lysis of autologous melanoma cells by tumour-infiltrating lymphocytes: association with clinical response. J Natl Cancer Inst 83: 932

    PubMed  Google Scholar 

  2. Alexander JP, Kudah S, Melsop KA, Hamilton TA, Edinger MG, Tubbs RR, Sica D, Tuason L, Klein E, Bukowski RM, Finke JH (1993) T-cells infiltrating renal cell carcinoma display a poor proliferative response even though they can produce interleukin 2 and express interleukin 2 receptors. Cancer Res 53: 1380

    PubMed  Google Scholar 

  3. Baars JW, Fonk JCM, Scheper RJ, Von Blomberg-van der Flier BME, Bril H, Van der Valk P, Pinedo HM, Wagstaff J (1992) Treatment with tumour infiltratign lymphocytes and interleukin-2 in patients with metastatic melanoma. A pilot study. Biotherapy 4: 289

    PubMed  Google Scholar 

  4. Bakker ABH, Schreurs MWJ, de Boer AJ, Kawakami Y, Rosenberg SA, Adema GJ, Figdor CG (1994) Melanocyte lineagespecific antigen gp100 is recognized by melanoma-derived tumour-infiltrating lymphocytes. J Exp Med 179: 1005

    PubMed  Google Scholar 

  5. Barnd DL, Lan MS, Metzgar RS, Finn OJ (1989) Specific, major histocompatibility complex-unrestricted recognition of tumour-associated mucins by human cytotoxic T cells. Proc Natl Acad Sci USA 86: 7159

    PubMed  Google Scholar 

  6. Barth RJ Jr, Mulé JJ, Spiess PJ, Rosenberg SA (1991) Interferon gamma and tumour necrosis factor have a role in tumour regressions mediated by murine CD8+ tumour-infiltrating lymphocytes. J Exp Med 173: 647

    PubMed  Google Scholar 

  7. Chen L, Linsley PS, Hellström KE (1993) Costimulation of T cells for tumour immunity. Immunol Today 14: 483

    PubMed  Google Scholar 

  8. Ebert EC, Roberts AI, O'Connell SM, Robertson FM, Nagase H (1987) Characterization of an immunosuppressive factor derived from colon cancer cells. J Immunol 138: 2161

    PubMed  Google Scholar 

  9. Finke JH, Zea AH, Stanley J, Longo DL, Mizoguchi H, Tubbs RR, Wiltrout RH, O'Shea JJ, Kudoh S, Klein E, Bukowski RM Ochoa AC (1993) Loss of T-cell receptor zeta chain and p56Ick in T-cells infiltrating human renal cell carcinoma. Cancer Res 53: 5613

    PubMed  Google Scholar 

  10. Finke JH, Rayman P, Hart L, Alexander JP, Edinger MG, Tubbs RR, Klein E, Tuason L, Bukowski RM (1994) Characterization of tumour-infiltrating lymphocyte subsets fom human renal cell carcinoma: specific reactivity defined by cytotoxicity, interferongamma secretion, and proliferation. J Immunother 15: 91

    Google Scholar 

  11. Flens MJ, Mulder WMC, Bril H, Von Blomberg van de Flier MBE, Scheper RJ, Van Lier RAW (1993) Efficient expansion of tumour-infiltrating lymphocytes from solid tumours with combined CD3 and CD28 monoclonal antibodies. Cancer Immunol Immunother 37: 323

    PubMed  Google Scholar 

  12. Geppert TD, Davis LS, Gur H, Wacholtz MC, Lipsky PE (1990) Accessory cell signals involved in T-cell activation. Immunol Rev 111: 5

    Google Scholar 

  13. Guillou PJ, Ramsden CW, Somers SS, Sedman PC (1989) Suppression of the generation of lymphokine-activated killer (LAK) cells by serum-free supernatants of in vitro maintained tumour cell lines. Br J Cancer 59: 515

    PubMed  Google Scholar 

  14. Hom SS, Schwartzentruber DJ, Rosenberg SA, Topalian SL (1993) Specific release of cytokines by lymphocytes infiltrating human melanomas in response to shared melanoma antigens. J Immunother 13: 18

    PubMed  Google Scholar 

  15. Hom SS, Rosenberg SA, Topalian SL (1993) Specific immune recognition of autologous tumour by lymphocytes infiltrating colon carcinomas: analysis by cytokine secretion. Cancer Immunol Immunother 36: 1

    PubMed  Google Scholar 

  16. Hoskin CW, Reynolds T, Blay J (1994) Colon adenocarcinoma cells inhibit anti-CD3-activated killer cell induction. Cancer Immunol Immunother 38: 201

    PubMed  Google Scholar 

  17. Ioannides CG, Fisk B, Jerome KR, Irimura T, Taylor Wharton J, Finn OJ (1993) Cytotoxic T cells from ovarian malignant tumours can recognize polymorphic epithelial mucin core peptides. J Immunol 150: 3693

    Google Scholar 

  18. Jerome KR, Domenech N, Finn OJ (1993) Tumour-specific cytotoxic T cell clones from patients with breast and pancreatic adenocarcinoma recognize EBV-immortalized B cells transfected with polymorphic epithelial mucin complementary DNA. J Immunol 151: 1654

    PubMed  Google Scholar 

  19. Jerome KR, Barnd DL, Bendt KM, Boyer CM, Taylor-Papadimitriou J, McKenzie IFC, Bast RC Jr, Finn OJ (1991) Cytotoxic T-lymphocytes derived from patients with breast adenocarcinoma recognize an epitope present on the protein core of a mucin molecule preferentially expressed by malignant cells. Cancer Res 51: 2908

    PubMed  Google Scholar 

  20. June CH, Bluesone JA, Nadler LM, Thompson CB (1994) The B7 and CD28 receptor families. Immunol Today 15: 321

    PubMed  Google Scholar 

  21. Kawakami Y, Zakut R, Topalian SL, Stötter H, Rosenberg SA (1992) Shared human melanoma antigens. Recognition by tumour-infiltrating lymphocytes in HLA-A2.1-transfected melanomas. J Imm 148: 638

    PubMed  Google Scholar 

  22. Kawakami Y, Eliyahu S, Delgado CH, Robbins PF, Rivoltini L, Topalian SL, Miki T, Rosenberg SA (1994) Cloning of the gene coding for a shared human melanoma antigen recognized by autologous T cells infiltrating into tumour. Proc Natl Acad Sci USA 91: 3515

    PubMed  Google Scholar 

  23. Kradin RL, Kurnick JT, Lazarus DS, Preffer FI, Dubinett SM, Pinto CE, Gifford J, Davidson E, Grove B (1989) Tumour-infiltrating lymphocytes and interleukin-2 in treatment of advanced cancer. Lancet 338: 577

    Google Scholar 

  24. Kuppner MC, Hamou MF, Sawamura Y, Bodmer S, De Tribolet N (1989) Inhibition of lymphocyte function by glioblastoma-derived transforming growth factor beta2. J Neurosurg 71: 211

    PubMed  Google Scholar 

  25. Liu Y, Linsley PS (1992) Costimulation of T-cell growth. Curr Opin Immunol 4: 265

    PubMed  Google Scholar 

  26. Miescher S, Whiteside TL, Carrel S, Fliedner V von (1986) Functional properties of tumour-infiltrating and blood lymphocytes in patients with solid tumours: effect of tumour cells and their supernatants on proliferative responses of lymphocytes. J Immunol 136: 1899

    PubMed  Google Scholar 

  27. Miescher S, Whiteside TL, Moretta L, Fliedner V von (1987) Clonal and frequency analysis of tumour-infiltrating T-lymphocytes from human solid tumours. J Immunol 138: 4004

    PubMed  Google Scholar 

  28. Miescher S, Stoeck M, Qiao L, Barras C, Barrelet L Fliedner V von (1988) Preferential clonogenic deficit of CD8-positive T-lymphocytes infiltrating human solid tumours. Cancer Res 48: 6992

    PubMed  Google Scholar 

  29. Nakagomi H Petersson M, Magnusson I, Juhlin, C, Matsuda M, Mellstedt H, Taupin J-L, Vivier E, Anderson P, Kiessling R (1993) Decreased expression of the signal-transducing zeta chains in tumour-infiltrating T-cells and NK cells of patients with colorectal carcinoma. Cancer Res53: 5610

    PubMed  Google Scholar 

  30. Nijhuis EWP, Van der Wiel-Van Kemenade E, Figdor CG, Van Lier RAW (1990) Activation and expansion of tumour-infiltrating lymphocytes by anti-CD3 and anti-CD28 monoclonal antibodies. Cancer Immunol Immunother 32: 245

    PubMed  Google Scholar 

  31. Ochalek T, Von Kleist S (1993) Study of the susceptibility of human colorectal tumour explants to LAK-cell lysis: comparison of various cytotoxicity tests. J Clin Lab Anal 7: 155

    PubMed  Google Scholar 

  32. Okadome M, Saito T, Tsukamoto N, Sano M, Kamura T, Nakano H (1991) Potential of human lymph node cells for antitumour activity mediated by interferon gamma. Cancer 68: 2378

    PubMed  Google Scholar 

  33. Pandolfino M-C, Viret C, Gervois N, Guilloux Y, Davodeau F, Diez E, Jotereau F (1992) Specificity, T cell receptor diversity and activation requirements of CD4+ and CD8+ clones derived from human melanoma-infiltrating lymphocytes. Eur J Immunol 22: 1795

    PubMed  Google Scholar 

  34. Ransom JH, Pelle BA, Hubers H, Keynton LM, Hanna MG Jr, Pomato N (1993) Identification of colon tumour-associated antigens by T-cell lines derived from tumour infiltrating lymphocytes and peripheral-blood lymphocytes from patients immunized with an autologous tumour cell/Bacillus Calmette-Guérin vaccine. Int J Cancer 54: 734

    PubMed  Google Scholar 

  35. Remacle-Bonnet MM, Pommier GJ, Kaplanski S, Rance RJ, Depieds RC (1976) Inhibition of normal allogeneic lymphocyte mitogenesis by a soluble inhibitor extracted from human colonic carcinoma. J Immunol 117: 1145

    PubMed  Google Scholar 

  36. 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 (1988) Use of tumour-infiltrating lymphocytes and interleukin-2 in the immunotherapy of patients with metastatic melanoma. N Engl J Med 319: 1676

    PubMed  Google Scholar 

  37. Shimizu U, Iwatsuki S, Herberman RB, Whiteside TL (1993) Effects of cytokines on in vitro growth of tumour-infiltrating lymphocytes obtained from human primary and metastatic liver tumours. Cancer Immunol Immunother 32: 280

    Google Scholar 

  38. Stoeck M, Miescher S, Qiao L, Capasso P, Barras C, Fliedner V von (1990) Stimulation of FACS-analysed CD4+ and CD8+ human tumour-infiltrating lymphocytes with ionomycin+phorbol-12,13-dibutyrate does not overcome their proliferative deficit. Clin Exp Immunol 79: 105

    PubMed  Google Scholar 

  39. Svennevig JL, Lunde OC, Holter J, Björgsvik D (1984) Lymphoid infiltration and prognosis in colorectal carcinoma. Br J Cancer 49: 375

    PubMed  Google Scholar 

  40. Tefany FJ, Barnetson R StC, Halliday GM, McCarthy SW, McCarthy WH (1991) Immunocytochemical analysis of the cellular infiltrate in primary regressing and non-regressing malignant melenoma. J Invest Dermatol 97: 197

    PubMed  Google Scholar 

  41. Thompson CB, Linsten T, Ledbetter JA, Kunkel SL, Young HA, Emerson SG, Leiden JM, June CH (1989)CD28 activation pathway regulates the production of multiple T-cell-derived lymphokines/cytokines. Proc natl Acad Sci USA 86: 1333

    PubMed  Google Scholar 

  42. Topalian SL, Solomon D, Avis FP, Chang AE, Freerksen DL, Linehan WM, Lotze MT, Robertson CN, Seipp CA, Simon P, Simpson CG, Rosenberg SA (1988) Immunotherapy with patients with advanced cancer using tumour infiltrating lymphocytes and recombinant IL-2: A pilot study. J Clin Oncol 6: 839

    PubMed  Google Scholar 

  43. Traversari C, Van der Bruggen P, Luescher IF, Lurquin C, Chomez P, Van Pel A, De Plaen E, Amar-Costesec A, Boon T (1992) A nonapeptide encoded by human gene MAGE-1 is recognized on HLA-A1 by cytolytic T lymphocytes directed against tumour antigen MZ2-E. J Exp Med 176: 1453

    PubMed  Google Scholar 

  44. Viret C, Davodeau F, Guilloux Y, Bignon J-D, Semana G, Breathnach R, Jotereau F (1993) Recognition of shared melanoma antigen by HLA-A2-restricted cytolytic T cell clones derived from human tumour-infiltrating lymphocytes. Eur J Immunol 23: 141

    PubMed  Google Scholar 

  45. Vose BM, Vánky F, Klein E (1977) Human tumour-lymphocyte interaction in vitro. V. Comparison of the reactivity of tumour-infiltrating, blood and lymph-node lymphocytes with autologous tumour cells. Int J Cancer 20: 895

    PubMed  Google Scholar 

  46. Whiteside TL, Miescher S, Hurlimann J, Moretta L, Fliedner V von (1986) Separation, phenotyping and limiting dilution analysis of T-lymphocytes infiltrating human solid tumours. Int J Cancer 37: 803

    PubMed  Google Scholar 

  47. Whiteside TL, Parmiani G (1994) Tumour-infiltrating lymphocytes: their phenotype, functions and clinical use. Cancer Immunol Immunother 39: 15

    PubMed  Google Scholar 

  48. Wiebke EA, Lotze MT, Rosenberg SA (1987) Tumour cell susceptability to lysis: marked increase in lysis by tumour-infiltrating lymphocytes following target stimulation with interferon gamma and tumour necrosis factor alpha — implications for immunotherapy. Surg Forum 38: 436

    Google Scholar 

  49. Wysocki LJ, Sato VL (1978) “Panning” for lymphocytes: a method for cell selection. Proc Natl Acad Sci USA 75: 2844

    PubMed  Google Scholar 

  50. Zocchi MR, Poggi A, Crosti F, Tongiani S, Rugarli C (1992) Signalling in human tumour infiltrating lymphocytes: the CD28 molecule is functional and is physically associated with CD45RO molecule. Eur J Cancer 28A: 749

    PubMed  Google Scholar 

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Authors and Affiliations

  1. Department of Pathology, Free University Hospital, De Boelelaan 1117, 1081 HV, Amsterdam, The Netherlands

    Wilhelmina M. C. Mulder, Marij J. Stukart, Micha Roos, Rik J. Scheper & Elisabeth Bloemena

  2. Central Laboratory of the Netherlands Red Cross Blood Transfusion Service, Amsterdam, The Netherlands

    René A. W. van Lier

  3. Department of Oncology, Free University Hospital, Amsterdam, The Netherlands

    John Wagstaff

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  1. Wilhelmina M. C. Mulder
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Mulder, W.M.C., Stukart, M.J., Roos, M. et al. Culture of tumour-infiltrating lymphocytes from melanoma and colon carcinoma: Removal of tumour cells does not affect tumour-specificity. Cancer Immunol Immunother 41, 293–301 (1995). https://doi.org/10.1007/BF01517217

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  • DOI: https://doi.org/10.1007/BF01517217

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