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Clonal analysis and in situ characterization of lymphocytes infiltrating human breast carcinomas


T lymphocytes were isolated from tumor biopsies in 13 patients with breast carcinomas. Immunohistology with monoclonal antibodies confirmed the presence of mononuclear cell infiltrates composed primarily of T lymphocytes in all tumors studied. While the proportion of T lymphocytes expressing the T4 or the T8 surface marker varied from tumor to tumor as determined by morphometric analysis, T8+ cells were more numerous than T4+ cells in 8/12 breast tumors studied. Relatively few T cells (<10% in 11/12 tumors) were in an activated state as judged by the surface expression of HLA-DR antigens or the receptor for interleukin-2 (IL-2). In 1 case 20% of the infiltrating mononuclear cells were expressing the IL-2 receptor. The tumor infiltrating lymphocytes (TIL) recovered from 10 tumors were cloned in a microculture system that permits proliferation of nearly 100% of normal peripheral blood T lymphocytes (PBL-T). In contrast to normal and autologous PBL-T, frequencies of proliferating T lymphocyte precursors (PTL-P) were depressed (<0.01) in 7/10 TIL preparations indicating a decreased responsiveness of TIL to phytohemagglutinin at the single-cell level. The frequency of PTL-P was noticeably higher in 2 cases (0.03 and 0.09) and close to normal in 1 case (0.39).

A total of 170 clones were expanded in vitro and analyzed for different functional capabilities. Most of these clones expressed the T4+/T8-phenotype (73%) and strikingly 53% of these T4+/T8− clones were cytolytic in a lectin-dependent assay, a functional subset which is uncommon among normal PBL-T. Some clones (10%) lysed allogeneic breast tumor cells (MCF7). Only 15% of the clones displayed natural killer activity. Among the cytolytic clones, 17 of 31 tested were also IL-2 producers irrespective of the T4 or T8 phenotype. Our results show that human mammary carcinomas contain many infiltrating T cells with cytolytic potential. Interestingly, among the proliferating cytolytic T cell clones (56% of the microcultures), many expressed the T4+/T8− phenotype. These findings may indicate that the in situ cytolytic reaction (against unknown antigens) is associated preferentially with class II antigens.

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  1. 1.

    Anichini A, Fossati G, Parmiani G (1985) Clonal analysis of cytotoxic T-lymphocyte response to autlogous human metastatic melanoma. Int J Cancer 35:683

  2. 2.

    Bhan AK, DesMarais CL (1983) Immunohistologic characterization of major histocompatibility antigens and inflammatory cellular infiltrate in human breast cancer. J Natl Cancer Inst 71:507

  3. 3.

    Bøyum A (1974) Separation of blood leukocytes, granulocytes and lymphocytes. Tissue Antigens 4:269

  4. 4.

    Brunner KT, MacDonald HR, Cerottini JC (1981) Quantitation and clonal isolation of cytolytic T lymphocyte precursors selectively infiltrating murine sarcoma virus-induced tumors. J Exp Med 154:362

  5. 5.

    De Vries JE, Spits H (1984) Cloned human cytotoxic T lymphocyte (CTL) lines reactive with autologous melanoma cells. I. In vitro generation, isolation and analysis to phenotype and specificity. J Immunol 132:510

  6. 6.

    Elston CW, Gresham GA, Rao GS, Zebro T, Haybittle JL, Houghton J, Kearney G (1982) The Cancer Research Campaign (Kings/Cambridge) trial for early breast cancer- clinicopathologic aspects. Br J Cancer 45:655

  7. 7.

    Eremin O, Coombs RRA, Ashby J (1981) Lymphocytes infiltrating human breast cancers lack K-cell activity and show low levels of NK-cell activity. Br J Cancer 44:166

  8. 8.

    Ferrini S, Biassoni R, Moretta A, Bruzzone M, Micolin A, Moretta L (1985) Clonal analysis of T lymphocytes isolated from ovarian carcinoma ascitic fluid. Phenotypic and functional characterization of T-cell clones capable of lysing autologous carcinoma cells. Int J Cancer 36:337

  9. 9.

    Fisher ER, Gregorio RM, Fischer B, Richmond C, Vellios F, Sommers SC (1975) The pathology of invasive breast cancer. Cancer 36:1

  10. 10.

    Göttlinger HG, Rieber P, Gokel JM, Lohe KJ, Riethmüller G (1985) Infiltrating mononuclear cells in human breast carcinoma: predominance of T4+ monocytic cells in the tumor stroma. Int J Cancer 35:199

  11. 11.

    Hamlin IME (1968) Possible host resistance in carcinoma of the breast, a histological study. Br J Cancer 22:383

  12. 12.

    Holmes EC (1985) Immunology of tumor infiltrating lymphocytes. Ann Surg 201:158

  13. 13.

    Hurlimann J, Saraga P (1985) Mononuclear cells infiltrating human mammary carcinomas. Immunohistological analysis with monoclonal antibodies. Int J Cancer 35:753

  14. 14.

    Introna A, Allavena P, Biondi A, Colombo N, Villa N, Mantovani A (1983) Defective natural killer activity within human ovarian tumors: low numbers of morphologically defined effectors present in situ. J Natl Cancer Inst 70:21

  15. 15.

    Jacobson S, Flerlage ML, McFarland HF (1985) Impaired measles virus-specific cytotoxic T cell responses in multiple sclerosis. J Exp Med 162:839

  16. 16.

    Jerrels RT, Dean JH, Richardson GK, McCoy JL, Herberman RB (1978) Role of suppressor cells in depression of in vitro lymphoproliferative responses of lung cancer and breast cancer patients. J Natl Cancer Inst 61:1001

  17. 17.

    Keller SE, Ioachim H, Pearse T, Siletto DM (1976) Decreased T lymphocytes in patients with mammary cancer. Am J Clin Pathol 65:445

  18. 18.

    Klein E, Svedmyr E, Mikael J, Vanky F (1977) Functional studies on tumor-infiltrating lymphocytes in man. Isr J Med Sci 13:747

  19. 19.

    Knuth A, Danowski B, Oettgen HF, Old LJ (1984) T-cell-mediated cytotoxicity against autologous malignant melanoma: Analysis with interleukin-2-dependent T-cell cultures. Proc Natl Acad Sci USA 81:3511

  20. 20.

    Landegren U (1984) Measurement of cell numbers by means of the endogenous enzyme hexosaminidase. Applications to detection of lymphokines and cell surface antigens. J Immunol Methods 67:379

  21. 21.

    Mandeville R, Lamoureux G, Legault-Poisson S, Poisson R (1982) Biological markers and breast cancer. A Multiparametric study. II. Depressed Immune Competence. Cancer 1001:1280

  22. 22.

    McCluskey DR, Roy AD, Abram WP, Martin WMC (1983) T lymphocyte subsets in the peripheral blood of patients with benign and malignant breast disease. Br J Cancer 47:307

  23. 23.

    Miescher S, Whiteside TL, Carrel S, von Fliedner V (1986) 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 136, 5:1899

  24. 24.

    Moretta A (1985) Frequency and surface phenotype of human T lymphocytes producing interleukin-2. Analysis by limiting dilution and cell cloning. Eur J Immunol 15:148

  25. 25.

    Moretta A, Colombatti M, Chapuis B (1981) Human spleen as a source of T cell growth factor. Clin Exp Immunol 44:262

  26. 26.

    Moretta L, Mingari MC, Sekaly PR, Moretta A, Chapuis B, Cerottini JC (1981) Surface markers of cloned human T cells with various cytolytic activities. J Exp Med 154:569

  27. 27.

    Moretta A, Pantaleo G, Moretta L, Cerottini JC, Mingari MC (1983) Direct demonstration of the clonogenic potential of every human peripheral blood T cell. Clonal analysis of HLADR expression and cytolytic activity. J Exp Med 157:743

  28. 28.

    Moy PM, Holmes EC, Golub SH (1985) Depression of natural killer cytotoxic activity in lymphocytes infiltrating human pulmonary tumors. Cancer Res 45:57

  29. 29.

    Mukherji B, MacAlister TJ (1983) Clonal analysis of cytotoxic T cell response against human melanoma. J Exp Med 158:240

  30. 30.

    Romagnani S, Maggi E, Parronchi P, Macchia D, Del Prete GF, Rossi-Ferrini PL, Ricci M, Moretta L (1986) Clonal analysis of T lymphocytes in spleens from patients with Hodgkin's disease. Frequent occurrence of unusual T-4 positive cells which co-express cytolytic activity and production of interleukin-2. Int J Cancer 37:343

  31. 31.

    Rowe DJ, Beverley PCL (1984) Characterization of breast cancer infiltrates using monoclonal antibodies to human leukocyte antigens. Br J Cancer 49:149

  32. 32.

    Slocum HK, Zlatko P, Pavelic P, Youcef M, Rustum M, Creaven PJ, Karakousis C, Takity H, Greco WR (1981) Characterization of cells obtained by mechanical and enzymatic means from human melanoma, sarcoma and lung tumors. Cancer Res 41:1428

  33. 33.

    Stein JA, Adler A, Etrom S, Maor M (1979) Immunocompetence, immunosuppression and human breast cancer: an analysis of their relationship by known parameters of cell-mediated immunity in well-defined clinical stages of disease. Cancer 38:1171

  34. 34.

    Taswell C (1981) Limiting dilution assays for the determinations of immunocompetent cell frequencies. I. Data analysis. J Immunol 126:1614

  35. 35.

    Tötterman TH, Parthenais E, Häyry P, Timonen T, Saksela E (1980) Cytological and functional analysis of inflammatory infiltrates in human malignant tumors. III. Further functional investigations using cultured autochthonous tumor cell lines and freeze-thawed infiltrating inflammatory cells. Cell Immunol 55:219

  36. 36.

    Vose BM (1982) Separation of tumor and host cell populations from human neoplasms. In: REID., (ed.), Methodologic Surveys, vol II: 45, Ellis Harwood Chichester

  37. 37.

    Vose BM (1982) Quantiation of proliferative and cytotoxic precursor cells directed against human tumours: Limiting dilution analysis in peripheral blood and at the tumour site. Int J Cancer 30:135

  38. 38.

    Vose BM, Moore M (1985) Human tumor-infiltrating lymphocytes: A marker of host response. Semin Hematol 22:27

  39. 39.

    Vose BM, White W (1983) Tumour-reactive lymphocytes stimulated in mixed lymphocyte and tumour culture. Clonal analysis of effector cells in cytotoxic and proliferative assays. Cancer Immunol Immunother 15:227

  40. 40.

    Vose BM, Vanky 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

  41. 41.

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

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Correspondence to Vladimir von Fliedner.

Additional information

Fogarty International Fellow of NIH, 1984–1985; on leave from Dept of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, Pa, USA

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Whiteside, T.L., Miescher, S., Hurlimann, J. et al. Clonal analysis and in situ characterization of lymphocytes infiltrating human breast carcinomas. Cancer Immunol Immunother 23, 169–178 (1986).

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  • Breast Carcinoma
  • Tumor Infiltrate Lymphocyte
  • Human Breast Carcinoma
  • Killer Activity
  • Breast Tumor Cell