Research Article Lymphoid cell in®ltration during breast cancer growth: A syngeneic rat model XU DONG ZHANG, GRANT DAVID SCHILLER, PETER GRANTLY GILL and BRENDON JOHN COVENTRY Department of Surgery, University of Adelaide, North Terrace, Adelaide, South Australia, Australia Summary The systematic study of potential alterations in lymphoid in®ltrates during tumour growth is extremely limited in humans. Therefore, development of a model utilizing a spontaneously arising mammary adenocarcinoma in Dark Agouti rats was adopted for the study of the dynamics of lymphoid cell in®ltration during tumour development. Syngeneic rats were inoculated with tumour cell suspensions and the tumours were resected from 5 to 15 days. Serial sections were immunohistochemically stained using a panel of monoclonal antibodies. Irrespective of tumour age, ED2 (macrophages) and W3/25 (CD4)-positive cells were the most prominent cell in®ltrates in tumours. There were no signi®cant dierences in cell counts for any marker between 8-day and 15-day tumours. However, in 5-day tumours there were signi®cantly fewer macrophages, OX19 + T cells, W3/25 + cells, OX8 + (CD8) cells and OX62 + dendritic cells. Interleukin-2 receptor a chain expression was low at all examined stages of tumour growth, indicating a lack of tumour in®ltrating lymphocyte (TIL) activation and/or possible TIL anergy. B cell staining was absent in all tumours, negating the possibility of these cells mediating coregulatory signals for TIL activation in the micro-environment of established tumours. The results parallel previous immunohistochemical ®ndings in humans, suggesting that a dysfunctional local immune response in breast cancer may be determined very early during tumour development. Key words: breast cancer, immunohistochemistry, lymphoid cells, syngeneic rat model. Introduction In®ltration of mononuclear in¯ammatory cells into human breast carcinomas has been well documented, and a pro- nounced lymphocyte in®ltrate has been correlated with an improved prognosis. 1,2 The predominant lymphoid cell in- ®ltrates in human breast cancer are known to be macro- phages and CD3 + T lymphocytes consisting of a mixture of CD4 + and CD8 + cells. 3 These cells are located mainly in the stroma around islands of tumour cells, and less frequently within tumour islands. However, despite the presence of lymphoid cells within the tumour micro-envi- ronment, clinically observed tumours continue to grow apparently unabated. It has been proposed that tumour in®ltrating lymphocytes (TIL) may be de-activated, or an- ergic, because early activation markers such as CD69, 38, 43 and CD45R0 are expressed, while little or no IL-2 and IL-2 receptor (IL-2R) is observed. 3,4 Consistent with this are studies showing that isolated breast cancer TIL exhibit poor proliferative responses to mitogen or IL-2 in vitro, in comparison to peripheral blood lymphocytes. 5 It is unclear whether an anti-tumour immune response is rendered completely ineective or whether an ongoing im- mune response is proceeding at an insucient rate to pro- duce tumour regression. Mechanisms for failure of full lymphocyte activation causing a poorly sustained or inef- fective immune response include: aberrant MHC class I and II antigen expression, defects in antigen processing and presentation, lack of costimulatory signals and induction of local tolerance by antigen excess or immunosuppressive factors liberated by tumour cells. 6±8 It is not clear at present at what stage during tumour growth `anergy' develops, or whether there is a critical tumour cell number after which the immunological cells become functionally suppressed. Examination of naturally occurring human breast cancers during very early development is precluded because tu- mours at these stages are usually not clinically detectable. Thus, little is known about changes in lymphoid cell com- position and distribution during very early breast cancer growth in humans. Observations of pre-invasive, in situ carcinomas in humans have shown that lymphocytes, den- dritic cells and macrophages accumulate on the stromal side of the basement membrane of ductal epithelial structures containing cytologically malignant cells (B Coventry pers. obs.). This suggests that lymphoid cells respond to antigens or chemo-attractant cytokines associated with malignant cells early in tumour development. A syngeneic animal model using a spontaneously arising, serially passaged rat mammary adenocarcinoma provides a means to investigate the development of lymphoid cell in®ltrates of tumours in more dynamic Correspondence: BJ Coventry, Department of Surgery, Univer- sity of Adelaide, Royal Adelaide Hospital, North Terrace, Adel- aide, SA 5000, Australia. Email: <bcoventry@medicine.adelaide.edu.au> Received 16 February 1998; accepted 20 August 1998. Immunology and Cell Biology (1998) 76, 550±555