Int. J . Cancer: 49, 317-319 (1991) 0 1991 Wiley-Liss, Inc.
Publication of the International Union Against Cancer Publication de I’Union lnternationale Contre 1e Cancer
LETTER TO THE EDITOR Dear Sir,
Spontaneous rosetting of human T lymphocytes with breast cancer cells Spontaneous rosetting of T lymphocytes to Reed-Sternberg cells has been observed in vitro and in vivo (Stuart et al., 1977; Payne et al., 1977; Morris and Stuart, 1984) and is thought toplay a major role in immunosurveillance. The occurrence of this phenomenon in Hodgkin’s lymphoma was thought to be unique (Payne et al., 1977; Stuart et al., 1977) and has not been reported in other neoplastic diseases. The molecular pathways mediating this interaction have been defined (Sanders et al., 1988a). As part of a study on the expression and regulation of T-cell adhesion surjiace ligands, CD58 (LFA-3) and CD54 (ICAM-I)on various cancer lines, we repeatedly observed that the breast-cancer cell line MCF-7 spontaneously forms rosettes with freshly isolated human T lymphocytes irrespective of tissue type. We have studied cell lines from other neoplasia, such as cervical, endometrial and prostate cancer, and found that these cells also spontaneously rosette with human T lymphocytes. The data suggest that the previously reported finding of T-cell rosetting with Reed-Sternberg cells is a common occurrence in neoplastic disease, and may in part explain the often observed lymphocytic infiltrate on tumour histology. The adhesion and cell-surface molecules involved in this interaction were investigated by the rosette assay and monoclonal antibody (MAb) inhibition studies. The breast-cancer cell line MCF-7 donated by ICRF was grown in RPMI 1640 with 10% FCS. Human T lymphocytes were purified from peripheral-blood mononuclear cells of normal donors by rosetting with 2-aminoethylisothiouronium-bromide-hydrochloromide-treated sheep erythrocytes. MAb TS2l9 to CD58 and MAb 84HI 0 to CD54 (Makgoba et al., 1988) were donated by Dr P. Mannoni (Institute J . Paoli-I Calmette, Marseille, France). MAb MHM23, spec@ for the p chain of LFA-I was donated by Dr J . Hildreth (Johns Hopkins University, Baltimore, MD). Anti-ILI MAb was donated by Dr R . Quinone and Dr R . Gress (National Cancer Institute, NIH, Bethesda, MD). All MAbs were used as purified immunoglobulin. For the rosette assay, MCF-7 cells were harvested and resuspended to I X I@ ml-’, and donor T lymphocytes suspended at I X lo7 ml- in RPMI 1640; 100 pl of each suspension were added to a test tube and 10 pg of the purified MAb added. Tubes were centrifuged at 300 g for 30 sec and incubated at 37°C for 1 hr. Cells were gently resuspended and rosettes counted on a wet mount slide. Rosettes, defined as 3 or more lymphocytes adhering to an MCF-7 cell, were scored by an observer blinded to the antibody treatment. The number of cells per condition was 300. Our initial observation (Fig. I ) showed that the breast-cancer cell line MCF-7 forms rosettes with autologous T lymphocytes. Similar results were obtained from cell lines of other tumours, such as cervical, prostate and endometrium (data not shown). A well described characteristic of such rosettes is that they form spontaneously with autologous or allogeneic T lymphocytes without subsequent cell lysis of the malignant cells (Payne et al., 1977; Stuart et al., 1977). The mechanism and pathophysiological significance of this rosetting phenomenon have remained unclear, although it has been thought to be important in immunosurveillance (Sanders et al., 1988). Recent studies have identified 2 pathways of antigen-independent adhesion used by T lymphocytes (Shaw et al., 1986). T-cell CD2 is a receptor for target-cell CD58 (Selvaraj et al., 1987; Makgoba et al., 1987), and T cell CDllaICD18 is a receptor for target CD54 (Rothlein el al., 1986; Makgoba et al., 1988; Marlin et al., 1987; Simmons et al., 1988) and ICAM-2 (Staunton et al., 1989). Other recent studies have demonstrated that the expression of these ligands correlates with antigen-specijic T-cell recognition in Burkitt’s lymphoma (Gregory et al., I988a. b). We investigated the possible involvement of these previously described adhesion pathways in the process of MCF-71T cell rosetting, using the MCF-7 line in a way similar to that in studies using the Reed-Sternberg cell line, L.428 (Sanders et al., 1988). The MCF-7 cell line, derived from a breast-cancer patient, has the morphological characteristics of freshly isolated breastcancer cells. MCF-7 cells express high levels of cell-surjiace CD.58 and CD54 and do not express CD2 or CDllaICD18, while peripheral-blood T lymphocytes in contrast express high levels of CDllaICD18 and CD2 and very moderate levels of CD58 or CD54 (Sanders et al., 1988). The amount of CD54 expressed by these cells varies. In contrast, the amount of CD58 remains constant. MAb-blocking studies showed that MAb to CD58 alone inhibited MCF-71T cell rosettes by 90% (Fig. 2 ) . MAb to CDllaICDI8 and CD54 inhibited rosetting minimally. MAb to ILI did not inhibit rosette formation. These results indicate that the spontaneous rosetting of autologous T lymphocytes which was described as being unique to Hodgkin’s disease occurs in other neoplastic conditions such as breast cancer. Results with other cancers such as cervical, endometrial and prostate carcinoma suggest that this process is common following neoplastic transformation. The adhesion is mediated by the same molecular pathways of T-cell adhesion which mediate the L.428lT cell interaction. Our results indicate that MCF-7IT cell rosetting, like the L428lT cell rosetting, is a manifestation of exaggerated antigen-independent adhesion mediated predominantly by T-cell CD2 receptor (TIIIE rosette receptor) binding to its ligand CD.58 on the MCF-7 cells and to a lesser extent by CDllalCD18-CD54 interaction. Such adhesion, to a lesser degree, is characteristic of normal T lymphocytes binding with a variety of targets. The exaggerated or normal antigen-independent adhesion seen in MCF-7 celllT cell rosetting and other neoplastic cell lines may reflect alterations in cell-surface adhesions proteins associated with malignant transformation. The
’
318
KENNETT ET AL.
FIGURE1 - Spontaneous rosettes of peripheral-blood T cells with MCF-7 cells. (a) Typical rosette formation with a single MCF-7 cell. (b) A cluster of MCF-7 cells with rosetting lymphocytes. Mab INHIBITION OF MCF-7/ROSElTES
1
2
3
4
5
ANTIBODY INHIBITION
FIGURE2 - MAb inhibition of T-cell rosettes with the MCF-7 cell line.
reason why some but not all CD58 molecules mediate adhesion remains unclear. It is possible that the unidentified posttranslational modification such as glycosylation of surface proteins associated with neoplastic transformation may lead to high-affinity forms of CD58 andlor CD54 and the subsequent increase in adhesion. The MCF-7 celllT cell rosetting is not a manifestation of antigen-specific T-cell mediated anti-tumour immunity, since it is not blocked by CD3 MAb (Sanders et al., 1987) and occurs with all unprimed allogeneic T cells so far tested. The data also strongly suggest that the phenomenon of spontaneous rosetting of T lymphocytes with neoplastic cells is more common than previously thought. Yours sincerely, R . KENNETT, R . SETH and M.W. MAKGOBA' Division of Molecular Endocrinology, Royal Postgraduate Medical School, Hammersmith Hospital, Du Cane Road, London, W12 OHS, U K .
April 24, 1991. ACKNOWLEDGEMENTS
The authors thank Drs. R. Gress, J . Hildreth, P . Mannoni and R. Quinones for contributing the various MAbs, Mr. K. Ferguson for reviewing the manuxript and support to R.K. M . W.M.'s research is supported by the MRC and the Royal Society.
IT0 whom correspondence and reprint requests should be addressed
SPONTANEOUS ROSETTING OF T LYMPHOCYTES WITH CANCER CELLS
319
REFERENCES GREGORY, C.D., MURRAY, R.J., EDWARD,C.F. and RICKINSON, A.B., Down-regulation of cell-adhesion molecules LFA-3 and ICAM-1 in Epstein-Barr-virus-positive Burkitt’s lymphocytes underlies tumor-cell escape from virus-specific T-cell surveillance. J . exp. Med., 167, 18111124 (1988~). GREGORY,C.D., RICKINSON, A.B., DE RIE, M.A., V A N SCHIINDEL, G.M.W. and MIEDEMA,F., Adhesion molecules, immunosurveillance, and B/cell malignancies. Lancet, I, 248 (19886). MAKGOBA, M.W., SANDERS, M.E., GALE,E., LUCE, G.E.G., DUSTIN, M.L., SPRINGER,T.A., CLARK,E.A., MANNONI,P. and SHAW,S., ICAM-1, a ligand for LFA-1-dependent adhesion of B, T and myeloid cells. Nature (Lond.), 331, 8 6 8 8 (1988). MAKGOBA, M.W., SHAW,S., GUGEL,E.A. and SANDERS, M.E., Human T-cell rosetting is mediated by LFA-3 on autologous erythrocytes. J . Immunot., 138, 3587-3589 (1987). MARLIN,S.D. and SPRINGER,T.A., Purified intercellular adhesion molecule- 1 (ICAM- l ) is a ligand for lymphocyte-function-associated antigen- l (LFA-I). Cell, 51, 813-819 (1987). PAYNE,S.V., JONES,D.B. and WRIGHT,D.H., Reed-Stemberg cell/ lymphocyte interaction. Lancer, 11, 768-769 (1977). ROTHLEIN,R., DUSTIN,M.L., MARLIN,S.D. and SPRINGER, T.A., A human intercellular adhesion molecule (ICAM-I) distinct from LFA-I. J. Immunol., 137, 1270-1274 (1986).
SANDERS,M.E., MAKGOBA, M.W. and SHAW,S., Human naive and memory T cells: reinterpretation of helper-inducer and suppressor-inducer subsets. Immunol. Today, 9, 195-199 (1988). SANDERS,M.E., MAKGOBA, M.W., SUSSMAN,E.H., LUCE, G.E.G., J. and SHAW,S., Molecular pathways of adhesion in spontaCOSSMAN, neous rosetting of T lymphocytes to Reed-Stemberg cells. Cancer Res., 48, 37-40 (1987). P., PLUNKETT, M.L., DUSTIN,M., SANDERS, M.E., SHAW,S. SELVARAI, and SPRINGER, T.A., The T-lymphocyte glycoprotein CD2 (LFA-2/Tll/ E-rosette receptor) binds the cell-surface ligand LFA-3. Nature (Lond.), 326, 40WOO (1987). R., GRESS,R.E., SPRINGER,T.A. SHAW,S . , LUCE,G.E.G., QUINONES, and SANDERS, M.E., Two antigen-independent adhesion pathways used by human cytotoxic T-cell clones. Nature (Load.), 323, 262-264 (1986). SIMMONS, D., MAKGOBA, M.W. and SEED,B., ICAM, an adhesion ligand of LFA-1, is homologous to the neural-cell adhesion molecule NCAM. Nature (Lond.), 331, 624-627 (1988). STAUNTON, D.E., DUSTIN,M.L. and SPRINGER, T.A., Functional cloning of ICAM-2, a cell-adhesion ligand for LFA-1 homologous to ICAM-1. Nature (Lond.), 339, 61-64 (1989). STUART, A.E., WILLIAMS, A.R. W. and HABESHAW, J.A., Rosetting and other reactions of the Reed-Stemberg cell. J . Pathol., 122, 81-90 (1977).
Publication of the International Union Against Cancer Publication de I’Union lnternationale Contre 1e Cancer
LETTER TO THE EDITOR Dear Sir,
Spontaneous rosetting of human T lymphocytes with breast cancer cells Spontaneous rosetting of T lymphocytes to Reed-Sternberg cells has been observed in vitro and in vivo (Stuart et al., 1977; Payne et al., 1977; Morris and Stuart, 1984) and is thought toplay a major role in immunosurveillance. The occurrence of this phenomenon in Hodgkin’s lymphoma was thought to be unique (Payne et al., 1977; Stuart et al., 1977) and has not been reported in other neoplastic diseases. The molecular pathways mediating this interaction have been defined (Sanders et al., 1988a). As part of a study on the expression and regulation of T-cell adhesion surjiace ligands, CD58 (LFA-3) and CD54 (ICAM-I)on various cancer lines, we repeatedly observed that the breast-cancer cell line MCF-7 spontaneously forms rosettes with freshly isolated human T lymphocytes irrespective of tissue type. We have studied cell lines from other neoplasia, such as cervical, endometrial and prostate cancer, and found that these cells also spontaneously rosette with human T lymphocytes. The data suggest that the previously reported finding of T-cell rosetting with Reed-Sternberg cells is a common occurrence in neoplastic disease, and may in part explain the often observed lymphocytic infiltrate on tumour histology. The adhesion and cell-surface molecules involved in this interaction were investigated by the rosette assay and monoclonal antibody (MAb) inhibition studies. The breast-cancer cell line MCF-7 donated by ICRF was grown in RPMI 1640 with 10% FCS. Human T lymphocytes were purified from peripheral-blood mononuclear cells of normal donors by rosetting with 2-aminoethylisothiouronium-bromide-hydrochloromide-treated sheep erythrocytes. MAb TS2l9 to CD58 and MAb 84HI 0 to CD54 (Makgoba et al., 1988) were donated by Dr P. Mannoni (Institute J . Paoli-I Calmette, Marseille, France). MAb MHM23, spec@ for the p chain of LFA-I was donated by Dr J . Hildreth (Johns Hopkins University, Baltimore, MD). Anti-ILI MAb was donated by Dr R . Quinone and Dr R . Gress (National Cancer Institute, NIH, Bethesda, MD). All MAbs were used as purified immunoglobulin. For the rosette assay, MCF-7 cells were harvested and resuspended to I X I@ ml-’, and donor T lymphocytes suspended at I X lo7 ml- in RPMI 1640; 100 pl of each suspension were added to a test tube and 10 pg of the purified MAb added. Tubes were centrifuged at 300 g for 30 sec and incubated at 37°C for 1 hr. Cells were gently resuspended and rosettes counted on a wet mount slide. Rosettes, defined as 3 or more lymphocytes adhering to an MCF-7 cell, were scored by an observer blinded to the antibody treatment. The number of cells per condition was 300. Our initial observation (Fig. I ) showed that the breast-cancer cell line MCF-7 forms rosettes with autologous T lymphocytes. Similar results were obtained from cell lines of other tumours, such as cervical, prostate and endometrium (data not shown). A well described characteristic of such rosettes is that they form spontaneously with autologous or allogeneic T lymphocytes without subsequent cell lysis of the malignant cells (Payne et al., 1977; Stuart et al., 1977). The mechanism and pathophysiological significance of this rosetting phenomenon have remained unclear, although it has been thought to be important in immunosurveillance (Sanders et al., 1988). Recent studies have identified 2 pathways of antigen-independent adhesion used by T lymphocytes (Shaw et al., 1986). T-cell CD2 is a receptor for target-cell CD58 (Selvaraj et al., 1987; Makgoba et al., 1987), and T cell CDllaICD18 is a receptor for target CD54 (Rothlein el al., 1986; Makgoba et al., 1988; Marlin et al., 1987; Simmons et al., 1988) and ICAM-2 (Staunton et al., 1989). Other recent studies have demonstrated that the expression of these ligands correlates with antigen-specijic T-cell recognition in Burkitt’s lymphoma (Gregory et al., I988a. b). We investigated the possible involvement of these previously described adhesion pathways in the process of MCF-71T cell rosetting, using the MCF-7 line in a way similar to that in studies using the Reed-Sternberg cell line, L.428 (Sanders et al., 1988). The MCF-7 cell line, derived from a breast-cancer patient, has the morphological characteristics of freshly isolated breastcancer cells. MCF-7 cells express high levels of cell-surjiace CD.58 and CD54 and do not express CD2 or CDllaICD18, while peripheral-blood T lymphocytes in contrast express high levels of CDllaICD18 and CD2 and very moderate levels of CD58 or CD54 (Sanders et al., 1988). The amount of CD54 expressed by these cells varies. In contrast, the amount of CD58 remains constant. MAb-blocking studies showed that MAb to CD58 alone inhibited MCF-71T cell rosettes by 90% (Fig. 2 ) . MAb to CDllaICDI8 and CD54 inhibited rosetting minimally. MAb to ILI did not inhibit rosette formation. These results indicate that the spontaneous rosetting of autologous T lymphocytes which was described as being unique to Hodgkin’s disease occurs in other neoplastic conditions such as breast cancer. Results with other cancers such as cervical, endometrial and prostate carcinoma suggest that this process is common following neoplastic transformation. The adhesion is mediated by the same molecular pathways of T-cell adhesion which mediate the L.428lT cell interaction. Our results indicate that MCF-7IT cell rosetting, like the L428lT cell rosetting, is a manifestation of exaggerated antigen-independent adhesion mediated predominantly by T-cell CD2 receptor (TIIIE rosette receptor) binding to its ligand CD.58 on the MCF-7 cells and to a lesser extent by CDllalCD18-CD54 interaction. Such adhesion, to a lesser degree, is characteristic of normal T lymphocytes binding with a variety of targets. The exaggerated or normal antigen-independent adhesion seen in MCF-7 celllT cell rosetting and other neoplastic cell lines may reflect alterations in cell-surface adhesions proteins associated with malignant transformation. The
’
318
KENNETT ET AL.
FIGURE1 - Spontaneous rosettes of peripheral-blood T cells with MCF-7 cells. (a) Typical rosette formation with a single MCF-7 cell. (b) A cluster of MCF-7 cells with rosetting lymphocytes. Mab INHIBITION OF MCF-7/ROSElTES
1
2
3
4
5
ANTIBODY INHIBITION
FIGURE2 - MAb inhibition of T-cell rosettes with the MCF-7 cell line.
reason why some but not all CD58 molecules mediate adhesion remains unclear. It is possible that the unidentified posttranslational modification such as glycosylation of surface proteins associated with neoplastic transformation may lead to high-affinity forms of CD58 andlor CD54 and the subsequent increase in adhesion. The MCF-7 celllT cell rosetting is not a manifestation of antigen-specific T-cell mediated anti-tumour immunity, since it is not blocked by CD3 MAb (Sanders et al., 1987) and occurs with all unprimed allogeneic T cells so far tested. The data also strongly suggest that the phenomenon of spontaneous rosetting of T lymphocytes with neoplastic cells is more common than previously thought. Yours sincerely, R . KENNETT, R . SETH and M.W. MAKGOBA' Division of Molecular Endocrinology, Royal Postgraduate Medical School, Hammersmith Hospital, Du Cane Road, London, W12 OHS, U K .
April 24, 1991. ACKNOWLEDGEMENTS
The authors thank Drs. R. Gress, J . Hildreth, P . Mannoni and R. Quinones for contributing the various MAbs, Mr. K. Ferguson for reviewing the manuxript and support to R.K. M . W.M.'s research is supported by the MRC and the Royal Society.
IT0 whom correspondence and reprint requests should be addressed
SPONTANEOUS ROSETTING OF T LYMPHOCYTES WITH CANCER CELLS
319
REFERENCES GREGORY, C.D., MURRAY, R.J., EDWARD,C.F. and RICKINSON, A.B., Down-regulation of cell-adhesion molecules LFA-3 and ICAM-1 in Epstein-Barr-virus-positive Burkitt’s lymphocytes underlies tumor-cell escape from virus-specific T-cell surveillance. J . exp. Med., 167, 18111124 (1988~). GREGORY,C.D., RICKINSON, A.B., DE RIE, M.A., V A N SCHIINDEL, G.M.W. and MIEDEMA,F., Adhesion molecules, immunosurveillance, and B/cell malignancies. Lancet, I, 248 (19886). MAKGOBA, M.W., SANDERS, M.E., GALE,E., LUCE, G.E.G., DUSTIN, M.L., SPRINGER,T.A., CLARK,E.A., MANNONI,P. and SHAW,S., ICAM-1, a ligand for LFA-1-dependent adhesion of B, T and myeloid cells. Nature (Lond.), 331, 8 6 8 8 (1988). MAKGOBA, M.W., SHAW,S., GUGEL,E.A. and SANDERS, M.E., Human T-cell rosetting is mediated by LFA-3 on autologous erythrocytes. J . Immunot., 138, 3587-3589 (1987). MARLIN,S.D. and SPRINGER,T.A., Purified intercellular adhesion molecule- 1 (ICAM- l ) is a ligand for lymphocyte-function-associated antigen- l (LFA-I). Cell, 51, 813-819 (1987). PAYNE,S.V., JONES,D.B. and WRIGHT,D.H., Reed-Stemberg cell/ lymphocyte interaction. Lancer, 11, 768-769 (1977). ROTHLEIN,R., DUSTIN,M.L., MARLIN,S.D. and SPRINGER, T.A., A human intercellular adhesion molecule (ICAM-I) distinct from LFA-I. J. Immunol., 137, 1270-1274 (1986).
SANDERS,M.E., MAKGOBA, M.W. and SHAW,S., Human naive and memory T cells: reinterpretation of helper-inducer and suppressor-inducer subsets. Immunol. Today, 9, 195-199 (1988). SANDERS,M.E., MAKGOBA, M.W., SUSSMAN,E.H., LUCE, G.E.G., J. and SHAW,S., Molecular pathways of adhesion in spontaCOSSMAN, neous rosetting of T lymphocytes to Reed-Stemberg cells. Cancer Res., 48, 37-40 (1987). P., PLUNKETT, M.L., DUSTIN,M., SANDERS, M.E., SHAW,S. SELVARAI, and SPRINGER, T.A., The T-lymphocyte glycoprotein CD2 (LFA-2/Tll/ E-rosette receptor) binds the cell-surface ligand LFA-3. Nature (Lond.), 326, 40WOO (1987). R., GRESS,R.E., SPRINGER,T.A. SHAW,S . , LUCE,G.E.G., QUINONES, and SANDERS, M.E., Two antigen-independent adhesion pathways used by human cytotoxic T-cell clones. Nature (Load.), 323, 262-264 (1986). SIMMONS, D., MAKGOBA, M.W. and SEED,B., ICAM, an adhesion ligand of LFA-1, is homologous to the neural-cell adhesion molecule NCAM. Nature (Lond.), 331, 624-627 (1988). STAUNTON, D.E., DUSTIN,M.L. and SPRINGER, T.A., Functional cloning of ICAM-2, a cell-adhesion ligand for LFA-1 homologous to ICAM-1. Nature (Lond.), 339, 61-64 (1989). STUART, A.E., WILLIAMS, A.R. W. and HABESHAW, J.A., Rosetting and other reactions of the Reed-Stemberg cell. J . Pathol., 122, 81-90 (1977).
