The Journal of Immunology, 2007,
179,
733
Copyright © 2007 by The American Association of Immunologists, Inc.
Response to Comment on "Characterization of Human Lung Tumor-Associated Fibroblasts and Their Ability to Modulate the Activation of Tumor-Associated T Cells"
Michael R. Nazareth,
Lori Broderick,
Michelle R. Simpson-Abelson,
Raymond J. Kelleher, Jr,
Sandra J. Yokota and
Richard B. Bankert
Department of Microbiology and Immunology, Witebsky Center for Microbial Pathogenesis and Immunology, University at Buffalo, State University of New York, Buffalo, NY 14214
We have two independently derived sets of new data that establish that B7-H1 is expressed by fibroblasts in the microenvironment of human non-small cell lung tumors. First, our flow cytometry data identify fibroblasts within disrupted lung tumor biopsy tissue through staining with a mAb specific for B7-H1 (clone MIH1). Using an Ab, SM1214P, which is specific for a 112-kDa molecule expressed on human fibroblasts (1, 3), we confirmed that the B7-H1+ cells were fibroblasts. We found that 42% of the tumor-derived SM1214P+ fibroblasts expressed B7-H1 on their cell surface. These B7-H1+SM1214P+ cells were, as expected, negative for CD45. Second, we demonstrated the presence of B7-H1 on tumor-associated fibroblasts by immunohistochemistry of formalin-fixed lung tumor tissues. Archived human lung tumor sections from our laboratory were stained in the laboratory of Dr. Lieping Chen (Johns Hopkins University, Baltimore, MD) using his anti-B7-H1 mAb clone 5H1, which has been used successfully to stain paraffin-embedded formalin-fixed human renal cell carcinomas after Ag retrieval (4). In four of four tumors, distinct membrane staining for B7-H1 was observed in tumor cell subsets (Fig. 1, A and B, asterisks). In two of four tumors, a portion of the cells with a fibroblast morphology, i.e., elongated cells with cytoplasmic extensions and flat/oval nuclei, stained positively with 5H1 (Fig. 1B, arrows). No staining of fibroblasts or tumor cells was observed with an isotype-matched control Ab (Fig. 1C). A trichrome stain of serial sections supports our assumption that these B7-H1+ cells were in fact fibroblasts (Fig. 1A). These data confirm our reported results that tumor-associated fibroblasts express B7-H1 on their surface in situ (5) and that this expression is not the result of cell culture as suggested by Drs. Ghebeh and Dermime (6). We recognize that the level of B7-H1 expression in the fibroblasts may vary from one tumor to another and that the level may be influenced by several different factors, including IFN-
, as we reported (5).
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FIGURE 1. Immunohistochemistry of human non-small cell lung tumor. Serial sections of a representative, formalin-fixed, human non-small cell lung tumor were stained with trichrome (A), B7-H1 (B), or an isotype control Ab (C). A, Trichrome staining of collagen emphasizes the fibroblasts, spindle-shaped cells with elongated nuclei (arrows). B, Anti-B7-H1 identifies a portion of positively-staining fibroblasts (arrows) and tumor cells that stain positively (asterisk). C, Immunohistochemistry with an isotype-control Ab confirms the specificity of the B7-H1 Ab in immunohistochemical staining of formalin-fixed, paraffin sections. Asterisks in each image identify nests of tumor cells in the tumor microenvironment. Magnification, x400.
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We are pleased that Drs. Ghebeh and Dermime (6) have expressed interest in our work and appreciate their comments, which stimulated us to look more closely for the B7-H1 expression in the tumor-associated fibroblasts before in vitro culture. However, their data that provoked their comments may be open to some questions for two reasons. First, the B7-H1 expression reported in their article was based upon comparatively weak cytoplasmic staining (not membrane staining as has been consistently reported for B7-H1 (4, 7)), and an isotype-matched control Ab was not included (8). Second, these authors failed to use any marker to positively identify their cells as fibroblasts either in vitro or in vivo.
The data presented in our paper extensively characterized primary cultures of tumor-associated fibroblasts phenotypically, and a main conclusion was that this heterogeneous population of cells is able to modulate the response of tumor-associated memory T cells to activation signals to the TCR (5). We establish here with this new data that B7-H1 is expressed on a portion of the tumor-associated fibroblast in situ. Therefore, this subset of cells in the tumor stroma represents a potential regulator of T cell function in the human lung tumor microenvironment.
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- Ghebeh, H., S. Dermime. 2007. Comment on "Characterization of human lung tumor-associated fibroblasts and their ability to modulate the activation of tumor-associated T cells.". J. Immunol. 179: 732[Free Full Text]
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- Ghebeh, H., A. Tulbah, S. Mohammed, N. Elkum, S. M. Amer, T. Al-Tweigeri, and S. Dermime. 2007. Expression of B7-H1 in breast cancer patients is strongly associated with high proliferative Ki-67-expressing tumor cells. Int. J. Cancer. In press.
