BMC Cancer 2008, 8:57 http://www.biomedcentral.com/1471-2407/8/57 A B C D E IFmigmuurneo4histochemical staining showing the expression of FOXP3, B7-H1 and PD-1 molecules by different subsets of TIL Immunohistochemical staining showing the expression of FOXP3, B7-H1 and PD-1 molecules by different sub- sets of TIL. Representative micrographs at × 530 magnification of (A) double staining of B7-H1 (brown color, membranous/ cytoplasmic) and FOXP3 (red color, nuclear) in an area rich in TIL of tumor section. (B) Double staining of PD-1 (brown color, membranous) and FOXP3 (red color, nuclear) in sections from the same tumor as in A. (C) double staining of FOXP3 (brown color, nuclear) and CD8 (red color, membranous). (D) double staining of PD-1 (brown color, membranous) and CD8 (red color, membranous). Solid arrows indicate CD8+/PD-1+ T lymphocytes and dashed arrow indicates a CD8-/PD-1+ T lym- phocyte. (E) Double staining of B7-H1 (brown color, membranous/cytoplasmic) and CD8 (red color, membranous). Solid arrow indicates a CD8+/B7-H1+ T lymphocyte and dashed arrow indicates a CD8-/B7-H1+ T lymphocyte. that FOXP3, B7-H1 and PD-1 molecules co-expressed in the TIL of the same tumor tissues may have synergistic effect in weakening the immune response of patients. IL- 2 may play a fundamental role in regulating the interac- tion between FOXP3+ Tregs, B7-H1+ TIL and PD-1+ TIL. B7- H1/PD-1 interaction contributes to induction of T lym- phocyte anergy [4] and this can be restored in the presence of IL-2 [49]. On the other hand, Tregs are dependent on IL- 2 for their expansion and function, and consumption of IL-2 present in the microenvironment may increase the inhibitory effect of B7-H1/PD-1 interaction [50]. We also investigated in the present study whether FOXP3, B7-H1 and PD-1 molecules are expressed by different T lymphocyte subsets. We have demonstrated that FOXP3+ Tregs is a separate population from PD-1+ and B7-H1+ TIL. Similar results have been recently reported in non-Hodg- kin's lymphoma by Yang et al [41] who showed that PD- 1 and B7-H1 expression were mainly found in a subset of non-Tregs T lymphocytes. The expression of FOXP3 was confirmed to be linked to CD4+ T lymphocytes as it has been reported by Roncador et al [51]. We have also con- firmed our previous findings showing that B7-H1 was expressed mainly in CD4+ T lymphocytes [18]. On the other hand, we have shown that PD-1 molecules were expressed mainly by CD8+ T lymphocytes. It is important to mention that exhausted CD8+ T lymphocytes have been reported to express the PD-1 molecule and its blockade restored the function of these T lymphocytes [52]. One of the most important findings of this study is the co- localization of immunosuppressive molecules expressed by different subsets of T lymphocytes in a group of high- risk breast cancer patients. Indeed, induction of immuno- suppressive molecules such as B7-H1, PD-1 and Tregs have been recently shown to counteract the anti-tumor effect of IL-12-based gene therapy in a transgenic mouse model of liver cancer [53]. Therefore, current interests in breast can- cer immunotherapy should be focused on designing immunological tools to block B7-H1 and its PD-1 ligand and to deplete Tregs in addition to cancer vaccination [54]. Blocking B7-H1 and PD-1 molecules with monoclonal antibodies or soluble ligands has been shown to enhance cancer immunity in animal models [55]. In addition, elimination of Tregs by an anti-CD25 mAb enhanced anti- tumor immunity and induced tumor regression in animal Page 9 of 12 (page number not for citation purposes)