BMC Cancer 2008, 8:57 http://www.biomedcentral.com/1471-2407/8/57 Background It is widely believed that dysfunction in the immune sys- tem of cancer patients allows the tumor cells to escape a process termed immunosurveillance [1-3]. A T lym- phocytes inhibitory molecule named B7-H1 (also called PD-L1) expressed by antigen presenting cells has been shown to induce T lymphocyte anergy and/or apoptosis after ligation to its T lymphocytes receptor PD-1 [4-7]. B7- H1 has been shown to be directly involved in the protec- tion of cancer cells from activated T lymphocytes [8]. Indeed, the expression of this molecule and its receptor has been described in several malignancies [9-17] where a strong link between its expression by the cancer cells and the patient clinicopathological status has been demon- strated in some of these malignancies [9,11,13,16]. We have shown previously that B7-H1 (PD-L1) is expressed in the tumor tissues of 50% of breast cancer patients and its expression was significantly associated with some impor- tant prognostic factors linked to high-risk patients [18]. Regulatory T cells (Tregs) are a subset of T lymphocytes that regulate the immune response by suppressing the prolifer- ation and cytokines production of effector T lymphocytes [19,20]. Tregs are thus important for protecting our body by suppressing auto-reactive T lymphocytes. FOXP3, a forkhead/winged helix transcription factor was found to be essential for the development and control of Tregs [21,22]. It has been used recently as a biomarker and a prognostic factor for malignant human tumor as reviewed in [23]. A direct link between the presence of Tregs and pro- gression of ovarian carcinoma has been demonstrated where human tumor FOXP3+ Tregs were found to suppress tumor specific immunity and contribute to reduced sur- vival of these patients [24]. In breast cancer, an increase in Tregs population both in peripheral blood and tumor tis- sues was also reported [25] and a recent study demon- strated a significant intratumoral infiltration of FOXP3+ Tregs in high-risk breast cancer patients and those at risk of late relapse [26]. Compelling evidences indicate a key role for the involve- ment of B7-H1 molecule in the development of Tregs. Induction of adaptive CD4+CD25+ Tregs which develop in the periphery from CD4+CD25- naïve T lymphocytes (in contrast to natural Tregs formed in the thymus) could not be formed in mice which are B7 -/- [27]. In addition, mice vascular endothelial cells were able to induce CD4+CD25+FOXP3+ Tregs only in the presence of B7-H1 [28]. Another study has demonstrated the involvement of B7-H1 as an essential element for the induction of Tregs after intra-tracheal delivery of an alloantigen [29]. Fur- thermore, B7-H1 was found to affect the function of Tregs in which its blockade decreased the inhibitory effect of such cells [30] and blockade of B7-H1/PD-1 interactions abrogated Tregs-mediated immunoregulation [31]. Although these studies have demonstrated the interaction between B7-H1 molecule and Tregs in animal models their co-existence in cancer patients was not evaluated. This is the first study to investigate the correlation between FOXP3+ Tregs and TIL-expressing B7-H1 and PD-1 in breast cancer patients. We have shown that their co-infil- tration is strongly associated with high-risk prognostic fac- tors and those T lymphocytes expressing FOXP3, B7-H1 and PD-1 molecules are of different T lymphocytes sub- sets. Methods Patients and samples collection This study was conducted in accordance with Helsinki Declaration and all patients signed a consent form approved by the Research Ethics Committee of King Faisal Specialist Hospital and Research Center (KFSH&RC). The study was approved by the Research Advisory Council (RAC) of KFSH&RC (RAC# 2030 034). Breast cancer specimens were collected from primary tumors of 68 patients including the 44 patients reported in our previous study [18] (median age 44 years) who were seeking treatments and had to undergo surgery (breast conservative surgery or total mastectomy) at KFSH&RC from 2003 to 2006. From the selected patients, 6 patients were removed from the study as they had no detectable TIL. Normal breast tissues were also obtained from 2 healthy women undergoing a plastic surgery and designated as BP. Upon excision of tissues by a surgeon, an anatomical pathologist obtained sample of the tumor tissue, denoted T, and an adjacent normal breast tissue from the same breast having the tumor, denoted N. Tis- sues from both T and N were processed as described before [18]. Briefly, they were fixed in formalin and embedded in paraffin for routine histopathological anal- ysis while other piece was snap frozen in liquid nitrogen, preserved at -80°C and sectioned using a cryostat. Immunohistochemistry Routine formalin-fixed, paraffin-embedded hospital tests were evaluated by immunohistochemistry for Her/2neu, Estrogen receptor, and progesterone receptor status as described before [18] while immunohistochemistry stain- ing of frozen tissue sections were carried out as follows: (a) Single staining B7-H1 and PD-1 detection were carried out as described previously on fresh cryogenic sections with slight modifi- cations [18,32] in which a very brief fixation in 2% para- formaldehyde (Fisher scientific) in PBS at RT (4 minutes for B7-H1 and 10 minutes for PD-1) was initially applied to improve the morphology of section. Briefly, sections were incubated for 15 minutes in 0.3% hydrogen perox- ide solution and 0.1% sodium azide (Sigma, Saint Louis, Page 2 of 12 (page number not for citation purposes)