BMC Cancer 2008, 8:57 http://www.biomedcentral.com/1471-2407/8/57 MO, USA). Sections were blocked with 10% goat serum (DAKO, Denmark) for 30 minutes followed by B7-H1 antibody (MIH1 clone, ebioscience, San Diego, CA, USA) or PD-1 antibody (J116 clone, ebioscience,) diluted at 1:50 overnight. After washing, sections were stained for 30 minutes at RT, with Labeled Polymer (EnVision+) horse- radish peroxidase (HRP) detection kit (DAKO). Colors were developed using DAB (Novocastra, Newcastle upon Tyne, UK) or AEC (Sigma) and sections were counter- stained with instant hematoxylin (Shandon, Pittsburgh, PA, USA). (b) Double staining Sections were washed in PBS, incubated with paraformal- dehyde for 4 minutes and blocked in H2O2 and goat serum as described above. The primary and secondary antibodies were added as follows: For CD3/FOXP3, CD8/ FOXP3, CD8/PD-1 or CD8/B7-H1 double staining the 2 primary antibodies from two different species, mouse anti-FOXP3 (236A/E7 clone, ebioscience) diluted at 1:100, mouse anti-B7-H1 (clone MIH1, ebioscience) diluted at 1:25 or mouse anti-PD-1 antibody (J116 clone, ebioscience) diluted at 1:50 were mixed with rabbit anti- CD3 (Dako) at 1:50 dilution or rabbit anti-CD8 (Abcam, Cambridge, UK) at 1:75 dilution, together in 10% human AB serum (Cambrex) and incubated for 2 h at RT or over- night at 4°C. Sections were washed 3 times in PBS and incubated for 30 minutes at RT with a secondary anti- body: swine anti-rabbit AP (Dako) diluted at 1:50 in a ready-to-use polymer (Envision+, Dako) linked to HRP. After washing 3 times with PBS, substrates were added starting with fuschin red (Dako) followed by DAB (Novo- castra). Slides were counterstained with instant hematox- ylin (Shandon). FOXP3 and B7-H1 double staining were carried out as described above except the primary antibod- ies used were rabbit anti-FOXP3 (polyclonal, Abcam) at 1:500 dilution mixed with mouse anti-B7-H1 (clone MIH1, ebioscience) at 1:25 dilution and overnight incu- bation at 4°C. FOXP3 and PD-1 double staining were started with a single staining of PD-1 as before, however after color development with DAB, sections were boiled for 3 minutes in an antigen retrieval citrate solution (pH = 6.0) to remove previously attached antibodies. FOXP3 antigen was then stained with mouse anti-FOXP3 anti- body (236A/E7 clone, ebioscience) diluted at 1:100 for 2 hours at RT followed by goat anti-mouse IgG1 AP (South- ern Biotech, Birmingham, Alabama, USA) for 30 minutes at RT. The color for FOXP3 staining was developed with Fuschin Red (Dako) and sections were counterstained with instant hematoxylin (Shandon). Interpretation of stained tumor sections The B7-H1 and PD-1 staining were scored by an anatom- ical pathologist (AT) as described previously [18]. FOXP3+ T lymphocytes were scored in 5–10% increments as a per- centage of total CD3+ staining cells in several high magni- fication fields. Intratumoral FOXP3+/CD3+ cells were only considered and counted while intrastromal FOXP3+ cells were disregarded. FOXP3+ cells with moderate or high intensity (++ or +++) were considered Tregs while weak staining (+) were ignored. Due to the co localization of the CD3 and PD-1 molecules in the T lymphocyte mem- branes, double staining of PD-1 or B7-H1 and CD3 were assumed based on single-staining of sequential slides. The cut off point for positive and negative for FOXP3, B7-H1 and PD-1 was 5% (5% of total CD3+ T-lymphocytes). Statistical Analysis Statistical analyses were used to determine the association between the B7-H1 expression in TIL or FOXP3+ Tregs infil- tration and the patients' clinico-pathological parameters. Nominal parameters were analyzed using the fisher exact test. Comparisons of the independent variables with the dependent variable were performed using a simple logis- tic regression analysis. Significance was defined as the probability of a type one error of < 5%, and 95% confi- dence intervals were included. The software package SAS was used for these analyses. Results Frequencies of Tregs, B7-H1+ and PD-1+ T lymphocytes in normal and breast cancer tissues In order to determine the frequencies of Tregs, B7-H1 and PD-1 expressing-T lymphocytes in normal breast tissues we stained sections obtained from breast patients under- going plastic surgery (BP) as well as normal breast tissues adjacent to breast cancer tumors (N) for the expression of FOXP3, B7-H1 and PD-1 molecules. There were a few T lymphocytes in both N and BP in which FOXP3+ Tregs rep- resented <5% of the total CD3+ TIL (Figure 1A). Similarly, T lymphocytes in normal tissues were negative for B7-H1 (Figure 1B). However, PD-1+ T lymphocytes were abun- dant in normal tissues with up to 30% of CD3+ cells co- expressing the PD-1 molecule (Figure 1C). In contrast to normal breast tissues, 56% of breast cancer patients had a higher Tregs frequency in their tumor tissues in which 5–50% of CD3+ TILs were Tregs (Figure 1D). Similarly, B7- H1 was expressed in TILs of 54% of breast cancer patients' tumor tissues in which 5–80% of TILs express this mole- cule. B7-H1 expression was not restricted to T lym- phocytes as 30% of breast cancer patients showed also B7- H1 expression in their tumor cells (Figure 1E). This result was also described in our previous study [18]. The PD-1 molecule was expressed in TILs in 60% of the patients in which 5–70% of TIL express this molecule (Figure 1F). The expression pattern of the PD-1 molecule was mem- branous and restricted to T lymphocytes only. Both intratumoral and intrastromal FOXP3+CD3+ cells were present in the patients tissues (Figure 2A) and the Page 3 of 12 (page number not for citation purposes)