BMC Cancer 2008, 8:57 http://www.biomedcentral.com/1471-2407/8/57 A B Intrastromal Intratumoral Intense Medium Weak FImigmuurneo2histochemical staining showing intratumoral and intrastromal FOXP3+ Tregs in breast cancer tissues Immunohistochemical staining showing intratumoral and intrastromal FOXP3+ Tregs in breast cancer tissues. Representative micrographs at × 530 magnification of CD3+ (red color membranous expression) and FOXP3+ (brown color nuclear expression) TIL of (A) intrastromal (left) and intratumoral (right) sections. Solid arrow indicates a CD3+/FOXP3+ Treg cell and dashed arrow indicates a CD3+/FOXP3- cell. (B) Intratumoral FOXP3+ TIL with different staining intensity (Intense, Medium, and weak). Cells were counterstained with hematoxylin. We have found that B7-H1+, PD-1+ TIL and FOXP3+ Tregs were significantly associated with similar bad prognostic factors. Therefore, we further investigated the correlation between B7-H1+ TIL or PD-1+ TIL and FOXP3+ Tregs infil- tration in breast tumors. There was a highly significant correlation between the expression of B7-H1 in TIL and intratumoral FOXP3+ Tregs (P < .0001) as shown by ANOVA test (Figure 3A) and by linear regression analysis (P < .004 and R2 = 0.3) (Figure 3B). There was also a sig- nificant correlation between FOXP3+ Tregs infiltration and PD-1 expression in TIL (P = .007) as shown by ANOVA (Figure 3C). Correlation of combined FOXP3, and B7-H1 expression in TIL with clinicopathological parameters of patients We next tested whether a combination of FOXP3 and B7- H1 molecules will have a synergistic effect on the correla- tion with the patients' clinicopathological parameters. We used a combination of the two factors; FOXP3+ Tregs and B7-H1 expression in TIL in the presence of PD-1 expres- sion (only patients with B7-H1-expressing TIL were con- sidered positive when PD-1+ TIL were present in the same tissue). The combined molecules maintained a significant correlation with the same prognostic factors (Table 2). Expression of FOXP3, B7-H1 and PD-1 molecules by different subsets of TIL We next asked whether FOXP3, PD-1 and B7-H1 mole- cules are expressed by different T lymphocyte subsets. Double-staining on 6 selected samples which had high FOXP3+ Tregs, high B7-H1+ TIL and PD-1+ TIL was per- formed. The selections were based on sections that had a good morphology to make the interpretation easier. Dou- ble-staining assays showed that B7-H1 and FOXP3 mole- cules were generally expressed by different T lymphocyte subsets (most B7-H1+ TIL were FOXP3- while only very few FOXP3+ Tregs co-express the B7-H1 molecule). In addi- tion, the distribution pattern of B7-H1+ TIL was different from that of FOXP3+ Tregs; Tregs were single cells distributed over the section of the tumor tissues while B7-H1+ TIL aggregated in clumps (Figure 4A). Similarly, PD-1 and FOXP3 molecules were expressed by different T lym- phocyte subsets (Figure 4B). Furthermore, double stain- ing assays demonstrated that FOXP3 and CD8 were Page 5 of 12 (page number not for citation purposes)