B7-H1 molecule increases the apoptosis of tumor-reactive T lymphocytes and reduces their immunogenicity. Breast cancer is the second most common cause of mortality after lung cancer. Direct evidence linking B7-H1 with cancer has been shown in several malignancies; however, its expression in breast cancer has not been investigated. We used immunohistochemistry to investigate the expression of the B7-H1 molecule in 44 breast cancer specimens and to study its correlation with patients' clinicopathological parameters. The expression of B7-H1 was shown in 22 of 44 patients and was not restricted to the tumor epithelium (15 of 44, 34% in tumor cells), but was also expressed by tumor-infiltrating lymphocytes (TIL; 18 of 44, 41%). Interestingly, intratumor expression of B7-H1 was significantly associated with histologic grade III-negative (P = .012), estrogen receptor-negative (P = .036), and progesterone receptor-negative (P = .040) patients. In addition, the expression of B7-H1 in TIL was associated with large tumor size (P = .042), histologic grade III (P = .015), positivity of Her2/neu status (P = .019), and severe tumor lymphocyte infiltration (P = .001). Taken together, these data suggest that B7-H1 may be an important risk factor in breast cancer patients and may represent a potential immunotherapeutic target using monoclonal antibody against the B7-H1 molecule.

The T-cell inhibitory molecule PD-L1 (B7-H1, CD274) is expressed on tumor cells of a subset of breast cancer patients. However, the mechanism that regulates PD-L1 expression in this group of patients is still not well-identified. We have used loss and gain of function gene manipulation approach, multi-parametric flow cytometry, large scale gene expression dataset analysis and immunohistochemistry of breast cancer tissue sections. Induction of epithelial to mesenchymal transition (EMT) in human mammary epithelial cells upregulated PD-L1 expression, which was dependent mainly on the activation of the PI3K/AKT pathway. Interestingly, gene expression signatures available from large cohort of breast tumors showed a significant correlation between EMT score and the PD-L1 mRNA level (p < 0.001). Strikingly, very strong association (p < 0.0001) was found between PD-L1 expression and claudin-low subset of breast cancer, which is known to have high EMT score. On the protein level, significant correlation was found between PD-L1 expression and standard markers of EMT (p = 0.005) in 67 breast cancer patients. Importantly, specific downregulation of PD-L1 in claudin-low breast cancer cells showed signs of EMT reversal as manifested by CD44 and Vimentin downregulation and CD24 upregulation. We have demonstrated a bidirectional effect between EMT status and PD-L1 expression especially in claudin-low subtype of breast cancer cells. Our findings highlights the potential dual benefit of anti-PD-L1 particularly in this subset of breast cancer patients that will likely benefit more from anti-PD-L1 targeted therapy as well as in monitoring biological changes upon treatment.

The expression of PD‐L1 in breast cancer is associated with estrogen receptor negativity, chemoresistance and epithelial‐to‐mesenchymal transition (EMT), all of which are common features of a highly tumorigenic subpopulation of cancer cells termed cancer stem cells (CSCs). Hitherto, the expression and intrinsic role of PD‐L1 in the dynamics of breast CSCs has not been investigated. To address this issue, we used transcriptomic datasets, proteomics and several in vitro and in vivo assays. Expression profiling of a large breast cancer dataset (530 patients) showed statistically significant correlation (p < 0.0001, r = 0.36) between PD‐L1 expression and stemness score of breast cancer. Specific knockdown of PD‐L1 using ShRNA revealed its critical role in the expression of the embryonic stem cell transcriptional factors: OCT‐4A, Nanog and the stemness factor, BMI1. Conversely, these factors could be induced upon PD‐L1 ectopic expression in cells that are normally PD‐L1 negative. Global proteomic analysis hinted for the central role of AKT in the biology of PD‐L1 expressing cells. Indeed, PD‐L1 positive effect on OCT‐4A and Nanog was dependent on AKT activation. Most importantly, downregulation of PD‐L1 compromised the self‐renewal capability of breast CSCs in vitro and in vivo as shown by tumorsphere formation assay and extreme limiting dilution assay, respectively. This study demonstrates a novel role for PD‐L1 in sustaining stemness of breast cancer cells and identifies the subpopulation and its associated molecular pathways that would be targeted upon anti‐PD‐L1 therapy.

Introduction: B7-H1 (PD-L1, CD274) is a T cell inhibitory molecule expressed in many types of cancer, leading to immune escape of tumor cells. Indeed, in previous reports we have shown an association of B7-H1 expression with high-risk breast cancer patients. Methods: In the current study, we used immunohistochemistry, immunofluorescence and Western blot techniques to investigate the effect of neoadjuvant chemotherapy on the expression of B7-H1 in breast cancer cells. Results: Among tested chemotherapeutic agents, doxorubicin was the most effective in downregulating cell surface expression of B7-H1 in vitro. These results were validated in vivo in a xenograft mouse model, as well as in murine heart tissue known to constitutively express B7-H1. The doxorubicin-dependent cell surface downregulation of B7-H1 was accompanied by an upregulation of B7-H1 in the nucleus. This re-distribution of B7-H1 was concurrent with a similar translocation of phosphorylated AKT to the nucleus. Inhibition of the PI3K/AKT pathway abrogated the doxorubicin-mediated nuclear up-regulation of B7-H1, suggesting an involvement of PI3K/AKT pathway in the nuclear up-regulation of B7-H1. Interestingly, siRNA knock down of B7-H1 lead to an increase in spontaneous apoptosis, as well as doxorubicin-induced apoptosis, which indicates an anti-apoptotic role for B7-H1 in breast cancer cells. The novel discovery of B7-H1 expression in the nuclei of breast cancer cells suggests that B7-H1 has functions other than inhibition of T cells. Conclusions: Our findings explain the previously reported immunomodulatory effect of anthracyclines on cancer cells, and provide a link between immunoresistance and chemoresistance. Finally these results suggest the use of dual combinatorial agents to inhibit B7-H1 beside chemotherapy, in breast cancer patients.

Background: Recent studies have demonstrated a direct involvement of B7-H1, PD-1 and FOXP3 molecules in the immune escape of cancer. B7-H1 is an inhibitory molecule that binds to PD-1 on T lymphocytes, while FOXP3 is a marker for regulatory T cells (T_regs). We have previously demonstrated the association of B7-H1-expressing T infiltrating lymphocytes (TIL) with high-risk breast cancer patients while other studies reported the involvement of FOXP3+ T_regsas a bad prognostic factor in breast tumors. Although the co-existence between the two types of cells has been demonstratedin vitroand animal models, their relative infiltration and correlation with the clinicopathological parameters of cancer patients have not been well studied. Therefore, we investigated TIL-expressing the B7-H1, PD-1, and FOXP3 molecules, in the microenvironment of human breast tumors and their possible association with the progression of the disease.Methods: Using immunohistochemistry, tumor sections from 62 breast cancer patients were co-stained for B7-H1, PD-1 and FOXP3 molecules and their expression was statistically correlated with factors known to be involved in the progression of the disease.Results: A co-existence of B7-H1⁺T lymphocytes and FOXP3⁺T_regswas evidenced by the highly significant correlation of these molecules (P< .0001) and their expression by different T lymphocyte subsets was clearly demonstrated. Interestingly, concomitant presence of FOXP3⁺T_regs, B7-H1⁺and PD-1⁺TIL synergistically correlated with high histological grade (III) (P< .001), estrogen receptor negative status (P= .017), and the presence of severe lymphocytic infiltration (P= .022).Conclusion: Accumulation of TIL-expressing such inhibitory molecules may deteriorate the immunity of high-risk breast cancer patients and this should encourage vigorous combinatorial immunotherapeutic approaches targeting T_regsand B7-H1/PD-1 molecules.

Objective— Severe heatstroke is a leading cause of morbidity and mortality during heat waves. The pathogenesis of tissue injury, organ failure, and death in heatstroke is not well understood. Methods and Result— We investigated the pathways of heatstroke-induced tissue injury and cell death in anesthetized baboons ( Papio hamadyras ) subjected to environmental heat stress until core temperature attained 42.5°C (moderate heatstroke; n=3) or onset of severe heatstroke (n=4) signaled by a fall in systolic blood pressure to <90 mm Hg and rise in core temperature to 43.1±0.1°C. Three sham-heated animals served as controls. Light and electron microscopy revealed widespread hemorrhage and thrombosis, transmural migration of leukocytes, and microvascular endothelium injury in severe heatstroke. Immunohistology and ultrastructural analysis demonstrated increased staining of endothelial von Willebrand factor (vWF), tissue factor (TF), and endothelial leukocyte-platelet interaction. Extensive apoptosis was noted in spleen, gut, and lung, and in hematopoeitic cells populating these organs. Double-labeling studies colocalized active caspase-3 and TF with apoptotic cells. Findings in sham-heated animals were unremarkable. Conclusion— These data suggested that microvascular injury, thrombosis, inflammation, and apoptosis may play an important role in the pathogenesis of heatstroke injury.