Background
It is widely believed that dysfunction in the immune system of cancer patients allows the tumor cells to escape a process termed immunosurveillance [
1‐
3]. A T lymphocytes 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 protection 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 demonstrated 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 important prognostic factors linked to high-risk patients [
18].
Regulatory T cells (T
regs) are a subset of T lymphocytes that regulate the immune response by suppressing the proliferation and cytokines production of effector T lymphocytes [
19,
20]. T
regs 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 T
regs [
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 T
regs and progression of ovarian carcinoma has been demonstrated where human tumor FOXP3+ T
regs were found to suppress tumor specific immunity and contribute to reduced survival of these patients [
24]. In breast cancer, an increase in T
regs population both in peripheral blood and tumor tissues was also reported [
25] and a recent study demonstrated a significant intratumoral infiltration of FOXP3+ T
regs in high-risk breast cancer patients and those at risk of late relapse [
26].
Compelling evidences indicate a key role for the involvement of B7-H1 molecule in the development of T
regs. Induction of adaptive CD4
+CD25
+ T
regs which develop in the periphery from CD4
+CD25
- naïve T lymphocytes (in contrast to natural T
regs 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
+ T
regs 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 T
regs after intra-tracheal delivery of an alloantigen [
29]. Furthermore, B7-H1 was found to affect the function of T
regs in which its blockade decreased the inhibitory effect of such cells [
30] and blockade of B7-H1/PD-1 interactions abrogated T
regs-mediated immunoregulation [
31]. Although these studies have demonstrated the interaction between B7-H1 molecule and T
regs in animal models their co-existence in cancer patients was not evaluated. This is the first study to investigate the correlation between FOXP3+ T
regs and TIL-expressing B7-H1 and PD-1 in breast cancer patients. We have shown that their co-infiltration is strongly associated with high-risk prognostic factors and those T lymphocytes expressing FOXP3, B7-H1 and PD-1 molecules are of different T lymphocytes subsets.
Discussion
The immune system use intricate balance between positive and negative signals to protect the body from foreign agents while preventing autoimmunity [
33]. This balance seems to be disturbed in various pathological conditions resulting in either inhibition of the immune system allowing invasion by tumor cells, or stimulation of the immune system to cause autoimmune diseases. For example, although interaction of B7-H1, a coinhibitory molecule, with its PD-1 ligand is important for prevention of autoimmunity [
34], tumor cells use this interaction as a mechanism of immune evasion [
35]. Furthermore, while the presence of T
regs is important for suppression of host immune response to prevent autoimmune diseases [
36], tumor cells can recruit T
regs to inhibit anti-tumor immunity in cancer patients [
37].
We have shown previously an abundant expression of B7-H1 molecule in high-risk breast cancer patients [
18] who have highly proliferative tumor cells [
32] and suggested the involvement of this molecule in the progression of the disease. In the present report we expanded our study to investigate the co-involvement of T
regs with B7-H1 in the immune evasion of breast cancer. Although the interaction between the two types of cells has been demonstrated
in vitro and animal models, their relative infiltration and correlation with the clinicopathological parameters of cancer patients have not been well studied. In this study, we investigated FOXP3+ T
regs and TIL-expressing B7-H1 and its ligand PD-1 in breast cancer patients. We have used FOXP3 as a detection marker for T
regs as it became recently a biomarker for studying T
regs in malignant human cancers [
23]. We have found that FOXP3
+ T
regs are abundant in tumor tissues of 56% of breast cancer patients but absent in normal tissues adjacent to the tumors. FOXP3
+ T
regs infiltrating tumors have been reported by other studies [
25,
26,
38]. Abundant accumulation of T
regs in tumor tissues might be due to the induction of CD4
+CD25
+ T
regs from peripheral CD4
+CD25
- T lymphocytes [
39,
40] and/or migration of T
regs from other parts of the body to the tumor area by chemotactic factors like CCL22 [
24,
41]. This hypothesis is supported by increase of T
regs in peripheral blood of cancer patients [
25,
42].
Standard prognostic factors linked to high-risk breast cancer patients include: young age, large tumor size, high histological grade, positive lymph node metastasis and negative hormonal receptors status [
43]. We have demonstrated that FOXP3
+ T
regs infiltrating tumor tissues correlates significantly with important bad prognostic factors: large tumor size (
P = .029), high histological grade (
P < .001) and estrogen receptor negative status (
P = .010). Similar findings have been recently reported by Bates
et al showing an association between high T
regs numbers, and high histological grade and estrogen receptor negative status [
26]. Estrogen receptor negative breast tumors are generally associated with poor prognosis which is usually attributed to the association with high proliferative rate and lack of differentiation [
44], that are features of progenitors of stem-like cells. Indeed it has been shown recently that stem cells obtained from both normal and cancer breast tissues lack the estrogen receptor [
45]. Such stem cells were found to produce TGFβ1 [
45] which is known to induce T
regs [
46]. The association of FOXP3
+ T
regs with bad prognostic factors may suggest a contribution of T
regs infiltrating breast cancer tissues to tumor escape from the immune system as their depletion lead to tumor rejection in animal models [
47]. We have also confirmed the B7-H1 expression in TIL and its correlation with bad prognostic factors reported in our previous study [
18]. The association of B7-H1 expression in TIL and FOXP3
+ T
regs infiltration with the same bad prognostic factors suggests that both molecules correlate with each other. Indeed, there was a highly significant correlation between the expression of FOXP3
+ T
regs and B7-H1 (
P < .0001).
We have also found that PD-1 expression in TIL correlated with bad prognostic factors. PD-1 has been reported to be expressed in normal tissues to regulate self-reactive T cell responses [
48]. In the present study PD-1 was expressed in up to 30% of T lymphocytes from normal breast tissues while up to 80% of TIL were PD-1
+. The abundant expression of PD-1 in TIL together with B7-H1 (absent in normal tissues) seen in the present study may contribute to downregulation of the patients' immune response [
4,
6]. Furthermore, combination of FOXP3
+ T
regs, B7-H1
+ TIL and PD-1
+ TIL were found to be highly associated with patients who had high histological grade III (
P < .001) and estrogen receptor negative tumors (
P < .007) suggestive of synergistic contribution to bad prognosis. It seems 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 interaction between FOXP3
+ T
regs, B7-H1
+ TIL and PD-1
+ TIL. B7-H1/PD-1 interaction contributes to induction of T lymphocyte anergy [
4] and this can be restored in the presence of IL-2 [
49]. On the other hand, T
regs 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+ T
regs is a separate population from PD-1
+ and B7-H1
+ TIL. Similar results have been recently reported in non-Hodgkin's lymphoma by Yang
et al [
41] who showed that PD-1 and B7-H1 expression were mainly found in a subset of non-T
regs 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 confirmed 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 immunosuppressive molecules such as B7-H1, PD-1 and T
regs 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 cancer immunotherapy should be focused on designing immunological tools to block B7-H1 and its PD-1 ligand and to deplete T
regs 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 T
regs by an anti-CD25 mAb enhanced anti-tumor immunity and induced tumor regression in animal models [
47]. Furthermore, elimination of T
regs by IL-2-conjugated to diphtheria-toxin (ONTAK) enhanced vaccine-mediated anti-tumor immunity in cancer patients [
56]. Interestingly, a very recent study, in an animal model of renal cell carcinoma, has shown that only triple treatment consisting of tumor vaccine, B7-H1 blockade, and T
regs depletion can result in a complete tumor regression and long lasting protective immunity [
57] supporting the use of triple therapy treatment of cancer patients.
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
HG designed the study, carried out the immunohistochemistry for B7-H1, coordinated the work and wrote the manuscript. EB carried out immunohistochemistry of PD-1, FOXP3, and CD8 molecules. AT (anatomical pathologist) read and interpreted the sections. NE (statistician) carried out the statistical analysis. TA (medical oncologist) participated in conceiving the study and provided the clinical data. SD (principal investigator) wrote the proposal, conceived, supervised the study, and wrote the manuscript. All authors read and approved the final manuscript.