FOXP3+ Tregs and B7-H1+/PD-1+ T lymphocytes co-infiltrate the tumor tissues of high-risk breast cancer patients: Implication for immunotherapy

Hazem Ghebeh, Eman Barhoush, Asma Tulbah, Naser Elkum, Taher Al-Tweigeri, Said Dermime, Hazem Ghebeh, Eman Barhoush, Asma Tulbah, Naser Elkum, Taher Al-Tweigeri, Said Dermime

Abstract

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 (Tregs). 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+ Tregs as a bad prognostic factor in breast tumors. Although the co-existence 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. 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+ Tregs was 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+ Tregs, 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 Tregs and B7-H1/PD-1 molecules.

Figures

Figure 1
Figure 1
Immunohistochemical staining of FOXP3+ Tregs B7-H1 +and PD-1+ in T lymphocytes of breast tissues. Representative micrographs at × 530 magnification of (A&D) CD3/FOXP3 double staining (red color, membranous for CD3 and brown nuclear color for FOXP3 expression). (B&E) B7-H1 single staining (brown color, membranous/cytoplasmic expression). (C&F) PD-1 single staining (brown color, membranous expression). Upper panel (A-C) is sections for normal breast duct and lower panel (D-F) is sections for infiltrating ductal carcinoma of the breast.
Figure 2
Figure 2
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.
Figure 3
Figure 3
Correlations between FOXP3+ Tregs, B7-H1+ TIL and PD-1+ TIL. (A) statistical analysis of FOXP3+ Tregs infiltration in tumor tissues with B7-H1+ TIL, as analyzed with ANOVA of three groups of breast cancer patients with different FOXP3+ Tregs infiltration (Low = 0 to 4%, Medium = 5 to 14% and High 15% and above of total CD3+ TIL), (B) their linear correlation analysis and (C) statistical analysis of FOXP3+ Tregs infiltration with PD-1+ TIL in breast cancer as analyzed with ANOVA of three groups of breast cancer patients with different FOXP3+ Tregs infiltration (Low = 0 to 4%, Medium = 5 to 14% and High 15% and above of total CD3+ TIL).
Figure 4
Figure 4
Immunohistochemical staining showing the expression of FOXP3, B7-H1 and PD-1 molecules by different subsets of TIL. Representative micrographs at × 530 magnification of (A) double staining of B7-H1 (brown color, membranous/cytoplasmic) and FOXP3 (red color, nuclear) in an area rich in TIL of tumor section. (B) Double staining of PD-1 (brown color, membranous) and FOXP3 (red color, nuclear) in sections from the same tumor as in A. (C) double staining of FOXP3 (brown color, nuclear) and CD8 (red color, membranous). (D) double staining of PD-1 (brown color, membranous) and CD8 (red color, membranous). Solid arrows indicate CD8+/PD-1+ T lymphocytes and dashed arrow indicates a CD8-/PD-1+ T lymphocyte. (E) Double staining of B7-H1 (brown color, membranous/cytoplasmic) and CD8 (red color, membranous). Solid arrow indicates a CD8+/B7-H1+ T lymphocyte and dashed arrow indicates a CD8-/B7-H1+ T lymphocyte.

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