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

Ghebeh H, Barhoush E, Tulbah A, Elkum N, Al-Tweigeri T, Dermime S - BMC Cancer (2008)

Bottom Line: B7-H1 is an inhibitory molecule that binds to PD-1 on T lymphocytes, while FOXP3 is a marker for regulatory T cells (Tregs).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).

View Article: PubMed Central - HTML - PubMed

Affiliation: Tumor Immunology Section, King Faisal Specialist Hospital and Research Center, P,O, Box 3354, Riyadh 11211, Saudi Arabia. hghebeh@kfshrc.edu.sa

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.

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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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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.

Mentions: We next asked whether FOXP3, PD-1 and B7-H1 molecules 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 performed. The selections were based on sections that had a good morphology to make the interpretation easier. Double-staining assays showed that B7-H1 and FOXP3 molecules 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 addition, 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 lymphocyte subsets (Figure 4B). Furthermore, double staining assays demonstrated that FOXP3 and CD8 were mainly expressed by two different T lymphocyte subsets (Figure 4C) and only very few CD8+ TIL (< 5%) were FOXP3+. Therefore, it seems that most FOXP3+ Tregs described here are of the CD4+ subset since they were restricted to CD3+ T lymphocytes. On the other hand PD-1 molecule was mainly expressed by CD8+ T lymphocytes with only few CD8- population were positive for PD-1 (Figure 4D). B7-H1 was mainly expressed by CD8- T lymphocytes (Figure 4E) consistent with our previous observation of its expression by CD4+ T lymphocytes [18].


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

Ghebeh H, Barhoush E, Tulbah A, Elkum N, Al-Tweigeri T, Dermime S - BMC Cancer (2008)

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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Related In: Results  -  Collection

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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.
Mentions: We next asked whether FOXP3, PD-1 and B7-H1 molecules 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 performed. The selections were based on sections that had a good morphology to make the interpretation easier. Double-staining assays showed that B7-H1 and FOXP3 molecules 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 addition, 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 lymphocyte subsets (Figure 4B). Furthermore, double staining assays demonstrated that FOXP3 and CD8 were mainly expressed by two different T lymphocyte subsets (Figure 4C) and only very few CD8+ TIL (< 5%) were FOXP3+. Therefore, it seems that most FOXP3+ Tregs described here are of the CD4+ subset since they were restricted to CD3+ T lymphocytes. On the other hand PD-1 molecule was mainly expressed by CD8+ T lymphocytes with only few CD8- population were positive for PD-1 (Figure 4D). B7-H1 was mainly expressed by CD8- T lymphocytes (Figure 4E) consistent with our previous observation of its expression by CD4+ T lymphocytes [18].

Bottom Line: B7-H1 is an inhibitory molecule that binds to PD-1 on T lymphocytes, while FOXP3 is a marker for regulatory T cells (Tregs).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).

View Article: PubMed Central - HTML - PubMed

Affiliation: Tumor Immunology Section, King Faisal Specialist Hospital and Research Center, P,O, Box 3354, Riyadh 11211, Saudi Arabia. hghebeh@kfshrc.edu.sa

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.

Show MeSH
Related in: MedlinePlus