Preferential accumulation of regulatory T cells with highly immunosuppressive characteristics in breast tumor microenvironment

Azharuddin Sajid Syed Khaja, Salman M Toor, Haytham El Salhat, Issam Faour, Navid Ul Haq, Bassam R Ali, Eyad Elkord, Azharuddin Sajid Syed Khaja, Salman M Toor, Haytham El Salhat, Issam Faour, Navid Ul Haq, Bassam R Ali, Eyad Elkord

Abstract

Immunosuppressive cells such as regulatory T cells (Tregs) have an ambiguous role in breast cancer prognosis, with studies reporting both positive and negative correlations between Treg infiltration and prognosis. This discrepancy could be due to the different immunosuppressive molecules present in these cells. In the present study, we phenotypically characterize different Treg subsets infiltrating the tumor microenvironment (TME), compared to adjacent normal tissue and peripheral blood of primary breast cancer (PBC) patients. We report that the majority of tumor-infiltrating CD4+ and CD8+ T cells have terminally exhaustive phenotype as assessed by CD39 and PD-1 expressions. We also show that Tregs are accumulated in breast TME compared to normal tissue. Further characterization of Tregs showed that these are mainly FoxP3+Helios+ and express high levels of CTLA-4 and PD-1. This preferential accumulation of FoxP3+Helios+ Treg subset with co-expression of different immune inhibitory molecules might have a negative effect on breast cancer prognosis. Taken together, our results suggest that breast tumor cells might utilize Tregs, and different suppressive pathways involving CD39, PD-1 and CTLA-4 molecules in creating an immune-subversive environment for them to survive, and a dual blockade of these immunosuppressive molecules might be considered as an effective method in breast cancer treatment.

Keywords: FoxP3; helios; immune checkpoint receptors; primary breast cancer; regulatory T cells.

Conflict of interest statement

CONFLICTS OF INTEREST

The authors declare no conflicts of interest.

Figures

Figure 1. T-cell infiltration in normal and…
Figure 1. T-cell infiltration in normal and tumor tissues in primary breast cancer
(A). Representative images of immunohistochemical staining of tumor-infiltrating CD3+ T cells in formalin-fixed paraffin embedded breast non-tumor (NT) and tumor tissues (TT). (B). Freshly isolated immune cells infiltrating NT (NILs) and TT (TILs) from 11 PBC patients were stained with 7AAD, CD45, CD3, CD4 and CD8 antibodies for identification of T cells and their subsets. Representative flow cytometric plots of surface staining from one cancer patient are shown. 7AAD dye was used to gate live cells, followed by lymphocyte identification by CD45 and CD3 stainings. Different subsets of T cells were then characterized using CD4 and CD8 antibodies. (C). Scatter plots showing the differences in tissue-infiltrating CD45+CD3−, CD45+CD3+, CD4+ and CD8+ cells between NILs and TILs.
Figure 2. Phenotypic characteristics of CD4 +…
Figure 2. Phenotypic characteristics of CD4+ T cells in NILs and TILs
Freshly isolated NILs and TILs were stained for CD3, CD4, CD25, PD-1, CD39 and LAP surface markers and their relative frequencies were calculated in CD4+ T cells. (A). Representative flow cytometric plots for these markers in NILs and TILs from a cancer patient. (B). NILs and TILs from one cancer patient showed exceptionally high levels of LAP expression. (C). Scatter plots showing the differences in CD25, PD-1, CD39 and LAP between NILs and TILs.
Figure 3. Phenotypic characterization of CD3 +…
Figure 3. Phenotypic characterization of CD3+CD4− (CD8+) T cells in NILs and TILs
Freshly isolated NILs and TILs were stained for CD3, CD4, CD25, PD-1 and CD39 surface markers. Live cells were gated using 7AAD dye. Levels of CD25, PD-1 and CD39 markers were calculated in CD8+ T cells. (A). Representative flow cytometric plots for these markers in NILs and TILs from a cancer patient are shown. (B). Scatter plots comparing the differences between these surface markers in CD8+ T cells between NILs and TILs. (C). Scatter plots showing the differences in these markers between CD4+ and CD8+ populations in TILs.
Figure 4. Expression of PD-1/CD39 in CD4…
Figure 4. Expression of PD-1/CD39 in CD4+ and CD8+ T cells in NILs and TILs
Representative flow cytometric plots for co-expression of PD-1/CD39 in CD4+ (A) and CD8+ (B) T cells. Scatter plots comparing PD-1/CD39 co-expression between NILs and TILs in CD4+ (C) and CD8+ (D) T cells.
Figure 5. Expression of PD-1/CTLA-4 in CD4…
Figure 5. Expression of PD-1/CTLA-4 in CD4+ and CD8+ T cells
PBMC from HD and PBC patients, NILs and TILs were stained for CD3, CD4 and PD-1 surface markers. After fixation and permeabilization, cells were stained for intracellular CTLA-4. Live cells were gated using Fixable Viability Dye 660. Representative flow cytometric plots showing PD-1 and CTLA-4 co-expression in CD4+ T cells (A) and whisker plots (B) showing differences in their expression in HD-PBMC, PBC-PBMC, NILs and TILs. (C). Pie charts show the relative percentages of PD-1 and CTLA-4 co-expression in CD4+ T cells. (D). Representative flow cytometric plots for co-expression of PD-1/CTLA-4 in CD8+ cells in NILs and TILs are shown.
Figure 6. FoxP3 and Helios expression in…
Figure 6. FoxP3 and Helios expression in CD4+ T cells
PBMC from HD and PBC patients, NILs and TILs were stained for CD3 and CD4 antibodies followed by intracellular staining for FoxP3 and Helios. Live cells were first gated using Fixable Viability Dye 660. (A). Representative flow cytometric plots of FoxP3 staining from one cancer patient are shown. (B). Scatter plot showing the differences in frequencies of FoxP3+ Tregs between different samples. (C). IHC staining of FoxP3+ expression in one NT and TT samples. (D). Flow cytometric plots of FoxP3 and Helios co-expression in CD4+ T cells from different samples and whisker plots (E) showing differences in various FoxP3 and Helios-expressing Treg subsets. (F). Non-parametric Spearman's test showing correlations between FoxP3 and Helios expressions in NILs and TILs. (G). Pie charts show the relative percentages of different FoxP3 and Helios Treg subsets.
Figure 7. PD-1 and CTLA-4 expression in…
Figure 7. PD-1 and CTLA-4 expression in different FoxP3 and Helios Treg subsets
(A). Non-parametric Spearman's test showing correlations between CTLA-4 and FoxP3, and CTLA-4 and Helios expressions in TILs. (B). Representative flow cytometric plots showing PD-1 and CTLA-4 expression in different FoxP3/Helios Treg subsets from HD-PBMC, PBC-PBMC, NILs and TILs. (C). Whisker plots comparing the levels of PD-1+CTLA4+ cells in FoxP3−Helios+, FoxP3+Helios+ and FoxP3+Helios− Treg subsets within different samples.

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