Abstract P4-04-11: Preferential expression of the chemokine receptor 8 (CCR8) on regulatory T cells (Treg) infiltrating human breast cancers represents a novel immunotherapeutic target
Authors: Plitas G , Konopacki C , Wu K , Paula B , Morrow M , Rudensky A 
Published: May 2016

Treg cells are identified by the expression of the transcription factor FoxP3 and preserve immune homeostasis by the establishment and maintenance of peripheral tolerance. This suppressive function however, limits anti-tumor immune responses and represents a critical obstacle to immunotherapy. Safely targeting Treg cells will require selective elimination of tumor infiltrating Treg cells, as systemic depletion will lead to immune related adverse events. We hypothesize that differential gene expression analysis of Treg cells isolated from human breast tumors and peripheral blood will identify a tumor specific means to target Treg cells for the immunotherapy of breast cancer.

Methods: Tumor infiltrating lymphocytes were isolated from fresh operative specimens of patients undergoing surgery for primary invasive breast carcinoma. Lymphocytes were also isolated from normal breast tissue and peripheral blood buffy coat. T cell subsets including Treg cells were isolated by fluorescent activated cell sorting and analyzed by RNAseq. Mixed bone marrow chimeric mice were generated by reconstituting irradiated immunodeficient mice with a mixture of CCR8-/- + FoxP3-/- or CCR8+/+ + FoxP3-/- bone marrow, thus creating mice with Treg cells lacking CCR8 and controls.

Results: We found that Treg cells are more prevalent in breast tumors as opposed to normal breast tissue regardless of the biologic subtype of breast cancer (p&lt;0.05). Gene expression profiling of Treg cells and CD4 T cells isolated from tumor or blood revealed a distinct tumor Treg cell gene signature. This signature was enriched for cytokine binding and chemokine receptor GO categories (FDR&lt;0.005). Specifically, we identified CCR8 to be differentially and robustly expressed on tumor infiltrating Treg cells. This was validated by flow cytometry on over 50 primary breast cancers where the mean florescence intensity of CCR8 on Treg cells was at least twice that observed on conventional CD4 T cells (p&lt;0.05). CCR8 expression on Treg cells also significantly correlated with higher-grade cancers (p&lt;0.05). Using a data set generated by the Cancer Genome Atlas, we found that a high CCR8/FOXP3 gene expression ratio is strongly associated with worse disease free and overall survival of breast cancer (p&lt;0.001) patients while FOXP3 gene expression level alone does not predict disease outcome. To investigate the role of CCR8 expression on Treg cells in a preclinical murine model of mammary carcinogenesis, we implanted syngeneic polyoma middle-T antigen-driven breast cancer cells in the mammary fat pads of mixed bone marrow chimeric mice in which Treg cells lack CCR8 expression. CCR8 deficiency in Treg cells significantly decreased primary tumor progression and distant metastases without any overt immunopathology (p&lt;0.05).

Conclusions: Treg cells infiltrate human breast cancers and suppress anti-tumor immune responses. Our results demonstrate that CCR8 is selectively expressed by human breast cancer infiltrating Treg cells. Targeting CCR8 represents a promising immunotherapeutic approach for the treatment of patients with breast cancer. Depleting CCR8 antibodies are currently in development for additional preclinical and human studies.

Citation Format: Plitas G, Konopacki C, Wu K, Paula B, Morrow M, Rudensky A. Preferential expression of the chemokine receptor 8 (CCR8) on regulatory T cells (Treg) infiltrating human breast cancers represents a novel immunotherapeutic target. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P4-04-11.