Abstract A73: The selective recruitment and retainment of regulatory T cells in human colorectal cancer.
Authors: Ward Stephen T. , Hepburn Elizabeth A. , Li Ka-kit , Curbishley Stuart M. , Hejmadi Rahul K. , Ismail Tariq , Bicknell Roy , Rot Antal , Adams David H. 
Published: August 2014

Background: Colorectal cancer (CRC) is the third most common malignancy in the United States and United Kingdom and deadly in one third of patients. Tumor lymphocytic infiltration is associated with improved survival and analysis of some lymphocyte subsets has revealed that their prognostic ability rivals the TNM tumor staging system.

Regulatory T cells (Tregs) are known to be enriched in CRC and it is postulated that they promote immunological tumor escape by suppression of effector T cell responses. Little is known however about the signals controlling entry of Tregs into CRC.

Methods: Matched CRC, distal colonic tissue, draining lymph node and blood were obtained from patients undergoing resection of CRC having given informed consent. The study was approved by the Local Research and Ethics Committee. Tumor-infiltrating lymphocytes were isolated from fresh tissue and phenotyped for chemokine receptors and various other markers using multicolor flow cytometry.

The presence of tissue chemokines was analyzed using real-time PCR and Western blotting. CD3+ cells were isolated from tumor tissue and placed in a transwell system to measure chemotaxis in response to various chemokines. Migrated and non-migrated T cells were phenotyped to establish differential migration of T cell subsets. CD4+CD127lowCD25+ T cells were isolated by fluorescent-activated cell sorting from tumor tissue and incubated with fluorescent-labeled responder cells in standard suppression assays.

Peripheral blood lymphocytes were co-cultured with tumor supernatant and effects on lymphocyte phenotype and proliferation were analyzed by flow cytometry.

Results: The proportion of T cells with a suppressive phenotype (Treg, CD4+CD127lowCD25+) was significantly increased in CRC compared to matched distal colon. More than 95% of this cell population expressed the transcription factor, foxp3.

The chemokine receptor CCR5 was found to be markedly upregulated on Treg compared to other effector T cells and in CRC compared to distal colon. CCR4 and to a lesser extent, CCR6, were also upregulated on Treg compared to other T cells. The ligands for CCR5 (CCL3, CCL4 and CCL5) were overexpressed in CRC tissue compared to matched colon. CCL4 was found to localize to the tumor endothelium. The ligands for other chemokine receptors were not overexpressed in the tumor tissue compared to the colon and were therefore not investigated further.

Tumor-resident Treg migrated in response to CCR5 ligands and blockade of CCR5 inhibited this migration. Co-culture experiments demonstrated that CCR5 was upregulated on peripheral blood lymphocytes, and especially Treg, in mixed lymphocyte reactions. This effect that could be augmented by the addition of tumor supernatant.

Conclusion: Conditions exist to actively recruit CCR5+ Treg into colorectal cancer tissue in humans. CCR5 upregulation is also promoted by the tumor microenvironment that may provide a tumor-retention signal. CCR5 inhibition may prove to be a novel immunotherapy for colorectal cancer by blocking the selective recruitment and/or egress of suppressive Treg, thereby promoting an anti-tumor immune response.

Citation Format: Stephen T. Ward, Elizabeth A. Hepburn, Ka-kit Li, Stuart M. Curbishley, Rahul K. Hejmadi, Tariq Ismail, Roy Bicknell, Antal Rot, David H. Adams. The selective recruitment and retainment of regulatory T cells in human colorectal cancer. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr A73.