Löfroos et al. Eur J Med Res (2017) 22:40 DOI 10.1186/s40001-017-0283-8 European Journal of Medical Research RESEARCH Open Access Colorectal cancer‑infltrating T lymphocytes display a distinct chemokine receptor expression profle Ann‑Britt Löfroos1,2†, Mohammad Kadivar2†, Sabina Resic Lindehammer1,2 and Jan Marsal1,2,3* Abstract Background: T lymphocytes exert important homeostatic functions in the healthy intestinal mucosa, whereas in case of colorectal cancer (CRC), infltration of T lymphocytes into the tumor is crucial for an efective anti-tumor immune response. In both situations, the recruitment mechanisms of T lymphocytes into the tissues are essential for the immunological functions deciding the outcome. The recruitment of T lymphocytes is largely dependent on their expression of various chemokine receptors. The aim of this study was to identify potential chemokine receptors involved in the recruitment of T lymphocytes to normal human colonic mucosa and to CRC tissue, respectively, by examining the expression of 16 diferent chemokine receptors on T lymphocytes isolated from these tissues. Methods: Tissues were collected from patients undergoing bowel resection for CRC. Lymphocytes were isolated through enzymatic tissue degradation of CRC tissue and nearby located unafected mucosa, respectively. The expres‑ sion of a broad panel of chemokine receptors on the freshly isolated T lymphocytes was examined by fow cytometry. Results: In the normal colonic mucosa, the frequencies of cells expressing CCR2, CCR4, CXCR3, and CXCR6 dif‑ fered signifcantly between ­CD4+ and ­CD8+ T lymphocytes, suggesting that the molecular mechanisms mediating T lymphocyte recruitment to the gut difer between ­CD4+ and ­CD8+ T lymphocytes. In CRC, the frequencies of cells expressing CCR2 and CXCR5 were signifcantly lower in both the ­CD4+ and ­CD8+ T lymphocyte populations compared to unafected colonic mucosa, and the frequency of ­CCR9+ cytotoxic T lymphocytes was signifcantly decreased in CRC tissue. Conclusions: With regard to the normal gut mucosa, the results suggest that the molecular mechanisms mediating T lymphocyte recruitment difer between ­CD4+ and ­CD8+ T lymphocytes, which are important for understanding gut homeostasis. Importantly, T lymphocytes from CRC compared to normal colonic tissue displayed a distinct chemokine receptor expression profle, suggesting that mechanisms for recruitment of T lymphocytes to CRC tissue are skewed compared to normal colonic mucosa. Understanding these mechanisms could help in developing new strategies in cancer immunotherapy and to optimize already available alternatives such as immune checkpoint inhibitors. Keywords: Chemokine receptor, T lymphocyte, Colorectal cancer, Mucosa, Homing Background death in Western countries [1]. Typically, the patient Colorectal cancer (CRC) is the third most common type raises an adaptive immune response towards the tumor, of cancer in adults and the second leading cause of cancer giving rise to tumor-infltrating T lymphocytes (TIL) [2, 3]. Te lymphocytes are recruited from the blood cir- *Correspondence: [email protected] culation to the tumor site and infltrate the tumor mass; †Ann-Britt Löfroos and Mohammad Kadivar have contributed equally to a sequential process directed by adhesion molecules, this work chemokines, and chemokine receptors. 3 Department of Gastroenterology, Skane University Hospital, 22185 Lund, Sweden Chemokines constitute a large family (48 known Full list of author information is available at the end of the article human members) of small (7–10 kDa) peptides that are © The Author(s) 2017. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Löfroos et al. Eur J Med Res (2017) 22:40 Page 2 of 10 classifed according to the position of their conserved and non-infamed colonic mucosal T lymphocytes in cysteine residues into four groups: CXC, CC, C, and humans [22–26]. CX3C [4, 5]. Based on their function, they are referred to Cancer tissue is typically infltrated by efector T lym- as homeostatic, infammatory, or both [6]. Chemokines phocytes and the presence of these TILs has been corre- mediate their efects by binding to 7-transmembrane lated with improved clinical outcome in diferent human G-protein coupled receptors expressed on target cells [4]. cancers, including CRCs [2, 3, 27, 28]. In a study by Tere are 18 identifed human chemokine receptors (and Galon et al., patients with high frequencies of infltrating 5 additional atypical receptors), grouped according to the CD3+ T lymphocytes within their colorectal tumors had type of ligand they bind, and have been named CXCR1 a better 5-year survival rate (73%) than those with low through 6, CCR1 through 10, CX3CR1, and XCR1 [7]. numbers of CD3 + T lymphocytes (30%) [29]. Further- Chemokines are mainly known for their ability to direct more, it has been shown that both CD4 + and CD8 + efec- leukocyte recruitment and migration under both homeo- tor T lymphocytes may have anti-tumor properties and static and infammatory conditions [6, 8, 9]. Homeostatic independently correlate with improved outcome [30–32]. chemokines (constitutively expressed) are important Tus, TILs and their ability to localize to CRCs consti- for several physiological processes such as embryogen- tute attractive targets for cancer immunotherapy. Indeed, esis, hematopoiesis, and lymphocyte trafcking [8, 10], new anti-cancer therapies, such as ipilimumab which is a whereas the expression of infammatory chemokines is blocking antibody to cytotoxic T lymphocyte-associated induced upon pathological events, such as infammation protein-4 (CTLA-4), are associated with increased infl- and cancer growth [8, 11]. Furthermore, in cancer biol- tration of T lymphocytes into the tumor tissue and signif- ogy, chemokines play a role in a number of additional icantly increased survival rates [33]. Despite the fact that processes, such as tumor cell growth/survival, metastatic these studies recognize the protective efect of infltrating spreading, and angiogenesis [11–13]. T lymphocytes against cancer and that the initial studies Te colonic mucosa is lined by a single-cell layer of regarding TILs were published almost 30 years ago, the columnar epithelium sitting on a laminin/collagenous molecular mechanisms whereby TILs are recruited into basement membrane. Underlying this is a layer of loose tumors remain elusive [34, 35]. connective tissue called lamina propria (LP), which in A number of chemokines, such as CCL2–4, CXCL1, turn is demarcated by a thin smooth muscular layer CXCL5, and CXCL8–10, have been shown to be present (muscularis mucosae). Te formation of CRC is thought at elevated levels in the CRC microenvironment com- to start through inappropriate epithelial proliferative and pared to normal tissues [36, 37]. However, it remains antiapoptotic activity leading to formation of adenomas, unclear which chemokine receptors T lymphocytes use to which evolve into pre-invasive carcinoma in situ [14]. infltrate into the tumor mass. Te previous studies have Pre-invasive CRCs eventually acquire the ability to invade presented data for a few of the known chemokine recep- through the submucosa and muscularis propria, and tors regarding the expression on TILs in CRC (i.e., CCR2, fnally to metastasize [14]. Te progression from normal CCR4–7, and CXCR3) [38–43]. Te results of a recently epithelium through adenoma to colorectal carcinoma is published study showed that CXCR3 was expressed on characterized and driven by multiple accumulated muta- signifcantly fewer CD4 + and CD8+ T lymphocytes in tions of cancer genes [15]. tumor tissue compared to the unafected tissue [44]. In In normal colonic mucosa, efector lymphocytes that contrast, there was a higher frequency of CD4 + T lym- localize to the epithelial compartment (intraepithelial phocytes expressing CCR4 in the tumor tissue [44]. lymphocytes; IEL) are primarily CD8 + T lymphocytes. Te aim of the current study was to identify potential Interestingly, IEL have been implicated in the control of chemokine receptors involved in T lymphocyte recruit- intestinal epithelial cell growth/turnover and in sensing ment to human normal colonic mucosa and to CRC and eliminating cancerous or injured epithelial cells [16, tissue. We examined the expression of 16 chemokine 17]. In contrast, the LP contains mainly CD4+ T lympho- receptors on CD4+ and CD8+ T lymphocytes in normal cytes (lamina propria lymphocytes; LPL) at an approxi- colonic mucosa as compared with CRC tissue. Te results mate CD4/CD8 ratio of 2:1 at homeostatic conditions showed that CD4+ and CD8+ T lymphocytes in unaf- and is thought to be important in maintaining a tolero- fected colonic mucosa have distinct chemokine receptor genic environment [18]. Colonic T lymphocyte homing is profles, and furthermore that T lymphocytes from unaf- less studied than homing to the small intestine, but likely fected colonic tissue as compared with CRC tissue difer requires either integrin α4β7 or α4β1 [19–21]. In addi- in their chemokine receptor