IMMUNOLOGY ORIGINAL ARTICLE A distinct chemokine axis does not account for enrichment of Foxp3+ CD4+ T cells in carcinogen-induced fibrosarcomas Beatrice Ondondo,* Summary Emily Colbeck, Emma Jones, The frequency of CD4+ Foxp3+ regulatory T (Treg) cells is often signifi- Kathryn Smart, Sarah N. Lauder, cantly increased in the blood of tumour-bearing mice and people with James Hindley, Andrew Godkin, cancer. Moreover, Treg cell frequencies are often higher in tumours com- Bernhard Moser, Ann Ager and pared with blood and lymphoid organs. We wished to determine whether Awen Gallimore certain chemokines expressed within the tumour mass selectively recruit Institute of Infection Immunity and Treg cells, thereby contributing to their enrichment within the tumour- Biochemistry, Cardiff University School of Medicine, Cardiff, UK infiltrating lymphocyte pool. To achieve this goal, the chemokine profile of carcinogen-induced fibrosarcomas was determined, and the chemokine À receptor expression profiles of both CD4+ Foxp3 and CD4+ Foxp3+ T cells were compared. These analyses revealed that the tumours are charac- terized by expression of inflammatory chemokines (CCL2, CCL5, CCL7, CCL8, CCL12, CXCL9, CXCL10 and CX3CL1), reflected by an enrich- À ment of activated Foxp3 and Foxp3+ T cells expressing T helper type 1- + doi:10.1111/imm.12430 associated chemokine receptors. Notably, we found that CXCR3 T cells Received 02 July 2014; revised 01 December were significantly enriched in the tumours although curiously we found 2014; accepted 01 December 2014. no evidence that CXCR3 was required for their recruitment. Instead, À *Present address: Nuffield Department of CXCR3 marks a population of activated Foxp3 and Foxp3+ T cells, Medicine, The Jenner Institute (ORCRB), which use multiple and overlapping ligand receptor pairs to guide their University of Oxford, Oxford, OX3 7DQ, UK migration to tumours. Collectively, these data indicate that enrichment of + Correspondence: Awen Gallimore, Institute Foxp3 cells in tumours characterized by expression of inflammatory of Infection and Immunity, Cardiff chemokines, does not occur via a distinct chemokine axis, thus selective University School of Medicine, Cardiff CF14 chemokine blockade is unlikely to represent a meaningful therapeutic 4XN, UK. strategy for preventing Treg cell accumulation in tumours. Email: [email protected] Senior author: Awen Gallimore. Keywords: chemokines; regulatory T cells; tumour immunology. b Introduction support a role for transforming growth factor- in pro- moting their proliferation in tumours and in tumour- Tumours are known to employ a number of mechanisms draining lymph nodes (TDLN)9,10 and also for driving À to evade host immune responses. One such mechanism is conversion of conventional CD4+ Foxp3 T cells (Tconv suppression of otherwise potent T cells,1 natural killer cells) into CD4+ Foxp3+ Treg cells.11 In a previous study cells,2 natural killer T cells3 and dendritic cells4 by whereby the carcinogen methylcholanthrene (MCA) was CD4+ Foxp3+ regulatory T (Treg) cells. It has been shown used to induce fibrosarcomas in mice, we examined the in human tumours and in experimental animal models of T-cell receptor (TCR) repertoires of Tconv cells and Treg cancer that the frequency of Treg cells is significantly cells and found that the repertoires of tumour-infiltrating increased in peripheral blood, lymph nodes and spleens Tconv and Treg cells were distinct. This lack of TCR and that even higher frequencies are found within overlap observed between the two populations argues – tumours.5 7 strongly against the hypothesis that Treg cell enrichment Several mechanisms that may account for enrichment in tumours occurs through conversion of Tconv cells into of Treg cells are reviewed in ref. 8. There is evidence to Treg cells.12 Another possibility is that intra-tumoural Abbreviations: Ct, threshold cycle; NDLN, tumour non-draining lymph nodes; TDLN, lymph nodes draining the tumour 94 ª 2014 The Authors. Immunology Published by John Wiley & Sons Ltd., Immunology, 145, 94–104 This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. CXCR3+ T cells are enriched in carcinogen-induced tumours 2 Treg cell enrichment occurs through selective recruitment (RT ProfilerTM PCR array for Mouse inflammatory cyto- – of Treg cells via tumour-expressed chemokines.13 16 There kines and receptors; PAMM-011; SABiosciences) to detect is however a general lack of comparative data on the and quantify gene expression levels. Samples were run in chemokine receptor expression profiles of Tconv versus a 96-well plate in an ABI 7900HT FAST Block instrument Treg cells, limiting the understanding of whether a single (Applied Biosystems, Foster City, CA) using a two-step or multiple chemokine(s) can selectively promote Treg cycling programme as follows: step 1, 95° for 10 min; cell recruitment. step 2, 95° for 15 seconds, followed by 60° for 1 min; In the study described herein, we conducted a broad step 2 was repeated for 40 cycles. Data were analysed analysis of chemokine expression by MCA-induced fibro- using the DDCt method. Specific mRNA expression levels sarcomas and a side-by-side analysis of Foxp3+ and for each gene were normalized as a ratio relative to À Foxp3 CD4+ T cells in terms of their phenotype and expression of internal control housekeeping genes, namely migratory capacity. The study describes delineation of the glucuronidase b (Gusb), hypoxanthine guanine phospho- chemokine profile of MCA-induced tumours as well as ribosyl transferase 1 (Hprt1), heat-shock protein 90 000 a the chemokine receptors expressed by both Tconv and cytosolic class B member 1 (Hsp90ab1), glyceraldehyde-3- Treg cells. This information was subsequently used to test phosphate dehydrogenase (Gapdh) and cytoplasmic the hypothesis that the tumour chemokine profile allows b-actin (Actb). for selective accumulation of Treg cells, thereby contrib- uting to immunosuppression within the tumour microen- Lymphocyte isolation vironment. Spleens and lymph nodes were disrupted by mashing l Materials and methods through a 40- m nylon cell strainer (BD Biosciences, San Jose, CA) using a sterile 2-ml syringe plunger. Tumours were excised and chopped into pieces using scalpel blades. Mice The pieces were then mashed with a syringe plunger and Six- to eight-week-old female C57BL/6 (Thy1.1) mice and the resulting cell suspension was filtered through 70-lm Foxp3-GFP transgenic mice, obtained from Professor nylon cell strainers (BD Biosciences). The cell suspensions Alexander Rudensky,17 were housed under specific patho- were then centrifuged before red blood cells lysis using gen-free conditions. All experiments were conducted in ammonium–chloride–potassium lysis buffer (Gibco, compliance with UK Home Office regulations. Grand Island, NY). Tumour induction Flow cytometry and antibodies Mice were anaesthetized and injected subcutaneously (in Mononuclear cells isolated from spleens, lymph nodes the hind leg) with 400 lg of 3-methylcholanthrene and tumours were first stained with a dead cell marker (MCA; Sigma-Aldrich, St Louis, MO) in 100 ll of olive (LIVE/DEAD Fixable Aqua stain; Invitrogen) according oil. Tumours occurred between 80 and 150 days after to the manufacturer’s instructions. Cells were then injection. Tumour-bearing mice were killed before the washed and stained for cell surface markers and chemoki- tumours reached 1Á5 cm in diameter. ne receptors. Stained cells were washed, fixed and acquired on a FACS Canto II flow cytometer (BD Bio- sciences). Data analysis was performed using SUMMIT 4.3 RNA extraction and quantitative RT-PCR software (Dako, Glostrup, Denmark). The antibodies used Spleen, lymph node and tumour tissues were snap frozen were as follows: anti-CD4-Pacific Blue antibody (BioLeg- in liquid nitrogen and stored at À80° until needed. Total end, San Diego, CA), goat polyclonal antibody to CCR1 RNA was extracted using TRIzol reagent (Invitrogen, (Santa Cruz Biotechnology, Santa Cruz, CA) followed Carlsbad, CA) under the manufacturer’s specification. with phycoerythrin (PE)-conjugated rabbit polyclonal The quantity and quality of RNA were determined using antibody to goat IgG (Abcam, Cambridge, UK), rabbit the Agilent 2100 bioanalyzer (Agilent Technologies, Santa monoclonal antibody to CCR2 (Abcam) followed with Clara, CA), and only high-quality samples with an RNA PE-conjugated donkey polyclonal antibody to rabbit IgG integrity number ≥ 8 were used for quantitative RT-PCR. (Abcam), anti-CCR4-PE-Cy7 (BioLegend),anti-CCR3- Five micrograms of total RNA (equivalent to 50 ng RNA Alexa Fluor 647 (BioLegend), biotin-conjugated anti- per gene) was used for first-strand cDNA synthesis and CCR5 antibodies followed with streptavidin-PerCP-Cy5.5 elimination of contaminating genomic DNA, performed (both from eBioscience, San Diego, CA), anti-CXCR3- using the RT2 First strand kit (SABiosciences, Frederick, APC (eBioscience, San Diego, CA), anti-CXCR4-APC MD) as specified by the manufacturer. The cDNA was (BD Biosciences), rabbit polyclonal antibody to anti- used in a highly validated quantitative RT-PCR array CX3CR1 (Abcam) followed with PE-conjugated donkey ª 2014 The Authors. Immunology Published by John Wiley & Sons Ltd., Immunology, 145, 94–104 95 B. Ondondo et al. polyclonal antibody to rabbit IgG (Abcam). Foxp3 (Fig. 1b), thereby indicating that Foxp3+ T cells are not À expression was detected by