The effects of CCR5 inhibition on regulatory T-cell recruitment to colorectal cancer

Abstract

Background: Regulatory T cells (Treg) are enriched in human colorectal cancer (CRC) where they suppress anti-tumour immunity. The chemokine receptor CCR5 has been implicated in the recruitment of Treg from blood into CRC and tumour growth is delayed in CCR5-/- mice, associated with reduced tumour Treg infiltration.

Methods: Tissue and blood samples were obtained from patients undergoing resection of CRC. Tumour-infiltrating lymphocytes were phenotyped for chemokine receptors using flow cytometry. The presence of tissue chemokines was assessed. Standard chemotaxis and suppression assays were performed and the effects of CCR5 blockade were tested in murine tumour models.

Results: Functional CCR5 was highly expressed by human CRC infiltrating Treg and CCR5(high) Treg were more suppressive than their CCR5(low) Treg counterparts. Human CRC-Treg were more proliferative and activated than other T cells suggesting that local proliferation could provide an alternative explanation for the observed tumour Treg enrichment. Pharmacological inhibition of CCR5 failed to reduce tumour Treg infiltration in murine tumour models although it did result in delayed tumour growth.

Conclusions: CCR5 inhibition does not mediate anti-tumour effects as a consequence of inhibiting Treg recruitment. Other mechanisms must be found to explain this effect. This has important implications for anti-CCR5 therapy in CRC.

Figure 1

(A) Representative flow cytometry data of surface CCR5 expression by Tconv (blue) and Treg (red), isolated from CRC, compared with isotype-matched control antibody (grey, IMC). (B) Mean fold change in relative quantification of mRNA expression of Foxp3, CCR5, KLF2 and CREB1 to GAPDH by Treg relative to Tconv, isolated from CRC (n=5). Error bars represent the standard error of the mean. Asterisks indicate statistically significant differences (Wilcoxon signed-rank test). (C) Double immunohistochemistry of FFPE CRC sections, stained for Foxp3 (black nuclear stain) and CCR5 (red cytoplasmic stain). Two representative samples are shown (top and bottom) of 4 samples stained. Two magnifications are shown (left, magnification × 100 and right, magnification × 400) (left, magnification × 100) and at higher magnification (right, × 400). Open arrows indicate double-positively stained cells. Abbreviation: CRC=colorectal cancer; FFPE=formalin-fixed paraffin-embedded.

Figure 2

(A) Relative quantification of chemokine mRNA expression to GUS and IPO8 by tissue type. Capped lines indicate statistically significant differences (Wilcoxon signed-rank test). (B) Western blots probing for CCL3, CCL4 and GUS in eight samples of CRC with matched distal colon of standard protein concentration (2 mg BSA per ml). (C) Semi-quantitative measurement of CCL3, CCL4 and CCL5 levels by ELISA in eight samples of CRC with matched distal colon of standard protein concentration (2 mg BSA per ml). Capped lines indicate statistically significant differences (Wilcoxon signed-rank test). (D) Immunohistochemistry of CCL4 in frozen sections of CRC (representative of four cases), magnification × 200 (top) and × 400 (bottom). Asterisks indicate positive CCL4 staining of the tumour endothelium. Abbreviations: CRC=colorectal cancer; GUS=ß-glucuronidase; IPO8=Importin-8; DC=distal colon; BSA=bovine serum albumin.

Figure 3

(A) Left: percentage expression of HELIOS by CRC-isolated Tconv and matched Treg, measured by flow cytometry (n=4). Right: mean percentage methylation of the TSDR locus by cell type. Error bars represent the data range (n=4). Capped lines indicate statistically significant differences (Mann-Whitney test). (B) Representative data from a Treg suppression assay: peripheral blood responder T cells (Tresp) were cultured alone (top left) or with Treg Suppression Inspector beads (Miltenyi Biotec Ltd, UK) (top right). Tresp were then cultured with CRC-isolated CCR5^low Treg, CCR5^high Treg or CD4⁺CD25⁻ (Tconv) cells at given ratios. Percentage in top right of each histogram represents the percentage proliferation of Tresp. (C) Transwell migration assay of CRC-isolated Treg (left) and Tconv (right), pre-treated with either 1 μM Maraviroc or DMSO-vehicle, to media alone or media containing 20 ng ml⁻¹ recombinant CCL4 (n=6 per group). (D) The Treg and Tconv fraction of lymphocytes migrating across the transwell. Capped lines indicate statistically significant differences between groups (Wilcoxon signed-rank test). Abbreviations: CRC=colorectal cancer; DMSO=dimethyl sulphoxide.

Figure 4

(A) In vitro proliferation assay of CT26 and B16-F10 cells cultured with different concentrations of met-RANTES (CT26), TAK-779 (CT26) and UK-484900 (B16-F10) in triplicate. Error bars represent 95% confidence intervals about the mean. (B, C) Median percentage and MFI expression of surface CCR5 by tumour-isolated Treg (CD4⁺Foxp3⁺), Tconv (CD4⁺Foxp3⁻) and CD8⁺ cells, from wild-type BALB/c mice (CT26 tumours) and hCCR5KI mice (B16-F10 tumours) treated with 10 days of PBS (n=10 per group). Error bars represent the IQR about the median. Capped lines indicate statistically significant differences (Wilcoxon signed rank test). Abbreviations: MFI=median fluorescent intensity; PBS=phosphate-buffered saline.

Figure 5

Tumour growth kinetics as measured by BLI for CT26 tumours in wild-type BALB/c mice (A) and B16-F10 tumours in hCCR5KI mice (D), treated with injections of PBS (controls) or CCR5 antagonists. Tumour growth kinetics by calliper measurements for CT26 tumours in wild-type BALB/c mice (B) and B16-F10 tumours in hCCR5KI mice (E), treated with injections of PBS (controls) or CCR5 antagonists. CT26 (C) and B16-F10 (F) tumour weights post resection in mice treated with PBS (controls) or CCR5 antagonists. Error bars represent the standard error about the mean. Asterisks indicate statistically significant differences compared with the control group (Mann-Whitney). Capped lines indicate statistically significant differences between groups (Mann-Whitney). Abbreviations: BLI=bioluminescent imaging; PBS=phosphate-buffered saline.

Figure 6

The proportion of tumour-isolated CD4⁺ cells with a Treg (CD4⁺Foxp3⁺) phenotype, the Treg proportion, in BALB/c mice (A) and hCCR5KI mice (B), treated either with PBS (controls) or CCR5 antagonists. (C) Mean tumour tissue and serum levels of CCL3, CCL4, CCL5 and VEGF, measured by semi-quantitative ELISA, in hCCR5KI mice, treated either with PBS (controls) or UK-484900 (n=5 per group). Error bars represent the standard error of the mean. Capped lines indicate statistically significant differences (Mann-Whitney). Abbreviations: hCCR5KI=human CCR5 knock-in; PBS=phosphate-buffered saline.