Dual role of tumour-infiltrating T helper 17 cells in human colorectal cancer

Abstract

Background: The immune contexture predicts prognosis in human colorectal cancer (CRC). Whereas tumour-infiltrating CD8+ T cells and myeloid CD16+ myeloperoxidase (MPO)+ cells are associated with favourable clinical outcome, interleukin (IL)-17-producing cells have been reported to correlate with severe prognosis. However, their phenotypes and functions continue to be debated.

Objective: To investigate clinical relevance, phenotypes and functional features of CRC-infiltrating, IL-17-producing cells.

Methods: IL-17 staining was performed by immunohistochemistry on a tissue microarray including 1148 CRCs. Phenotypes of IL-17-producing cells were evaluated by flow cytometry on cell suspensions obtained by enzymatic digestion of clinical specimens. Functions of CRC-isolated, IL-17-producing cells were assessed by in vitro and in vivo experiments.

Results: IL-17+ infiltrates were not themselves predictive of an unfavourable clinical outcome, but correlated with infiltration by CD8+ T cells and CD16+ MPO+ neutrophils. Ex vivo analysis showed that tumour-infiltrating IL-17+ cells mostly consist of CD4+ T helper 17 (Th17) cells with multifaceted properties. Indeed, owing to IL-17 secretion, CRC-derived Th17 triggered the release of protumorigenic factors by tumour and tumour-associated stroma. However, on the other hand, they favoured recruitment of beneficial neutrophils through IL-8 secretion and, most importantly, they drove highly cytotoxic CCR5+CCR6+CD8+ T cells into tumour tissue, through CCL5 and CCL20 release. Consistent with these findings, the presence of intraepithelial, but not of stromal Th17 cells, positively correlated with improved survival.

Conclusions: Our study shows the dual role played by tumour-infiltrating Th17 in CRC, thus advising caution when developing new IL-17/Th17 targeted treatments.

Keywords: CANCER IMMUNOBIOLOGY; COLORECTAL CANCER; IMMUNE RESPONSE; INFLAMMATORY MEDIATORS; T LYMPHOCYTES.

Figure 1

Tumour infiltration by interleukin-17 (IL-17)-producing cells does not predict survival in colorectal cancer (CRC). IL-17 expression was evaluated by immunohistochemistry on a tissue microarray (TMA) including 1151 cases of primary CRC. (A) Representative pictures of IL-17 staining I (see ‘Materials and methods’). Numbers of IL-17+ cells per punch are indicated. (B) Distribution of IL-17+ cells within the epithelial or stromal fraction of healthy colonic tissues (Ctr) or tumour samples (T). Statistical significance was assessed by χ² test. (C) Kaplan–Meier curves illustrating overall survival (OS, left panel) and relapse-free survival (RFS, right panel) probability according to IL-17+ cell density. Numbers of deaths/total cases within each category are indicated. Statistical significance was assessed by log-rank test. (D) Numbers of IL-17+ cells within CRC cases characterised by low or high infiltration of CD16+, MPO+ and CD8+ cells, according to cut-off scores identified by receiver operating characteristic curve analysis, as described in ‘Materials and methods’. Statistical significance was assessed by χ² test. MPO, myeloperoxidase.

Figure 2

Colorectal cancers (CRC)-infiltrating interleukin (IL)-17+ cells are polyfunctional Th17. Single cell suspensions from freshly excised clinical specimens of CRC and corresponding tumour-free colonic mucosa (Ctr) and peripheral blood mononuclear cells from healthy donors (PBMC HD) or patients with CRC (PBMC Pz), were incubated with phorbol 12-myristate 13-acetate (PMA)/ionomycin/brefeldin for 5 h. Surface staining for specific cell population markers and intracellular staining for Foxp3 and cytokines was then performed. (A) Representative flow cytometric analysis of CRC infiltrates stained for IL-17 and the indicated cell-specific markers. Tumour-infiltrating cells are gated based on physical parameters, as defined by analysis of autologous PBMC. (B) Frequencies of cells positive for the indicated markers within gated IL-17+ cells. Means are indicated by bars. Numbers of samples evaluated for each marker are indicated in parentheses. (C) Frequencies of IL-17+ cells on gated CD3+ T cells obtained from PBMC HD or PBMC CRC and single cell suspensions from freshly excised clinical specimens of Ctr and corresponding CRC. Means are indicated by bars. Numbers of samples evaluated for each marker are indicated in parentheses. Statistical significance was assessed by t test. (D) Representative flow cytometric analysis of intracellular cytokine staining on gated CRC-infiltrating CD3+CD4+ cells. (E) Frequencies of cells positive for the indicated cytokines gated on CD3+CD4+ IL-17+ T cells. Means are indicated by bars. Numbers of samples evaluated for each cytokine are indicated in parentheses. GM-CSF, granulocyte-macrophage colony stimulating factor; IFN, interferon; TNF, tumour necrosis factor.

Figure 3

Colorectal cancers (CRC)-Th17 mediate protumorigenic effects in an interleukin (IL)-17-dependent manner. Endothelial cells (EC), tumour-associated stromal cells (TASC) and CRC cell lines (LS180, COLO205) were conditioned for 24 h with CRC-Th17 supernatants untreated (Th17 SN) or pretreated with anti-IL-17 neutralising antibodies (Th17+ anti-IL-17). Vascular endothelial growth factor (VEGF) (A) or IL-6 (B) release was measured in culture supernatants by ELISA. Statistical significance was analysed by one-way analysis of variance. Data refer to experimental triplicates from two independent experiments performed with supernatants from two different clones. Ctr, healthy colonic tissue.

Figure 4

Colorectal cancers (CRC)-infiltrating Th17 favour recruitment and activation of neutrophils. (A) Migration of neutrophils, purified from blood of healthy donors, towards control medium (Ctr), supernatants of Th17 clones expanded from CRC-infiltrating cells (Th17 SN) or Th17 supernatants pre-treated with anti-interleukin (IL)-17 (Th17 SN+ anti-IL-17), anti-IL-8 (Th17 SN+ anti-IL-8), or anti-granulocyte-macrophage colony stimulating factor (anti-GM-CSF antibodies) (Th17 SN+ anti-GM-CSF), was evaluated after 90 min incubation by flow cytometry. Data refer to experimental triplicates from three independent experiments performed with supernatants from three different clones. Means±SD are depicted. Statistical significance was assessed by one-way analysis of variance (ANOVA). (B) Myeloperoxidase (MPO) release by neutrophils exposed to control medium (Ctr), Th17 supernatants (Th17 SN) or Th17 supernatants pretreated with anti-IL-17, IL-8 or GM-CSF antibodies was assessed after 4 h incubation by ELISA. Means±SD are depicted. Data refer to experimental triplicates from three independent experiments performed with supernatants from three different clones. Statistical significance was assessed by one-way ANOVA.

Figure 5

Th17 cells favour recruitment of CD8+ T cells by triggering chemokine release from endothelial cells (EC). (A) Peripheral blood cells (PB, n=7) and tumour cell suspensions from patients with colorectal cancer (CRC) (n=12) were surface stained for CD8 in combination with the indicated chemokine receptors. Left panels: representative flow cytometric analysis. Right panels: percentages of CD8+ cells expressing the indicated chemokine receptors. Means±SD are depicted. (B) The expression of the indicated chemokine genes was assessed on CRC samples (n=21) by quantitative PCR. Expression levels relative to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) are depicted. (C) Endothelial, tumour and stromal cells were sorted from cell suspensions derived from CRC specimens by flow cytometry, based on CD31, EpCAM and CD90 expression, respectively. mRNA levels of the indicated chemokine genes were assessed in sorted cells by quantitative PCR. Gene expression levels relative to GAPDH are depicted. Data refer to analysis of one representative sample out of four. (D) Chemokine release by HMEC cells untreated (EC) or exposed overnight to recombinant interleukin-17 (rIL-17; 50 ng/mL) (EC+IL-17) or to Th17 clone supernatants (EC+Th17 SN), was measured by ELISA. Chemokine content in Th17 SN was also assessed as control. Data refer to experimental triplicates from three experiments performed with three different clones from one patient. Means±SD are depicted. Statistical significance was assessed by one-way analysis of variance (ANOVA). (E) Migration of CD8+ T cells, purified from peripheral blood mononuclear cells (PBMC) of healthy donors, towards supernatants of HMEC cells untreated (EC), or exposed to rIL-17 (50 ng/mL) (EC+IL17) or Th17 supernatants (EC+Th17), was assessed after 90 min of incubation by flow cytometry. Migration towards Th17 supernatants (Th17 SN) was also assessed as control. Data refer to experimental triplicates from three experiments performed with three different clones from one patient. Means±SD are depicted. Statistical significance was assessed by one-way ANOVA. (F and G) Chemokine contents (F) and cytotoxic T lymphocyte (CTL) chemoattraction capacity (G) of supernatants from HMEC cells untreated (EC), exposed overnight to Th17 supernatants (EC+Th17 SN), or exposed to Th17 supernatants pretreated with anti-IL-17 (EC+Th17+anti-IL-17) or anti-tumour necrosis factor α (anti-TNF-α) antibodies (EC+Th17+anti-TNF-α). Data refer to three experiments performed with supernatants of Th17 bulk populations derived from three different samples. Means±SD are depicted. Statistical significance was assessed by one-way ANOVA.

Figure 6

Th17 cells directly attract cytotoxic CD8+ T cells. (A) Migration of CD8+ T cells towards control medium (Ctr) or supernatants of two different colorectal cancer (CRC)-Th17 clones (Th17.15 SN and Th17.71 SN), was assessed after 90 min incubation by flow cytometry. (B) Chemokine release by Th17 clones (Th17.15 and Th17.71), activated with plate bound anti-CD3 and soluble anti-CD28 antibodies for an overnight period, was measured by ELISA. Means±SD of experimental triplicates are depicted. (C) Migration of CD8+ T cells towards Th17 clone supernatants depleted of CCL5 (Th17 SN−CCL5), CCL20 (Th17 SN−CCL20) or both (Th17 SN−CCL5−CCL20), relative to control (migration towards untreated Th17 supernatants). (D) Migration of CD8+ T cells towards Th17 clone supernatants (Th17 SN) or towards low or high doses of recombinant CCL20 (300 and 1000 ng/mL, respectively) and CCL5 (60 and 200 ng/mL, respectively). (A–D) Means±SD from experimental triplicates are depicted. One representative experiment out of two is shown. Statistical significance was assessed by one-way analysis of variance (ANOVA). (E) Representative flow cytometric analysis of CRC infiltrates stained for CD8 and CCR6 markers (left panel). Frequencies of CCR6+CD8+ T cells were measured by flow cytometry in peripheral blood mononuclear cells of healthy donors (PBMC HD n=7) or patients with CRC (PBMC Pz n=12) and in single cell suspensions from freshly excised clinical specimens of tumour-free colonic mucosa (Ctr) and corresponding CRC (n=12). Means are indicated by bars. Statistical significance was assessed by t test. (F) Left and middle panels: representative flow cytometric analysis of CRC-infiltrating CD8+ T cells stained for CCR6, CCR5 and TIA-1. Right panel: frequencies of CCR6+ CCR5+ cells within TIA-1- or TIA-1+ CRC-infiltrating CD8+ T cells (n=7).

Figure 7

Th17 cells promote recruitment of cytotoxic CD8+ T cells into colorectal cancer (CRC) tissues. (A) CRC-Th17-infiltrating engineered tumour tissues (see online supplementary figure S10) were left untreated or were activated by adding CytoStim to the perfusion medium. Culture media were collected 20 h later and chemokine contents were assessed by ELISA. Statistical significance was assessed by Mann–Whitney test. (B and C) Three hours after Th17 activation, the perfusion in the bioreactor was stopped and CD8+ T cells were added to the system. After an overnight period, scaffolds were removed and tumour infiltration by CD8+ T cells was evaluated by immunofluorescence analysis upon staining with CD4- and CD8-specific antibodies (B) and by flow cytometry upon staining of single cell suspensions with EpCAM-, CD4- and CD8-specific antibodies (C). Percentages of CD8+ cells in tumour tissues infiltrated by resting or activated Th17 cells are reported. Means±SD from three experimental replicates performed with one Th17 clone are depicted. Statistical significance was assessed by Mann–Whitney test. (D) CSFE+CD8+ T cells were adoptively transferred in tumour bearing mice alone or together with equal numbers of CRC-Th17 (four mice/condition). Absolute numbers of CD8+CSFE+ T cells were evaluated by flow cytometry upon staining of tumour and Th17 cells with anti-EpCAM and anti-CD4 antibodies, respectively. Statistical significance was assessed by Mann–Whitney test.

Figure 8

Intra-epithelial localisation of interleukin (IL)-17+ cells correlates with prolonged relapse-free survival (RFS) survival. Kaplan–Meier curves illustrating RFS probability according to infiltration by IL-17+ cells within the epithelial (left panel) or stromal (right panel) compartment. Numbers of deaths/total cases within each category are indicated. Statistical significance was assessed by log-rank test.