Immune cell infiltration in colorectal cancer effectively predicts clinical outcome. IL22, produced by immune cells, plays an important role in inflammatory bowel disease, but its relevance in colorectal cancer remains unclear. Here, we addressed the prognostic significance of IL22+ cell infiltration in colorectal cancer and its effects on the composition of tumor microenvironment. Tissue microarrays (TMA) were stained with an IL22-specific mAb, and positive immune cells were counted by expert pathologists. Results were correlated with clinicopathologic data and overall survival (OS). Phenotypes of IL22-producing cells were assessed by flow cytometry on cell suspensions from digested specimens. Chemokine production was evaluated in vitro upon colorectal cancer cell exposure to IL22, and culture supernatants were used to assess neutrophil migration in vitro. Evaluation of a testing (n = 425) and a validation TMA (n = 89) revealed that high numbers of IL22 tumor-infiltrating immune cells were associated with improved OS in colorectal cancer. Ex vivo analysis indicated that IL22 was produced by CD4+ and CD8+ polyfunctional T cells, which also produced IL17 and IFNγ. Exposure of colorectal cancer cells to IL22 promoted the release of the neutrophil-recruiting chemokines CXCL1, CXCL2, and CXCL3 and enhanced neutrophil migration in vitro. Combined survival analysis revealed that the favorable prognostic significance of IL22 in colorectal cancer relied on the presence of neutrophils and was enhanced by T-cell infiltration. Altogether, colorectal cancer–infiltrating IL22-producing T cells promoted a favorable clinical outcome by recruiting beneficial neutrophils capable of enhancing T-cell responses.

Colorectal cancer is the third most common cause of cancer-related death worldwide (1). Infiltration of immune cells into colorectal cancer tumors predicts clinical outcome more effectively than tumor–node–metastasis staging (2). Infiltration by cytotoxic T cells, Th type 1 cells, T regulatory cells, and neutrophils associates with favorable outcome in human colorectal cancer (3–6), whereas the role of IL17-producing T cells (Th17) is still debated (2, 7).

IL22 is a cytokine of the IL10 family produced by different cell types of the innate immune system, including group 3 innate lymphoid cells (ILC3), and of the adaptive immune system, including Th17, naïve CD4+ T cells polarized upon exposure to TNFα and IL6, also known as Th22 cells (8), and CD8+ T cells (9, 10). IL22 receptors, including IL22R αchain and IL10Rβ chain, are uniquely expressed by keratinocytes and a variety of epithelial cells, including intestinal cells (11). IL22 plays key roles in wound healing and tissue repair, and in the maintenance of the “barrier” functions of skin and of intestinal and bronchial epithelial layers (12–14). In these anatomic regions, IL22 synergizes with IL17 and TNFα to promote the expression of proteins involved in host defense (15–17) and innate immunity against bacterial infections. IL22 also induces epithelial cell proliferation and upregulation of genes encoding prosurvival molecules (18–21), and may protect the liver, intestine, and lungs from tissue destruction (19–24). Interestingly, IL22 also plays a role in the maintenance of host–microbiota symbiosis (25).

The fact that IL22 promotes inflammation and concomitantly prevents tissue destruction is intriguing. Notably, exogenous IL22 delivery is sufficient to promote inflammation in mice (26, 27). The functional relevance of IL22 depends on specific tissue microenvironment. For example, in the absence of IL17, IL22 promotes tissue repair in the lung (18). In the colon, IL22 protects against experimental chronic colitis and promotes intestinal wound healing following acute intestinal injury (28). However, IL22 has also been suggested to play a role in the pathogenesis of a variety of human diseases, including psoriasis, arthritis, and inflammatory bowel disease (IBD; refs. 13, 29–31).

There is a paucity of data about the prognostic significance of IL22 in cancer (12, 32–34), in particular its role in human colorectal cancer remains unclear. In murine models, “uncontrolled” IL22 production promotes colorectal cancer development (33), possibly by direct effects on stem cells (35) or by enhancing cancer cell proliferation (36). Accordingly, in a murine model of colon cancer induced by administration of pathogenic Helicobacter hepaticus and carcinogenic azoxymethane in immunodeficient animals, IL22 produced by innate lymphoid cells mediates a protumorigenic effect via Stat3 activation in epithelial cells (37, 38). IL22 produced by human tumor-infiltrating lymphocytes also promotes colorectal cancer cell proliferation in vivo in a xenograft model (37). On the other hand, IL22 plays a key role in the control of genotoxic damage induced by carcinogens in colon epithelial stem cells, thereby limiting mutagenesis and cancer outgrowth (39).

Most of these studies have addressed direct effects of IL22 on tumor cells, whereas its potential ability to condition the colorectal cancer tumor microenvironment has not been explored in comparable detail (40). Prognostic significance of IL22 expression in human colorectal cancer, the overwhelming majority (>90%) of which is not associated to IBD or to clinically relevant chronic colitis, has not been investigated in large cohorts of patients. To fill this knowledge gap, in this study we used two tissue microarrays (TMA), collectively including >500 clinically annotated colorectal cancer specimens, to investigate the impact of IL22 on clinical outcome and its possible influence on tumor microenvironment composition.

TMA construction

The TMAs were constructed at the Pathology Biobank at the University Hospital of Basel (Basel, Switzerland). Unselected, nonconsecutive, formalin-fixed, paraffin-embedded primary colorectal cancer tissue blocks were used as donor blocks. Tissue cylinders with a diameter of 1 mm were punched from morphologically representative areas of each donor block and brought into one recipient paraffin block (30 × 25 mm), using the TMA GrandMaster (TMA-GM; 3D-Histech Ltd, Sysmex AG) technology. Each punch was derived from the center of the tumor in an area with no necrosis so that each TMA spot consisted of more than 50% tumor cells. Approval by the local ethics committee (Ethik Kommission beider Basel, EKBB) for the use of this clinically annotated TMA was obtained in advance, as stated in previous publications (6, 7, 41).

Clinicopathologic features

Clinicopathologic data for patients included in the TMAs were collected retrospectively in a nonstratified and nonmatched manner. The larger TMA set was a subset of a previously published TMA (42) including patients undergoing surgery from 1987 to 1996. The validation TMA was built with surgical specimens of patients who underwent surgery at the University Hospital of Basel (Basel, Switzerland) in the years from 2007 to 2012. Clinicopathologic characteristics are listed in Supplementary Tables S1 and S2. Overall survival (OS) was defined as primary endpoint. Available follow-up data for the testing and validation cohort had a mean event-free follow-up time of 115 and 36 months, respectively.

IHC

IHC IL22 staining was assessed on two TMAs consisting of 538 and 100 colorectal cancer specimens, respectively. After excluding samples for which the tissue punch was absent or had poor staining quality, 425 and 89 colorectal cancers samples, respectively, were available. Staining was performed using the BenchMark ULTRA IHC System (Ventana Medical Systems, Inc.), following the manufacturer's instructions, with an IL22-specific mAb (Creative Diagnostic #DCABH-2900, clone JNH9G22F3, dilution 1:100) and iVIEW-DAB as chromogen. Immunoreactivity was scored as number of tumor-infiltrating IL22+ immune cells by experienced pathologists (L. Tornillo and L. Terracciano). IL17 and CD66b staining protocols have been reported in previous studies (6, 7).

Clinical specimen collection and processing

Freshly excised clinical specimens were collected from patients undergoing surgical treatment at University Hospital of Basel (Basel, Switzerland) and St. Claraspital (Basel, Switzerland). Informed written consent was obtained from all patients whose specimens were analyzed for this study.

Tumor or healthy tissue fragments were snap frozen for RNA extraction or enzymatically digested (2 mg/mL collagenase IV, Worthington Biochemical Corporation and 0.2 mg/mL DNAse I, Sigma-Aldrich, for 1 hour at 37°C) to obtain single-cell suspensions. In addition, peripheral blood mononuclear cells from healthy donors were isolated by density gradient centrifugation (Histopaque-1077, Sigma-Aldrich; 400 × g for 30 minutes at room temperature, without break). Use of human samples in this study was approved by local ethical authorities (Ethikkommission Nordwest und Zentralschweiz, EKNZ 2014–388).

The Cancer Genome Atlas analysis

Gene expression data [as fragments per kilobase of transcript per million mapped reads values (FPKM)] from 597 colorectal cancer samples and 51 normal colorectal mucosa specimens, were obtained from The Cancer Genome Atlas (TCGA) Genomics Data Commons harmonized data portal using TCGA biolinks R package (43). Clinical information regarding 597 colorectal cancers (see Supplementary Table S3) was retrieved from the Human Protein Atlas (44). After normalization, expression (FPKM values) of genes encoding IL22 and a panel of cytokines, chemokines, and immune markers was retrieved and correlations between immune markers and patients' OS were evaluated.

Flow cytometry and cell sorting

Cell suspensions obtained from colorectal cancer and tumor-free colonic mucosa were incubated with 50 ng/mL phorbol 12-myristate 13-acetate, 1 μg/mL ionomycin, and 5 μg/mL Brefeldin A (Sigma-Aldrich) for 5 hours. Cells were fixed with the Intracellular Fixation and Permeabilization Buffer Set (eBioscience), following the manufacturer's instructions, and surface stained with fluorochrome-conjugated antibodies specific for human CD3 (clone SK7), CD4 (clone SK3), CD8 (clone SK1), and CD56 (clone B159), all from BD Biosciences. Following permeabilization, intracellular staining with antibodies specific for human IFNγ (BD Bioscience, clone 25723.11), IL22 (R&D Systems, clone 142928), and IL17 (eBioscience, clone 64DEC17) was performed. Cells were analyzed by FACSCalibur or Fortessa Flow Cytometers (BD Biosciences). Primary colorectal cancer cells were sorted from tumor cell suspensions by magnetic microbeads conjugated to EpCAM-specific Antibodies (MACS MicroBeads, Miltenyi Biotec, catalog no. 130–061–101), following the manufacturer's instructions. Cell purity was >97%, as evaluated by flow cytometry. Data were analyzed using FlowJo Software (Tree Star).

Real-time reverse transcription PCR assays

Total RNA was extracted from stored colorectal cancer tissues or sorted cell populations using NucleoSpin RNA Kit (Macherey-Nagel, catalog no. 740955.50), following the manufacturer's instructions, and quantified by Spectrophotometry (NanoDrop, Thermo Fisher Scientific). RNA was reverse transcribed using the Moloney Murine Leukemia Virus Reverse Transcriptase (M-MLV RT, Invitrogen). Quantitative PCR was performed in the ABI prism 7700 sequence detection system, using SYBR Green (Roche) or TaqMan Universal Master Mix, No AmpErase UNG (Applied Biosystems), and commercially available primer sequences. All primers are listed in Supplementary Table S4. Each gene was assayed in duplicate wells, using 20 ng template each. Expression of individual genes was analyzed by using the 2−ΔΔCt method (45), as relative to the expression of GAPDH house-keeping gene.

Cell lines

LS180, HT29, Colo205, HCT15, SW480, SW620, and DLD-1 human colorectal cancer cell lines were purchased from the European Collection of Cell Cultures (period 2013–2015), and immediately stored in liquid nitrogen. Cells used for individual experiments were thawed from original cryopreserved aliquots and maintained in culture, for a maximum of 10 passages, in RPMI1640 (Gibco) or, for HT29 in McCoy's 5A Medium (Sigma-Aldrich) or, for SW480 and SW620 in L15 Medium (Leibovitz, Sigma-Aldrich), supplemented with 10% FBS, GlutaMAX-I, and Kanamycin (Gibco). Absence of Mycoplasma contamination in cultured cells was verified by PCR testing prior to investigations.

Chemokine induction in colorectal cancer cell lines

Colorectal cancer cells from LS180 and HT29 established cell lines were plated in 24-well plates (Sigma-Aldrich, 3.5 × 105 cells/well in 0.5 mL) in culture media and then incubated with 10 and 100 ng/mL concentrations of IL22 (R&D Systems) at 37°C. After 4 hours, chemokine expression was assessed by quantitative PCR.

ELISA

Chemokine content in culture supernatants was assessed by ELISA using CXCL1 and CXCL3 DuoSet ELISA (R&D Systems, catalog nos. DY275 and DY801, respectively), following the manufacturer's instructions. Results were collected by Spectrophotometer (BioTek Instruments) using the SoftMax Pro 6 software.

Cell proliferation assay

Colorectal cancer cells from LS180 and HT29 established cell lines were plated in 24-well plates (Sigma-Aldrich, 105 cells/well in 0.5 mL) in RPMI1640 and McCoy's 5A Medium (Sigma-Aldrich), respectively, and then incubated with 10 or 100 ng/mL recombinant human IL22 (R&D Systems, catalog no. 782-IL/CF) for 4 days at 37°C. Cell proliferation was quantified by CyQUANT Cell Proliferation Assay (Thermo Fisher Scientific, catalog no. C7026), following the manufacturer's instructions.

Migration assay

CD8+ T cells and neutrophils were sorted from peripheral blood of healthy donors by Magnetic Microbeads (MACS MicroBeads, Miltenyi Biotec, catalog no. 130-045-201, and EasySep Human Neutrophil Isolation Kit, Stemcell Technologies, catalog no. 17957, respectively), according to the manufacturer's instructions, to a purity of >98%, as confirmed by flow cytometry. Chemotaxis assays were performed using 96-well transwell plates with 5-μm pore size membranes (Corning Costar). Supernatants from LS180 or HT29 cells, left untreated or treated overnight with 10 or 100 ng/mL of IL22, were added to the bottom chambers (250 μL/well). In specific experiments, colorectal cancer cell line supernatants were depleted of CXCL1 and/or CXCL3, prior to use, by using specific capture antibodies (R&D Systems, clone 20326, catalog no. MAB275 and clone 49801, catalog no. MAB160, respectively). CD8+ T cells and neutrophils (1.5 × 104 cells/well in 80 μL) were placed in the top chamber and allowed to migrate for 60 minutes at 37°C. The number of cells that migrated into the bottom chamber was quantified by flow cytometry. The extent of cell migration was expressed as a migration index, calculated as number of cells migrated toward supernatants/number of cells migrated toward control medium.

Statistical analysis

IL22+ tumor-infiltrating immune cells were counted on each of the 425 and 89 colorectal cancer plus 16 nonmalignant cores, respectively. After having proven an association between the number of IL22+ infiltrating cells and OS by univariate Cox regression, an optimal threshold was estimated by regression tree analysis (rpart Statistical Package Software R package, version 3.4.1, 2017-06-30). The obtained threshold was found to be almost equal to the median value. Subsequently, continuous values were dichotomized subdividing half of the collective as colorectal cancer with low or high IL22 immune infiltration. χ2 or Fisher exact tests were used to determine the association between IL22 infiltration and clinicopathologic discrete features, as well as the Wilcoxon signed rank-sum test for comparison with continuous values. Survival curves were depicted according to the Kaplan–Meier method and compared with the log-rank test. Moreover, individual survival curves were compared one by one and the P values were adjusted according to the Benjamini–Hochberg method, which controls the FDR and the expected proportion of false discoveries among the rejected hypotheses (Survminer R package).

IL17+ and CD66b+ cells were evaluated as reported previously (6, 7). Upon staining with antibodies specific for IL17 (goat polyclonal anti-human IL17, R&D Systems) or CD66b (BioLegend, clone G10F5), numbers of positive cells per punch were scored by experienced pathologists. By regression tree analysis (rpart package), cut-off values for both markers, IL17 and CD66b, were set at 10 cells per punch. After dichotomization, Kaplan–Meier curves were plotted, and compared by log-rank test.

The assumption of proportional hazards was verified for all markers by analyzing the correlation of Schoenfeld residuals and the ranks of individual failure times. Any missing clinicopathologic information was assumed to be missing at random.

All P values were two-sided and considered significant at P < 0.05. Analyses were performed by using the Statistical Package Software R (version 3.4.1, 2017-06-30, http://www.r-project.org or higher) and GraphPad Prism 7 Software (GraphPad Software).

High density of IL22+ cells is associated with favorable prognosis in human colorectal cancer

IL22 protein expression was first evaluated by IHC on a testing TMA set including 425 primary colorectal cancer specimens (Supplementary Table S1). As expected, positive staining was clearly detected on infiltrating immune cells. However, a more diffuse staining was also detectable on tumor cells (Fig. 1A). To clarify this issue, IL22 gene expression was investigated in colorectal cancer cells isolated from established cell lines with consistently negative results (Supplementary Fig. S1), suggesting that colorectal cancer cell positivity might be due to the IL22 fraction bound to its specific receptor on epithelial cells.

Figure 1.

High density of IL22+ cells is associated with favorable prognosis in human colorectal cancer. A, Representative pictures of colorectal cancers with low (left) or high (right) infiltration by IL22+ immune cells (scale bar, 50 μm). B and C, Kaplan–Meier curves depicting the probability of OS of patients dichotomized according to low or high number of IL22+ colorectal cancer–infiltrating immune cells in the test TMA (n = 425) and in the validation TMA (n = 89). Statistical significance was assessed by log-rank test. H, high; L, low.

Figure 1.

High density of IL22+ cells is associated with favorable prognosis in human colorectal cancer. A, Representative pictures of colorectal cancers with low (left) or high (right) infiltration by IL22+ immune cells (scale bar, 50 μm). B and C, Kaplan–Meier curves depicting the probability of OS of patients dichotomized according to low or high number of IL22+ colorectal cancer–infiltrating immune cells in the test TMA (n = 425) and in the validation TMA (n = 89). Statistical significance was assessed by log-rank test. H, high; L, low.

Close modal

Therefore, we focused our analysis on tumor-infiltrating immune cells. IL22+ cells were counted in each TMA core. IL22+ cells were detectable within both normal colonic tissues and colorectal cancer, although in the latter case at a significantly higher density (P = 0.039). Observed continuous values ranged between 0 and 300 cells per core with median and mean values of 20 and 39 cells per core, respectively. Median value was used to dichotomize tumor specimens into IL22-low and -high groups. Representative examples of colorectal cancer displaying low and high infiltration are displayed in Fig. 1A. As summarized in Table 1, the presence of high numbers of IL22+ cells was slightly associated with lower T stage (P = 0.043). Instead, no significant association was found with histologic subtype (P = 0.494), lymph node metastases (P = 0.059), tumor grade (P = 0.255), vascular invasion (P = 0.099), tumor border configuration (P = 0.151), and microsatellite instability (P = 0.452).

Table 1.

Correlation of IL22+ cell density with clinicopathologic features in n = 425 colorectal cancer specimens.

IL22 lowIL22 high
CharacteristicsN = 221(100%)N = 204(100%)Pa
Age 
 Years (median, mean) 71, 69.5 (40–90) 71, 70.5 (40–96) P = 0.318 
Sex 
 Female 117 (52.9) 104 (51.0) P = 0.687 
 Male 104 (47.1) 100 (49.0)  
Diameter 
 mm (median, mean) 50, 52.4 (10–170) 50, 51.0 (7–160) P = 0.527 
Tumor location 
 Left sided 136 (61.5) 138 (67.6) P = 0.167 
 Right sided 85 (38.5) 65 (31.9)  
Histologic subtype 
 Mucinous (4.1) 19 (9.3) P = 0.494 
 Nonmucinous 212 (95.9) 185 (90.7)  
pT stage 
 pT1 (3.2) (3.9) P = 0.043b 
 pT2 26 (11.8) 32 (15.7)  
 pT3 140 (63.3) 138 (67.6)  
 pT4 39 (17.6) 21 (10.3)  
pN stage 
 pN0 99 (44.8) 112 (54.9) P = 0.059c 
 pN1 64 (29.0) 51 (25.0)  
 pN2 48 (21.7) 35 (17.2)  
Tumor grade 
 G1 (3.2) (1.0) P = 0.255 
 G2 193 (87.3) 185 (90.7)  
 G3 11 (5.0) 12 (5.9)  
Vascular invasion 
 Absent 150 (67.9) 155 (76.0) P = 0.099 
 Present 62 (28.1) 44 (21.6)  
Tumor border 
 Pushing 54 (24.4) 64 (31.4) P = 0.151 
 Infiltrating 156 (70.6) 135 (66.2)  
PTL inflammation 
 Absent 167 (75.6) 145 (71.1) P = 0.162 
 Present 45 (20.4) 54 (26.5)  
Microsatellite stability  
 Deficient 30 (13.6) 33 (16.2) P = 0.452 
 Proficient 191 (86.4) 171 (83.8)  
5-year survival rate 
(95% CI) 0.43 0.36–0.50 0.58 0.51–0.65 P = 0.004 
IL22 lowIL22 high
CharacteristicsN = 221(100%)N = 204(100%)Pa
Age 
 Years (median, mean) 71, 69.5 (40–90) 71, 70.5 (40–96) P = 0.318 
Sex 
 Female 117 (52.9) 104 (51.0) P = 0.687 
 Male 104 (47.1) 100 (49.0)  
Diameter 
 mm (median, mean) 50, 52.4 (10–170) 50, 51.0 (7–160) P = 0.527 
Tumor location 
 Left sided 136 (61.5) 138 (67.6) P = 0.167 
 Right sided 85 (38.5) 65 (31.9)  
Histologic subtype 
 Mucinous (4.1) 19 (9.3) P = 0.494 
 Nonmucinous 212 (95.9) 185 (90.7)  
pT stage 
 pT1 (3.2) (3.9) P = 0.043b 
 pT2 26 (11.8) 32 (15.7)  
 pT3 140 (63.3) 138 (67.6)  
 pT4 39 (17.6) 21 (10.3)  
pN stage 
 pN0 99 (44.8) 112 (54.9) P = 0.059c 
 pN1 64 (29.0) 51 (25.0)  
 pN2 48 (21.7) 35 (17.2)  
Tumor grade 
 G1 (3.2) (1.0) P = 0.255 
 G2 193 (87.3) 185 (90.7)  
 G3 11 (5.0) 12 (5.9)  
Vascular invasion 
 Absent 150 (67.9) 155 (76.0) P = 0.099 
 Present 62 (28.1) 44 (21.6)  
Tumor border 
 Pushing 54 (24.4) 64 (31.4) P = 0.151 
 Infiltrating 156 (70.6) 135 (66.2)  
PTL inflammation 
 Absent 167 (75.6) 145 (71.1) P = 0.162 
 Present 45 (20.4) 54 (26.5)  
Microsatellite stability  
 Deficient 30 (13.6) 33 (16.2) P = 0.452 
 Proficient 191 (86.4) 171 (83.8)  
5-year survival rate 
(95% CI) 0.43 0.36–0.50 0.58 0.51–0.65 P = 0.004 

Abbreviations: CI, confidence interval; PTL, peritumoral lymphocytic.

aCorrelation between the IL22-low and IL22-high subgroup. Percentage may not add to 100% due to missing values of some variables.

bOverall difference in subgroup analysis due to T4.

cOverall difference in subgroup analysis due to N0.

Survival analysis at 5 years showed that patients with tumors characterized by high numbers of IL22+ infiltrating cells had significantly higher survival probability than those with tumors displaying low IL22+ cell numbers [OS, 58%; confidence interval (CI), 51%–65.0% vs. 43%; CI, 36%–50%; P = 0.004; Table 1].

Kaplan–Meier survival curve analysis revealed that the favorable prognostic effect of IL22+ cell infiltration remained constant over time (P = 0.0003; Fig. 1B). The positive prognostic impact of high IL22+ cell infiltration was confirmed upon staining of a TMA with an independent validation cohort (Supplementary Table S2) of 89 patients with colorectal cancer (P = 0.005; Fig. 1C). In this second cohort, for which data regarding adjuvant treatment were also available, multivariate analysis showed that IL22 retained an impact on OS irrespective of adjuvant chemotherapy (P = 0.02; Supplementary Table S5).

IL22 was expressed by polyfunctional T cells predictive of prolonged survival

We next investigated phenotypes and functions of colorectal cancer–infiltrating IL22+ cells. Flow cytometric analysis of single-cell suspensions obtained from colorectal cancer clinical specimens revealed that IL22+ cells were mostly found within conventional CD3+ T cells and expressed CD4 and, to lower percentages, CD8 molecules (Fig. 2A and B). Expression of IL22 at the single-cell level, based on intracellular cytokine staining, was not significantly increased in cells infiltrating tumors as compared with those present in normal tissues (median fluorescence intensity within gated CD3+ IL22+ cells in tumors vs. nontumor tissues: 54 ± 32 vs. 53 ± 47; P = 0.88 and mean fluorescence intensity within CD3+ IL22+ cells in tumors vs. nontumor tissues: 125 ± 97 vs. 85 ± 63; P = 0.15). However, a large fraction of IL22+ cells also showed production of additional cytokines, such as IL17 and IFNγ, thus indicating that they were polyfunctional T cells (Fig. 2C), as reported previously (7, 46). Notably, this subset of IL22-producing cells showed a trend toward an increase in colorectal cancer as compared with control tissues (Fig. 2C).

Figure 2.

IL22 is expressed by conventional polyfunctional T cells. Single-cell suspensions obtained from freshly excised clinical specimens of colorectal cancer and tumor-free colonic tissues were surface stained with antibodies specific for CD3, CD4, CD8, and CD56 and intracellularly stained for IL17 and IFNγ, and analyzed by flow cytometry. A, Representative phenotypic analysis. B, Percentages of cells positive for the indicated marker or cytokine within the IL22+ gate (n ≤ 15). C, Percentages of IL17+ or IFNγ+ cells within the IL22+ cell gate in colorectal cancer (CRC TISSUE) and corresponding tumor-free colonic tissues (CTRL TISSUE). Means and SDs are indicated by bars. Statistical significance was assessed by the Wilcoxon signed rank test.

Figure 2.

IL22 is expressed by conventional polyfunctional T cells. Single-cell suspensions obtained from freshly excised clinical specimens of colorectal cancer and tumor-free colonic tissues were surface stained with antibodies specific for CD3, CD4, CD8, and CD56 and intracellularly stained for IL17 and IFNγ, and analyzed by flow cytometry. A, Representative phenotypic analysis. B, Percentages of cells positive for the indicated marker or cytokine within the IL22+ gate (n ≤ 15). C, Percentages of IL17+ or IFNγ+ cells within the IL22+ cell gate in colorectal cancer (CRC TISSUE) and corresponding tumor-free colonic tissues (CTRL TISSUE). Means and SDs are indicated by bars. Statistical significance was assessed by the Wilcoxon signed rank test.

Close modal

Consistent with their “Th17-like” phenotype, numbers of infiltrating IL22+ cells were significantly higher within colorectal cancer characterized by high infiltration by IL17+ cells (Supplementary Fig. S2A). In TCGA cohort, IL22 gene expression significantly correlated with that of IL17 gene (r = 0.578; P < 0.0001, see Table 2). In the TMA cohort, tumors displaying high densities of both IL22+ and IL17+ cells were characterized by enhanced survival probability (Supplementary Fig. S2B).

Table 2.

Correlations between expression of IL22 gene and selected cytokine/chemokine genes in TCGA cohort (n = 597).

Spearman correlation coefficientP
IL17A 0.578 <0.00001 
CXCL2 0.447 <0.00001 
CXCL1 0.401 <0.00001 
CXCL3 0.393 <0.00001 
CXCL8 0.152 0.0002 
Spearman correlation coefficientP
IL17A 0.578 <0.00001 
CXCL2 0.447 <0.00001 
CXCL1 0.401 <0.00001 
CXCL3 0.393 <0.00001 
CXCL8 0.152 0.0002 

IL22 stimulated colorectal cancer cells to release neutrophil-recruiting chemokines

TMA staining data indicated that colorectal cancer infiltration by IL22-producing T cells was associated with improved survival. This was an unexpected finding, because in murine colorectal cancer models, IL22 has mostly been shown to play a direct protumorigenic role, by enhancing tumor cell proliferation (35).

Indeed, we found that human colorectal cancer cells do express IL22 receptors, that is, IL22 Rα and IL10Rβ chains (Supplementary Fig. S3A and S3B), thus potentially responding to direct IL22-mediated effects. However, when we evaluated the direct effects of IL22 on LS180 and HT29 colorectal cancer cell lines, we did not observe any significant impact of IL22 on colorectal cancer cell proliferation in vitro (Supplementary Fig. S3C and S3D).

In contrast, IL22 treatment consistently increased gene expression of CXCL1, CXCL2, CXCL3 neutrophil-recruiting chemokines in two different colorectal cancer cell lines (Fig. 3A; Supplementary Fig. S4A). CXCL1 and CXCL3 protein release was also detectable in culture supernatants (Fig. 3B; Supplementary Fig. S4B), and enhanced neutrophil migration in vitro was accordingly observed (Fig. 3C).

Figure 3.

IL22 enhances the release of neutrophil-recruiting chemokines by colorectal cancer cells. Colorectal cancer cells from the LS180 cell line were treated with IL22 (10 or 100 ng/mL), as indicated. A, After 4 hours, expression levels of genes encoding the indicated chemokines were analyzed by qRT-PCR, using GAPDH as reference gene. Means and SD from five independent experiments are shown. Statistical significance was assessed by two-way ANOVA test (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001). B, After an overnight culture, CXCL1 and CXCL3 chemokine contents in culture supernatants were measured by ELISA. Means and SD from three independent experiments are shown. Statistical significance was assessed by Mann–Whitney test (*, P < 0.05; **, P < 0.01). C, Migration of neutrophils, isolated from healthy donors, toward culture supernatants from LS180 cells, untreated (CTRL) or exposed to the indicated doses of IL22 for an overnight period, was assessed in the presence or absence of CXCL3- and CXCL1-blocking reagents. Means and SD from three independent experiments are shown. Statistical significance was assessed by one-way ANOVA test (*, P < 0.05). D, Expression of CXCL1, CXCL2, and CXCL3 genes was evaluated in primary colorectal cancer cells sorted, based on EpCAM expression, from cell suspensions obtained upon enzymatic digestion of fresh colorectal samples. IL22 gene expression was assessed in corresponding whole colorectal cancer tissues, and, using the median of detected values as cutoff, tumors were classified as IL22 high or IL22 low. Expression of the indicated chemokines genes in colorectal cancer cells from IL22-high versus IL22-low tumors is depicted. E, Expression of the neutrophil marker CD66b in whole colorectal cancer tissues of IL22-high versus IL22-low tumors. All indicated P values were assessed by Mann–Whitney test (*, P < 0.05; **, P < 0.01).

Figure 3.

IL22 enhances the release of neutrophil-recruiting chemokines by colorectal cancer cells. Colorectal cancer cells from the LS180 cell line were treated with IL22 (10 or 100 ng/mL), as indicated. A, After 4 hours, expression levels of genes encoding the indicated chemokines were analyzed by qRT-PCR, using GAPDH as reference gene. Means and SD from five independent experiments are shown. Statistical significance was assessed by two-way ANOVA test (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001). B, After an overnight culture, CXCL1 and CXCL3 chemokine contents in culture supernatants were measured by ELISA. Means and SD from three independent experiments are shown. Statistical significance was assessed by Mann–Whitney test (*, P < 0.05; **, P < 0.01). C, Migration of neutrophils, isolated from healthy donors, toward culture supernatants from LS180 cells, untreated (CTRL) or exposed to the indicated doses of IL22 for an overnight period, was assessed in the presence or absence of CXCL3- and CXCL1-blocking reagents. Means and SD from three independent experiments are shown. Statistical significance was assessed by one-way ANOVA test (*, P < 0.05). D, Expression of CXCL1, CXCL2, and CXCL3 genes was evaluated in primary colorectal cancer cells sorted, based on EpCAM expression, from cell suspensions obtained upon enzymatic digestion of fresh colorectal samples. IL22 gene expression was assessed in corresponding whole colorectal cancer tissues, and, using the median of detected values as cutoff, tumors were classified as IL22 high or IL22 low. Expression of the indicated chemokines genes in colorectal cancer cells from IL22-high versus IL22-low tumors is depicted. E, Expression of the neutrophil marker CD66b in whole colorectal cancer tissues of IL22-high versus IL22-low tumors. All indicated P values were assessed by Mann–Whitney test (*, P < 0.05; **, P < 0.01).

Close modal

Increased expression of genes encoding T-cell–recruiting chemokines, such as CCL22, CXCL9, and CXCL11, was also boosted by IL22 in LS180, but not in HT29 cells (Fig. 3A; Supplementary Fig. S4A).

IL22+ T cells displayed cross-talk with neutrophils in colorectal cancer samples

On the basis of these findings, we hypothesized that a cross-talk between tumor and beneficial immune cell populations could underlie the favorable prognostic significance of IL22+ cell infiltration in colorectal cancer.

Consistent with our in vitro data, we found a significant positive correlation between expression of IL22 gene and that of CXCL1 (r = 0.401; P < 0.00001), CXCL2 (r = 0.447; P < 0.00001), and CXCL3 (r = 0.393 and P < 0.00001) genes in colorectal cancer specimens from TCGA database (Table 2). Upon ex vivo analysis on primary colorectal cancer cells sorted from clinical specimens, we found that expression of genes encoding neutrophil-recruiting chemokines was significantly enhanced in tumor cells derived from samples displaying high IL22 expression as compared with those purified from tumors displaying no or low IL22 expression (Fig. 3D). Accordingly, increased CD66b expression, consistent with higher neutrophil densities, was detected in IL22-high versus IL22-low tumors (Fig. 3E).

In our TMA cohort, we observed that high neutrophil densities, as defined by CD66b marker–specific staining, were significantly (P < 0.00001) associated with higher IL22+ colorectal cancer–infiltrating cell numbers (Fig. 4A) and high IL22 gene expression (Supplementary Fig. S5A).

Figure 4.

The positive prognostic effect of IL22+ cells depends on tumor-infiltrating neutrophils and on their interplay with T cells. A, Distribution of CD66b+ cells according to the low or high IL22+ colorectal cancer–infiltrating cells. B, Kaplan–Meier curves depicting the probability of OS in the entire collective (gray line) and in patients stratified according to combinations of IL22+ and CD66b+ cell density in the overall testing TMA (n = 356 instead of 425 because of CD66b missing values). C, Kaplan–Meier curves depicting the probability of OS in patients with tumors characterized by IL22+-high density in the entire collective (gray line) and stratified according to combinations of CD66b+ and CD3+ cell infiltration (n = 156 instead of 221 because of missing CD66+ and/or CD3+ values). H, high; L, low.

Figure 4.

The positive prognostic effect of IL22+ cells depends on tumor-infiltrating neutrophils and on their interplay with T cells. A, Distribution of CD66b+ cells according to the low or high IL22+ colorectal cancer–infiltrating cells. B, Kaplan–Meier curves depicting the probability of OS in the entire collective (gray line) and in patients stratified according to combinations of IL22+ and CD66b+ cell density in the overall testing TMA (n = 356 instead of 425 because of CD66b missing values). C, Kaplan–Meier curves depicting the probability of OS in patients with tumors characterized by IL22+-high density in the entire collective (gray line) and stratified according to combinations of CD66b+ and CD3+ cell infiltration (n = 156 instead of 221 because of missing CD66+ and/or CD3+ values). H, high; L, low.

Close modal

The positive prognostic significance of high IL22+ cell infiltration in colorectal cancer was lost in the absence of CD66b+ cells, thus indicating that the beneficial effect of IL22-producing cells required neutrophil recruitment (Fig. 4B). A similar trend (P = 0.09) was also detectable in TCGA data (Supplementary Fig. S5B).

Neutrophils costimulate antigen-driven activation of colorectal cancer–infiltrating CD8+ T cells (6). Remarkably, the presence of high CD8+ or CD3+ cell infiltration, in addition to IL22+ and CD66b+ cells, further enhanced their prognostic significance (Fig. 4C; Supplementary Fig. S6).

Tumor infiltration by immune cell populations characterized by different cytokine production profiles heavily impacts clinical outcome of human colorectal cancer (3, 4, 47, 48); however, the role of immune cells producing IL22, a cytokine involved in tissue repair processes at mucosal surfaces, has remained elusive.

In experimental murine models, direct effects of IL22 on epithelial stem cells and tumor cells associate with tumor progression. IL22 also plays a role in IBD pathogenesis, potentially resulting in colorectal cancer outgrowth (37, 38). However, only ≤2% of sporadic human colorectal cancers are associated with clinically relevant IBD (49, 50). The prognostic significance of tumor infiltration by IL22+ cells in the majority of colorectal cancer cases, diagnosed in the absence of a clinically significant chronic colitis/inflammation, has not been thoroughly investigated. High expression of DOTL1, an IL22-induced methyltransferase, associates with poor patient survival (35). However, no data regarding the prognostic impact of IL22 gene or protein expression were provided.

This is the first study evaluating the impact of colorectal cancer–infiltrating IL 22+ cells on patient survival. Upon analysis of two independent cohorts including 425 and 89 primary colorectal cancer cases, respectively, we unexpectedly observed that high infiltration by IL22+ cells was associated with favorable prognosis. Evaluation at transcriptional level in TCGA cohort also provided data in-line with our findings of protein expression from TMA. Thus, these independent analyses concur in indicating IL22 expression as favorable predictive factor in human colorectal cancer.

Phenotypic analysis revealed that IL22-producing cells mostly comprised of polyfunctional Th17 and CD8+ T cells, producing IL17 and IFNγ, in addition to IL22. These findings are in-line with previous studies reporting IL22 production by Th17 cells in human sporadic colorectal cancers (35, 46). The role of IL17 and Th17 cells in colorectal cancer is still debated. IL17 is suggested to be protumorigenic in mice and negatively influences colorectal cancer prognosis in humans (51). However, colorectal cancer–infiltrating Th17 cells might play a dual role depending on their tissue localization (7). Here, we showed that IL17 was positively associated with favorable prognosis only in the presence of high density of IL22+ tumor-infiltrating cells, thus indicating a distinct role of polyfunctional Th17 cells as compared with cells producing IL17 only. On the other hand, in accordance with previous studies (35, 40), CD3 IL22 cells, possibly including ILCs, appeared to represent a minor fraction of human colorectal cancer–infiltrating IL22-producing cells. In a previously reported IBD-associated colorectal cancer mouse model (35), IL22-producing ILCs sustained tumor progression in immunodeficient animals. It is conceivable that in colorectal cancer subtypes driven by distinct pathogenesis, IL22 is produced by different cell populations, possibly endowed with distinct functional roles and prognostic significance.

The observed association between infiltration by IL22+ cells and improved prognosis contradicts previous studies showing a protumorigenic direct effect of IL22 on tumor cells (52, 53). Although we did not observe any significant effect when we evaluated the impact of IL22 on colorectal cancer cell proliferation in vitro according to published protocols (52, 53), it is possible that IL22 plays a dual role during different stages of the disease. While IL22 might contribute to tumor development during early phases of oncogenesis, in clinically detectable tumors with an established microenvironment, it might help to recruit beneficial immune cell populations.

Here, exposure of colorectal cancer cells to IL22 resulted in increased secretion of neutrophil-recruiting chemokines, including CXCL1, CXCL2, and CXCL3, and enhanced neutrophil migration in vitro. Consistently, in human colorectal cancer samples, expression of IL22 positively correlated with expression of neutrophil-recruiting chemokines in whole-tumor tissues and in purified tumor cells. This was associated with a higher expression of the CD66b neutrophil marker. The positive prognostic effect of IL22+ cells was dependent on the presence of neutrophils.

Neutrophils are key players in the immune response against infectious challenges. However, their role in tumor immunobiology has long been neglected, possibly due to important differences in the granulocyte compartment between humans and experimental mice (54). A number of studies indicate that neutrophils may exert antitumor effects, potentially of high clinical relevance (55). In previous works, we and others (6, 55–57) showed that neutrophil infiltration associates with favorable prognosis in colorectal cancer. In addition, colorectal cancer–associated neutrophils costimulate antigen-triggered tumor-infiltrating T cells (6). Our data further underline the critical prognostic relevance of neutrophil infiltration in colorectal cancer microenvironment and unravel a previously unsuspected ability of IL22 to shape its composition. By stimulating production of neutrophil-recruiting chemokines in tumor cells, IL22-producing T cells might favor colorectal cancer infiltration by neutrophils, ultimately enhancing T-cell activation and expansion, and favoring a more positive clinical outcome.

In this scenario, the nature of stimuli favoring differentiation and recruitment of IL22-producing T cells and their antigenic specificities remains to be clarified. Also, it must be noted that the magnitude of IL22-induced effects ultimately depends on responsiveness of cells from individual tumors. Indeed, expression and functionality of IL22 receptors on tumor cells or their chemokine production capacity are determined by their genetic and epigenetic make-up (3, 47). This likely accounts for the variability of IL22-mediated effect across individual cases and the possible lack of neutrophilic infiltration in spite of high IL22 expression.

In conclusion, upon analysis of a large cohort of colorectal cancer cases, we found an unexpected positive prognostic effect of tumor-infiltrating IL22-producing T cells and showed the capacity of IL22 to recruit beneficial neutrophils into the tumor microenvironment by triggering the production of specific chemokines by colorectal cancer cells. This knowledge may be exploited for more precise tumor prognostication and may suggest the development of innovative treatments aimed at enhancing colorectal cancer infiltration by beneficial immune cell populations.

No potential conflicts of interest were disclosed.

N. Tosti: Resources, data curation. E. Cremonesi: Resources, data curation. V. Governa: Data curation. C. Basso: Data curation, formal analysis, data acquisition for the revision. V. Kancherla: Formal analysis. M. Coto-Llerena: Data curation, formal analysis. F. Amicarella: Supervision, writing–review and editing. B. Weixler: Writing–review and editing. S. Däster: Writing–review and editing. G. Sconocchia: Funding acquisition, writing–review and editing. P.E. Majno: Writing–review and editing. D. Christoforidis: Writing–review and editing. L. Tornillo: Supervision, validation, writing–review and editing. L. Terracciano: Supervision, writing–review and editing. C.K.Y. Ng: Formal analysis, methodology, writing–review and editing. S. Piscuoglio: Formal analysis, supervision, validation, writing–review and editing. M. von Flüe: Writing–review and editing. G. Spagnoli: Conceptualization, supervision, writing–review and editing. S. Eppenberger-Castori: Data curation, formal analysis, writing–review and editing. G. Iezzi: Conceptualization, supervision, funding acquisition, methodology, writing–original draft. R.A. Droeser: Conceptualization, supervision, funding acquisition, methodology, writing–original draft.

The authors are grateful to Prof. Dr. Fabio Grassi (Institute of Research in Biomedicine, Bellinzona, Switzerland) and to Prof. Dr. Andreas Diefenbach (Charité - Universitätsmedizin Berlin, Germany) for critical comments and discussion. This study was supported by the Swiss Cancer League (KFS-3528-08-2014), the Werner and Hedy Berger-Janser – Foundation for cancer research, the “Stiftung zur Krebsbekämpfung,” and by the Swiss National Science Foundation (grant no. 310030_185234; to G. Iezzi). G. Sconocchia was supported by the Italian Association for Cancer Research grant N IG17120.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1.
Siegel
R
,
Ma
J
,
Zou
Z
,
Jemal
A
. 
Cancer statistics, 2014
.
CA Cancer J Clin
2014
;
64
:
9
29
.
2.
Fridman
WH
,
Zitvogel
L
,
Sautès-Fridman
C
,
Kroemer
G
. 
The immune contexture in cancer prognosis and treatment
.
Nat Rev Clin Oncol
2017
;
14
:
717
34
.
3.
Galon
J
,
Costes
A
,
Sanchez-Cabo
F
,
Kirilovsky
A
,
Mlecnik
B
,
Lagorce-Pages
C
, et al
Type, density, and location of immune cells within human colorectal tumors predict clinical outcome
.
Science
2006
;
313
:
1960
4
.
4.
Tosolini
M
,
Kirilovsky
A
,
Mlecnik
B
,
Fredriksen
T
,
Mauger
S
,
Bindea
G
, et al
Clinical impact of different classes of infiltrating T cytotoxic and helper cells (Th1, Th2, Treg, Th17) in patients with colorectal cancer
.
Cancer Res
2011
;
71
:
1263
71
.
5.
Frey
DM
,
Droeser
RA
,
Viehl
CT
,
Zlobec
I
,
Lugli
A
,
Zingg
U
, et al
High frequency of tumor-infiltrating FOXP3+regulatory T cells predicts improved survival in mismatch repair-proficient colorectal cancer patients
.
Int J Cancer
2010
;
126
;
2635
43
.
6.
Governa
V
,
Trella
E
,
Mele
V
,
Tornillo
L
,
Amicarella
F
,
Cremonesi
E
, et al
The interplay between neutrophils and CD8+ T cells improves survival in human colorectal cancer
.
Clin Cancer Res
2017
;
23
:
3847
58
.
7.
Amicarella
F
,
Muraro
MG
,
Hirt
C
,
Cremonesi
E
,
Padovan
E
,
Mele
V
, et al
Dual role of tumour-infiltrating T helper 17 cells in human colorectal cancer
.
Gut
2017
;
66
:
692
704
.
8.
Duhen
T
,
Geiger
R
,
Jarrossay
D
,
Lanzavecchia
A
,
Sallusto
F
. 
Production of interleukin 22 but not interleukin 17 by a subset of human skin-homing memory T cells
.
Nat Immunol
2009
;
10
:
857
63
.
9.
Qin
WZ
,
Chen
LL
,
Pan
HF
,
Leng
RX
,
Zhai
ZM
,
Wang
C
, et al
Expressions of IL-22 in circulating CD4+/CD8+ T cells and their correlation with disease activity in SLE patients
.
Clin Exp Med
2011
;
11
:
245
50
.
10.
Nograles
KE
,
Zaba
LC
,
Shemer
A
,
Fuentes-Duculan
J
,
Cardinale
I
,
Kikuchi
T
, et al
IL-22-producing “T22” T cells account for upregulated IL-22 in atopic dermatitis despite reduced IL-17-producing TH17 T cells
.
J Allergy Clin Immunol
2009
;
123
:
1244
52
.
11.
Wolk
K
,
Kunz
S
,
Witte
E
,
Friedrich
M
,
Asadullah
K
,
Sabat
R
. 
IL-22 increases the innate immunity of tissues
.
Immunity
2004
;
21
:
241
54
.
12.
Hernandez
P
,
Gronke
K
,
Diefenbach
A
. 
A catch-22: interleukin-22 and cancer
.
Eur J Immunol
2018
;
48
:
15
31
.
13.
Ouyang
W
,
O'Garra
A
. 
IL-10 family cytokines IL-10 and IL-22: from basic science to clinical translation
.
Immunity
2019
;
50
:
871
91
.
14.
Lindemans
CA
,
Calafiore
M
,
Mertelsmann
AM
,
O'Connor
MH
,
Dudakov
JA
,
Jenq
RR
, et al
Interleukin-22 promotes intestinal-stem-cell-mediated epithelial regeneration
.
Nature
2015
;
528
:
560
4
.
15.
Liang
SC
,
Tan
XY
,
Luxenberg
DP
,
Karim
R
,
Dunussi-Joannopoulos
K
,
Collins
M
, et al
Interleukin (IL)-22 and IL-17 are coexpressed by Th17 cells and cooperatively enhance expression of antimicrobial peptides
.
J Exp Med
2006
;
203
:
2271
9
.
16.
Aujla
SJ
,
Chan
YR
,
Zheng
M
,
Fei
M
,
Askew
DJ
,
Pociask
DA
, et al
IL-22 mediates mucosal host defense against Gram-negative bacterial pneumonia
.
Nat Med
2008
;
14
:
275
81
.
17.
Eyerich
S
,
Eyerich
K
,
Pennino
D
,
Carbone
T
,
Nasorri
F
,
Pallotta
S
, et al
Th22 cells represent a distinct human T cell subset involved in epidermal immunity and remodeling
.
J Clin Invest
2009
;
119
:
3573
85
.
18.
Sonnenberg
GF
,
Nair
MG
,
Kirn
TJ
,
Zaph
C
,
Fouser
LA
,
Artis
D
. 
Pathological versus protective functions of IL-22 in airway inflammation are regulated by IL-17A
.
J Exp Med
2010
;
207
:
1293
305
.
19.
Zenewicz
LA
,
Yancopoulos
GD
,
Valenzuela
DM
,
Murphy
AJ
,
Karow
M
,
Flavell
RA
. 
Interleukin-22 but not interleukin-17 provides protection to hepatocytes during acute liver inflammation
.
Immunity
2007
;
27
:
647
59
.
20.
Radaeva
S
,
Sun
R
,
Pan
HN
,
Hong
F
,
Gao
B
. 
Interleukin 22 (IL-22) plays a protective role in T cell-mediated murine hepatitis: IL-22 is a survival factor for hepatocytes via STAT3 activation
.
Hepatology
2004
;
39
:
1332
42
.
21.
Sugimoto
K
,
Ogawa
A
,
Mizoguchi
E
,
Shimomura
Y
,
Andoh
A
,
Bhan
AK
, et al
IL-22 ameliorates intestinal inflammation in a mouse model of ulcerative colitis
.
J Clin Invest
2008
;
118
:
534
44
.
22.
Zenewicz
LA
,
Yancopoulos
GD
,
Valenzuela
DM
,
Murphy
AJ
,
Stevens
S
,
Flavell
RA
. 
Innate and adaptive interleukin-22 protects mice from inflammatory bowel disease
.
Immunity
2008
;
29
:
947
57
.
23.
Pickert
G
,
Neufert
C
,
Leppkes
M
,
Zheng
Y
,
Wittkopf
N
,
Warntjen
M
, et al
STAT3 links IL-22 signaling in intestinal epithelial cells to mucosal wound healing
.
J Exp Med
2009
;
206
:
1465
72
.
24.
Simonian
PL
,
Wehrmann
F
,
Roark
CL
,
Born
WK
,
O'Brien
RL
,
Fontenot
AP
. 
γδ T cells protect against lung fibrosis via IL-22
.
J Exp Med
2010
;
207
:
2239
53
.
25.
Goto
Y
,
Obata
T
,
Kunisawa
J
,
Sato
S
,
Ivanov
II
,
Lamichhane
A
, et al
Innate lymphoid cells regulate intestinal epithelial cell glycosylation
.
Science
2014
;
345
:
1254009
.
26.
Liang
SC
,
Nickerson-Nutter
C
,
Pittman
DD
,
Carrier
Y
,
Goodwin
DG
,
Shields
KM
, et al
IL-22 induces an acute-phase response
.
J Immunol
2010
;
185
:
5531
8
.
27.
Zheng
Y
,
Danilenko
DM
,
Valdez
P
,
Kasman
I
,
Eastham-Anderson
J
,
Wu
J
, et al
Interleukin-22, a T(H)17 cytokine, mediates IL-23-induced dermal inflammation and acanthosis
.
Nature
2007
;
445
:
648
51
.
28.
Mizoguchi
A
. 
Healing of intestinal inflammation by IL-22
.
Inflamm Bowel Dis
2012
;
18
:
1777
84
.
29.
Sabat
R
,
Ouyang
W
,
Wolk
K
. 
Therapeutic opportunities of the IL-22-IL-22R1 system
.
Nat Rev Drug Discov
2014
;
13
:
21
38
.
30.
Mizoguchi
A
,
Yano
A
,
Himuro
H
,
Ezaki
Y
,
Sadanaga
T
,
Mizoguchi
E
. 
Clinical importance of IL-22 cascade in IBD
.
J Gastroenterol
2018
;
53
:
465
74
.
31.
Eyerich
K
,
Dimartino
V
,
Cavani
A
. 
IL-17 and IL-22 in immunity: driving protection and pathology
.
Eur J Immunol
2017
;
47
:
607
14
.
32.
Nagakawa
H
,
Shimozato
O
,
Yu
L
,
Takiguchi
Y
,
Tatsumi
K
,
Kuriyama
T
, et al
Expression of interleukin-22 in murine carcinoma cells did not influence tumour growth in vivo but did improve survival of the inoculated hosts
.
Scand J Immunol
2004
;
60
:
449
54
.
33.
Huber
S
,
Gagliani
N
,
Zenewicz
LA
,
Huber
FJ
,
Bosurgi
L
,
Hu
B
, et al
IL-22BP is regulated by the inflammasome and modulates tumorigenesis in the intestine
.
Nature
2012
;
491
:
259
63
.
34.
Zhuang
Y
,
Peng
LS
,
Zhao
YL
,
Shi
Y
,
Mao
XH
,
Guo
G
, et al
Increased intratumoral IL-22-producing CD4(+) T cells and Th22 cells correlate with gastric cancer progression and predict poor patient survival
.
Cancer Immunol Immunother
2012
;
61
:
1965
75
.
35.
Kryczek
I
,
Lin
Y
,
Nagarsheth
N
,
Peng
D
,
Zhao
L
,
Zhao
E
, et al
IL-22+CD4+ T cells promote colorectal cancer stemness via STAT3 transcription factor activation and induction of the methyltransferase DOT1L
.
Immunity
2014
;
40
:
772
84
.
36.
Sun
D
,
Lin
Y
,
Hong
J
,
Chen
H
,
Nagarsheth
N
,
Peng
D
, et al
Th22 cells control colon tumorigenesis through STAT3 and polycomb repression complex 2 signaling
.
Oncoimmunology
2015
;
5
:
e1082704
.
37.
Jiang
R
,
Wang
H
,
Deng
L
,
Hou
J
,
Shi
R
,
Yao
M
, et al
IL-22 is related to development of human colon cancer by activation of STAT3
.
BMC Cancer
2013
;
13
:
59
.
38.
Kirchberger
S
,
Royston
DJ
,
Boulard
O
,
Thornton
E
,
Franchini
F
,
Szabady
RL
, et al
Innate lymphoid cells sustain colon cancer through production of interleukin-22 in a mouse model
.
J Exp Med
2013
;
210
:
917
31
.
39.
Gronke
K
,
Hernández
PP
,
Zimmermann
J
,
Klose
CSN
,
Kofoed-Branzk
M
,
Guendel
F
, et al
Interleukin-22 protects intestinal stem cells against genotoxic stress
.
Nature
2019
;
566
:
249
53
.
40.
Crome
SQ
,
Nguyen
LT
,
Lopez-Verges
S
,
Yang
SYC
,
Martin
B
,
Yam
JY
, et al
A distinct innate lymphoid cell population regulates tumor-associated T cells
.
Nat Med
2017
;
23
:
368
75
.
41.
Weixler
B
,
Cremonesi
E
,
Sorge
R
,
Muraro
MG
,
Delko
T
,
Nebiker
CA
, et al
OX40 expression enhances the prognostic significance of CD8 positive lymphocyte infiltration in colorectal cancer
.
Oncotarget
2015
;
6
:
37588
99
.
42.
Zlobec
I
,
Karamitopoulou
E
,
Terracciano
L
,
Piscuoglio
S
,
Iezzi
G
,
Muraro
MG
, et al
TIA-1 cytotoxic granule-associated RNA binding protein improves the prognostic performance of CD8 in mismatch repair-proficient colorectal cancer
.
PLoS One
2010
;
5
:
e14282
.
43.
Colaprico
A
,
Silva
TC
,
Olsen
C
,
Garofano
L
,
Cava
C
,
Garolini
D
, et al
TCGA biolinks: an R/Bioconductor package for integrative analysis of TCGA data
.
Nucleic Acids Res
2016
;
44
:
e71
.
44.
Uhlen
M
,
Zhang
C
,
Lee
S
,
Sjöstedt
E
,
Fagerberg
L
,
Bidkhori
G
, et al
A pathology atlas of the human cancer transcriptome
.
Science
2017
;
357
:
eaan2507
.
45.
Livak
KJ
,
Schmittgen
TD
. 
Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT method
.
Methods
2001
;
25
:
402
8
.
46.
Doulabi
H
,
Rastin
M
,
Shabahangh
H
,
Maddah
G
,
Abdollahi
A
,
Nosratabadi
R
, et al
Analysis of Th22, Th17 and CD4+cells co-producing IL-17/IL-22 at different stages of human colon cancer
.
Biomed Pharmacother
2018
;
103
:
1101
16
.
47.
Bindea
G
,
Mlecnik
B
,
Tosolini
M
,
Kirilovsky
A
,
Waldner
M
,
Obenauf
AC
, et al
Spatiotemporal dynamics of intratumoral immune cells reveal the immune landscape in human cancer
.
Immunity
2013
;
39
:
782
95
.
48.
Fridman
WH
,
Pages
F
,
Sautes-Fridman
C
,
Galon
J
. 
The immune contexture in human tumours: impact on clinical outcome
.
Nat Rev Cancer
2012
;
12
:
298
306
.
49.
Munkholm
P
. 
Review article: the incidence and prevalence of colorectal cancer in inflammatory bowel disease
.
Aliment Pharmacol Ther
2003
;
18
:
1
5
.
50.
Vagefi
PA
,
Longo
WE
. 
Colorectal cancer in patients with inflammatory bowel disease
.
Clin Colorectal Cancer
2005
;
4
:
313
9
.
51.
De Simone
V
,
Pallone
F
,
Monteleone
G
,
Stolfi
C
. 
Role of TH17 cytokines in the control of colorectal cancer
.
Oncoimmunology
2013
;
2
:
e26617
.
52.
Song
B
,
Ma
Y
,
Liu
X
,
Li
W
,
Zhang
J
,
Liu
J
, et al
IL-22 promotes the proliferation of cancer cells in smoking colorectal cancer patients
.
Tumor Biol
2016
;
37
:
1349
56
.
53.
De Simone
V
,
Franzè
E
,
Ronchetti
G
,
Colantoni
A
,
Fantini
MC
,
Di Fusco
D
, et al
Th17-type cytokines, IL-6 and TNF-α synergistically activate STAT3 and NF-kB to promote colorectal cancer cell growth
.
Oncogene
2015
;
34
:
3493
503
.
54.
Hagerling
C
,
Werb
Z
. 
Neutrophils: critical components in experimental animal models of cancer
.
Semin Immunol
2016
;
28
:
197
204
.
55.
Ponzetta
A
,
Mantovani
A
,
Jaillon
S
. 
Dissecting neutrophil complexity in cancer
.
Emerg Top Life Sci
2017
;
1
:
457
70
.
56.
Galdiero
MR
,
Bianchi
P
,
Grizzi
F
,
Di Caro
G
,
Basso
G
,
Ponzetta
A
, et al
Occurrence and significance of tumor-associated neutrophils in patients with colorectal cancer
.
Int J Cancer
2016
;
139
:
446
56
.
57.
Ponzetta
A
,
Carriero
R
,
Carnevale
S
,
Barbagallo
M
,
Molgora
M
,
Perucchini
C
, et al
Neutrophils driving unconventional T cells mediate resistance against murine sarcomas and selected human tumors
.
Cell
2019
;
178
:
346
60
.