T Cell-derived Interleukin-22 Drives The Expression Of CD155 By Cancer Cells To Suppress NK Cell Function And Promote Metastasis
Oct 10, 2023
SUMMARY
Although T cells can exert potent anti-tumor immunity, a subset of T helper (Th) cells producing interleukin-22 (IL-22) in breast and lung tumors is linked to dismal patient outcomes. Here, we examined the mechanisms whereby these T cells contribute to disease. In murine models of lung and breast cancer, constitutional and T cell-specific deletion of Il22 reduced metastases without affecting primary tumor growth. Deletion of the IL-22 receptor on cancer cells decreases metastasis to a degree similar to that seen in IL-22-deficient mice. IL-22 induced high expression of CD155, which bound to the activating receptor CD226 on NK cells. Excessive activation led to decreased amounts of CD226 and functionally impaired NK cells, which elevated the metastatic burden. IL-22 signaling was also associated with CD155 expression in human datasets and with poor patient outcomes. Taken together, our findings reveal an immunosuppressive circuit activated by T cell-derived IL-22 that promotes lung metastasis.
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INTRODUCTION
The major hallmark of neoplastic progression and the primary cause of cancer-related mortality is the ability of cancer cells to disseminate to secondary sites and form metastases.1,2 The formation of metastasis can be prevented by immunosurveillance involving natural killer (NK), cytotoxic, and T helper (Th) 1 cells.3,4 In contrast, regulatory T cells (Treg), circulating monocytes, and Th cell-derived IL-17A form an immunosuppressive microenvironment, enabling immune escape and promoting metastases.4–6 Therefore, it is critical to identify signaling cascades that define the function of pro- vs. anti-tumorigenic immune cells.7,8 Interleukin-22 (IL-22) is a cytokine produced by Th17 and, in humans, also by the Th1 subset, known to promote cancer cell growth, enhance migration, protect from apoptosis, induce epithelial-to-mesenchymal transition, and sustain stemness of malignant cells.9–13 It also promotes early carcinogenesis, acting on precursor lesions or immature cancer stem cells.14–18 IL-22-producing cells, mainly Th cells, but also gamma delta (gd) T cells, invariant natural killer T (iNKT) cells, and innate lymphoid cells (ILCs), have been detected in primary cancer lesions.19–24 IL-22 is expressed at biologically relevant levels in breast, colon, lung, gastric, and hepatocellular carcinoma.9,11,12,25,26 In most studies, its expression is associated with a poor prognosis, higher disease stage, and faster tumor progression.13,22–24,27–29 IL-22 acts exclusively through the IL-22 receptor (IL-22R) comprised of two subunits, IL-22RA1 and IL-10RB.30,31 The action of secreted IL-22 is modulated by an inhibitor, the IL-22 binding protein (IL-22BP, IL-22RA2), a homolog of IL-22RA1, that is mainly produced by myeloid cells.32,33 Under steady-state conditions, IL-22 is an essential homeostatic cytokine at epithelial barriers such as the gut, lung, and skin.34,35 At these sites, IL-22 promotes protection, regeneration, and repair to sustain barrier integrity,36,37 and its absence exacerbates inflammation-induced carcinogenesis.13,38 Together, these data highlight the broad, context-dependent functions of IL-22 in both physiological and pathological conditions. Upon receptor binding, IL-22 triggers the Janus kinases Jak1 and Tyk2 to phosphorylate STAT3, STAT1, or STAT5, but IL-22 can initiate other downstream pathways, including the mitogen-activated protein kinases (MAPK) cascade or PI3K-AktmTOR signaling, depending on the cellular context.13,36,39–44 This diversity of signaling pathways is reflected by the multitude of physiological effects that have been associated with IL-22 signaling, including those described above, as well as protection from genotoxic damage and the induction of anti-bacterial peptides, mucus, pro- and anti-inflammatory cytokines, and chemokines.36,38,39,45,46 Cancer cells induce the production of IL-22 from Th cells in breast and lung cancer patients.12,47,48 NLRP3 inflammasome driven release of IL-1b induces IL-22 production from T cells in the tumor, and both IL-22+ Th cells and an NLRP3-IL-1b signature can be found in tumor samples of breast and lung cancer.48,49 Here, we set out to delineate a mechanism whereby IL-22 promotes breast and lung cancer progression. We found that IL-22 promotes metastasis spread to the lung, revealing a circuit wherein IL-22 mediated immunosuppression in the metastatic niche by promoting the expression of CD155 on cancer cells, which was associated with decreased expression of CD226 on NK cells and reduced interferon-g (IFNg) production. Clinical data indicate that activation of such pathways is linked to patient outcomes.
RESULTS
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IL-22 impacts disseminated cancer cells in syngeneic mouse models of lung and breast carcinoma
To understand the impact of the IL-22-IL-22R1 signaling axis on cancer progression, we analyzed syngeneic murine models of breast and lung carcinoma. As cancer patients mostly succumb to metastatic disease, we recapitulated this with phenotypically relevant models. We implanted either 4T1 breast cancer or Line-1 lung cancer cells subcutaneously (s.c.) in the right flank of wild-type mice (WT) and mice lacking IL-22 expression (Il22 / ) (Figure 1A). IL-22 did not impact the outgrowth of the primary tumors in either model (Figures 1B and 1C). Upon reaching pre-defined termination criteria (tumor >225mm2 or ulceration), the lungs were the main metastatic site in our model with occasional metastases found elsewhere (not shown). Il22 / mice demonstrated decreased metastatic dissemination of Line-1 and 4T1 cells to the lung compared with wild-type animals independently of the primary tumor size using different methods of metastasis detection (Figures 1B, 1C, and S1A). We have also observed a similar metastatic phenotype in an orthotopic model of breast cancer, where 4T1 cells were implanted into the mammary fat pad (Figure S2B). All used methods of blinded macroscopic counting, clonogenic assays, or histology showed high consistency in detecting lower metastatic burden in Il22 / animals (Figures 1B, 1C, and S1C). This implicates the pivotal role of IL-22 in the metastatic process. Next, we forced metastasis through intravenous cancer cell injection, which bypasses the need for tissue detachment and invasion (Figure 1D). Intravenous (i.v.) injections of both cell lines mirrored the phenotype in the subcutaneous model. We could indeed confirm a lower metastatic load in Il22 / mice (Figures 1E and 1F). These results indicate a specific role for IL-22 in disseminated cells in circulation. To discern strain-specific effects, we used an E0771 breast cancer model (Figure 1G).50,51 Intravenous injection of E0771- GFP cells revealed a diminished metastatic burden in Il22 / animals validated by flow cytometry (Figures 1H and S1D). Similarly, we observed a lower metastatic burden in the liver of mice when E0771-GFP cells were injected intrasplenically (Figure S1E). The entire left lung of E0771-GFP-injected mice was optically cleared following an iDISCO protocol to quantify metastases with light-sheet microscopy in situ (Figure 1I).52 Here, Il22 / mice exhibited a reduced propensity to develop metastases, whereas the size of the visualized metastases did not differ and had no specific pattern of their localization (Figure 1J). In summary, IL-22 acted on disseminated cancer cells, enabling breast and lung cancer metastases to the lung.
T cells are the relevant source of IL-22 in the metastatic niche in the lung
We previously identified CD4+ T cells as the main source of IL-22 in primary human lung tumors and bronchoalveolar lavage samples.12,47,48 To delineate the source of IL-22 in the lungs of our models, we intravenously injected E0771 cells into Foxp3mRFP Il17aGFPIl22sgBFP reporter animals and quantified IL-22+ cells using flow cytometry (Figures 2A and S2A).53 Populations were defined as CD4+ , CD8+ and double-negative (DN) (CD4 , CD8 ) ab T cells (CD3+ gdTCR NK1.1 ), gd T cells (CD3+ gdTCR+ NK1.1 ), and CD3+ NK1.1+ and CD3 NK1.1+ cells (Figures 2B and S2B). We observed an increase in the fraction of CD4+, CD8+ T cells, and NK1.1+ cells that produced IL-22 in the lungs of tumor-injected animals (Figures 2C, S2C, and S2D). Here, CD4+ and CD8+ T cells constituted the majority of IL-22-producing cells in tumor-bearing mice (Figure 2C). Moreover, we identified that such CD4+ T cells produced IL-22 but not IL-17A (Figure 2D). These cells had low CD44 expression, confirming their memory phenotype in line with our previous observations (Figure S2E).48 To further explore these findings across models, we used intracellular staining to assess the production of IL-22 in the Line-1 s.c. model, which yielded similar results except for a diminished fraction of CD8+ T cell IL-22 producers (Figures S2F–S2I). We could also identify that IL-22+ cells did not co-express IFNg, which sets apart mouse IL-22 producers from the Th1 subset (Figure 2G).49 Furthermore, we used confocal microscopy on precision-cut lung slices from reporter animals to interrogate their spatial distribution in the lungs of tumor-injected mice. Here, we could identify that such IL-22 and IL-17A producers localize almost exclusively to the metastatic foci (Figure 2E). As demonstrated by flow cytometry, we found no correlation between IL-22 and IL-17A production from the reporter cells in the metastatic foci, indicating that IL-22 and IL-17 are indeed produced by two different cellular subsets. We also confirmed that a large portion of IL-22-producing cells are CD4+ T cells (Figure 2E). As these data indicated a predominant role for T cells in IL-22 production, we generated an Il22floxCd4cre mouse with a conditional deletion of Il22 in all mature T cells (Figure 2F). When challenged with E0771-GFP cells, Il22floxCd4cre mice had a reduced propensity to develop metastases in the lung, reminiscent of the phenotype observed in the global Il22 / animals (Figure 2F). However, we could also confirm that cre-recombinase under the control of CD4 promotor completely abolished IL-22 production not only in CD4+, but also in CD8+ T cells isolated from the spleen of Il22floxCd4cre mice, and therefore this model could not be utilized to pinpoint a specific source of IL-22 (Figure S2J). To confirm the role of Th cells as the crucial source of IL-22 in our model, we transferred wild-type and Il22 / CD4+ T cells into Rag1 / II22/ animals that subsequently received E0771- GFP cells i.v. (Figure 2G). Here, we could confirm that IL-22 production by adoptively transferred CD4+ T cells is sufficient to promote lung metastases in our model but abolished when using CD4+ T cells isolated from II22 / animals (Figure 2G). Importantly, differences in metastasis were affected by T cell engraftment upon transfer (Figure S2K). In conclusion, we identified Th cells as a sufficient source of IL-22 that drives metastases in the lung of tumor-bearing mice, and we next sought to identify the relevant target cell.
Figure 1. IL-22-knockout reduces the number of lung metastases but does not affect tumor growth in syngeneic mouse models of lung and breast carcinoma
Figure 2. T cells are the primary source of IL-22 in the lung of tumor-bearing mice
The expression of IL-22RA1 on tumor cells is indispensable for the formation of metastasis
IL-22RA1 expression is restricted to non-hematopoietic cells and serves as a limiting factor for IL-22 signaling. To interrogate its influence on the metastatic phenotype, we generated a stable Il22ra1 deletion in 4T1 and Line-1 cells (Figure 3A). In line with our previous findings, tumor growth of 4T1 Il22ra1 cells was largely unaffected compared with 4T1 control cells (Figure 3B). However, mice that were injected with 4T1 Il22ra1 cells s.c. or i.v. had fewer macroscopic and clonogenic metastases in the lung compared with control 4T1 cells (Figures 3B and 3C). To confirm that this effect is not clone-dependent, we generated and analyzed three Line-1 Il22ra1 clones and tested them against three control clones, which yielded similar results (Figures 3D and 3E). This confirms that IL-22RA1-expressing cancer cells are the relevant target of IL-22 in driving lung metastases. To rule out the off-target effects of the methodology, we used IL-22BP to inhibit IL-22 signaling. We established 4T1 and Line-1 cell lines that constitutively secreted IL-22BP (Il22ra2+ ) (Figure S3A). When injected s.c., Line-1 Il22ra2+ cells grew comparably with the control at the implantation site, as previously seen in the Il22ra1 models and the Il22 / mice, although 4T1 Il22ra2+ cells grew slower (Figure S3B). Here, 4T1 Il22ra2+ cells had a largely reduced number of metastases when injected s.c. (Figure S3B and S3C). Despite greater variability, the Line-1 Il22ra2+ cells also formed fewer metastases when injected s.c. or i.v. (Figure S3D and S3E). Thus, IL-22 neutralization through IL-22BP largely mimicked the phenotype observed with Il22ra1 cells and Il22 / mice, corroborating the relevance of the cytokine for the metastatic process.
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IL-22 controls the outgrowth of tumor cells during the early stage of metastatic engraftment
To determine the role of IL-22 signaling during the dissemination process, we analyzed the kinetics of metastatic seeding in our models. For this, we injected 4T1-GFP cells i.v. and analyzed lungs at 12 and 48 h after injection (Figure S4A). We used confocal microscopy to quantify the numbers of GFP+ colonies (defined as cell clusters of >100 mm) and individual cells per mm2 of lung tissue (Figure S4B). We could not detect differences at 12 h after injection, indicating that the seeding might not be majorly affected by IL-22 (Figures S4C and S4D). However, at the 48-h time point, the numbers of GFP+ cells and colonies decreased in the lung of Il22 / mice (Figures S4C and S4D). This suggested a role for IL-22 in driving early metastasis in the lungs. To assess the rate of proliferation, we co-injected mice with 5-ethynyl-20 -deoxyuridine (EdU) 4 h before investigation at 12,24, 48 h, and 7 days after tumor injection (Figure S4E). Similar to microscopic counting, we did not detect differences in the numbers of GFP+ cells earlier than 48 h after injection (Figures S4F–S4G). In contrast, we could detect differences in EdU incorporation, which indicates a higher fraction of dividing cells only by day 7 after injection (Figure S4G). Thus, we reason that differences in lung tumor burden affected by IL-22 observed as early as 48 h are mediated by a mechanism independent of proliferation.
Figure 3. IL-22RA1 expression on tumor cells is indispensable for metastasis formation
IL-22 regulates the expression of CD155 on tumor cells and thereby promotes metastasis
Based on our findings that IL-22 acts on IL-22ra1+ cancer cells to promote metastasis, we performed bulk RNA sequencing of 4T1 cells treated with IL-22 to further delineate the underlying mechanism (GEO: GSE202314) (Figure 4A). We discovered 147 genes that were differentially regulated. Of these, the expression of 133 genes was increased, and 14 decreased upon IL-22 treatment (Figures 4B and S5A). We validated Pvr (poliovirus receptor, Pvr) as one of the most significantly increased targets using qPCR (Figure S5B). This is notable because CD155, the product of Pvr, is overexpressed in various cancers and possesses tumor-promoting properties, including metastasis.54–56 To confirm our findings, we evaluated the expression of CD155 in IL-22-stimulated 4T1, Line-1, and E0771 cells by flow cytometry (Figure 4C). We detected an increase in the expression of CD155 in all cell lines over 72 h (Figures 4D and 4E), but this effect was absent in cells lacking IL-22RA1 (Figure S5C). Next, we evaluated the impact of IL-22 on CD155 expression in E0771-GFP cells from the lungs of tumor-bearing mice (Figure 4F). Here, we could confirm that cells implanted into Il22 / mice had a lower expression of CD155, which correlated with a smaller fraction of E0771-GFP cells detected by flow cytometry (Figures 4G and 4H). To verify the role of CD155 in metastasis, we established Pvr Line-1 and 4T1 cell lines (Figures 4I, S5D, and S5G). While this had little effect on their capacity to grow subcutaneously (Figures S5E and S5F), it abolished the ability to form metastases in the lung (Figures 4J and S5H). We could reverse this process by constitutive CD155 expression in Pvr cells independently of IL-22-induced regulation (Pvr+ ).
Figure 4. IL-22 signaling increases the expression of CD155 on the surface of tumor cells and confers resistance to metastasis control
In this setting, we could induce metastases in the lungs of Il22 / mice, highlighting the link between these two molecules and their role as mediators of the metastatic process (Figures 4K and S5I).
CD155 on tumor cells is associated with decreased expression of CD226 on NK cells and reduced IFNg production
CD155 plays an intrinsic role in proliferation and adhesion in cancer cells,54–57 among others. We did not detect deficiencies in the proliferation of Line-1 Pvr cells in vitro (data not shown). Importantly, CD155 has a cell-extrinsic pro-metastatic role by binding to the immunomodulatory receptors CD96, CD226, or TIGIT on the surface of NK and T cells.54–56,58,59 To pinpoint the binding partners of CD155, we analyzed anti-tumor responses in the lungs of mice containing 4T1-luciferase+ cells (4T1-Luc) (Figure 5A). We confirmed using an in vivo imaging system (IVIS) that wild-type and Il22 / mice had similar seeding of tumor cells by day 5 after injection, and the differences in tumor burden increased over the course of two weeks (Figures 5B and 5C). Indeed, the defect of IFNg production by NK cells, but not other cell types, showcased the loss of humoral effector mechanisms (Figures 5D, 5E, and S6A) and correlated with higher tumor burden (Figure S6B). This effect was consistently found in the Line-1 s.c. model (Figures S6C–S6E). Scrutinized by chip cytometry,60 samples from 4T1-lung metastasis-bearing mice demonstrated increased expression of CD155 in the metastatic foci in WT but not in Il22 / animals (Figures 5F and S6F). We found higher infiltration of NK cells into the metastatic foci of Il22 / but not WT animals, suggesting higher activation and confirming the dependency on NK cells as anti-tumor effector cells (Figure 5F). CD226, but not TIGIT or CD96, was differentially expressed by NK cells and, to a lesser extent, by CD8+ T cells in the lungs of wild-type and Il22 / animals (Figures 5G and S6G–S6H). CD226 is a co-receptor essential for the activation of effector functions of NK and CD8 T cells. Therefore, we set out to explore its pathophysiological relevance in our model.61–64
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Blockade of CD226 abrogates the anti-metastatic phenotype of IL-22-deficient animals
Excessive CD155-mediated signaling present in the tumor microenvironment can induce internalization and degradation of CD226 in effector cells.61,63 To delineate the role of excessive CD155 expression on CD226 and subsequent anti-tumor response, we injected 4T1 control and Pvr+ cells i.v. in Il22/ mice, and two groups also received anti-CD226 blocking antibody (480.1) (Figure 6A). Both Pvr+ cells and CD226 blockade could similarly promote lung metastasis in Il22 / mice, and these effects did not synergize (Figure 6B). Similar to our previous observations in wild-type animals, this was sufficient to inhibit IFNg production by NK cells (Figure 6C). Finally, we detected a decrease in CD226 expression on NK cells in Pvr+-injected mice compared with control cells. This correlated with the decreased capacity of these NK cells to produce IFNg (Figure 6D). Furthermore, we interrogated the potential of agonistic TIGIT antibody (IG9) to inhibit NK cell activation in Il22 / animals and of CD96 blockade (3.3) to prevent the inhibition of NK cell function (Figure 6E). Neither the TIGIT agonist nor CD96 antagonist altered the number of metastases when compared with Il22 / animals that received control or Pvr+ 4T1 cells, respectively (Figure 6F). When activated, TIGIT could inhibit IFNg production from CD8+ T but not NK cells (Figure 6G). Thus, an IL-22- CD155 axis triggers decreased expression of CD226 in NK cells and renders them inert in the tumor microenvironment.
CD155 expression complements the IL-22 gene signature in breast and lung cancer patients
Finally, we assessed the clinical relevance of CD155 in the context of the IL-22-IL-22RA1 axis. CD155 expression alone is associated with unfavorable prognosis in a variety of cancer entities.54 We analyzed RNA sequencing data from the cancer genome atlas (TCGA) lung adenocarcinoma (TCGA: LUAD, n = 504) and HER2-positive patient samples from the invasive breast carcinoma (TCGA: BRCA, n = 110) datasets. We focused on key IL-22-related genes: IL22RA1, IL22RA2, IL10RB, and PVR. To stratify patient cohorts, we utilized agglomerative clustering, an unsupervised clustering method, resulting in three major clusters (Figures 7A and 7B). This revealed distinctive gene expression patterns: cluster 0 (IL22RA1hi, IL22RA2lo, IL10RBmed, PVRhi), cluster 1 (IL22RA1lo, IL22RA2hi, IL10RBhi, PVRlo), and cluster 2 (IL22RA1lo, IL22RA2lo, IL10RBlo, PVRmed) (Figure 7C). These clusters were evenly distributed in these two cohorts (Figure 7D). Patients in cluster 0 and LUAD dataset cluster 2 had worse survival than patients in cluster 1 (Figure 7E). Survival of clusters 1 and 2 did not differ in both cohorts (Figure 7E). Further, we calculated restricted mean survival times (RMST) for clusters 0 and 1 to quantify the difference in expected survival time until five years of follow-up, resulting in 361.18 days for LUAD and 93.23 days for BRCA (Figure 7F). Clusters 0 and 1 had differences in the frequency of pathologic disease stages within them in the LUAD but not in the BRCA cohort (Figure 7G). Importantly, such survival differences between clusters (IL22RA1hiPVRhi) and 1 (IL22RA1loPVRlo) mainly stem from patients diagnosed in the early (I and II), but not at advanced, stages of the disease (III and IV) (Figure S7A). To assess the impact of each gene on survival, we utilized Cox’s proportional hazards model. Both IL22RA1 (hazard ratio [HR] = 1.23) and PVR (HR = 1.28) impact survival, whereas IL22RA2 and IL10RB did not change the hazard in the LUAD cohort (Figure S7B). Moreover, only CD226 (p = 0.06), but not TIGIT or CD96, trended to influence the survival, in line with our findings in preclinical models (Figure S7B). We used the CIBERSORTx deconvolution algorithm on the LUAD cohort to assess whether gene expression patterns of our clusters have an impact on immune cell infiltration in patients.65 Interestingly, there is an increase in CIBERSORTx units for activated NK cells in cluster 1 when compared with clusters 0 and 2 in LUAD patients, whereas there were no differences in resting NK cells or activated CD4+ memory T cells (Figure S7C) with a similar trend in the BRCA cohort (Figure S7D). Together, these results from clinical cohorts demonstrate the relevance of a regulatory link between T cell-derived IL-22 and CD155.
Figure 5. CD226 expression is higher on NK cells in Il22–/– mice
DISCUSSION
In this study, we discovered a mechanism by which T cells produce IL-22 to promote lung metastasis in mouse models of lung and breast cancer. Mechanistically, T cells at the metastatic foci in the lung, predominantly CD4+, produce IL-22 that signals directly through its receptor expressed in cancer cells, promoting expression of the pro-metastatic molecule CD155.54-56,58,59 Despite the well-studied pan-cancer expression of CD155 and its intrinsic and extrinsic roles in cancer progression,54 the pathway responsible for CD155 regulation in malignant cells remains elusive.66–68 We demonstrated that IL-22 increased CD155 expression in lung and breast cancer cell lines in vitro and in vivo, whereas its constitutive expression enabled metastases in Il22 / mice, compared with control and deficient cells. Increased expression of CD155 in the tumor lung microenvironment led to a reduction of co-stimulatory molecule CD226 on NK cells, diminishing their localization to metastases and IFNg production, which correlated with higher tumor burden. In our previous studies, we detected an accumulation of IL-22-producing T cells in non-small-cell lung carcinoma (NSCLC) patient tumor samples.12,48 We have demonstrated that cancer cells trigger NLRP3-dependent secretion of IL-1b that induces such IL-22 production mainly from Th cells.18 Worth noting is that, in humans, due to the differences in the Th cell cytokine pro- files, IL-22 is also induced in Th1 cells. It is conceivable that this might impair the anti-tumor immune response. Importantly, such Th1 variants might partially explain cancer hyperprogression upon T cell activation following checkpoint blockade.69 In line with our previous observations, we confirmed the accumulation of IL-22-producing CD4+ and CD8+ T cells, but also NK1.1+ cells, in the metastatic niche in the lung of tumor injected animals. It is important to note that we observed strain-specific differences in the accumulation of such IL-22-producing CD8+ T cells across our models. However, abolishing the IL-22 production in total mature T cells was sufficient to recapitulate the effect we observed in IL-22-deficient animals.70 Also, the adoptive transfer of CD4+ T cells into Rag / II22 / mice was sufficient to induce lung metastases, pinpointing the paramount role of Th cells as a source of IL-22. Conversely, our previous findings suggest that despite an accumulation of CD8+ T cells in tumor samples, their contribution to the IL-22 pool is minor.48 In any case, the characterization of such CD8+ IL-22-producing T cells is essential to carefully evaluate their pro- or anti-tumor properties as new data on this subpopulation emerges.71,72 Also, the primary role of NK and NKT cells in tumor control outweighs their potential contribution to metastasis formation through IL-22, which is discerned in a Rag / II22 / adoptive transfer experiment using animals devoid of mature T cells but having functional NK cells.37 This hypothesis is further supported by our findings in the metastatic foci where NK cells were found more abundantly than cytotoxic T cells, highlighting NK cells as essential players in tumor control in the absence of IL-22. It is important to note that we focused on IL-22 producers at metastatic foci in the lung but did not consider their origin, clonality, or their distribution in blood or lymphoid organs. Moreover, various sources of IL-22 promote tumor progression in a context-dependent manner mediated by the pleiotropic action of this cytokine. Along these lines, similar IL-22-producing cells in various compartments (lung vs. spleen vs. lymph node) could differentially affect pro-tumoral phenotypes or have no function depending on the context, which will need to be investigated further. Cancer studies repeatedly report that IL-22 affects the development and growth of primary tumors and, eventually, neoplastic progression.26,73–76 This notion is mainly justified by the ability to promote migration, invasion, and stemness of cancer cells in vitro and thus promote metastasis formation.76,77 Importantly, ablation of IL-22 can alleviate the immunosuppressive microenvironment in a Kras-mutant model of lung carcinoma.14 In the current study, we demonstrated that the increased metastatic burden was a direct effect of IL-22 on disseminated IL-22RA1+ tumor cells, which resulted in increased colony outgrowth. Importantly, our data do not formally rule out an impact on non-tumor cells. As such, the influence of IL-22 on tumor cell dissemination through the intrinsic action of endogenously expressed IL-22R is extensively highlighted by Giannou et al.78
Figure 6. CD226 signaling is indispensable for IFNg production from NK cells
We demonstrated that Pvr is one of the genes with increased expression on cancer cells upon IL-22 treatment. In this context, CD155-deficient cells formed few metastases, both in wild type and Il22 / mice, and, importantly, the reintroduction of CD155 allowed us to reconstitute the metastatic phenotype. The intrinsic role of CD155 in cancer cells has been well studied and is known to affect seeding, tumor cell proliferation, and migration.56,57 However, Pvr cells did not display inhibited proliferation or migration in our hands, and the seeding of tumor cells was unaffected in Il22 / mice. Initially identified on antigen-presenting cells, CD155 serves as an extrinsic promotor of tumor progression that suppresses NK and T cell function by binding to CD96 and TIGIT on their surface and induces internalization and downregulation of CD226.56,61–64,79–85Due to its immunosuppressive function, CD155 in cancer and host cells exerts pro-metastatic properties and is proposed as a target for checkpoint inhibition blockade.61,86 NK cells receive a co-stimulatory signal from antigen-presenting cells via CD226.87 However, excessive stimulation of CD226 in the tumor microenvironment leads to internalization and degradation.63 This is typically counteracted by CD96 and TIGIT, which bind CD155 with a higher affinity.58 Interestingly, 4T1 cells are known to induce CD226 downregulation in tumor-infiltrating lymphocytes and suppress IFNg production.88 Here, we demonstrated that IL-22 deficiency preserved CD226 expression on NK and CD8+ T cells. However, only NK cells had dramatically higher IFNg production capabilities and inversely correlated with metastatic burden. Interestingly, activation of TIGIT signaling in our study inhibited IFNg production from CD8+ T, but not NK, cells and was not sufficient to increase the metastatic burden. Similarly, another receptor for CD155, CD96, was neither differentially regulated in Il22 / mice nor did inhibition thereof prevent metastasis, indicating that CD155 does not suppress NK cells via CD96 in our model. This highlights cell type-specific regulation of anti-tumor responses by CD155 and its various binding partners.
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The impact of CD155 on the prognosis and its role in the pathogenesis of lung, breast, colon, and other types of cancer is established.79–85 There is vast evidence on the prognostic relevance of IL-22 and its related genes in various cancer entities.10,21,27,75,89–91 However, some studies report no influence of IL-22 expression on survival.12 While these discrepancies might be due to the heterogeneity in patient populations, sampling, and reporting issues, many of these studies focus on a single gene related to IL-22 signaling. Here, we utilized agglomerative clustering to discern expression patterns of IL22RA1, IL22RA2, IL10RB, and PVR in LUAD and BRCA cohorts in TCGA and affiliate them with the clinical data. Here, we identified three patterns of expression of these genes: pattern 0 (IL22RA1hi, IL22RA2lo, PVRhi), pattern 1 (IL22RA1lo, IL22RA2hi, PVRlo), and pattern 2 (IL22RA1lo, IL22RA2lo, PVRmed). We identified that high expression of the IL22RA1 coincided with a high expression of PVR, which also translated into poor overall survival outcomes, particularly in patients diagnosed with early (I and II), but not advanced (III and IV), pathological stages, highlighting the stage-specific role of this mechanism. Conversely, high expression of IL22RA2, also known as IL-22BP, was correlated with lower PVR expression and better survival.18,34 The third pattern corresponded to all-low expression and represented immunologically cold tumors.92 Along these lines, CIBERSORTx deconvolution indicated that cluster 1 characterized by high IL22RA2 expression features a gene signature for activated, but not resting, NK cells compared with other clusters. CD226 expression is demonstrated to stratify patients for outcome in several NSCLC clinical trials.93 However, due to the dual way of pre- and post-translational regulation of CD226, expression is not always reflected in mRNA sequencing data.94 Hence, studies that focus on the post-translational regulation of CD226 evaluate its expression in clinical samples using antibody staining.63 Nevertheless, when interrogating the TCGA dataset concerning the relationship of CD155 binding partners to survival, only CD226 trended toward bettering prognosis (log (HR) = 0.24, p = 0.06), whereas TIGIT and CD96 demonstrated no correlation with survival. Importantly, tumor cells engineered to secrete IL-22BP formed fewer metastases, highlighting the potential of the IL-22 pathway for targeted therapeutic intervention. This could counteract tumor CD155 overexpression, as direct targeting of which remains challenging due to a complex network of co-receptors. Of further note, the long-term effects of IL-22 neutralization on metastasis are unknown but could have a direct impact on the therapeutic consideration of T cells or provide the rationale for IL-22 neutralization using antibodies with a beneficial safety profile, such as Fezakinumab (trial NCT01941537) or engineered IL-22 with structure-based design.39,95 In summary, we identified IL-22-induced CD155 overexpression on the tumor cells as a mechanism that benefits metastatic outgrowth. This essential role in prognosis stressed the potential of IL-22 as a therapeutic target in cancer. So far, the neutralization of IL-22 is proposed mainly as a strategy to treat autoimmune diseases.31,49 Our data on IL-22BP as a neutralizer of IL-22, which phenocopied the global IL-22 deficiency, underpinned the therapeutic potential for targeting the IL-22-IL-22R1 axis and should be further explored in preclinical and clinical studies.
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