br This transcriptional signature showed a number
This transcriptional signature showed a number of genes to be highly upregulated in KP lung-associated gd T Dynasore compared to splenic gd T cells (Figures 7D and 7E), and these data were further validated at the mRNA and/or protein levels (Figures S7B–S7D). In addition to IL-17A and IL-17F, gd T cells in the KP lungs strongly expressed other effector molecules that might contribute to tumor-promoting inflammation. For example, CXCL2 is a neutrophil chemoattractant, whereas PTGS2 (prostaglandin-endoperoxide synthase 2) is essential for the biosynthesis of prostaglandin, a potent inflammatory mediator.
Figure 5. Commensal Microbiota Are Important for the Differentiation and Activation of Tumor-Associated gd T Cells in the Lung
See also Figure S5.
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IL-1R1 and IL-23R expression were also elevated in gd T cells from tumor-bearing lungs (Figure 7E), indicative of their enhanced capability of responding to IL-1b and IL-23 induced by microbial products as compared to splenic gd T cells.
Among the top differentially expressed genes, IL-22 is an epithelial growth factor significantly upregulated in lung-tumor associated gd T cells. Although known to exert a tumor-promot-ing effect in colorectal cancer (Kirchberger et al., 2013; Wang et al., 2017), the role of IL-22 in lung cancer has not been well studied. We tested the effect of IL-22 receptor deletion in devel-oping lung cancer cells by introducing a lentivirally encoded sin-gle guide RNA (sgRNA) targeting IL-22RA1 in KP mice harboring a LSL-Cas9 allele. As shown in Figure 7, targeting IL-22RA1 led to a significant reduction in tumor burden (Figure 7F). Moreover, recombinant IL-22 stimulated tumor cell proliferation in vitro (Fig-ure S7E). As IL-22RA1 determines cellular sensitivity toward IL-22, we further examined IL22RA1 expression in human lung cancer patients and found IL-22RA1 expression was substan-tially elevated in LUAD samples as compared to normal lung tis-sues (Figures 7G and S7F). When patients were stratified by their levels of IL22RA1 expression, we found that those with high expression (top 30%) exhibited significantly worse survival as compared to the rest of the cohort (Figure 7H). These data are consistent with a role for IL-22 signaling in human lung cancer progression. Another top-scoring gene in the KP lung gd T cell signature was amphiregulin (Areg), a ligand for the EGF receptor (EGFR). Increased Areg levels have been correlated with a poor prognostic outcome and poor response to therapy in lung cancer patients (Busser et al., 2011). We observed a robust expression of Areg in lung tumor-associated gd T cells at both the mRNA and the protein levels (Figures S7B and S7D). KP tumor-derived cell lines showed dose-dependent increases in proliferation in response to recombinant Areg (Figure S7G). These findings, together with the data on the IL-22 pathway, provide strong evidence that microbiota-activated gd T cells may promote lung cancer progression by directly stimulating tumor cell prolif-eration, in addition to driving inflammation in the lung microenvironment.
The lung is a mucosal tissue exposed to a vast number of air-borne microbes and colonized by a diverse bacterial community in normal physiological conditions. Thus, the development of lung cancer may be influenced not only by the distal intestinal mi-crobiota indirectly through its regulation of the systemic immune
response, but also by direct interaction of the local airway micro-biota with developing tumor cells and the local immune system. Our findings demonstrate that lung tumor growth in the KP model is associated with increased total bacterial load and reduced bacterial diversity in the airway, suggesting that tumor-associ-ated barrier defects and airway obstruction might result in impaired bacterial clearance and altered growth conditions for microbes. Our data support a model in which this dysregulation of local microbiota stimulates tissue-resident gd T cells, which in turn produce IL-17 and other pro-inflammatory mediators to pro-mote neutrophil expansion and tumor cell proliferation. This may further potentiate inflammation and dysbiosis, establishing a vi-cious cycle that exacerbates tumor growth.