DCs are professional antigen presenting cells and can effectively present antigen determinants to activate T cells because they express high levels of co-stimulators and MHC molecules, such as CD80, CD86 and MHC II, particularly after activation [
22,
23]. In this study, we found that T24-exposed DCs displayed significantly reduced levels of CD86, HLA-DR, CD11c expression, and exhibited an impaired capacity to stimulate allogenic T cell proliferation, particularly for those exposed to the CXCL9-treated T24 cells. These data indicated that T24 inhibited the LPS-induced DC activation, consistent with previous studies [
24,
25] and support the notion that tumor cells can suppress the differentiation, trafficking and activation of DCs in a tumor microenvironment for their evasion from immunosurveillance [
26]. There is growing evidence that TADCs change their function from immunostimulatory to immunosuppressive types during the tumor progression [
27], which may be attributed to high levels of PD-L1/PD-1 expression in the tumor microenvironment [
3,
17,
18]. Interestingly, we found that TADCs (after co-cultured with T24 cells) produced significantly higher levels of CXCL9, but not IL-8, RANTES, MCP-1 and IP-10. Furthermore, treatment with the supernatants of cultured TADCs, or recombinant CXCL9 significantly up-regulated PD-L1 expression in T24 cells, which were abrogated by pre-treatment with CXCR3-specific antagonist AM487. In addition, treatment with CXCL9 significantly enhanced the activation of STAT3 and Akt in T24 cells. Such novel findings indicated that bladder cancer cells enhanced CXCL9 in TADCs, which significantly up-regulated PD-L1 expression in cancer cells by activating the CXCR3-related STAT3/Akt signaling. Because the PD-L1 is a potent inhibitor of antitumor T cell immunity the up-regulated PD-L1 expression in cancer cells should inhibit the activation and function of antigen presenting cells and effector T cells, promoting cancer evasion from antitumor responses. Given that anti-PD-1 and anti-PD-L1 are promising to treat some types of solid cancers the CXCL9/CXCR3/PD-L1 axis may be new therapeutic targets for intervention of bladder cancers. Therefore, our findings may provide new insights into the mechanisms underlying how tumors escape from immunosurveillance.
Many types of solid cancers have a complex chemokine network, which can hijack the chemokine system from immune cells and promote their own growth and metastasis in autocrine and paracrine manners [
28,
29]. For instance, some chemokines from tumor cells can alter DC’s phenotype and impair immune response [
26,
29,
30]. CXCR3 plays a crucial role in tumor microenvironment [
31] and CXCR3 over-expression can recruit more TADCs [
32]. Furthermore, CXCR3 can regulate the growth and metastasis of cancers [
33,
34]. High levels of CXCL9 and CXCL10 expression in lymph nodes can promote melanoma cell metastasis through the CXCR3 signaling [
35]. Up-regulated CXCR3 expression in primary and metastatic ovarian tumors was associated with a reduced progression-free survival and overall survival [
36]. It is well known that IFN-γ can induce the expression of ELR-negative CXC chemokines, such as CXCL9, and enhance PD-L1 expression [
37‐
40]. In this study, we found that CXCL9 secreted by TADCs enhanced PD-L1 expression in bladder cancer T24 cells, which was abrogated by the CXCR3 antagonist AMG487. In addition, we found that CXCL9 treatment up-regulated STAT3 and Akt activation in T24 cells. Such findings suggest that in a tumor environment, IFN-γ can through its IFNR1/2 receptors activate the JAK/STAT3 signaling to directly induce PD-L1 expression and enhance CXCL9 expression, which indirectly through its CXCR3 activates the STAT3 and Akt signaling to up-regulate PD-L1 expression in tumor cells [
21,
38,
41]. Actually, phosphorylated STAT1 and STAT3 dimer in tumor cytosol can bind to the PD-L1 promoter to induce PD-L1 expression [
42] while inhibition of STAT3 activity by STATTIC mitigates the CXCR3 activation-induced PD-L1 expression in tumor cells [
21]. Hence, STAT3 activation is crucial for inducing PD-L1 expression in tumor cells and inhibition of STAT3 activity may inhibit the CXCL9/CXCR3 signaling-induced PD-L1 expression, benefiting bladder cancer patients. Currently, the STAT3 inhibitors are being tested in clinical trials for bladder cancer [
43]. Given that up-regulated PD-L1 expression should attenuate antitumor T cell immunity, our findings may shed new lights in the feedback regulation of inflammatory IFN-γ responses on antitumor T cell immunity and tumor evasion from immunosurveillance.