IL-9 is shown by a growing body of data to be linked to lung cancer. Published findings are nonetheless incongruent (22, 23). In this study, LLC and CMT167 mouse models were utilized to explore the role of IL-9 in lung cancer. IL-9 inhibited tumor growth and enhanced anti-tumor CD8+ T cell immune responses in mice harboring IL-9R-expressing CMT167 cells, but no effect was evident in mice bearing LLC cells that lacked IL-9R expression. IL-9 therapy caused increased tumor-infiltrating DCs and elevated MHC-I surface expression in CMT167 tumors, which might explain the tumor suppression mediated by CD8+ T cells. Moreover, IL-9 could synergize with anti-PD-1 in CMT167 tumors.
This study is the first to discover IL-9R expression in CMT167 cells. LLC cells, on the contrary, did not express IL-9R, consistent with previous research (19). Despite the significance of IL-9R expression in tumor suppression, results in this study indicated that IL-9 signaling is not directly linked to either apoptosis or proliferation of the two murine lung cancer cell lines. This observation differs to previous reports that showed IL-9 could promote the proliferation and migratory activity of human lung cancer cell A549 (24). Likely, such discrepancies are due to the different cell lines being tested in our study versus others. Indeed, in melanoma, variations have been reported in different cell lines. Growth suppression was observed only in HTB-72 and SK-Mel melanoma cells upon direct IL-9 exposure, while no responses were identified in HTB-65 and CRL-11147 cells (20). Nonetheless critical involvement of IL-9 through signaling upon IL-9R binding has been illustrated in in vivo models and knockdown experiments. In vivo, IL-9 inhibited the growth of IL-9R-expressing CMT167 tumors but not LLC tumors. Importantly, significant upregulation of CD4+ and CD8+ T lymphocyte frequencies were observed in IL-9-treated CMT167 tumors, indicating the ability of IL-9 to induce T cell immunity. In contrast, in the LLC model, neither tumors nor splenocytes showed significant T cell alteration upon IL-9 treatment. The unresponsiveness of LLC tumors to IL-9 in this study may be different to previous investigations although reports are inconsistent (19, 22, 23, 25). We believe such disparities could mainly arise from the experimental design. Some studies focused on the role of Th9 cells in LLC tumor-bearing mice instead of IL-9 (22, 25). Th9 cells not only secrete IL-9 but also other cytokines including IL-21 and IL-10 (29, 30), which could also directly contribute to inhibition of tumor growth and metastasis (29). As a result, compared with IL-9 injection, the adoptive transfer of Th9 cells into LLC-bearing animals may lead to more complicated outcomes. Furthermore, concomitant administration of IL-9 (19) or direct inoculation of IL-9 transfected LLC cells (23) would not be able to specifically delineate existing versus therapeutic function of IL-9 during LLC tumor development. In this study, IL-9 therapy was initiated only after the full establishment of tumor mass at a palpable size. We believe this study design is more clinically relevant and translatable, and the therapeutic potential of IL-9 could be specifically addressed.
Augmented sensitivity of CMT167 to IL-9-mediated immunotherapy could be due to the intrinsic characteristics of cancer cells (IL-9R positive versus IL-9R negative) as well as extrinsic contributions from the immune compartments in the tumor microenvironment (28). Again, results in this study highlighted the importance of IL-9R and thus IL-9 signaling in triggering anti-cancer immunity since blocking the IL-9/IL-9R axis by IL-9R knockdown abrogated tumor inhibition and anti-tumor T cell responses in CMT167 cells. Although both CD4+ and CD8+ T cells were elevated in the CMT167 tumor, CD8+ T cells were demonstrated to mediate growth control of the CMT167 tumor by IL-9 therapy. We further explored the molecular underpinnings of increased intratumoral CD8+ T cell responses. The T cell activation markers, CD69 and CD25, exhibited no change on the CD8+ T cells from IL-9-treated and control CMT167 tumors, indicating that a major effect of IL-9 is to enhance CD8+ T cell infiltration or proliferation rather than boosting its activation. CXCR3 ligand (CXCL9/10/11) are crucial chemokines that recruit T lymphocytes into tumors (31), and IL-9 increased CXCL9 expression in primary astrocytes during experimental autoimmune encephalomyelitis (32). Nonetheless neither CXCL9, CXCL10, nor CXCL11 exhibited any noticeable alteration in CMT167 tumors treated with IL-9. Instead, IL-9 increased the proportion of DCs, particularly cDC1, in CMT167 tumors, and played pivotal roles in CD8+ T cell priming (27) and recruitment into tumors (33). In line with our study, IL-9-secreting Th9 cells have been shown to elicit strong anti-tumor cytotoxic T cell responses in a lung metastatic melanoma model by upregulating the recruitment of DCs in a Ccl20/Ccr6-dependent manner (18). Whether the increased intratumoral CD8+ T cell population is also a direct consequence of enhanced DC recruitment in an IL-9-treated CMT167 tumor warrants further investigation.
Th9 cells were discovered to boost MHC-I expression on cervical cancer cells (34).
Impaired antigen presentation induced by mutations or loss of heterozygosity of MHC-I are common mechanisms of immune evasion by solid tumors (28, 35). Our study appears to be the first to demonstrate enhanced MHC-I expression on CMT167 cells in response to IL-9 therapy. Blocking the IL-9/IL-9R axis in CMT167 cells eliminated MHC-I upregulation by IL-9, demonstrating that MHC-I elevation was induced directly by IL-9. CMT167 cells had negligible basal MHC-I surface expression in vitro but were dramatically induced by IFN-γ and further augmented upon IL-9 stimulation, indicating enhanced tumor immunogenicity induced by IL-9. IL-9 has been reported to activate Jak-STAT, ERK/MAPK (36, 37), and PI3K/Akt pathways (38). In this study, phosphorylation of ERK1/2 was elevated by IL-9 in CMT167 cells, suggesting that IL-9 may upregulate MHC-I expression by activating the ERK1/2 pathway. This is in line with a recent study showing that upregulated ERK pathway activation increased MHC-I expression in NSCLC cells (36).
Previous studies have shown that preexisting CD8+ T cells correlate positively with the success of ICB (39). Besides, enhancing MHC- I levels in cancer cells is a promising strategy to improve ICB efficacy (40). In addition to the enhanced CD8+ T cell proportion and increased MHC-I surface expression, IL-9 has been observed to upregulate PD-1 expression on CD8+ T cells and PD-L1 expression on cancer cells in a CMT167 tumor. The interaction between PD-L1 and PD-1 leads to T cell dysfunction and exhaustion (41), and PD-L1 expression was shown to be positively correlated with ICB responses (42), leading us to hypothesize that IL-9 may synergize with anti-PD-1 in CMT167 tumor-bearing mice. We show here that the combination treatment indeed significantly delayed tumor growth and enhanced the proportion of CD8+ T cells in CMT167 tumors, indicating a synergistic effect of IL-9 and anti-PD-1 in mice bearing CMT167 tumors. Given that only a small population of NSCLC patients could potentially benefit from anti-PD-1 ICB, results from this study provide experimental evidence for the use of IL-9 as an adjuvant therapy to PD-1 blockade.
Our study has limitations that future studies should address. First, we based our findings on murine lung cancer cell lines and subcutaneous models that may not fully represent the situation in human lung cancer patients. Before proceeding to clinical trials with IL-9, more research is needed on human lung cancer cell lines and/or humanized in vivo models. Second, we focused on MHC-I due to its direct interaction and indispensable role in mounting cytotoxic T cell immune responses in tumors. It is worth noting that our findings may aid in the treatment of malignancies only in which MHC-I is repressed by gene regulatory mechanisms, not in those whose MHC-I components are genetically deleted or HLA genes are lost due to heterozygosity. Other mechanisms that may also contribute to the enhanced CD8+ T cell responses, such as DC presentation activity and clonal expansion, need to be further studied. Third, increases in other immune cells such as CD4+ T cells and cDC1 were also observed upon IL-9 therapy. Future research should address the question of how these changes to other immune cell types are regulated and their potential contributions(s) to anti-cancer activities of IL-9 in CMT167 tumor. For instance, the possible involvement of cytokines and chemokines derived from both cancer cells and these immune cells in response to IL-9 therapy needs to be analysed. Finally, IL-9 is widely involved in immune diseases such as allergic asthma (43), thrombotic diseases (44, 45), and arthritis (38). Strategic and careful design of dose escalation and control of side effects are crucial if we intend to treat lung cancer patients with IL-9.
In summary, our findings indicate that IL-9 plays an important role in regulating cell-surface MHC-I expression and thereby promotes intratumoral CD8+ T cell infiltration or proliferation in CMT167 tumors. Our study is mechanistically new and has great translational potential. It suggests that IL-9 treatment can potentially be utilized in MHC-I-low lung cancer as an adjuvant to ICB therapy. In addition, IL-9R may be able to predict NSCLC patients' susceptibility to IL-9 therapy and ICB responses based on differences in IL-9R expression and responses in mice harboring LLC and CMT167 cells.