Background Checkpoint blockade therapy (CBT) can effectively treat lung tumors; however, only 30% of lung cancer patients respond to CBT [1]. Clinical data showed that patients with low T cell infiltration in the tumor microenvironment (TME) respond poorly to CBT [2]. Gaining insight into the molecular and immunological mechanism that impact the entry of CD8 T cells into the TME will enable the development of innovative therapeutic approaches that can be used in combination to effectively treat individuals with cancer. Using a T cell gene signature, we segregated NSCLC patients into T cell-infiltrated and non-T cell-infiltrated and found that Sox2 upregulation correlates with a lack of T cell infiltration. Here we aimed to investigate how high expression of Sox2 in tumors mediates immune evasion in NSCLC.

Methods We overexpressed Sox2 in a mouse lung adenocarcinoma cell line driven by KrasG12^D/+ and Tp53^-/-. SOX2-positive (KPS2) and control (KPCt) tumor cells were inoculated subcutaneously or via tail vein injection to induce lung metastasis. We treated tumors with anti-PD-L1 and anti-CTLA-4 blocking antibodies and analyzed for tumor burden. T cell infiltration was evaluated by fluorescence microscopy. KPCt and KPS2 cell lines were engineered to express the model antigen SIY to characterize tumor-specific T-cell responses. Regulatory T cells were targeted by systemic depletion using the Foxp3.DTR mouse model or via neutralization of DKK1 using the mDKN-01 antibody. Finally, we assessed the therapeutic benefit of combining anti-DKK1 treatment with CBT to treat SOX2 tumors.

Results We found that Sox2 overexpression induces resistance to CBT mediated by CD8 T cell exclusion from the tumor core. Analysis of tumor-reactive T cells indicated that T cell priming and differentiation into cytotoxic effector T cells were unaffected. However, cytotoxic CD8 T cells failed to infiltrate KPS2 tumors while enriched in the peritumoral regions. Interestingly, we found a high density of regulatory T cells in the peritumoral area. Depletion of regulatory T cells significantly improved the activation of the tumor vasculature and the infiltration of cytotoxic CD8 T cells into the tumor core. Furthermore, we showed that combining a neutralizing antibody against DKK1 with CBT significantly reduced the density of regulatory T cells in the TME, increased CD8 T cell infiltration, and improved tumor control.

Conclusions Our results show that tumor cell-intrinsic activation of Sox2 in NSCLC promotes immune evasion and contributes to immunotherapy resistance by retaining effector CD8 T cells outside of the tumor mass.

Acknowledgements This work was supported by NCI K99/R00 award, the Ludwig Center at MIT, the SITC-Nektar Therapeutics Equity and Inclusion in Cancer Immunotherapy Fellowship, and Leap Therapeutics Inc.

References

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2. Chen, D. S. & Mellman, I. Elements of cancer immunity and the cancer-immune set point. Nature 2017;541, 321−330, doi:10.1038/nature21349.

Ethics Approval All mouse experiments were approved by MIT’s Committee on Animal Care (CAC) – PHS Animal Welfare Assurance # D16–00078 (A3125–01).