1309 A STUB1-CHIC2 complex decreases IL-27Rα expression on CD8+T cells to restrain tumor immunity

Background

Immunotherapies have revolutionized the treatment of cancer, yet most patients do not respond to ICB, highlighting the need to identify novel regulators of tumor immunity for improving clinical responses. In vivo CRISPR-based loss-of-function screens are a powerful approach for identifying and prioritizing regulators of CD8⁺ T cell responses to tumors, while modeling the complex spatial, metabolic, cellular, and cytokine milieu in the tumor-draining lymph node and tumor.¹ These screens have been used to identify novel negative regulators of CD8⁺ T cells,^{2 3} but have been performed with in vitro preactivated CD8⁺ T cells, precluding the evaluation of genes that regulate CD8⁺ T cell priming and trafficking from the tumor-draining lymph node.^{4 5}

Methods

To identify regulators of CD8⁺ T cell priming and trafficking, we performed an 899-gene in vivo CRISPR screen interrogating murine naive CD8⁺ T cells responding to mice inoculated with B16-OVA melanoma cells. We compared gene KO and control CD8⁺ T cell functions by flow cytometry and tumor growth control capacity in multiple murine cancer models. We evaluated molecular mechanisms by mass spectrometry, flow cytometry of murine and human CD8⁺ T cells, and genetic epistasis experiments.

Results

Our in vivo screen in naive CD8⁺ T cells uncovered many novel regulators compared to published screens starting with activated CD8⁺ T cells. In particular, gRNAs targeting the E3 ubiquitin ligase, STUB1, were significantly enriched in the screen, suggesting targeting STUB1 may improve CD8⁺ T cell responses to tumors. We found that Stub1 KO CD8⁺ T cells expressed more functional markers relative to control cells, including Granzyme B, Perforin, and IFNγ. Moreover, Stub1 KO CD8⁺ T cells significantly attenuated tumor growth compared to control T cells in multiple murine models of cancer. We performed mass spectrometry on control and Stub1 KO CD8⁺ T cells and found that STUB1 ubiquitinates the adaptor protein CHIC2 and regulates the expression of multiple cytokine receptors, including IL27Rα. Mechanistically, we found that STUB1 physically interacts with CHIC2 to regulate cytokine receptor expression in mouse and human CD8⁺ T cells. Using genetic approaches, we showed that IL27Rα expression is essential for the accumulation of Stub1 KO or Chic2 KO CD8⁺ T cells in tumors and tumor growth control.

Conclusions

Together, these findings demonstrate that the STUB1-CHIC2 complex is a novel regulator of cytokine receptor expression in CD8⁺ T cells. These data provide the rationale for inhibiting this pathway to improve CD8⁺ T cell-mediated anti-tumor immunity.

References

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Consent

All animal studies were undertaken under the animal care guidelines from the Harvard Medical School Standing Committee on Animals and the National Institutes of Health (Protocol number: IS00000041-9). Human PBMCs were purchased from Research Blood Components, which obtained informed consent from all participants and was undertaken under IRB 120160613.