E3 Ubiquitin Ligase Cbl-B Deficient Cd8+T Cells Overcome Treg Cell-Mediated Suppression Through Ifn-Gamma And Induce Robust Anti-Tumor Immunity

Background Adoptive T cell therapy (ACT) is reaching its potential in multiple malignancies. However, anti-tumor T cell responses can be attenuated by suppressive cells in the tumor microenvironment, such as CD4+FoxP3+ regulatory T (Treg) cells. Depletion of Treg cells can be technically challenging in ACT and may be associated with unwanted adverse effects. Alternatively, studies suggest that specific modifications in T cell signaling network may render T cells resistant to regulation by Treg cells. Here, we investigated the role of Casitas B- Lineage Lymphoma-b (Cbl-b), an E3 ubiquitin ligase and a negative regulator of TCR signaling pathways, in rendering CD8+ T cells resistant to the effects of Treg cells to bolster ACT. Methods In vitro stimulated Cbl-b+/+ or Cbl-b-/- Thy1.1+ P14 TCR-transgenic CD8+ T cells were adoptively transferred into B16-gp33 melanoma-bearing Thy1.2+ FoxP3-GFP/DTR transgenic mice treated with or without diphtheria toxin (n = 15). Tumor size and overall survival were measured. Congenically labelled T cells from tumor, draining lymph node, and spleen were comprehensively profiled using flow cytometry. To further examine the biological mechanism of Treg resistance, we performed in vitro Treg suppression assays and RNA-sequencing. Results Adoptively transferred tumor-specific Cbl-b-/- effector CD8+ T cells mediated superior control over tumor growth and increased overall survival in comparison to the wild-type counterpart. Depletion of FoxP3+ cells increased the quantity and percentage of CD25+ 4-1BB+ expressing P14 Thy1.1+ CD8+ T cells in the tumor, whereas the effect of FoxP3+ cell depletion was negligible with Cbl-b deficient CD8+ T cells. Cbl-b deficiency also attenuated sensitivity to Treg cell-mediated suppression in vitro. Transcriptomic analyses suggested that Cbl-b regulates pathways associated with cytokine production and cellular proliferation. Specifically, hyper-secretion of IFN-γ by Cbl-b deficient CD8+ T cells attenuated suppression by Treg cells. In murine models of adoptive T cell therapy, Cbl-b deficient CD8+ T cells were less susceptible to suppression by Treg cells in the tumor through the effects of IFN-γ. Conclusions We demonstrate that adoptively transferred effector CD8+ T cells are susceptible to regulation by Treg cells in the tumor, and that ablation of Cbl-b abrogates Treg cell-mediated suppression. We highlight the therapeutic implications of targeting Cbl-b in the context of ACT. Acknowledgements We would like to thank Dr. Tak Mak and Dr. Naoto Hirano for their suggestions and insights for this project.