Diacylglycerol Kinase ζ (DGKζ) and Casitas b-Lineage Proto-Oncogene b–Deficient Mice Have Similar Functional Outcomes in T Cells but DGKζ-Deficient Mice Have Increased T Cell Activation and Tumor Clearance

Abstract

Targeting negative regulators downstream of the T cell receptor (TCR) represents a novel strategy to improve cancer immunotherapy. Two proteins that serve as critical inhibitory regulators downstream of the TCR are diacylglycerol kinase ζ (DGKζ), a regulator of Ras and PKC-θ signaling, and Casitas b-lineage proto-oncogene b (Cbl-b), an E3 ubiquitin ligase that predominantly regulates PI(3)K signaling. We sought to compare the signaling and functional effects that result from deletion of DGKζ, Cbl-b, or both (double knockout, DKO) in T cells, and to evaluate tumor responses generated in a clinically relevant orthotopic pancreatic tumor model. We found that whereas deletion of Cbl-b primarily served to enhance NF-κB signaling, deletion of DGKζ enhanced TCR-mediated signal transduction downstream of Ras/Erk and NF-κB. Deletion of DGKζ or Cbl-b comparably enhanced CD8+ T cell functional responses, such as proliferation, production of IFNγ, and generation of granzyme B when compared with WT T cells. DKO T cells demonstrated enhanced function above that observed with single knockout T cells after weak, but not strong, stimulation. Deletion of DGKζ, but not Cbl-b, however, resulted in significant increases in numbers of activated (CD44hi) CD8+ T cells in both non-treated and tumor-bearing mice. DGKζ-deficient mice also had enhanced control of pancreatic tumor cell growth compared to Cbl-b-deficient mice. This represents the first direct comparison between mice of these genotypes and suggests that T cell immunotherapies may be better improved by targeting TCR signaling molecules that are regulated by DGKζ as opposed to molecules regulated by Cbl-b.

Diacylglycerol kinase ζ limits IL-2-dependent control of PD-1 expression in tumor-infiltrating T lymphocytes

“…13 Our previous observations showing a correlation between enhanced IL-2 production and reduced PD-1 expression as a result of DGKζ depletion might be a plausible mechanism to explain the enhanced control of engrafted tumors observed in DGKζ-deficient mice. [28][29][30] Although the Jurkat cell line is a good platform in which to study cytokine production, these cells lack cytotoxic abilities. We, thus, moved to mouse models that facilitate the examination of the DGKζ modulation of antitumor T cell functions.…”

“…25 26 Compared with DGKα, DGKζ has additional functions that limit the PKCθ/PDK-1/AKT axis, that in turn regulates nuclear factor κ-lightchain-enhancer of activated B cells (NFκB) activation and mTOR metabolic control downstream of CD28 activation. 27 DGKζ-deficient mice show partial but clear resistance to orthotopically implanted tumors, [28][29][30] suggesting that DGKζ inhibition help to re-invigorate exhausted TIL and engineered CAR T cells. 31 32 In spite of its potential therapeutic value, there is limited information on the effects of DGKζ targeting in human T cells, partially as a result of the lack of isoform-specific inhibitors.…”

E3 ubiquitin ligase Casitas B lineage lymphoma-b and its potential therapeutic implications for immunotherapy

“…Deletion of Cbl‐b resulted in a substantial up‐regulation in CD8 + T cell functional responses, including proliferation, granzyme B production and infiltration into the tumor site by increasing the LFA‐1 when compared with wild‐type T cells [139]. Stromness et al .…”

Beyond the Cell Surface: Targeting Intracellular Negative Regulators to Enhance T cell Anti-Tumor Activity

“…Recently, our lab compared T cell activation and tumor clearance by targeting either Cbl-b or DGKζ in mice [241]. We observed that naïve CD8 + T cells from mice lacking either Cbl-b or DGKζ showed enhanced CD8 + T cell responses after in vitro stimulation when compared with wild type T cells, an effect that was enhanced with deletion of both genes.…”