Bernard, P.-L. | bioRxiv Science

bioRxiv2021

DOK1 and DOK2 regulate CD8+ T cell signaling and memory formation without affecting tumor cell killing

Targeting intracellular inhibiting proteins is a promising strategy to improve CD8+ T cell anti-tumor efficacy. DOK1 and DOK2 are CD8+ T cell inhibitory proteins that are targeted in this study in order to improve the activation and cytotoxic capacities of these cells. To evaluate the role of DOK-1 and DOK-2 depletion in physiology and effector function of T CD8+ lymphocyte and in cancer progression, a transgenic T cell receptor mouse model specific to melanoma antigen hgp100 (pmel-1 TCR Tg) was established. Depletion of both Dok1 and Dok2 did not affect the development, proliferation, mortality, activation and cytotoxic function of naive CD8+ T cells. However, after an in vitro pre-stimulation Dok1/Dok2 DKO CD8+ T cells had higher percentage of effector memory T cells and showed an increase in levels of pAKT and pERK upon TCR stimulation. Despite this improved TCR signaling, pre-stimulated Dok1/Dok2 DKO CD8+ T cells did not show any increase in their activation or cytotoxicity capacities against melanoma cell line expressing hgp100 in vitro. Altogether we demonstrate here a novel aspect of the negative regulation by DOK1 and DOK2 proteins in CD8+ T cells. In conclusion, DOK1 and DOK2 have an inhibitory role following long term T cell stimulations.

immunology↗

bioRxiv2021

CISH targeting in NK cells activates natural cytotoxicity receptor signaling and reduce cell exhaustion to unsilence primary anti-tumor response

Cytokine inducible SH2-containing protein (CISH) is a natural killer (NK) cell negative regulator of cytokine signaling pathway. To further understand CISH functions in NK cells, we developed a conditional Cish-deficient mouse model in NK cells (Cishfl/flNcr1Ki/+). We detected no developmental or homeostatic difference in NK cells. However, global gene expression of Cishfl/flNcr1Ki/+ NK cells compared to Cish+/+Ncr1Ki/+ NK cells revealed upregulation of pathways and genes associated with NK cell cycling and activation. We show that CISH does not only regulate interleukin-15 (IL-15) signaling pathways but also natural cytotoxicity receptors (NCR) pathways. Indeed, CISH protein expression level increases upon NCR triggering. Primed Cishfl/flNcr1Ki/+ NK cells display increased activation upon NCR stimulation. Cishfl/flNcr1Ki/+ NK cells display lower activation thresholds and Cishfl/flNcr1Ki/+ mice are more resistant to tumor metastasis. Remarkably, we found that Cishfl/flNcr1Ki/+ mice were also more resistant to primary breast cancer growth in addition to superior control of spontaneous tumor metastasis. CISH deletion favors NK cell accumulation to the primary tumor, optimizes NK cell killing properties and decreases TIGIT immune checkpoint receptor expression, limiting NK cell exhaustion. Finally, we argue that specifically enhancing NK cell function is sufficient to boost anti-tumor response to both primary and secondary tumor models. Using CRISPRi, we then targeted CISH in human NK-92 or primary NK cells. According to the results in our mouse model, CISH deletion favors NCR signaling and anti-tumor functions in human NK cells. Our results validate CISH as an emerging therapeutic target to enhance NK cell immunotherapy.

immunology↗