Jennifer Tran

Supplementary Figure 6. EphA2-CAR ML NK cells display enhanced functional responses against HPV positive and HPV negative HNSCC cell lines.

Supplementary Figure 4. Cetuximab does not impair in vitro growth of HNSCC cells or cause toxicity to mice in NSG xenograft models.

Supplementary Figure 7. EphA2-CAR-ML NK cells require intracellular CAR signaling and have antigen specific enhanced response to HNSCC.

Supplementary Figure 1. Memory-Like (ML) NK cells from normal donors exhibit improved ability to control HNSCC cell lines compared to conventional NK (cNK) cells.

Supplementary Figure 2. HNSCC cell lines and patient samples express multiple inhibitory and activating surface ligands.

Supplementary Table S1. Demographic and clinical data of advanced head and neck squamous cell carcinoma patients.

Supplementary Figure 5. Expression of EphA2 on target cells and gating strategy for CAR-expressing NK cells

Supplementary Figure 3. Individual blockade of DNAM-1, NKG2D and CD2 modestly impairs NK cell activity against HNSCC.

Natural killer (NK) cells are innate immune lymphocytes important for host viral and tumor immunity. We investigated the requirement for ATP citrate lyase (ACLY) in NK cell function using an inducible genetic mouse model. ACLY regulates the citrate-malate shuttle, generating cytosolic acetyl-coenzyme A that is primarily used for acetylation or lipid synthesis. ACLY-deficient NK cells upon IL-15 activation exhibited significant defects in glycolysis, proliferation, cytokine production, and cytotoxicity, without decreased intracellular lipids. Notably, ACLY deficiency specifically resulted in reduced NK cell responses to activating receptors associated with the adapter proteins DAP10 or DAP12. This is due to decreased DAP12 and increased DAP10 transcript and protein, coupled with epigenetic profiling that demonstrated altered histone acetylation of these genes in ACLY KO. Supplementation of ACLY-deficient NK cells with acetate was sufficient to overcome most functional defects, including restoring DAP10/12 expression and activating receptor function, emphasizing the importance of ACLY-generated cytosolic acetyl-coenzyme A for NK effector functions.

Mechanisms related to tumor evasion from NK cell-mediated immune surveillance remain enigmatic. Dickkopf-1 (DKK1) is a Wnt/β-catenin inhibitor, whose levels correlate with breast cancer progression. We find DKK1 to be expressed by tumor cells and cancer-associated fibroblasts (CAFs) in patient samples and orthotopic breast tumors, and in bone. By using genetic approaches, we find that bone-derived DKK1 contributes to the systemic DKK1 elevation in tumor-bearing female mice, while CAFs contribute to DKK1 at primary tumor site. Systemic and bone-specific DKK1 targeting reduce tumor growth. Intriguingly, deletion of CAF-derived DKK1 also limits breast cancer progression, without affecting its levels in circulation, and regardless of DKK1 expression in the tumor cells. While not directly supporting tumor proliferation, stromal-DKK1 suppresses NK cell activation and cytotoxicity by downregulating AKT/ERK/S6 phosphorylation. Importantly, increased DKK1 levels and reduced cytotoxic NK cells are detected in women with progressive breast cancer. Our findings indicate that DKK1 represents a barrier to anti-tumor immunity through suppression of NK cells.