Pamela Wong

The requirement for the T-box transcription factors (TF) T-bet and Eomes in innate lymphoid cells (ILCs) beyond their development is not well understood. Here, we generate an inducible, NKp46-specific T-bet knock-out (KO) model and compare it to corresponding Eomes KO and combined T-bet Eomes double KO mice to define T-box TFs requirement in the homeostasis and function of mature NK cell and other NKp46 ⁺ ILC. Inducible T-bet deletion reduces stage IV NK cell numbers in the spleen and tissues, while preserving NK cells in the bone marrow and lymph nodes. Liver ILC1 and small intestine lamina propria NKp46 ⁺ ILC3 are also lost upon T-bet deletion, indicating the requirement for continuous T-bet expression in these ILC types. Combined T-bet and Eomes KO leads to a rapid loss of NK cells, markedly greater than with individual T-bet or Eomes KO. Direct comparison of inducible T-bet and Eomes KO models reveals that Eomes is critical for host protection against murine cytomegalovirus, whereas T-bet is dispensable, despite loss of ILC1. These findings establish the tissue-specific and non-redundant roles for T-box TFs in NKp46 ⁺ ILCs homeostasis and response to viral infection.

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.

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.