Treatment with anti-CD49a mAb does not affect the body weight of the mice.

The limitation of wild-type interleukin-12 (IL-12) in its clinical application lies in its systemic activation, which results in severe toxicities. Here, we develop a fusion protein named αTIGIT-IL12 (T-12), which fuses the 13G6 (αTIGIT) antibody scFv fragment in tandem with IL-12. T-12 can selectively localize to the tumor site and concurrently target intratumoral natural killer (NK) and CD8+ T cells in vivo. T-12 demonstrated exceptional efficacy in reducing tumor burden across multiple tumor models in mice, dependent on NK and CD8+ T cells. T-12 preferentially activates tumor-infiltrating NK and CD8+ T cells over their peripheral counterparts, in contrast to wild-type IL-12. Compared with wild-type IL-12, T-12 exhibits greater safety upon systemic administration while treating tumor-bearing models, and the maximal tolerance dosage was elevated by up to about 100-fold. T-12 exhibits potent therapeutic efficacy in checkpoint-insensitive tumor models and metastatic tumor models. These findings underscore the potential of the T-12 fusion protein as a strategy in immunotherapy.

CD49a deficiency prevents the exhaustion of tumor-infiltrating CD8+ T cells in experimental lung metastasis model.

Conventional natural killer (cNK) cells are educated through inhibitory receptor engagement with MHC-I ligands, which endows them with potent cytotoxicity and robust IFN-γ production. Although type 1 innate lymphoid cells (ILC1s) exhibit analogous functionality, whether they undergo a similar education process remains unclear. Our study reveals that the inhibitory receptor CD200R educates ILC1s for optimal cytotoxic competence but is dispensable for IFN-γ production. Unlike cNK cells, ILC1 cytotoxicity remains intact in mice lacking MHC-I molecules, and IFN-γ production shows only a limited, context-dependent reduction. While CD200R selectively suppresses ILC1 cytotoxicity upon CD200 engagement in vitro, ILC1s from CD200R-deficient mice exhibit impaired degranulation and cytotoxic activity, with unaltered IFN-γ production. Analogous to cNK cell education, CD200R educates ILC1 cytotoxicity primarily through the WNT/β-catenin pathway. Importantly, the impaired effector functions of uneducated ILC1s could be restored by exposure to inflammatory cytokines. Overall, our study defines a critical role for the inhibitory receptor CD200R in educating ILC1s to achieve cytotoxic maturity, revealing a parallel yet distinct mechanism from cNK cell education.

Inducible CD49a knockout in NK cells prevents tumor growth and prolongs survival in experimental lung metastasis model.

CD49a deficiency in NK cells does not affect NK cells maturation.

Supplementary Figure S8. NK-92 cells with FBXO38 overexpression show enhanced antitumor activity in vivo

Abstract Type 1 innate lymphoid cells (ILC1) are abundant in the adult liver and are pivotal for immune surveillance and modulation, but the regulation of their maintenance and functionality remains underexplored. Here, we re-analyze published single-cell RNA- sequencing data and find increased expression of Asb2 in ILC1s from adult mouse livers. Conditional ablation of Asb2 in NKp46 + cells, depleting ASB2 in ILC1s and NK cells, in mice impairs ILC1 survival and reduces adult liver ILC1 numbers. Proteomics and bulk RNA-sequencing reveal enriched lipid metabolism pathways in ASB2-deficient ILC1s, concomitant with increased lipid storage. Importantly, pharmacological inhibition of lipid synthesis prevents the apoptosis of ASB2-deficient ILC1s in vitro. In a mouse model of colorectal cancer liver metastasis, we find increased ILC1 lipid storage, and conditional Asb2 deficiency in ILC1 cells exacerbates liver metastasis progression. Conversely, inhibiting lipid accumulation in wild-type colorectal cancer-bearing mice prolongs animal survival, potentially via promoting ILC1-mediated anti-tumor immunity. Thus, our study uncovers ASB2-regulated lipid metabolism as a gatekeeper for ILC1 homeostatic fitness and tumor surveillance, highlighting potential ILC1-based therapeutic strategies against liver tumors.

Supplemental Table 1. Antibodies used for flow cytometry in this study

Supplementary Figure S4. FBXO38 does not affect NK cell trafficking

Supplementary Figure S1. Down regulation of FBXO38 in IT NK cells

Frequencies of immune cell subsets in WT versus CD49a-KO mice.

Supplementary Figure S7. FBXO38 promotes human NK-92 cell proliferation in vitro

Supplementary Figure S6. FBXO38 deficiency increases the expression of suppressors of cell cycle program in TINK cells

Supplementary Figure S3. FBXO38 knockout does not affect spleen NK cell development and effector function

Supplementary Figure S5. FBXO38 is positively correlated with IL2B and IL2G in human TINK cells

CD49a deficiency prevents tumor growth and prolongs survival in various tumor models.

BACKGROUND:Hepatocellular carcinoma (HCC) features a complex tumor immune microenvironment (TIME) where natural killer cell exhaustion (NKEX) facilitates immune evasion. Its regulatory networks and prognostic value remain insufficiently understood. METHODS:We integrated single-cell (GSE149614) and bulk RNA-seq (TCGA-LIHC) data. Following Seurat-based clustering, we utilized CellChat and pySCENIC for communication and transcription factor analysis. NKEX-associated modules were identified via gene set variation analysis and weighted gene co-expression network analysis. A prognostic signature was developed using LASSO-Cox regression and validated in an external cohort (ICGC). CSF1 was prioritized for validation through clinical immunohistochemistry, siRNA knockdown, and Western Blotting. Candidate compounds identified via reverse network pharmacology were validated through CCK-8 assays, docking, western blotting and quantitative PCR. RESULTS:Single-cell analysis revealed pronounced NKEX and disrupted communication in HCC. A four-gene prognostic signature (AKR1B1, SMS, CSF1, CFL1) demonstrated robust predictive performance in both TCGA (1-year AUC: 0.759) and external validation cohorts. High-risk patients showed significantly poorer survival. CSF1 was markedly upregulated in HCC tissues; its silencing inhibited Huh7 cell migration and invasion while upregulating CXCL10 in Huh7 cells and CXCR3 in NK92 cells. Molecular docking and CCK-8 assays justified the dosage and identified punicalagin and evoden as potent inhibitors that significantly suppressed CSF1 expression at both mRNA and protein levels. CONCLUSION:Through integrated multi-omics and experimental validation, we characterized NKEX in HCC and established a robust prognostic signature. CSF1 emerged as a key immunomodulatory target. Punicalagin and evoden were identified as potential lead compounds to modulate the CSF1, offering a promising strategy to restore antitumor immunity in HCC.

Supplementary Figure S2. Tumor grown faster in cKO mice than Ncr1Cre/+ mice

CD49a is upregulated and associated with exhaustion of tumor-infiltrating CD8+ T cells.