What question did this study set out to answer?

This research aims to uncover the mechanisms of NK cell dysfunction in pancreatic cancer and identify targetable pathways for enhancing NK cell functionality.

May 30, 2026

Systematic modeling of tumor-microenvironment–mediated NK cell dysfunction in pancreatic cancer to identify KLRB1 as a target for cell engineering.

Key Points

This research aims to uncover the mechanisms of NK cell dysfunction in pancreatic cancer and identify targetable pathways for enhancing NK cell functionality.
Utilized two in vitro suppression models: human NK cell co-culture with cancer-associated fibroblasts and conditioned medium from PDAC organoids.
Conducted scRNA-seq to identify upregulated inhibitory receptors, particularly KLRB1.
Implemented CRISPR-Cas9 to knock out KLRB1 in human NK cells and assessed functionality in both in vitro and in vivo co-engagement models.
KLRB1 was significantly upregulated in NK cells exposed to both CAF co-culture and PDAC organoid CM.
KLRB1-KO NK cells exhibited higher degranulation (CD107a+) and increased IFN-γ production compared to wild type under suppression models.
In vivo, KLRB1-KO NK cells showed superior ability to control tumors, maintaining a transcriptionally active effector phenotype, unlike wild type cells that developed dysfunction.

Abstract

e14541 Background: Adoptively transferred natural killer (NK) cells fail in pancreatic ductal adenocarcinoma (PDAC) due to rapid functional suppression within the immunosuppressive tumor microenvironment (TME). A precise understanding of the dominant inhibitory mechanisms, especially those exerted by cancer-associated fibroblasts (CAFs) and soluble tumor-derived factors, is lacking. We hypothesized that a systematic, multi-model dissection of PDAC-mediated suppression could reveal conserved, targetable pathways for engineering resistant NK cells. Methods: To model the PDAC TME, we used two complementary in vitro suppression systems—co-culture of expanded human peripheral blood NK cells with patient-derived CAFs (cell-contact inhibition) and culture in conditioned medium from patient-derived PDAC organoids (soluble-factor inhibition)—and performed scRNA-seq to identify the most consistently upregulated inhibitory receptor, knocked it out in primary human NK cells using CRISPR–Cas9, and compared functional recovery of WT versus KO NK cells in both in vitro models and in an NSG mouse co-engrafted with human PDAC cells and CAFs. Results: scRNA-seq of NK cells exposed to either CAF co-culture or PDAC organoid CM revealed a shared and dominant upregulation of the inhibitory receptor KLRB1 (encoding CD161). CRISPR-mediated KLRB1-KO did not impair NK cell development or basal cytotoxicity. Critically, under both suppression models, KLRB1-KO NK cells maintained significantly higher levels of degranulation (CD107a+) and IFN-γ production compared to WT NK cells. Transcriptomically, KO cells preserved effector gene programs and exhibited attenuated induction of exhaustion signatures. In the in vivo co-engraftment model, adoptively transferred KLRB1-KO NK cells mediated superior tumor control. Single-cell analysis of NK cells recovered from tumors confirmed that KO cells maintained a higher frequency of a transcriptionally active effector cluster, whereas WT NK cells were predominantly driven into dysfunctional states. Conclusions: By systematically modeling both contact-dependent and soluble-mediated immunosuppression, we identify KLRB1 as a central, conserved "molecular brake" induced in NK cells by the PDAC TME. Its deletion generates "TME-resilient" NK cells with preserved effector function in situ. This work validates a discovery pipeline using complementary TME models and establishes KLRB1-KO as a promising strategy to engineer next-generation allogeneic NK cell products for overcoming microenvironmental suppression in solid tumors.

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