Background: Immune checkpoint blockade can reinvigorate pre-existing anti-tumor T cells, but it often fails in “cold” tumors such as pancreatic cancer, where T cells are poorly primed, excluded from the tumor, or absent altogether1,2. This creates a major challenge in the field: therapies designed to reinvigorate T cells may be ineffective when a productive T cell response has never been properly initiated. HPK1 is a promising druggable intracellular brake on immune activation, and most studies have focused on its role in restraining T cell receptor signaling3. However, HPK1 is also expressed in dendritic cells (DCs), the immune cells responsible for antigen presentation and T cell priming. We therefore asked whether releasing HPK1-mediated inhibition in DCs is required to convert an immune-excluded, checkpoint-resistant pancreatic tumor into one capable of productive T cell immunity.
Methods: We used preclinical pancreatic tumor models spanning T cell-low “cold” and T cell-high “hot” immune states to dissect how HPK1 loss converts resistant tumors into immune-responsive lesions. To determine whether HPK1 promotes anti-tumor immunity through T cells alone or through DC-mediated initiation, we used conditional HPK1-floxed mice crossed with lineage-specific Cre drivers, adoptive transfer and bone marrow chimera experiments. To identify the cellular interactions and signaling pathways required for tumor control, we integrated IBEX multiplex imaging and CITE-seq to link immune cell phenotype, spatial organization, DC-T cell interactions, and IFNγ-associated signaling niches within the tumor microenvironment (TME).
Results: In a checkpoint blockade-resistant pancreatic adenocarcinoma model, global HPK1 deficiency converted an immune-excluded “cold” tumor into one capable of robust anti-tumor immunity and complete regression after anti-PD-1 treatment. Mechanistic studies revealed that HPK1-deficient DCs, rather than T cells alone, were required to initiate this response, while T cells served as essential effectors and HPK1-deficient T cells further amplified tumor control. HPK1-deficient DCs showed enhanced antigen presentation, costimulatory activity, IFNγ responsiveness, and interaction with T cells. Spatial imaging identified pSTAT1-enriched immune niches in the TME, where DCs, IFNγ- producing T cells, and CXCL9-expressing cells clustered together. Disrupting this circuit through IFNγ neutralization or interfering with chemokine-dependent cluster formation
reduced DC maturation, impaired T cell recruitment, and diminished tumor control. Together, these findings show that HPK1-deficient DCs organize a localized IFNγ- dependent DC-T cell feedback circuit that overcomes a crucial limitation to induction of robust adaptive immunity and checkpoint resistance in “cold” pancreatic tumors.
Conclusion: Our study identifies HPK1-deficient DCs as essential initiators of anti-tumor immunity in checkpoint blockade-resistant pancreatic tumors. These DCs organize IFNγ- dependent immune niches that recruit and activate T cells, converting a “cold” TME into
one capable of productive anti-tumor immunity. This shifts the therapeutic focus of HPK1 targeting beyond T cells: drugs optimized only for T cell activation may miss a critical DC dependent mechanism needed to overcome resistance in “cold” tumors.