Feng Shao

Targeting innate immunity holds promise in cancer immunotherapy, particularly in improving checkpoint inhibitors. However, the use of agonists of the promising innate receptors TLRs and STING^1,2,3,4 is facing challenges. Here we examined the antitumour function of the α-kinase 1 (ALPK1) receptor for bacterial ADP-heptose (ADP-Hep)^5,6,7. Treatment of mice with ADP-Hep induced multiple proinflammatory factors including CXCL10 and CCL2, and stimulated Alpk1-dependent antitumour immunity. Mice bearing a gain-of-function ALPK1(T237M) disease variant⁸ also rejected grafted tumours. Using medicinal chemistry, we identified a more potent analogue, UDSP-Hep. In contrast to ADP-Hep, UDSP-Hep distinguished Alpk1 polymorphism, which correlates with mouse susceptibility to bacteria-associated colitis^9,10,11,12. UDSP-Hep exhibited a stronger Alpk1-mediated antitumour effect and synergized with checkpoint inhibitors. The effect required CD8⁺ T cells, dendritic cells (DCs) and macrophages, and was sensitive to antibodies that block CXCL10 or CCL2 function. ALPK1 agonists activated DCs for cross-presentation, promoting tumour-specific T cell expansion in the tumour-draining lymph nodes. ALPK1 has wider expression than STING in non-immune cells with a distinct inflammatory signature. UDSP-Hep is differentiated from STING agonists in stimulating tumour-cell antigen presentation, macrophage–DC cross-priming and protective memory T cell differentiation, and it does not induce T cell apoptosis. Our study elucidates the antitumour effect of ALPK1 agonism and suggests the potential of ALPK1 agonists in cancer immunotherapy.

Allergens that induce allergic airway inflammation are highly diverse, but they commonly activate type 2 immune responses^1,2. Airway epithelial cells are crucial in allergen sensing^3,4,5. However, the shared features among diverse allergens that elicit similar innate responses, and their epithelial detection mechanisms, remain poorly defined^1,2,6,7,8,9. Here we identify pore-forming proteins as one of the common stimuli of allergic airway inflammation and reveal their immune-activation mechanisms. Using the prevalent mould allergen Alternaria alternata as a model, we established an in vitro system to investigate type 2 innate immune sensing. A six-step biochemical fractionation identified Aeg-S and Aeg-L as the core immune-stimulatory components. Biochemical reconstitution and cryo-electron microscopy reveal that these proteins form 16- to 20-mer transmembrane pore complexes. Their cooperative perforation acts as a bona fide type 2 immune adjuvant to support antigen-specific T helper 2 and immunoglobulin E responses. Genetically engineered A. alternata strains that lack pore-forming activity do not induce allergic responses in mice. Furthermore, pore-forming proteins from various species, despite structural and membrane target differences, are sufficient to trigger respiratory allergies. Perforations in airway epithelial cells initiate allergic responses through two mechanisms: one triggers IL-33 release, and the other involves Ca²⁺ influx, which activates MAPK signalling and type 2 inflammatory gene expression. These findings provide insight into how type 2 immune responses detect common perturbations caused by structurally diverse stimuli. Targeting downstream signalling of epithelial perforation may open new avenues for treating respiratory allergies.

Plasma membrane integrity is vital for nearly all aspects of cell functioning¹. Mechanical forces can cause plasma membrane damage², but it is not known whether there are large molecules that regulate plasma membrane integrity under mechanical strain. Here we constructed a 384-well cellular stretch system that delivers precise, reproducible strain to cultured cells. Using the system, we screened 10,843 siRNAs targeting 2,726 multi-pass transmembrane proteins for strain-induced membrane permeability changes. The screen identified NINJ1, a protein recently proposed to regulate pyroptosis and other lytic cell death^3,4, as the top hit. We demonstrate that NINJ1 is a critical regulator for mechanical strain-induced plasma membrane rupture (PMR), without the need of stimulating any cell death programs. NINJ1 level on the plasma membrane is inversely correlated to the amount of force required to rupture the membrane. In the pyroptosis context, NINJ1 on its own is not sufficient to fully rupture the membrane, and additional mechanical force is required for full PMR. Our work establishes that NINJ1 functions as a bona fide determinant of membrane biomechanical properties. Our study also suggests that PMR across tissues of distinct mechanical microenvironments is subjected to fine tuning by differences in NINJ1 expression and external forces.

Plasma membrane integrity is vital not only for cell survival but also nearly all aspects of cell functioning. Mechanical stress can cause plasma membrane damage, but it is not known whether there are large molecules (proteins) that control plasma membrane integrity. Here we constructed a 384-well cellular stretch system that delivers precise, reproducible mechanical strain to adherent cells. Using the system, we screened 10,843 siRNAs targeting 2,726 multi-pass transmembrane proteins for stretch-induced membrane permeability changes. The screen identified NINJ1, a protein recently proposed to regulate pyroptosis and other lytic cell death, as the top hit. We demonstrate that NINJ1 is a critical regulator for mechanical force-induced plasma membrane rupture (PMR), without the need of stimulating any cell death programs. Low NINJ1 expression renders the membrane more resistant to stretching, while high expression of NINJ1 lowers the threshold of PMR under mechanical strain. NINJ1 level on the plasma membrane is inversely correlated to tension required to rupture the membrane. In the pyroptosis context, NINJ1 on its own is not sufficient to fully rupture the membrane, and additional mechanical stress is required for full PMR. Our work establishes that NINJ1 functions as a bona fide determinant of membrane biomechanical properties. Our study also suggests that PMR across tissues of distinct mechanical environments is subjected to fine tuning by differences in NINJ1 expression and external mechanical forces.

Bacterial small molecule metabolites such as adenosine-diphosphate- d - glycero -β- d - manno -heptose (ADP-heptose) and their derivatives act as effective innate immune agonists in mammals. We show that functional nucleotide-diphosphate-heptose biosynthetic enzymes (HBEs) are distributed widely in bacteria, archaea, eukaryotes, and viruses. We identified a conserved STT _R5 motif as a hallmark of heptose nucleotidyltransferases that can synthesize not only ADP-heptose but also cytidine-diphosphate (CDP)– and uridine-diphosphate (UDP)–heptose. Both CDP- and UDP-heptoses are agonists that trigger stronger alpha-protein kinase 1 (ALPK1)–dependent immune responses than ADP-heptose in human and mouse cells and mice. We also produced ADP-heptose in archaea and verified its innate immune agonist functions. Hence, the β- d - manno -heptoses are cross-kingdom, small-molecule, pathogen-associated molecular patterns that activate the ALPK1-dependent innate immune signaling cascade.