Slutter, B. | Search bioRxiv

bioRxiv2025

Probing immune signatures of conjugated pattern recognition receptor ligands identifies chimeras with adjuvant and antitumor activity

Pattern recognition receptor (PRR) ligands hold great promise as adjuvants and immunotherapeutics. Here, we demonstrate that chemical conjugation of PRR ligands results in synergistic immune response amplification inaccessible to unlinked agonist mixtures. To identify potent immune agonists, we synthesized conjugated PRR ligands incorporating distinct agonist pairings, each targeting two carefully selected PRRs. We used a phenotypic screen using human peripheral blood mononuclear cells (PBMCs) to single out chimeric PRR ligands capable of inducing robust immune response both in terms of cytokine response and cytotoxicity against cancer cells. Chimeric TLR4/TLR7 and TLR7/RIG-I ligands showed broad immune activation in vitro as well as enhancement of antigen-specific cellular and humoral responses in mice. Intratumoral delivery of chimeric TLR4/TLR7 ligand induced robust antitumor response in a syngeneic mouse B16F10 tumor model. These results demonstrate the profound effects that conjugation can have on immune response and support the use of conjugated PRR ligands as adjuvants/immunotherapeutics. ONE SENTENCE SUMMARYConjugated PRR ligands enhance immune activation, serve as potent adjuvants, and induce antitumor response in a mouse model. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=97 SRC="FIGDIR/small/647694v1_ufig1.gif" ALT="Figure 1"> View larger version (15K): org.highwire.dtl.DTLVardef@d2904corg.highwire.dtl.DTLVardef@1ffa772org.highwire.dtl.DTLVardef@14a3592org.highwire.dtl.DTLVardef@103320a_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2024

Investigating T cell Recruitment in Atherosclerosis using a novel Human 3D Tissue-Culture Model reveals the role of CXCL12 in intraplaque neovessels

BackgroundDevelopment of effective treatments for atherosclerosis requires new models that better predict the human immune response. Although T cells are abundant in human atherosclerotic lesions and play a key role in the pathogenesis, the mechanism involved in plaque infiltration remains ill defined. MethodsWe developed a three-dimensional tissue-culture model to study leukocyte recruitment to human atherosclerotic plaques. In this study, human atherosclerotic plaques obtained during carotid endarterectomy surgery were co-cultured with patient-matching T cells. Exogenous T cells were stained using a multi-factor staining strategy, which involved intracellular fluorescent cell tracker dyes combined with nuclear labels. Flow cytometry was used to assess the presence of the labeled cells within the plaques, and microscopic analysis was performed to examine their localization. ResultsFlow cytometry and microscopy cell-tracking analysis demonstrated that exogenous T cells successfully migrated into atherosclerotic plaques. Furthermore, infiltrated CD8+ T cells displayed a significant increase of CD69 expression, indicating their activation within the tissue. Blocking chemokine receptors, particularly CXCR4, significantly impaired T cell infiltration, demonstrating that exogenous CD8+ T cells invade plaques through chemotactic migration. Surprisingly, 3D microscopy combined with optical tissue clearing strategy revealed that CXCL12, the sole ligand of CXCR4, mainly accumulated in intraplaque neovessels. Single-cell RNA sequencing (scRNAseq) analysis further confirmed that endothelial cells from intraplaque neovessels were the primary source for CXCL12. Additionally, exogenous T cells were found within and in proximity to these neovessels, suggesting that the CXCL12/CXCR4 axis regulates T cell recruitment through intraplaque neovessels. ConclusionsOverall, these findings shed new light on the mechanism of action of CXCL12 in atherosclerosis and demonstrated the potential of the model to advance our understanding of leukocyte accumulation in human atherosclerosis and assist in testing novel pharmacological therapies.

molecular biology↗