Structure-Function Relationships of Heparan Sulfate-Based Neoproteoglycans as Selective Immunostimulatory Scaffolds

Heparan sulfate proteoglycans (HSPGs) play a critical role in regulating inflammatory responses. Their diverse functions arise from the distinct structural heterogeneity of heparan sulfate (HS) side chains. Deciphering these structural determinants offers a promising strategy for developing novel vaccine adjuvants and immunotherapeutic agents. Herein, we evaluated a panel of neoproteoglycans (neoPGs) functionalized with structurally defined HS oligosaccharides to identify ligands that selectively trigger cytokine responses in immune cells. Our results demonstrate that highly sulfated, L-iduronic acid-containing, N-sulfated HS-conjugated neoPGs, specifically H7 and H12 induced pro-inflammatory cytokines TNF-α, IL-1β, and IL-6 in human peripheral blood mononuclear cells (PBMCs), compared to their D-glucuronic acid analogs. Notably, cotreatment with lipopolysaccharide (LPS) did not result in a synergistic increase in cytokine levels, indicating that these HS neoPGs likely activate immune signaling independently of the LPS-mediated pathway. In contrast, similar experiments with murine macrophage cell line PMJ2-PC showed only modest cytokine induction, particularly with the H6 ligand, further supporting a cell-type specific and Toll-like receptor (TLR) independent mechanism of action. Collectively, these data identify structurally distinct HS domains that can function as cell-specific immunomodulators, offering new opportunities for the rational design of glycan-based adjuvants and therapeutic immune regulators.