Extracellular matrix proteins costimulate early CD40L expression on CD4⁺ T cells through distinct β1-integrin pathways

Objective To compare the costimulatory effects of different extracellular matrix (ECM) proteins on the early CD40 ligand (CD40L) expression in TCR-activated CD4⁺ T cells, and to elucidate the β1-integrin-dependent pathways specifically engaged by fibronectin (FN) and laminin (LN).Methods ECM proteins were plate-bound and cocultured with TCR-activated CD4⁺ T cells. CD40L expression was assessed by flow cytometry. Functional-domain were mapped using FN120 and FN40 fragments. Integrin-blocking antibodies (α5, α6, and β1) were used to define the signaling pathways. Daclizumab was applied to determine whether late CD40L expression depends on IL-2R signaling.Results FN and LN markedly enhanced early CD40L expression, whereas VN, CN and TSP1 showed no significant effects. FN activity was restricted to the FN120 fragment. FN- and LN-induced CD40L expression relied on α5β1 and α6β1 integrins, respectively. CD40L expression induced by FN or LN exhibited a sustained monophasic pattern and was independent of IL-2R signaling.Conclusion FN and LN promote CD40L expression through distinct β1-integrin heterodimers, revealing an ECM–integrin axis that regulates T-cell activation and offering mechanistic insights for targeted immunomodulation in chronic inflammatory microenvironments.