Functional profiling of porcine precision-cut lymph node slices as an ex vivo model for TLR-mediated and PCV2-specific immune responses

Introduction Precision-cut lymph node slices (PCLNS) provide a physiologically relevant ex vivo model to investigate lymphoid immune activation and vaccine adjuvant function while preserving native tissue architecture. In this study, we established for the first time porcine PCLNS and assessed the immunostimulatory capacity of PCLNS to Toll-like receptor (TLR) 7/8 agonist (Resiquimod; R848) alone or in combination with porcine circovirus type 2 (PCV2) or PCV2 virus-like particles (VLPs). Methods Lymph nodes (LNs) were collected from PCV2-vaccinated pigs. PCLNS were generated using a Krumdieck tissue slicer and cultured in vitro with R848, PCV2, VLPs and their combinations Supernatants were collected daily for cytokine quantification and PCV2-specific antibody detection. Flow cytometry and immunofluorescence imaging were performed to examine B cell responses. Results PCLNS maintained viability and preserved key histological structures over a five-day culture period, although progressive follicular depletion developed across conditions, potentially due to yet suboptimal culture condition. Phenotypic profiling revealed that R848 induces stronger B cell activation, including increased Ki-67 expression and plasma cell expansion. These cellular responses were accompanied by rapid and selective induction of IL-10, IL-12 and TNF following R848 stimulation. Daily supplementation with B cell survival factors (CD40L, BAFF and IL-21) failed to prevent germinal centre (GC) collapse despite modest effects on viability. Stimulation of PCLNS with PCV2 elicited detectable PCV2-specific IgG but not IgM, with the strongest humoral and cytokine responses observed following combined PCV2 and R848 stimulation, indicating a potential synergistic enhancement of recall-like immune response. In contrast, VLPs induced minimal antibody responses even with R848 co-stimulation. Collectively, these findings establish PCLNS as a tractable platform for assessing adjuvant-driven immune activation and demonstrate that TLR7/8 engagement seems to be a superior driver of B cell proliferation, plasma cell differentiation and pro-inflammatory cytokine production compared to other B cell survival factors, in porcine lymphoid tissue.