Development and application of an optimized human PBMC-derived lymphoid organoid model for immune evaluation of recombinant TeNT-Hc protein

To establish a lymphoid organoid model based on human peripheral blood mononuclear cells (PBMCs) and characterize the antigen-specific immune responses induced by the recombinant tetanus neurotoxin heavy chain (TeNT-Hc) protein based on this model.

Using viability and apoptosis of in vitro cultured PBMCs as indicators, the effects of key parameters, including different organoid culture methods, extracellular matrix,serum components and seeding densities, on the construction of lymphoid organoid model were compared. Recombinant TeNT-Hc protein was used as the model antigen to stimulate the organoids. Histological staining, flow cytometry and enzyme-linked immunosorbent assay (ELISA) were applied to evaluate the formation of germinal center-like structures, dynamic changes in specific immune cell subsets, and levels of antibody secretion.

Under the optimized culture conditions, the cell viability of the constructed lymphoid organoids was 65.1% after 16 days of culture. Stimulation of lymphoid-like organoids with the model antigen resulted in marked activation and differentiation of B cell and T cell subsets, accompanied by the expansion of follicular helper T cell subsets. Germinal center-like structures were observed on day 8. Lymphoid organoids mounted B and T cell immune responses upon antigen stimulation, and antigen-specific antibody secretion was detected by day 6. PBMC-derived organoids from five healthy volunteers trended towards immune activation in response to antigen stimulation while retaining inter-individual differences in immune response intensity.

This established PBMC-derived lymphoid organoid model can effectively induce antigen-specific immune responses and possesses the dual ability to conduct standardized analysis and reflect the immune heterogeneity of the population. It provides a novel research tool for screening of vaccine candidate antigens and for the exploration of humoral immune mechanisms.