Phenotypic and functional characteristics of splenocytes in tetanus toxoid-hyperimmunized Balb/c mice is influenced by the context of tetanus toxoid application

Abstract

Purpose/Objective: The hyperimmunization with tetanus toxoid (TTd) induces protective TTd-specific as well as autoreactive b2-glycoprotein I (b2GPI)-specific immune responses in BALB/c mice. The overall immune response characteristics, especially its pathogenic potential, depended on adjuvants applied prior and in combination with TTd. Beside structural homology between TTd and b2GPI, tolerance toward b2GPI could be impaired by adjuvants acting as polyclonal stimulators. In order to clarify the impact of adjuvants, phenotypic and functional analyses of immune system cells within spleen were done upon immunization completion. Materials and methods: Non- or CFA-pretreated BALB/c mice were immunized with TTd (3 · 100 lg/dose; 2-week intervals) mixed with alum or 2.5M glycerol. Ex vivo analyses of CD3, CD4, CD8, CD19, CD 25, CD27 and mIgM expression on age-matched control and immunized mice’s splenocytes were done by flow cytometry. Changes in TLR2, TLR4 and TLR9 expression were assessed indirectly, by measuring cytokine production, following in vitro stimulation of splenocytes with appropriate agonist. Results: TTd-immunization diminished CD27 expression on T cells implying on their differentiation into potent effector cells. T cell activation (increase in CD25 expression and the raise of percentage of CD4+ CD8+ CD3+ ) and B cell activation (rise in percentage of CD19+ CD25+ cells and the increase of mIgM density) occurred in all immunized mice, being more intensive in CFA-pretreated groups. Irrespective to the applied immunization protocol, statistically significant rise in abundance of CD4- CD8- cells (often cited as cells having suppressive potential) within T cell pool was registered too. Differences in cytokines production (IL4, IL10, IFNc) registered upon in vitro stimulation with peptidoglycan, LPS and CpG ODN implied on context-dependant modulation of TLR2, TLR4 and TLR9 expression on splenocytes. Conclusions: TTd-hyperimmunization promoted concomitant rise in abundance of activated cells and the cells that have suppressive potential. This could be regarded as an attempt of the system to retain control. Imbalance in percentages and activities between activated cells and those having suppressive potential, highly influenced by the context of TTd application, is most likely the cause for the observed pathology appearance after TTd hyperimmunization.