Background: Dysregulated germinal center (GC) reactions and aberrant B-T cell interactions drive autoantibody production and tissue inflammation across rheumatic diseases such as rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). Conventional animal models and PBMC assays incompletely recapitulate human GC biology and antigen-specific responses, limiting translational confidence.

Objectives: We combined a human tonsil organoid platform that reconstructs GC-like microenvironments with a flow-cytometry-based immune synapse (IS) assay to quantify how therapeutics modulate B-T cell cooperation, activation, and effector function.

Methods: Tonsil organoids were stimulated with inactivated influenza vaccine (IIV) to elicit antigen-specific B cell responses. Longitudinal profiling captured B cell maturation (CD38/CD27), class switching (surface IgA/IgG), antigen-specific antibody secretion, and follicular helper T cell (Tfh) phenotypes. Pharmacologic perturbation with mTOR and JAK inhibitors benchmarked the platform for immunomodulator screening. In parallel, a multiplex flow cytometry IS assay quantified B-T cell doublets (CD3 ⁺ CD20 ⁺ ) and downstream T cell activation/proliferation (e.g., CD25, CTV dilution) alongside B cell cytotoxicity, using blinatumomab (anti-hCD19×CD3) as a reference engager to demonstrate concentration-dependent IS formation, CD4 ⁺ /CD8 ⁺ T cell activation, and targeted B cell lysis across donors.

Results: IIV stimulation induced a pre-GC (CD38 ⁺ CD27 ⁻ ) to plasmablast (CD38 ⁺⁺⁺ CD27 ⁺ ) transition, dynamic class-switch changes in IgA/IgG compartments, Tfh modulation, and antigen-specific antibody secretion. mTOR and JAK inhibition attenuated GC maturation and antibody output, validating the system’s pharmacologic sensitivity. The IS assay resolved robust, dose-responsive increases in B-T cell doublets, T cell activation/proliferation, and B cell killing with blinatumomab, establishing quantitative readouts of B-T cell synapse biology. Together, these platforms delineate complementary mechanisms: GC/Tfh dynamics in organoids and proximal B-T synapse formation and effector function in the IS assay. This enables MoA-specific signatures for rheumatology-relevant modalities (e.g., JAK/mTOR inhibitors, BTK inhibitors, BAFF/APRIL axis blockers, CD20/CD19 depletion, IL-6R/TNF blockade, and co-stimulation inhibitors) and supports ranking by effects on GC output, class switching, Tfh phenotypes, synapse formation, and immunotoxicity.

Conclusions: A paired tonsil organoid + IS framework provides a human, ex vivo, decision-enabling toolkit to screen and mechanistically profile therapeutics targeting autoimmune rheumatic disease. By capturing mechanism-consistent, dose-responsive pharmacodynamics across GC maturation and B-T synapse function, this approach improves translatability, supports lead prioritization and combination design, and may enhance prediction of clinical success when benchmarked against standards of care.

REFERENCES: [1] Wagar, L.E., Salahudeen, A., Constantz, C. M., Wendel B. S. et al. (2021). Nature Medicine 27, 125-135. doi:10.1038/s41591-020-01145-0.

Acknowledgments: NIL.

Disclosure of Interests: None declared.