B cell

Autoimmunity Assays

Antibody production (Total IgG)
Memory B cell Antibody Isotypes
Naïve B cells Isotype switching
B cell proliferation/activation/cytokine secretion
Antigen presentation

Tonsillar re-aggregate model

Tonsils are a secondary lymphoid tissue rich in B cell subsets and the specialized, anatomically restricted T follicular helper (Tfh) subset of CD4 T cells. Tonsils provide an accessible source of cells for those interested in B cell immunology, Tfh biology and vaccine development. The tonsillar cell re-aggregate model provides functional insight into human germinal center biology, allowing measurement of key features including naive B cell differentiation, plasma cell/plasmablast differentiation, and antibody production.

**Figure** A. Schematic representation of tonsil re-aggregate model. Cells are seeded at high density in 96 well plates; unstimulated (US) or stimulated with quadrivalent inactivated influenza vaccine (**IIV**) in the presence of vehicle or reference inhibitors such as Rapamycin. B cell readouts include B cell phenotyping, B cell proliferation and hemagglutinin (HA)-specific IgG by ELISPOT or ELISA.

**Figure B.** Flow plots representing B cell subpopulations. C. Bar graphs showing frequencies of each B cell subpopulation.

**Figure D**. Flow plots representing total B cell proliferation. E. Bar graphs showing frequencies of divided B cells. Gating strategy: CD45+ > Viable > FSC vs SSC > Singlets > CD4-CD8- > CD19hi/lo

**Figure** F. HA-specific IgG production on day 7 measured by ELISpot. Representative images from one donor (left) and bar graph showing spot forming units (SFU) per 200,000 cells from 3 pre-selected IIV responsive donors run in duplicate wells (right). G. HA-specific IgG secretion on day 12 by ELISA. Representative dilution curves from one donor

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