FIGURE 3.

Activation Induced Marker induction by GC Tfh cells. (A) Experimental design for human tonsillar AIM assay (B) Representative flow cytometry plots of CD25⁺OX40⁺ upregulation by live GC Tfh cells (CXCR5^hiPD-1^hiCD45RA⁻CD4⁺) from 3 different tonsils following 18 hours stimulation with 1μg/mL Staphylococcal enterotoxin B (SEB). (C) Median CD25⁺OX40⁺ expression by live GC Tfh cells following stimulation with SEB. Data are from 14 samples from 2 independent experiments. The response by antigen-specific cells was background subtracted for each donor. (D) Comparison of Strep-specific GC Tfh by ICS (TNF⁺CD40L⁺) and AIM (CD25⁺OX40⁺). The % Strep-specific GC Tfh responses were background subtracted. Data are from 9 samples from 2 independent experiments. The response by antigen-specific cells was background subtracted for each donor. (E) CD25⁺OX40⁺ GC Tfh cells are also PD-L1⁺. Representative FACS plots of CD25⁺PD-L1⁺ co-expression following stimulation with either Strep or SEB. Strep-specific PD-L1⁺CD4⁺ GC Tfh cells (black contour plot) were overlaid onto Strep-specific CD25⁺OX40⁺ GC Tfh cells (grey dots). SEB responsive PD-L1⁺CD4⁺ GC Tfh cells (black contour plot) were overlaid onto SEB responsive CD25⁺OX40⁺ GC Tfh cells (grey dots). Data are from 14 samples from 2 independent experiments. (F). Representative flow cytometry overlay plots demonstrating CD25⁺OX40⁺ cells (black dots) within each sorted population (grey dots) for each condition (Strep-stimulated, and SEB-stimulated). This is representative of 3 independent experiments consisting of 7 donors.