Pd 1 bv421

LNs were stained and optically cleared as previously described (80 (link)). Volume staining was performed using the following antibodies/dilutions: B220-eFluor 615 (ThermoFisher, clone RA3–6B2) 1:80; CD4-Alexa Fluor 488 (BioLegend, clone RM4–5) 1:80, PD1-BV421 (BioLegend, clone RMP1–30) 1:80, FoxP3-eFluor 570 (ThermoFisher, clone FJK-16s) 1:50, BCL6-Alexa Fluor 647 (BD Biosciences, clone K112–91) 1:50; CD35-BV510 (BD Biosciences, clone 8C12) 1:300. Confocal imaging was performed on an inverted Leica SP8 microscope using a 40× 1.3 NA oil immersion objective. 3D image stacks were analyzed using Imaris 9.2.1 (Bitplane). 3D image stacks were analyzed using Imaris 9.6.0. (Bitplane). GC surfaces were delineated as BCL6⁺ volumes at a smoothing resolution of 10μm. Within GCs, T cells were delineated as CD4⁺ volumes at a smoothing resolution of 0.569μm. Among GC-T cells, GC-T_FH cells were identified as BCL6⁺ PD1⁺ T cells. To determine the relevant BCL6⁺ and PD1⁺ cutoffs, three separate 0.333*10⁶ μm³ control volumes were sampled from the B cell follicle (not intersecting any GCs) in each image, T cells were segmented as CD4⁺ volumes, and the 95^th–99^th percentiles of BCL6 and PD1 expression among these non-GC-T cells were calculated. From these delineations of GC regions and GC-T_FH cells, the volume of each GC and the density of GC-T_FH cells were calculated.

Surface markers were evaluated using combinations of fluorochrome-conjugated monoclonal antibodies that were each titrated individually for their optimal stain index. PBMCs were stained at 10 million cells/mL in 200uL PBS. All PBMCs were incubated for 10 minutes with an amine-reactive viability dye (LIVE/DEAD Aqua, Invitrogen), washed twice, and then stained for 15 minutes at room temperature with combinations of monoclonal antibodies. For ex vivo phenotyping, cells were stained with CD3-AF700 (UCHT1, BD), CD4-PECy5 (RPA-T4, BD), CD8-APC-AF750 (3B5, Invitrogen), CD45RO-PETR (UCHL1, Beckman Coulter), CCR7-BV421 (150503, BD), CXCR5-AF488 (RF8B2, BD), PD-1-PE (EH12.2H7, BioLegend), CD14-V500 (M5E2, BD), and CD19-V500 (HIB19, BD). In vitro phenotyping was performed with combinations of CD3-AF700, CD4-PECy5, CD8-APC-AF750, CD45RO-PETR, CXCR5-AF488, CD14-V500, CD19-V500, ICOS-PE (DX29, BD), CD40L-PE (TRAP1, BD) and PD-1-BV421 (EH12.2H7, BioLegend). All PBMCs were washed twice after staining, fixed with 2% paraformaldehyde, and analyzed on a BD LSR Fortessa (BD Biosciences) at the VMC Flow Cytometry Shared Resource.
Flow cytometry data was analyzed using BD Biosciences FACSDiva Software. In all experiments, forward and side scatter were used to identify lymphocytes and from that population non-viable, CD14+, CD19+, CD8+ cells were excluded from further analysis (Fig. S1).