Cd4 alexa fluor 488

Tumor tissues were digested using Mouse Tumor Dissociation kits (Miltenyi) according to the manufacturer’s protocols. Isolated cells were stained for viability using the Aqua Amine fixable live dead dye (Thermo Fisher) using standard protocols. After viability staining, cells were resuspended in FACS buffer (PBS with 0.5% BSA and 2 mM EDTA) and Fc blocked using mouse or human TrueStain FcX (Biolegend). For analysis stains, cells were incubated for 30 minutes at 4C with the following anti-mouse antibodies from Thermo Fisher: CD90.2 (Thy-1.2) SuperBright 645, clone 53-2.1; CD4-AlexaFluor 488, clone GK1.5, CD8a-SuperBright 780, clone 53-6.7; CD3e-PE-Cy5, clone 145-2C11; B220-PE-Cy5, clone RA3-6B2; CD19-PE-Cy5, clone 1D3; CD11c-PE-eFluor610, clone N418; CD45-AlexaFluor 700, clone 30-F11; MHCII-APC-eFluor 780, clone M5/114.15.2; antibodies from Biolegend: NK1.1-PE-Cy5, clone PK136; F4/80-AlexaFluor488; XCR-1-PE, clone ZET; anti-mouse CD80-APC, clone 16-10A1; CD86-Bv785, clone GL-1, PD-1-PE, clone 29F.1A12; LAG-3-PE-Cy7, clone C987W; anti-human CD40-Bv421, clone 5C3; antibodies from BD: anti-mouse CD40-Bv421, clone 3/23; anti-mouse CD172a-Bv605 (Sirpα), clone P84. Samples were analyzed using an Attune NxT flow cytometer (Thermo Fisher) and data was analyzed using FlowJo 10 (TreeStar).

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.

Harvested PBMCs were stained for viability (Live/Dead Fixable stain, Invitrogen), washed and stained for surface markers, and washed for fixation and permeabilization. To detect intracellular CD154 and cytokines, cells were fixed in 4% paraformaldehyde (Electron Microscopy Services, Hatfield, PA) and treated with permeabilization buffer (eBioscience, San Diego, CA) before staining with labeled antibodies. For FoxP3 staining, cells were processed using the FoxP3/Transcription Factor Staining Buffer Set (eBioscience) before staining with antibodies. Antibody panels used for surface and cytoplasmic staining are shown in Tables E1–E4. Stained cells were subsequently analyzed on a LSR Fortessa (BD Biosciences). For Treg sorting or depletion, harvested PBMCs were stained with CD4 Alexa Fluor 488 (Clone OKT4, BioLegend, San Diego, CA), CD25 Alexa Fluor 700 (Clone BC96, BioLegend), and CD127 PE-Cy7 (Clone eBioRDR5, eBioscience). Cells were sorted on the FACS Aria IIu (BD Biosciences).