Bcl6 alexa fluor647

Abs for flow cytometric analysis were purchased from eBioscience or BioLegend (San Diego, CA) unless otherwise noted: CD4 APCeFlour780 (GK1.5), CD44 FITC (IM7), CD45.1 allophycocyanine (A20), Ly6C PEcy7 (HK1.4), CXCR5 purified (2G8), KLRG1 PE (2F1 KLRG1), CD127 FITC (SB/199), CD122 PE (TM-b1), PD-1 FITC and allophycocyanine (RMP1-30), CCR7 AlexaFluor488 (4B12), CD62L FITC (MEL-14), IFN-γ FITC and PerCPCy5.5 (XMG1.2), TNF-α FITC (MP6-XT22), IL-2 PE (JES6-5H4), GzmB PE (GB12, Invitrogen, Carlsbad, CA), T-bet Alexa Fluor488 (4B10, Santa Cruz Biotechnology, Santa Cruz, CA), GATA3 PE (TWAJ), RORγt allophycocyanine (AFKJS-9), Bcl6 Alexa Fluor647 (K112-91, BD Biosciences, San Jose, CA), eomes PE (Dan11mag), Foxp3 FITC (FJK-16s), biotinconjugated Affinipure goat anti-rat IgG (H+L) (Jackson ImmunoResearch, West Grove, PA) and streptavidin PE. Samples were analyzed using MacsQuant in the Dartlab core facility or Accuri flow cytometers. Data were analyzed using FlowJo software (Tree Star, Ashland, OR) or Accuri software (BD Biosciences).

The following antibodies were used: CD4-AlexaFluor 700, CXCR5-Biotin, ICOS-PECy7, Bcl-6-AlexaFluor 647, CD27-APCH7, CD45RA-PE, CD19-APC or V450, IgD-FITC, IL-21-AlexaFluor 647, IFN-γ-PECy7, IL-10-PE, Streptavidin-PE-TexasRed (BD Biosciences), CD38-PerCPeFluor710, IL-6-FITC, (Biolegend). Neutralizing antibodies specific for human IL-6 and IL-21R and isotype controls from R&D Systems.

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.