Rf8b2

Frozen lymph node sections 4 μm thick were fixed in 1% paraformaldehyde (Sigma, St. Louis, MO), stained for 1 hour with rat anti-CXCR5 (RF8B2, BD Bioscience), mouse anti-CD4 (RPA-T4, BD Bioscience) and rabbit anti-CD20 (Abcam, Cambridge, MA), then treated for 30 min with secondary antibodies AF488 donkey anti-rat, AF647 chicken anti-rabbit and AF594 goat anti-mouse (Invitrogen, Grand Island, NY). Stained slides were viewed on a Leica DM5000B fluorescent microscope and 10 to 15 randomly selected areas were imaged using Qwin Leica FW4000 software (Leica). Follicular and extrafollicular tissue areas within the images were defined morphologically by CD20 staining, and percentages of CXCR5+, CD4+, and CXCR5+CD4+ tissue area determined by quantitative image analysis (QWin Pro; v.3.4.0, Leica).

Cryopreserved PBMC samples from pretreatment, cycles 1–4 (weeks 3–12) were thawed and stained with master mix of antibodies for surface stains including CD4 (Biolegend, OKT4), CD8 (ebioscience, RPA-T8), 2B4 (Beckman Coulter, IM2658), CD45RA (Biolegend, HI100), TIM-3 (F38-2E2), LAG-3 (Enzo, ALX-804-806B-C100), CXCR5-BV421 (BD, RF8B2) and CD27 (BD, L128) and intracellular stains for FOXP3 (BD, 259D/C7), CTLA-4 (BD, BNI3), Eomes (ebioscience, WD1928), T-bet (Biolegend, 4B10), GzmB (Life Tech, GB11), TCF-1-AlexaFluor647 (Biolegend, 7F11A10) and Ki67 (BD, B56). Permeabilization was performed using the FOXP3 Fixation/Permeabilization Concentrate and Diluent kit (eBioscience). PD-1 on post pembro specimens was detected using anti-human IgG4 PE (Southern Biotec). Pretreatment samples were pretreated with 25 μg ml⁻¹ pembro in vitro for 30 min at 37 °C, washed twice and stained with standard antibody mix. Cells were resuspended in 1% paraformaldehyde until acquisition on a BD Biosciences LSR II cytometer and analysed using FlowJo (Tree Star).