Fig 2: An EBV+ pre-GC activated state with hallmarks of T-bet+ memory B cells develops in a subset of LCLs. (A) AP-eMBC cluster cells within one in-house LCL dataset exhibits an atypical memory B cell (atMBC) phenotype prone to plasma differentiation. (B) Upregulation of genes encoding receptors for IL-21, IFNG, and TLR7 ligands within identified LCL atMBCs coincide with the AP-eMBC phenotype. (C) Model experimental design to stimulate plasma cell differentiation from AP-eMBC/atMBCs within LCLs. (D) Growth analysis of LCLs treated with 2 µg/mL TLR7 agonist R848 (blue), 10 ng/mL IL-21 (orange), or both (green) versus control treatment (0.1% DMSO, red). Data are presented as mean +/- standard deviation cell counts with intra-replicate normalization to Day 0 across 3 biological replicates per condition. Statistically significance of differences (Day 3 IL-21 vs. DMSO, p = 0.04 and Day 3 R848+IL-21 vs. DMSO, p = 0.016) were calculated using Welch’s t test (two-tailed, paired). (E) Representative CD23 (FCER2) expression in LCLs at Day 4 by treatment group. (F) CD27 and IgD staining of LCLs at Day 4 by treatment group to evaluate the frequency of EBV-infected double-negative (DN) B cells (IgD-/CD27-). (G) Gating strategy to identify DN B cell-derived plasma cells (IgD-/CD27-/CD23-/CD38+/CD138++) and quantification by treatment relative to control treated LCLs at Day 4. Statistical significance was evaluated by Welch’s two-tailed t-test.