Fig 2: LCL subsets exhibit co-expression of genes associated with distinct GC phenotypes. (A) Gene module scores for mantle zone (MZ), dark zone (DZ), DZ cycling, light zone (LZ), plasmablast (PB), and antibody-secreting cell (ASC) states across tonsils and LCLs. Trajectories depict the path starting at the MZ phenotype and progressing through DZ, DZ cycling, LZ, post GC PB, and Late ASC. Blue trajectories depict the core GC and red trajectories represent exits from this dynamic. [MZ module = CCR6, CD22, CD69, FCRL4, FCRL5, BANK1, MARCH1; DZ module = AICDA, FOXO1, CXCR4, AURKC, IL2RB; DZ cycling module = TCF3, EZH2, CCND3, E2F2, TP53, PLK4, BRCA1; LZ module = NFKB1, NFKB2, CD80, CD83, CD86, BCL2A1, EBI3, CD40, CR2, MIR155HG, ACKR3, MYO1C, MYC; PB module = PRDM1, XBP1, MZB1, TNFRSF17, CD27, CD38; Late ASC = SDC1] (B) Detailed example of a GC-like cell paths in GM12878. Violin plots depicting key gene expression are presented to highlight the core GC dynamic (blue trajectory and box) as well as terminal differentiation to Late ASCs (red trajectory and box). The bifurcation point between ASC and GC re-entry trajectories is associated with cluster-resolved expression of IRF4 and PRDM1, with lower expression of these genes associated with perpetual GC re-entry and higher expression associated with Late ASCs.

Fig 3: SLE patient T-bethi BDN cells rapidly differentiate in ASCs in the absence of BCR stimulation.(a–d) ASC generation from SLE BN cells requires early but transient BCR activation. Cartoon (a) depicting in vitro stimulation conditions to activate sort-purified T-betlo BN cells from SLE patients. BN cells were stimulated for 3 days with R848, cytokines (IFN?, IL-2, IL-21, BAFF) ± anti-Ig (Step 1) and then washed and recultured for an additional 3 days with the same stimuli ± anti-Ig (Step 2). Cells were analyzed by flow cytometry on day 6 (b–d). Phenotypic characterization (b) of IgDnegCD27neg BDN cells in cultures containing anti-Ig for all 6 days showing expression of T-bet, CD11c, FcRL5, CD21 and CXCR5 by the T-bethi BDN subset. The frequency (c) and number (d) of CD38hiCD27+ ASCs in cultures lacking anti-Ig (-,-), containing anti-Ig for all 6 days (+,+) or exposed to anti-Ig for the first 3 days only (+,-). (e) SLE patient T-bethi BDN cells rapidly differentiate in ASCs. Purified SLE B cell subsets (T-betlo BN, T-betlo CD11cnegCXCR5+ CD27negIgDneg DN1 memory cells, T-betlo CD27+ memory B cells (Bmem) and T-bethi CD11c+CXCR5neg DN2 cells) were stimulated with cytokines (IFN?, IL-21, IL-2, BAFF) and R848 for 2.5 days then counted and transferred to anti-IgG ELISPOT plates for 6 hr. The frequency of IgG ASCs derived from each B cell subset is shown. See Figure 8—figure supplement 1 for gating strategy to purify B cell subsets from SLE patients. Data shown in (c–d) are from a single SLE individual and are representative of 2 independent experiments. Data reported in (e) are representative of 3 independent experiments using B cells sorted from 3 different SLE donors. Statistical analyses were performed using one-way ANOVA with Tukey’s multiple comparison test (d–e). P values *<0.05, **<0.01, ***<0.001, ****<0.0001.

Fig 4: EBV+ activated and differentiated cell fractions spontaneously recover parental LCL heterogeneity in culture. (A) Growth curves from ICAM-1Hi/CD27Lo, ICAM-1Hi/CD27Lo, and ICAM-1Hi/CD27Lo LCL fractions after sorting. Data from each day are presented as the mean number of cells normalized to initial population size at day 0 (error bars = standard deviation, n = 3 LCLs per fraction). (B) Representative time-resolved staining for ICAM-1 and CD27 in sorted fractions capture recovery of parental line phenotypic heterogeneity. (C) Fraction-resolved quantification of cell growth and phenotype distribution over six days in culture after sorting.

Fig 5: 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.