Fig. 5: Generation of α-PD-1 mAb secreting LLPCs from engineered human primary B cells upon transfer into NSG mice.

a The strategy of LLPCs differentiation from engineered primary B cells upon transfer into NSG mice and 5 months of reconstitution. b Representative example of the spleen and bone marrow from humanized NSG mice. The identification of three subsets were shown as follows: mature B cells (CD19⁺), PBs (CD19⁻CD38⁺CD138⁻), and putative LLPCs (CD19⁻CD38⁺CD138⁺). The proportion of mature B cells, PBs and LLPCs are shown on the right panel. Results are the mean ± SEM from three individual mice. c The ELISpot assay results of the α-PD-1 mAb secreting subsets are shown from a representative experiment. The numbers of spot-forming cells/10⁴ cells are presented on the right panel as the mean number ±SEM from three separate experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ns, no significant difference; one-way ANOVA with Tukey’s post hoc tests (b, c) were used. d The concentrations of α-PD-1 mAb were monitored in serum after the transfer of engineered B cells every 15 days for 5 months. Results from three independent donors were combined. Data are represented as mean ± SEM. e Selected “PC-related genes” are shown from three NSG mice sorted for B cells and LLPCs. Heatmaps showed the z-score normalized expression of the differentially expressed genes involved in the “PC-related gene” signature. RNA expression levels are indicated with a red/blue scale for high and low expression levels, respectively. f GSEA plots showed the enrichment genes of differentiation from B cells compared with plasma cells. The plot of running enrichment score (RES) is shown in green (top). Vertical bar (in black) in the middle indicate a gene within the differentiation gene set. The correlation of gene expression with subclusters is shown on the bottom. g–l Ratio of expression (log₂ fold) in LLPCs to that in B cells for genes encoding transcription factors (g), cell cycle (h), protein folding and metabolism (i), immune response and B-cell differentiation (j), apoptosis (k), and autophagy and ER stress response (l). Data are pooled from three mice.