Fig 2: Long-term antitumor efficacy of engineered LLPCs in combinations with targeted inhibitors.a The strategy of investigating long-term efficacy of LLPCs combined with trametinib (Tra) and dabrafenib (Dab) treatment against human A375 melanoma. Mice treated only with vehicle (PBS, pH 7.0) were used as the control. Trametinib and dabrafenib were administrated every 2 days for 10 days. Nivolumab was given three times every week. b Tumor growth curves. Treatments began at day 10. Removing of inhibitors is marked by the black arrow. Representative graph of two repetitions of this experiment is shown. Data are presented as mean ± SEM. *P < 0.05, **P < 0.01; one-way ANOVA with Tukey’s post hoc test (b) was used. c The tumor infiltrating hCD3+ and hCD8+ lymphocytes. The melanoma biopsies were formalin fixed and processed for immunohistochemistry analysis at day 42. Anti-human CD3 antibody and anti-human CD8 antibody were used for primary staining (scale bar, 100 µm). d Representative flow cytometric analysis of LLPCs proportion in the spleen and representative immunofluorescence images indicating the engineered LLPCs population in the bone marrow. Human CD138 is shown in red, human CD90 in green, and DAPI-stained nuclei in blue. Scale bar, 10 µm.

Fig 3: Generation of a-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 a-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 a-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.

Fig 4: IL-38 expression in human tonsils. (A) Tonsil sections were stained for CD138 (blue), Pan-Cytokeratin (PanCK, green), and IL-38 (red). Nuclei were counter-stained with DAPI. The magnification shows CD38 expression in CD138+ plasma cells. (B) Tonsil sections were stained for CD20 (green) and IL-38 (red). Nuclei were counter-stained with DAPI. Representative images of 4 donors are shown. Scale bars: 100 µm (20 µm in the magnified section of (A)).