Panel A: A diagram illustrates the workflow for mRNA transcriptional analysis from pooled digital spatial transcriptomics data acquired from FFPE tissue sections. Panel B: Two heatmaps are color-coded to represent different cell types, including B memory, B naive, endothelial cells, fibroblasts, macrophages, mast cells, monocytes (classic and non-classic), neutrophils, NK cells, pDCs, plasma cells, TCD4 memory, TCD4 naive, TCD8 memory, TCD8 naive, and Treg cells. Panel C: Violin plots summarize the major cell populations, including B naive, endothelial, macrophages, CD4 T naive, fibroblasts, and neutrophils, comparing wild-type (WT) and SEPTIN6 mutant samples. Panel D: A scatter plot compares gene expression between the SEPTIN6 mutant and control marrow, with log-fold change plotted against average log-expression. Genes with high expression in plasma cells are highlighted within a dotted circle. Panel E: A diagram shows the experimental setup where human CD34 positive cells are transduced with lentivirus and injected into NBSGW mice. The bi-directional lentiviral vector diagram illustrates the expression of wild-type (WT) or genetic mutation (GM) SEPTIN6 protein or empty vector control. Panel F: Two scatter bar plots display the percentage of GFP positive cells determined by flow cytometry for common lymphoid progenitors (CLP) and early B cell progenitors, comparing empty vector control, SEPTIN6 WT, and SEPTIN6 GM conditions.
Mutant SEPTIN6 BM tissue has plasma cells and mature neutrophils, despite reduced early lymphoid progenitors. (A) Diagram of workflow for mRNA transcriptional analysis from pooled digital spatial transcriptomics (DSP) data acquired from FFPE tissue sections prepared from BM biopsy #3 from our proband (III.d) versus age- and gender-matched control. (B and C) (B) Cellular deconvolution estimates for the proportions of different cell types in each ROI were performed for each sample, and (C) the major populations are summarized. (D) Pooled regions (n = 49 ROIs per subject) were analyzed for gene expression and log fold change by log expression plotted to compare the SEPTIN6 mutant versus control marrow. The dotted circle highlights genes with high expression in plasma cells. (E) Human CD34+ cells were transduced with lentivirus, and 2 days after transduction, 1 million cells were retro-orbitally injected into NBSGW mice. Diagram of the bidirectional LV to express a WT or GM SEPTIN6 (*428Qext*9) protein (5) or empty vector. Transduced cells express an eGFP reporter cis-linked with shmiR to restrict endogenous SEPTIN6 expression compared with an empty vector control. (F) BM was isolated at 4 mo after transfer and the percent GFP+ determined by flow cytometry for each progenitor population. CLPs were gated Lin−CD34+CD38+CD19−CD10+ CD45RA+CD135− (left), and early B cell progenitors were gated Lin−CD34+CD38+CD19+CD10+ (right). Results are pooled animals from 7 human donors across two experiments. *P < 0.05, unpaired student t test. A schematic was generated in BioRender. EV, empty vector; GM, genetic mutant; shmiR, short hairpin miRNA. (mDC: myeloid dendritic cells; pDC: plasmacytoid dendritic cell; Treg: regulatory T cell; WPRE: Woodchuck Hepatitis Virus Posttranslational Regulatory Element).