Mb1-Cre deletion of Bcl11a identifies a CLP-derived subset of pDCs

(A) Representative FACS plots of pDC (gated as B220+ PDCA1+) and B cell (gated as B220+ PDCA1-) percentages in the bone marrow (BM) of Bcl11a F/F mb1-Cre mice (cKO) and littermate controls. (B) Quantification of pDC and B cell populations in the BM of Bcl11a F/F mb1-Cre mice (cKO) and littermate controls as cells/100,000 cells. (C-D) Flow cytometric analysis of BM and spleens of recipient mice 8 weeks post BM transplantation. BM was transferred from either BCL11A -sufficient reporter control mice (mb1-Cre-YFP) or BCL11A-deficient cKO mice (Bcl11a F/F mb1-Cre-YFP) into lethally irradiated C57BL/6J recipients. (E) B cell numbers after BM transplantation in BM and spleens of recipient mice. (F) pDC numbers after BM transplantation in BM and spleens of recipient mice. (G) Comparison of YFP+ pDC percentages in the spleen and BM of recipient mice post BM transplantation. Student’s t-test was used for all statistical comparisons. Error bars=mean±s.d. The results are representative of two experiments each containing 3-4 mice per group.

CLP-derived B-pDC are licensed for T cell activation

(A-B) Comparison of MHCII MFI in YFP+ and YFP- pDCs from Mb1-Cre-YFP reporter mice via flow cytometric analysis. ***p=0.0005 (C-D) Mb1-Cre-YFP mice were injected with 50 ug/ml (100 ul) of CpG:ODN or control GpC:ODN and analyzed via flow cytometry for splenic pDC numbers/100,000 cells (***p<0.0001). (E) Flow cytometry quantification of YFP- pDC and B-pDCs (YFP+) fold change in cell numbers upon CpG:ODN in vivo challenge (**p=0.002142 for YFP negative pDC expansion; ***p= 0.00023 for B-pDC expansion, and *p=0.0151 for CpG stimulated B-pDC and YFP- pDC number comparison). (F-G) Fold change in CD86+CD89+ cell numbers for each pDC population in mice restimulated with CpG:ODN; (B-pDCs, ***p<0.0001; YFP- pDCs, **p=0.026). Difference in CD86+CD89+ cell numbers between stimulated B-pDCs and YFP- pDCs was also significant, **p=0.0034 (H) In vitro TLR9 engagement of B-pDC or pDC for ELISA against IFN-α orIL-12p40. (****p<0.0001 for both cytokines) (I) T cells were magnetically isolated from wildtype C57BL/6J mouse splenocytes using MACs columns, labelled with CSFE, and then cultured (2.5×104/well) alone or with CpG:ODN activated B-pDCs or pDCs (5×103/well) for 6 d. (J) The percentage of CFSE-negative CD3+ T cells in co-cultures were significantly higher in B-pDC compared to pDC (****p<0.0001). Student’s t-test was used for all statistical comparisons. Error bars=mean±s.d. The results are representative of three independent experiments each containing at least four mice per group.

Single cell RNA-seq analysis of mouse PDCA1+ bone marrow cells

(A) UMAP generated by Seurat clustering analysis. Cluster identities were assigned using the top 5 consensus CIPR identity scores. (B) Heatmap of the top 5 DEGs from each cluster. After Seurat clustering, cell reads were subsetted to include only cells classified as B cells, pDCs, and B-pDCs. DEGS were generated for the new data subset and markers associated with (C) mature B cells (D) classical pDCs were plotted as violin plots.

Single cell trajectory analysis of Prog1, B cells, and B-pDC clusters

(A) Single cells belonging to clusters Prog1, B cells, and B-pDCs were ordered and plotted as a function of pseudotime based on uniquely expressed markers using the unsurpevised Monocle dpFeature. Cluster Prog1 was classified as the root of the trajectory given its high expression of pre-pro B cell and stem cell associated markers. DEGs of interest were plotted as a function of pseudotime in Prog1 (B), B cells (C), or pDCs (D) using Monocle. Branch Y_112 represents the expression kinetic trend of the B-pDC cluster (green), while branch Y_83 represents the expression kinetic trend in expression of B cells according to branched expression analysis modeling, or BEAM.

Identification of Axl+ non-canonical pDCs in mice

(A) Violin plots depicting relative expression of conserved “non canonical AXL+ DCs” associated markers in mouse pDC clusters. (B) BM scRNA-seq data was subsetted to include only pDCs and Prog2 subsets. We used Monocle3 for partition-based learning of cell developmental trajectories. Louvain partitions were generated from single cell reads and their developmental trajectory was inferred using SimplePPT (top right quadrant). Pseudotime across clusters is shown in the (lower bottom quadrant).

Progenitor population analysis for mb1-cre driven YFP expression.

We analyzed YFP expression in BM progenitors to determine when and where mb1-Cre is active (representative plots shown). (A) Lineage negative cells were analyzed to examine LSK Hematopoietic progenitor (Lin-, Sca-1+, c-Kit+), MPP (LSK, Flt3int), and LMPP (LSK, Flt3hi), populations. Virtually all cells were YFP-. (B) Myeloid Dendritic Progenitors (MDP; Lin-, IL7Rα-, Flt3+, c-kithi, Sca1-) and (C) Common Dendritic Cell Progenitors (CDP; Lin-, IL7Ra-, Flt3+, c-kitlo, Sca1-, CD115+) were also negative for YFP expression, while (D) Common Lymphoid Progenitors (CLP; Lin-, IL7Ra+, Flt3+, c-kitlo, Sca1lo) contained YFP+ cells. Error bars=mean±s.d. The results are representative of three independent experiments each containing at least three mice per group.

Cell distributions in the spleen of adoptively transferred recipient mice.

Both Bcl11a F/F mb1-Cre+ and Mb1-Cre-YFP adoptively transferred recipient mice reconstituted other splenic cell types in normal numbers, including BM macrophages (CD11b+ F4/80+), granulocytes (Gr-1+ CD11b+) and splenic cDCs (CD11c+ CD11b+ B220-). Total T cells (CD3+ B220-) as well as CD4+ and CD8+ subsets were significantly increased in number in proportion to B/pDC cell loss. Student’s t-test was used for all statistical comparisons. The results are representative of two independent experiments each containing 3-4 mice per experimental group. Error bars=mean±s.d

Transcriptional analysis identifies two populations of pDC in mice: myeloid-derived classical pDC and CLP-derived B-pDC.

(A) Bone marrow pDC (B220+ PDCA1+ CD11cint CD11b-) were sorted based on expression of YFP. Four mice were pooled for each isolated RNA sample, for a total of three pDC and three B-pDC groups from 12 mice. RT-PCR was used to confirm that Bcl11a, Bst2, SpiC, and Id2 expression match RNA-seq trends between pDC and B- pDC (B) RNA-seq was performed for gene expression analysis of pDC vs B-pDC and 220/23,946 genes (∼1%, left) were significantly differentially expressed (q value < 0.05, right heatmap, Log2 expression difference displayed). (C) Gene Set Enrichment Analysis (GSEA). Normalized enrichment score (NES) and false discovery rate q-values(FDR);FDR≤.25isconsideredsignificant56. GO Term or Panther-derived pathways identified by DAVID analysis of ∼220 differentially expressed genes between the pDC and B- pDC subsets28, 29. Among the top B-pDC DEGs were Lyz1, Ccr3, Cd86, Cd83, Id2, Axl, Siglec1, and Cd81. (D) Scatter plot comparisons of all genes with Reads Per Kilobase of transcript, per Million mapped reads (RPKM) >1. Correlations of pDC vs B-pDC (R2 value = 0.8959), B cell vs. pDC R2 value = 0.4145) and B cell vs. B-pDC (R2 value = 0.404) are indicated.

Imaging flow cytometry as a tool to quantify AXL expression in pDC populations.

(A-B) Representative Imaging flow cytometry of manuscript gating scheme used to define B cells and pDCs and histograms for SIGLEC-H, AXL and CD19 in B cells and pDCs (C) Representative Imaging flow cytometry plots of AXL+ pDCs (here alternatively gated as SIGLEC-H+ PDCA1+) in Mb1-Cre-YFP reporter mice bone marrow cells (n=5). (D) Complete statistics table generated with IDEAS for Image Stream analysis including non-stained wildtype controls. (E) Comparison of AXL expression in YFP- and YFP+ (B-pDCs) (p<0.0001, unpaired t tests). The results are representative of two independent experiments each containing at least 3 mice per group.

Confirmation of unbiasedly identified cell cluster identities.

Seurat generated violin plots of genes associated with distinct cell populations including mature B cells, pDCs, granulocytes, pre-pDCs, NK cells, monocytes, macrophages, and myeloid progenitor cells. Plot identities were initially determined by running the package CIPR (Cluster Identity Predictor).

Expression of early B cell receptor genes in B-pDCs.

(A) Monocle was used to plot select early B cell genes as function of branched pseudotime for Prog1, B Cells, and B-pDC populations. (B) Plasma cell contamination in Mb1+ pDCs (B220+ PDCA1+) was ruled out by flow cytometric analysis of TACI and CD138 in BM of 6 C57BL/6J mice.

Transcriptional heterogeneity of Axl+ murine pDC populations.

(A) Dot plot depicting expression prevalence of select “AXL+ DC” associated markers from literature select and pDC DEGs for pDC subsets. (B) Correlation between expression of AXL+ DC associated markers and Axl in non-canonical mouse pDC subsets.

Top CIPR Assigned Identity Scores for Seurat Cluster DEGs.

CIPR IDs were generated using the top 1000 DEGS from each cluster. The top 5 consensus CIPR ID generated was used to rename Seurat clusters.

DEG comparison between progenitor clusters.

The top 50 differentially expressed features of Prog1 in relation to Prog2 were calculated using Seurat using non-parametric Wilcoxon rank sum test. In blue are upregulated genes and in red are downregulated genes.