Research ArticleLYMPHOCYTES

Lin28b controls a neonatal to adult switch in B cell positive selection

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Science Immunology  27 Sep 2019:
Vol. 4, Issue 39, eaax4453
DOI: 10.1126/sciimmunol.aax4453
  • Fig. 1 Establishing a positive correlation between CD5 expression and self-reactivity in BCR WT B-1 cells.

    (A) Flow cytometric analysis of peritoneal cavity (PerC) B cells 16 weeks after transplantation of E14.5 FL or ABM LinSca1+cKit+ (LSK) HSPCs into lethally irradiated CD45.1+CD45.2+ congenic recipients. Lineage panel for E14.5 FL LSK: Ter119B220Gr1CD3e; ABM LSK: Ter119B220Gr1CD11bCD3e; PerC B cells: Ter119Gr1CD3e. (B) CD5neg, CD5low, CD5int, and CD5hi gating strategy for WT adult PerC B-1 cells (LinCD19+CD43+CD23). (C) Histogram overlay showing post sort analysis of the populations defined in (B). (D) Relative distribution of IgHM CDR3 sequences as determined by high-throughput VDJseq of the indicated populations. (E) Gini index of IgHM CDR3 sequence reads in sorted CD5neg, CD5low, CD5int, and CD5hi populations (left to right). (F) Stacked bar graph indicates the combined frequency of IGHV-11 (white) and IGHV-12 (gray) containing IgHM CDR3 sequence reads. (G) Frequency of PtC liposome–reactive cells in the indicated B-1 populations as assessed by FACS. *P ≤ 0.05, ***P ≤ 0.001, ****P ≤ 0.0001 (n = 9 from three experiments). (H) Frequency of CDR3 containing ≥1 N-nucleotide additions at the N1 and N2 junctions combined. VDJseq data (B to F and H) are representative of two biological and technical replicates.

  • Fig. 2 Lin28b promotes the positive selection of CD5+ ImmB cells in neonatal mice.

    (A) Representative histogram overlay of CD5 surface expression on 2-day-old NBM and 4-month-old ABM small pre-B (LinCD19+CD93+IgMCD43CD24highFSClow) and ImmB cells (ImmB) (LinCD19+CD93+IgM+CD24high). Lower panel shows the CD5 fluorescence intensity IQRs of the same populations. Q1, 25th percentile; Q3, 75th percentile; M, median; FMO, fluorescence minus one staining control. (B) CD5 levels on PtC liposome–reactive and total ImmB cells from 2-day-old WT NBM. (C) Histogram overlay of CD5 levels on 2-day-old NBM and ABM of the indicated genotypes. Lower panel shows the CD5 IQR. (D) Representative FACS plots showing the frequency of PerC CD5+ B-1 cells from 10-day-old mice. (E) Quantification of data shown in (D) (n = 4 to 7 from three experiments). (F) Quantification of relative forward scatter (FSC-A) of ImmB from mice of the indicated genotypes and ages. Data are shown as a value relative to the FSC-A of a representative Lin28b+/+ 2-day-old sample (n = 3 to 7 from three experiments). ns, not significant. **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. (G) Representative histogram overlays of CD5 surface expression on WT BM ImmB of the indicated ages.

  • Fig. 3 Ectopic expression of Lin28b is sufficient to support enhanced B cell positive selection in the adult.

    (A) Representative histogram overlay of CD5 surface expression on ABM small pre-B cells and ImmB cells from tet-Lin28b and WT ABM mice fed a DOX diet for at least 10 days. Lower panel shows the CD5 IQR of the populations in the upper panel. (B) Quantification of relative FSC-A of pro-B, pre-B, and ImmB cells from tet-Lin28b ABM. The ratio of tet-Lin28b/WT cells from each experiment was calculated and plotted (n = 5 from three experiments). Statistics of the ratios against the value 1 were performed. ***P ≤ 0.001, ****P ≤ 0.0001. (C) Sort strategy for isolation of the 20% highest and lowest CD5-expressing ABM ImmB cells from DOX-fed tet-Lin28b mice. Sorted cells were transplanted intraperitoneally (I.P.) into untreated RAG1KO recipient mice on a normal diet. (D) Frequency of PerC CD5+ B-1 cells 3 weeks after transplantation (n = 3 from two experiments). (E) Representative CD5 surface expression on PerC B-1 cells 3 weeks after transplantation. (F) Representative PtC liposome reactivity of PerC B-1 cells.

  • Fig. 4 Lin28b alters the efficiency of overall B cell selection.

    (A) Schematic showing the cellular barcoding setup of pro-B cells. Tet-Lin28b pro-B cells (1 × 105) (LinCD19+IgMCD93+cKit+CD43+CD24loCD25) were sorted from untreated mice and transduced with Barcode-GFP-LV. Transduced cells were divided into two equal parts that were individually adoptively transferred into DOX-fed and untreated Rag1KO recipients, respectively. Barcode representation of mature splenic B cell progeny (CD19+CD93GFP+) was assessed 2 weeks after adoptive transfer. I.V., intravenously. (B) Stacked bars show representative read frequencies of individual barcodes retrieved. Number on top of bars indicates the number of unique barcodes detected in both PCR technical replicates (for details, see fig. S3). (C) Enumeration of barcodes retrieved (n = 5 from two experiments). Significance was tested using a paired t test. (D) Representative dot plots showing splenic mature B cells 2 weeks after transfer. (E) Histogram overlay shows representative CD5 expression of splenic mature B-1 (GFP+CD19+CD93CD43+CD23) and follicular B-2 subsets (GFP+CD19+CD93CD43CD23+). (F) Kinetics of EdU label progression during B cell development after administration of a single pulse of EdU into tet-Lin28b and WT mice fed a DOX diet for at least 10 days before labeling. Labeling of the indicated populations at the indicated time points was assessed by FACS (n = 3 to 5 per data point from two to three experiments). Error bars show the SD of the mean. *P ≤ 0.05.

  • Fig. 5 Lin28b promotes B cell positive selection by activating the c-Myc transcriptional program.

    (A) RNA-seq of sorted ABM ImmB cells from WT and tet-Lin28b mice after 2 weeks of DOX diet (n = 3). Volcano plot shows differential gene expression analysis (DESeq). (B) Normalized DESeq counts for the indicated genes. (C) Unsupervised GSEA for significantly enriched gene sets among the hallmark gene set collection from the Molecular Signature Database (45) (left) and top enriched gene sets (right). (D) Leading edge plots show enrichment analysis of the indicated gene sets. Top 100 predicted let-7 targets are extracted from TargetScan7.2 (83). (E) Normalized DESeq counts and qPCR analysis of Myc transcript levels in ABM ImmB cells from WT or tet-Lin28b mice (n = 3). (F) Western blot analysis of c-Myc and Lin28b transgene protein levels in sorted ABM ImmB cells. (G) Histogram showing c-Myc protein expression as measured by intracellular FACS in 3-day-old NBM and ABM ImmB cells from the indicated genotypes. (H) Western blot analysis showing c-Myc protein expression in 3-day-old NBM and spleen cells from the indicated genotypes. (I) RNA-seq of WT (n = 3) and Lin28b−/− (n = 4) 3-day-old NBM. Left: Top enriched hallmark gene sets. Right: Leading edge plot showing a depletion of c-Myc targets in Lin28b-deficient ImmB cells based on DESeq analysis.

  • Fig. 6 Ectopic Lin28b functionally replaces CD19 in B cell development and maintenance.

    (A) Representative FACS plots showing frequency of splenic B cell adult mice of the indicated genotypes fed a DOX diet for 3 weeks (n = 5 to 7 from three experiments). (B) Quantification (left) and absolute B cell numbers (right) in spleen of mice from (A). (C) CD5 levels on ABM ImmB cells from (A). (D) Representative plots of κ and λ light chain usage in ABM ImmB cells. (E) Quantification of (D) (n = 4 to 9 from three experiments). (F) Representative Western blot of pPDK1, pGSK3b, c-Myc, human Lin28b (tg-Lin28b), pS6, and b-actin levels in magnetically lineage-depleted (CD3Gr1CD11bTer119) splenic B cells (n ≥ 3). (G) Representative histogram of pS6 levels in ABM ImmB cells from WT and tet-Lin28b mice (n ≥ 3). (H) Proposed CD19/c-Myc/Lin28b positive feedback loop in neonatal and adult mice.

  • Fig. 7 Tet-Lin28b–induced positive selection during adult B lymphopoiesis produces fully functional CD5+ B-1 cells.

    (A) Schematic showing the DOX-STOP experimental setup. A total of 10,000 LSK HSPCs were sorted from the indicated donors and transplanted into sublethally irradiated Rag1KO recipients. Recipients were kept on a DOX diet for 4 weeks, allowing for transient B-1 cell output by tet-Lin28b HSPCs, followed by a normal diet for 12 weeks (fig. S6). (B) Gating strategy for FACS sorting of PerC CD5+ B-1 cells isolated from untransplanted adult (16-week-old) WT mice (nB-1) or the indicated reconstituted recipients 16 weeks after transplantation. (C) Frequency of PerC CD5+ B-1 cells 16 weeks after transplantation (n = 7 to 14 from three experiments). (D) A total of 2 × 104 sorted CD5+ B-1 cells from the indicated mice were competitively transferred by intraperitoneal injection along with congenic nB-1 competitors at a 1:2 ratio. Bar graph shows donor:competitor recovery ratio 12 weeks after transfer (n = 2 to 6 from two experiments). (E) Histogram showing CD5 expression levels of transferred nB-1, FLB-1, ABMB-1, and L28B-1 cells and their congenic competitors. (F) Frequency of CDR3 containing one or more N-nucleotide additions at the N1and N2 VDJ junctions combined. n = 3 to 7 biological replicates from three experiments. *P ≤ 0.05, **P ≤ 0.01, ****P ≤ 0.0001. Error bars show the SD of the mean. (G) Graphical summary. Our data put forward a unified model for the augmented B-1 cell output early in life. Lin28b-dependent ImmB cell positive selection early in life endows B-1 cells with their hallmark characteristics, including a stable CD5 expression profile and long-term fitness. Ectopic Lin28b in adult life can efficiently induce B-1 cell positive selection on the backdrop of a highly diverse adult type BCR junctional diversity. Adult-derived B-1 cells that develop independently of Lin28b are functionally comparable in terms of IL-10 and spontaneous IgM secretion but display reduced CD5 expression and impaired long-term fitness. We propose that Lin28b-mediated enhancement of B cell tonic signaling licenses self-reactive B-1 cell output during the neonatal period when the risk for harmful autoreactivity is constrained by limited junctional diversity, thereby contributing to an important layer of natural antibody-mediated immunity.

Supplementary Materials

  • immunology.sciencemag.org/cgi/content/full/4/39/eaax4453/DC1

    Fig. S1. Gating strategies for developing B cells.

    Fig. S2. Cell cycle analysis of tet-Lin28b ABM pre-B and ImmB subsets.

    Fig. S3. Determination of the minimal DOX treatment window during B cell maturation required for efficient CD5+ B-1 cell output from tet-Lin28b ABM.

    Fig. S4. Barcode filtering and analysis strategy.

    Fig. S5. Differentially expressed genes within top enriched Hallmark gene sets.

    Fig. S6. Genetic evidence implicating Lin28b in the CD19/PI3K/c-Myc pathway.

    Fig. S7. Transient tet-Lin28b expression of ABM progenitors promotes the output of functionally competent CD5+ B-1 cells.

    Table S2. Antibodies used in this study.

    Data file S1. Table S1: Raw data supplement.

    Data file S2. VDJseq PerC B-1 CD5 slices.

    Data file S3. DESeq2 normalized counts for adult tet-Lin28b and WT ImmB RNA-seq.

    Data file S4. DESeq2 normalized counts for neonatal Lin28b+/+, Lin28b−/− ImmB RNA-seq.

  • Supplementary Materials

    The PDF file includes:

    • Fig. S1. Gating strategies for developing B cells.
    • Fig. S2. Cell cycle analysis of tet-Lin28b ABM pre-B and ImmB subsets.
    • Fig. S3. Determination of the minimal DOX treatment window during B cell maturation required for efficient CD5+ B-1 cell output from tet-Lin28b ABM.
    • Fig. S4. Barcode filtering and analysis strategy.
    • Fig. S5. Differentially expressed genes within top enriched Hallmark gene sets.
    • Fig. S6. Genetic evidence implicating Lin28b in the CD19/PI3K/c-Myc pathway.
    • Fig. S7. Transient tet-Lin28b expression of ABM progenitors promotes the output of functionally competent CD5+ B-1 cells.
    • Table S2. Antibodies used in this study.

    Download PDF

    Other Supplementary Material for this manuscript includes the following:

    • Data file S1 (Microsoft Excel format). Table S1: Raw data supplement.
    • Data file S2 (.gz format). VDJseq PerC B-1 CD5 slices.
    • Data file S3 (Microsoft Excel format). DESeq2 normalized counts for adult tet-Lin28b and WT ImmB RNA-seq.
    • Data file S4 (Microsoft Excel format). DESeq2 normalized counts for neonatal Lin28b+/+, Lin28b−/− ImmB RNA-seq.

    Files in this Data Supplement:

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