Journal: Nature Immunology

Article Title: Somatic deficiency of the human E3 ubiquitin ligase CBL in leukocytes impairs B cell but not T cell development and function

doi: 10.1038/s41590-025-02381-7

Figure Lengend Snippet: a , Counts of peripheral B cells in individuals with CBL -LOH over time compared with the healthy range. Data from eight participants are shown. Healthy ranges are from pediatric clinical recommendations. b , IgG levels in individuals with CBL -LOH (P1–P8) over time compared with the healthy range. Data from eight participants are shown. Healthy ranges are from pediatric clinical recommendations. c , Quantification of the indicated B cell subsets in the peripheral cryopreserved mononuclear cell blood of HDs, heterozygous HDs and individuals with CBL -LOH of the indicated ages as determined by mass cytometry; controls 0–3 years old ( n = 2), controls 4–15 years old ( n = 9), controls 16–100 years old ( n = 28), pediatric participants (LOH) ( n = 5), adult participants (LOH) ( n = 2), heterozygous individuals ( n = 3). Data are shown as mean ± s.d. The statistical significance of differences was assessed in multiple two-sided Mann–Whitney tests, with correction for multiple testing; ** P < 0.005 and *** P < 0.0005. y.o., years old. d , Frequency of B cell subsets in cryopreserved PBMCs from HDs and individuals with CBL -LOH as determined by flow cytometry; HDs ( n = 13), individuals with CBL -LOH ( n = 5). Data are shown as mean ± s.d. The statistical significance of differences was assessed using multiple two-sided Mann–Whitney tests, with correction for multiple testing; * P < 0.05 and ** P < 0.005. e , CD5, CD9, CD21 and CD38 expression on transitional B cells of HDs (black) and individuals with CBL -LOH (red) as determined by flow cytometry; HDs ( n = 19), individuals with CBL -LOH ( n = 6). Data are shown as mean ± s.d. The statistical significance of differences was assessed by multiple two-sided Mann–Whitney tests, with correction for multiple testing; * P < 0.05, ** P < 0.005 and **** P < 0.00005. f , Percentage of CD21 hi cells among transitional B cells of HDs (black) and individuals with CBL -LOH (red) as determined by flow cytometry; HDs ( n = 9), individuals with CBL -LOH ( n = 5). Data are shown as mean ± s.d. The statistical significance of differences was assessed by Mann–Whitney test; *** P < 0.0005; gMFI, geometric mean fluorescence intensity.

Article Snippet: 162Dy , CD21 , REA940 , 130-124-315 , Miltenyi Biotec.

Techniques: Mass Cytometry, MANN-WHITNEY, Flow Cytometry, Expressing, Fluorescence

Journal: Nature Immunology

Article Title: Somatic deficiency of the human E3 ubiquitin ligase CBL in leukocytes impairs B cell but not T cell development and function

doi: 10.1038/s41590-025-02381-7

Figure Lengend Snippet: ( a ) Quantification of B-cell subsets in cryopreserved PBMCs from healthy donors, heterozygous carriers, and CBL-LOH patients of the indicated ages by mass cytometry. Controls 0–3 y.o. (n = 2); controls 4–15 y.o. (n = 9); controls 16–100 y.o. (n = 28); pediatric CBL-LOH (n = 5); adult CBL-LOH (n = 2); heterozygous individuals (n = 3). Mean ± s.d. Statistical significance was assessed using two-sided Mann–Whitney tests with correction for multiple testing. ( b ) Frequency of B-cell subsets in healthy donors (n = 10) and CBL-LOH patients (n = 4) by flow cytometry. Mean ± s.d. Significance was evaluated with two-sided Mann–Whitney tests adjusted for multiple comparisons. **p < 0.005. ( c ) MFI of B-cell markers on CD21 lo and transitional B cells from healthy donors (n = 6) and patients P1–3 (n = 3). Lines show means. ( d–f ) Modeling the CBL ΔExon8 variant in primary human CD34⁺ HSPCs. ( d ) Agarose gel electrophoresis of PCR products from AAVS1 or CBL loci 72 h after nucleofection; representative of >5 biological replicates. ( e ) Editing efficiency using sgRNA pair 1+2 by NGS, showing ~80% of the ~400 bp exon-8 deletion. ( f ) Western blot of CBL protein levels after CBL or AAVS1 editing. ( g ) Quantification of three biological replicates from (f). Mean ± s.d. ( h ) NGS quantification of exon-8 deletions for all three guide pairs (n = 2 biological replicates). Bars show means. ( i ) Editing efficiencies at days 8 and 21 in differentiation cultures by NGS. Mean ± s.d. from three biological replicates. ( j–m ) Bulk RNA-seq of AAVS1- and CBL-edited CD19⁺CD10⁺CD20 low HSPC-derived B-cell progenitors. ( j ) Gene-set enrichment analysis showing significantly enriched (red) or depleted (blue) pathways (NES: normalized enrichment score). ( k,l ) Differential expression of leading-edge genes in the ( k ) Hallmark G2–M checkpoint and ( l ) Hallmark mTORC1 signaling gene sets (Z-transformed normalized counts). ( m ) Transcriptional overlap between CBL-edited and PI3K GOF progenitors; Venn diagrams show shared significantly up- or downregulated genes, with overlap significance by binomial test.

Article Snippet: 162Dy , CD21 , REA940 , 130-124-315 , Miltenyi Biotec.

Techniques: Mass Cytometry, MANN-WHITNEY, Flow Cytometry, Variant Assay, Agarose Gel Electrophoresis, Western Blot, RNA Sequencing, Derivative Assay, Quantitative Proteomics, Transformation Assay