Journal: Kidney International Reports

Article Title: Early-Stage B-cells Predict Relapse After Rituximab Treatment in Patients With Membranous Nephropathy

doi: 10.1016/j.ekir.2026.106365

Figure Lengend Snippet: Reconstitution of B-cells over time. To characterize B-cell subsets, peripheral blood mononuclear cells were stained with fluorochrome-conjugated monoclonal antibodies (BD Biosciences) directed against the following antigens: CD45 (V500-C), CD3 (APC), CD19 (APC-H7), CD27 (BV421), IgD (PE), CD38 (BV711), CD4 (BV605), and CD8 (PE), as well as 7-AAD (Miltenyi Biotech). The stained cells were then analyzed using multicolor flow cytometry (BD FACS Lyric). The subsets of gated CD19 + cells were identified based on surface marker expression as follows: naïve (CD19+CD27-IgD+), nonswitched memory (CD19+CD27+IgD+), switched memory (CD19+CD27+IgD-), and double negative (CD19+CD27-IgD-); and within the CD19+CD38++ population, we distinguished transitional cells (CD19+CD38++CD27-IgD+), plasmablasts (CD19+CD38++CD27+IgD-), and double-negative CD38 + cells (CD19+CD38++CD27-IgD-). B-cell subsets were expressed as a percentage of the total lymphocyte count. (a) B-cell subpopulations were assessed in 39 patients at baseline and in 36 patients at 3 (M3), 6 (M6), 9 (M9), 12 (M12), and 18 (M18) months following the first rituximab infusion. The 3 patients who did not receive treatment were excluded from the follow-up. Complete depletion of B-cells was observed in all patients at M3 post-rituximab for all B-cell subpopulations. CD19 + cells reappeared 6 months after rituximab infusion. Naive cells re-emerged the most among B-cells, followed by CD38 + cells, transitional cells and finally memory cells. Data are shown as mean values (dots). (b–k) B-cell subpopulations at baseline and at subsequent time points were compared between relapsing patients ( n = 8) and nonrelapsing patients ( n = 19); the 2 patients who received additional anti-CD20 infusions were excluded from subsequent analyses. Data are shown as medians and interquartile range (IQR). P -values were calculated by comparing the median values of each cell subpopulation between relapsing and nonrelapsing patients using a nonparametric, unpaired Mann–Whitney U test.

Article Snippet: To characterize B-cell subsets, peripheral blood mononuclear cells were stained with fluorochrome-conjugated monoclonal antibodies (BD Biosciences) directed against the following antigens: CD45 (V500-C), CD3 (APC), CD19 (APC-H7), CD27 (BV421), IgD (PE), CD38 (BV711), CD4 (BV605), and CD8 (PE), as well as 7-AAD (Miltenyi Biotech).

Techniques: Staining, Bioprocessing, Flow Cytometry, Marker, Expressing, MANN-WHITNEY

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: 144Nd , CD38 , HIT2 , 3144014B , Fluidigm.

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 , b , Defective B cell maturation in the BM of individuals with CBL -LOH. Flow cytometry staining of cryopreserved BM mononuclear cells of HDs and participants P1, P2 and P3 with CBL -LOH. a , Representative flow staining of CD20 versus CD10 expression levels on CD34 − CD19 + cells in BM samples. b , Quantification of these subsets for HDs ( n = 8), individuals with CBL -LOH ( n = 3) and individuals with PIK3CD GOF ( n = 3). The line shows the mean of the data points. Statistical significance was assessed using multiple two-sided Mann–Whitney tests corrected for multiple testing; * P < 0.05. c , d , In vitro differentiation of control AAVS1 -edited and CBL -edited CD34 + HSPCs toward B cell identity. c , Flow cytometry staining for CD10 and CD20 among CD19 + cells in differentiation cultures after 3 weeks of coculture. d , Quantification of B cell ‘subsets’ based on flow cytometry marker expression (pre-BI, CD10 + CD20 − ; pre-BII, CD10 + CD20 + ; immature B, CD10 + CD20 ++ ; mature B, CD10 − CD20 ++ ) in this culture in control and two CBL -edited reactions. The lines show the means of three biological replicates, except for AAVS1 single guide RNA (sgRNA) 1, where two replicates are shown. Statistical significance was assessed using multiple two-sided Mann–Whitney tests corrected for multiple testing; ** P < 0.005. e , Transcriptional overlap between CBL -edited and PI3K GOF HSPC-derived B cell progenitors. Gene set enrichment analysis for PI3K GOF gene signatures in CBL Ub LOF samples is shown; NES, normalized enrichment score. No correction for multiple testing was performed for the two binomial tests. f , Quantitative genotyping by amplicon sequencing of B cell subsets and monocytes in individuals with CBL -LOH and HDs, as well as parents of P1–P3; HDs ( n = 3), individuals with CBL -LOH ( n = 5). Data are shown as mean ± s.d. g , CD38 staining intensity of primary B cell subsets of pediatric individuals with CBL -LOH compared with age-matched HDs; HDs ( n = 9), individuals with CBL -LOH ( n = 5). 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. h , Rate of apoptosis following stimulation with daratumumab of CD19 + cells from in vitro differentiation cultures of control and CBL -edited HSPCs. Data show the mean of three technical replicates. The experiment is representative of three biological replicates. i , Rate of apoptosis following stimulation with daratumumab of control and CBL Y371C KI REH cells. Each dot represents one biological replicate; n = 7. The statistical significance of differences was assessed using multiple paired, two-sided t- tests, with correction for multiple testing. j , Western blot of control and CBL Y371C KI REH cells following stimulation with monoclonal anti-CD38 (daratumumab) for the indicated times (min).

Article Snippet: 144Nd , CD38 , HIT2 , 3144014B , Fluidigm.

Techniques: Flow Cytometry, Staining, Expressing, MANN-WHITNEY, In Vitro, Control, Marker, Derivative Assay, Amplification, Sequencing, Western Blot

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 ) Levels of sCD40L, APRIL and BAFF in the plasma of CBL-LOH patients (n = 6) and healthy donors (n = 14). Line shows the mean of the displayed datapoints (one point per individual assessed). ( b ) Quantification of the production of sCD40L, APRIL and BAFF by PBMCs of CBL-LOH patients (n = 3) and healthy donors (n = 5). Line shows the mean of the displayed datapoints (one point per individual assessed). ( c-f ) Impact of sCD40L levels on B cell differentiation in vitro using CD34+ HSPCs. ( c,d ) B cell output at day 21 of co-culture of CD34+ HSPCs from two healthy donors. The MS5 co-culture was supplement with IL-7 (20 ng/mL) and the indicated doses of sCD40L. Mean of dots that represent technical replicates. ( e,f ) Quantification of B cell subsets based on CD10 and CD20 marker expression. Cells were treated as in ( c,d ). Mean ± s.d. ( g ) IL-21 production by sorted CD4 + naïve and memory T cells upon the indicated stimuli. Mean ± s.d. ( h ) Surface staining of CD38 expressed on CD19+ HSPC-derived B cell progenitors edited with the indicated sgRNAs. Mean ± s.d. of three biological replicates. ( i ) Surface staining of CD38 expressed on HEK293T, REH and BJAB cells as determined by flow cytometry. Mean ± s.d. of three biological replicates. ( j ) CBL Y371C KI BJAB (n = 9) cells are not more resistant than WT (n = 5) cells to BCR-induced apoptosis. Mean ± s.d. of clones over three independent experiments.

Article Snippet: 144Nd , CD38 , HIT2 , 3144014B , Fluidigm.

Techniques: Clinical Proteomics, Cell Differentiation, In Vitro, Co-Culture Assay, Marker, Expressing, Staining, Derivative Assay, Flow Cytometry, Clone Assay

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-f ) Western blots of wildtype and CBL Y371C KI REH cells upon CD38 crosslinking with daratumumab for the indicated time periods (min/h). ( b,c, e,f ) Shows quantifications of three biological replicates. Mean ± s.d. Statistical significance was assessed with Mann Whitney tests. *p<0.05. ( g,h ) ERK phosphorylation upon CD38 crosslinking in gene-edited HSPC-derived CD19+ B progenitors. Total CD19+ ( g ) or CD19+CD20high ( h ) B progenitors were sorted from co-cultures edited at the AAVS1 or CBL locus. Cells were stimulated for 15 min with daratumumab, followed by fixation, permeabilization and intracellular staining for phosphorylated ERK (pERK). n = 2 biological replicates. Bars show the mean.

Article Snippet: 144Nd , CD38 , HIT2 , 3144014B , Fluidigm.

Techniques: Western Blot, MANN-WHITNEY, Phospho-proteomics, Derivative Assay, Staining