Journal: Communications Medicine

Article Title: Multi-layered deep immune profiling, SARS-CoV-2 RNAemia and inflammation in unvaccinated COVID-19 individuals with persistent symptoms

doi: 10.1038/s43856-025-00832-8

Figure Lengend Snippet: a Proportion of naïve B cells (CD21 + CD27 - ), activated memory B cells (MBCs) (CD21 - CD27 +/- ), and classical MBCs (CD21 + CD27 + ) expressed within the total of non-antibody secreting B cells (CD19 + CD3 - CD20 - CD38 +/− ). b Frequency (%) of classical MBCs (CD21 + CD27+). c Proportion of total MBCs (activated and classical) expressing IgM (IgD-IgM+) or IgD (IgD+IgM−), and IgG or IgA within the pool of IgD-IgM- MBCs. d Frequency (%) of total MBCs (activated and classical) expressing IgA (IgD-IgM-IgG-IgA+). e Frequency (%) of RBD-specific (RBD-Biotin-Streptavidin-PE-Vio770+RBD-Biotin-Streptavidin-PE+) B cells expressed on the total of B cells (CD3-CD19+). f Frequency (%) of total MBCs (activated and classical) expressing CD24. Stacked graphs: descriptive graph showing the proportions within each group at each time point, statistics is shown in the dot-plots; r ed/blue dots: individual values; red/blue lines connecting median values at T0 and T1; longitudinal analyses (T1 compared to T0): Wilcoxon signed-rank test ( P values in red/blue); cross-sectional analyses (at each time point): Mann–Whitney U test (P values in black); EPS− (T0, T1): n = 7, 10; EPS+ (T0, T1): n = 10, 10; dotted lines: median of 7 pre-pandemic healthy controls. Statistical significance at p -value < 0.05.

Article Snippet: The following biotinylated antibodies were used: goat anti-human kappa and lambda light chain for total antibodies (Bethyl Laboratories, Inc.), rabbit monoclonal anti-human IgM and IgA (Abnova), mouse anti-human IgG1 (BD Biosiences) and IgG3 (Southern Biotech); followed by avidin-HRP (ThermoFischer Scientific) for 30 min at RT.

Techniques: Expressing, MANN-WHITNEY

Journal: Nature Communications

Article Title: The nutrient-sensing Rag-GTPase complex in B cells controls humoral immunity via TFEB/TFE3-dependent mitochondrial fitness

doi: 10.1038/s41467-024-54344-5

Figure Lengend Snippet: A , B B cells were stimulated with LPS/IL-4/BAFF and cultured with no amino acids (No AA), essential amino acids (EAA), or full amino acids (Full AA) ( A ), or indicated concentrations of amino acids ( B ) overnight. CD98, CD86, p-4EBP1, p-S6, and FSC-A levels were measured by flow cytometry. For CD98, CD86, p-S6, and FSC-A levels, n = 5 for each group. n = 3 for each group in p-4EBP1 expression. C – F Tamoxifen was administered to animals intraperitoneally daily for 4 consecutive days. Splenic B cells were purified 7 days after the last tamoxifen injection and stimulated with LPS/IL-4/BAFF. C Expression of p-4EBP1, p-S6, CD98, and FSC-A was measured by flow cytometry after overnight activation. Cre ER control (WT) ( n = 4), Cre ER Rraga fl/fl Rragb fl/fl ( n = 4), Cre ER Rptor fl/fl ( n = 4). D Expression of p-4EBP1, p-S6, p-S6K, AID, and LAMP1 was measured by immunoblot. β-actin was used as the loading control. Arrow indicates non-specific bands. Data represents 3 independent experiments. E B cells were labeled with CellTrace violet (CTV) and stimulated with indicated stimuli for 72 h. CTV dilution was measured by flow cytometry. F Expression of IgG1 and CTV dilution were examined by flow cytometry. Right, summary of the percentages of divided cells and IgG1 + B cells. WT ( n = 8), Cre ER Rraga fl/fl Rragb fl/fl ( n = 8), Cre ER Rptor fl/fl ( n = 4). G Rapamycin was added at 24 h after B cell activation with LPS/IL-4/BAFF. IgG1 expression was examined by flow cytometry at 72 h after activation. Right, summary of the percentages of IgG1 + B cells. The numbers indicate the fold differences of average IgG1 + percentages between WT and RagA/RagB deficient B cells. WT ( n = 5), Cre ER Rraga fl/fl Rragb fl/fl ( n = 4). Error bars represent mean ± SEM. ns, not significant. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001, one-way ANOVA ( A , B , and F ), two-tailed/unpaired Student’s t -test ( G ), or two-way ANOVA ( C ). Source data are provided as a file.

Article Snippet: Plates were washed twice with washing buffer (0.05% Tween 20 in PBS), blocked with 5% blocking protein (Bio-Rad) at 37 °C for 1 h, washed twice, and incubated with indicated sera samples at 37 °C for 1.5 h. Horseradish peroxidase (HRP)-conjugated secondary antibodies: anti-mouse IgG1, anti-mouse IgG2b, anti-mouse IgG2c, and anti-mouse IgG3 (all from SouthernBiotech), or anti-mouse IgM (Bethyl laboratories) were developed at 37 °C for 1 h after washing with a buffer for four times.

Techniques: Cell Culture, Flow Cytometry, Expressing, Purification, Injection, Activation Assay, Control, Western Blot, Labeling, Two Tailed Test

Journal: Nature Communications

Article Title: The nutrient-sensing Rag-GTPase complex in B cells controls humoral immunity via TFEB/TFE3-dependent mitochondrial fitness

doi: 10.1038/s41467-024-54344-5

Figure Lengend Snippet: A – E Tamoxifen was administered daily for 4 consecutive days followed by NP-OVA/alum immunization on day 11. A Expression of GL-7 and Fas in splenic B cells from μMT:Cre ER (WT), μMT:Cre ER Rraga fl/fl Rragb fl/fl , and μMT:Cre ER Rptor fl/fl mice. Right, summary of the percentages and numbers of GC B cells. B Expression of CXCR4 and CD86 in GC B cells. Right, summary of the ratio between dark zone (DZ) (CXCR4 + CD86 − ) and light zone (LZ) (CXCR4 − CD86 + ) cells. C Expression of CD138 and B220 in spleens. Right, summary of the percentages and numbers of CD138 + cells. D ELISA measurement of serum NP 23 -specific antibodies. WT ( n = 7), μMT:Cre ER Rraga fl/fl Rragb fl/fl ( n = 8), and μMT:Cre ER Rptor fl/fl ( n = 7). E ELISA measurement of serum NP 2 -specific antibodies. WT ( n = 5), μMT:Cre ER Rraga fl/fl Rragb fl/fl ( n = 5), and μMT:Cre ER Rptor fl/fl ( n = 7). F Expression of p-S6 and p-4EBP1 in fresh or overnight activated splenic B cells. G Heatmap of the mRNA expression of indicated genes in mouse follicular (FO), marginal zone (MZ), and GC B cells. H – L NP-OVA was administered to WT ( n = 11), Aicda Cre Rraga fl/fl Rragb fl/fl ( n = 10), and Aicda Cre Rptor fl/fl ( n = 6) mice. H Expression of GL-7 and Fas in splenic B cells. Right, summary of the GC B cell percentages. I Expression of NP and IgG1 in splenic B cells. Right, summary of the percentages of NP + IgG1 + GC B cells. J Expression of CXCR4 and CD86 on GC B cells. Right, summary of the DZ/LZ ratios. K Expression of CD138 and B220 on splenic lymphocytes. Right, summary of the percentages of CD138 + cells. L Expression of CD138 and IgG1 in plasmablasts. Right, summary of the percentages of IgG1 + plasmablasts. ELISA measurement of serum anti-NP 2 ( M ) and anti-NP 23 ( N ) antibodies, WT ( n = 13), Aicda Cre Rraga fl/fl Rragb fl/fl ( n = 8), and Aicda Cre Rptor fl/fl ( n = 8). Data in graphs represent mean ± SEM. ns, not significant. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001, one-way ANOVA ( A – C , F , H , I – L ), two-way ANOVA ( D , E , M and N ). Source data are provided as a file.

Article Snippet: Plates were washed twice with washing buffer (0.05% Tween 20 in PBS), blocked with 5% blocking protein (Bio-Rad) at 37 °C for 1 h, washed twice, and incubated with indicated sera samples at 37 °C for 1.5 h. Horseradish peroxidase (HRP)-conjugated secondary antibodies: anti-mouse IgG1, anti-mouse IgG2b, anti-mouse IgG2c, and anti-mouse IgG3 (all from SouthernBiotech), or anti-mouse IgM (Bethyl laboratories) were developed at 37 °C for 1 h after washing with a buffer for four times.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay

Journal: Nature Communications

Article Title: The nutrient-sensing Rag-GTPase complex in B cells controls humoral immunity via TFEB/TFE3-dependent mitochondrial fitness

doi: 10.1038/s41467-024-54344-5

Figure Lengend Snippet: A RNA sequencing was performed on 72-h activated B cells. Gene set enrichment analysis (GSEA) was conducted using differentially expressed genes (DEGs) with KEGG lysosome pathway plotted. B The enrichment of putative TFEB target genes. C RNA sequencing was performed on sorted germinal center (GC) B cells. Venn diagram of three DEG comparisons was presented. D – F GSEA was performed using the DEGs between indicated genotypes with KEGG lysosome pathway enrichment between indicated genotypes presented. G qRT-PCR of Lamp1 , Rragd , and Flcn expression. H Flow cytometry of LAMP1 expression, n = 5 mice per group. I Immunoblot of Raptor, RagA, and TFEB expression. Cytosolic and nuclear proteins were isolated from activated B cells. Lamin-B, nuclear control, tubulin, cytosol control. WT ( n = 5), Cre ER Rraga fl/fl Rragb fl/fl ( n = 7), and Cre ER Rptor fl/fl ( n = 4). J Expression of TFEB at indicated time-points ( n = 5 for each condition). Right, summary of relative TFEB expression (normalized to fresh). K qRT-PCR of Lamp1 , Rragd , and Flcn expression at indicated time-points. n = 4 for each condition. L , M Published scRNAseq dataset (E-MTAB-9478) was reanalyzed with gene signatures in naïve, marginal zone (MZ), GC B cells, and plasma cells presented ( L ). Gene signatures in GC B cells at different time points ( M ). N Transmission electron microscopy (TEM) of activated B cells. Arrows indicate mitochondria surrounded by double-layer structures. Scale bar, 2 μm. O GFP and mCherry expression with Mito-QC transduction. Right, summary of the normalized mCherry percentages (Relative to mCherry percentage in WT). WT ( n = 8), and Cre ER Rraga fl/fl Rragb fl/fl ( n = 10). P IgG1 expression on B cells stimulated in the presence of indicated inhibitors. Right, summary of IgG1 + B cell percentages. WT ( n = 7), Cre ER Rraga fl/fl Rragb fl/fl ( n = 6). Data represents at least 3 ( G – K , O , P ) and 2 ( N ) independent experiments. Data in graphs represent mean ± SEM. ns, not significant. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001, one-way ANOVA ( G – K ), two-tailed/unpaired Student’s t -test ( O and P ). Source data are provided as a file.

Article Snippet: Plates were washed twice with washing buffer (0.05% Tween 20 in PBS), blocked with 5% blocking protein (Bio-Rad) at 37 °C for 1 h, washed twice, and incubated with indicated sera samples at 37 °C for 1.5 h. Horseradish peroxidase (HRP)-conjugated secondary antibodies: anti-mouse IgG1, anti-mouse IgG2b, anti-mouse IgG2c, and anti-mouse IgG3 (all from SouthernBiotech), or anti-mouse IgM (Bethyl laboratories) were developed at 37 °C for 1 h after washing with a buffer for four times.

Techniques: RNA Sequencing, Quantitative RT-PCR, Expressing, Flow Cytometry, Western Blot, Isolation, Control, Clinical Proteomics, Transmission Assay, Electron Microscopy, Transduction, Two Tailed Test

Journal: Nature Communications

Article Title: The nutrient-sensing Rag-GTPase complex in B cells controls humoral immunity via TFEB/TFE3-dependent mitochondrial fitness

doi: 10.1038/s41467-024-54344-5

Figure Lengend Snippet: A qRT-PCR of Tfeb , Rragd , Rragc , and Lamp1 expression. GFP + B cells were sorted from vector, WT TFEB, and Ca TFEB transduced B cells. B Expression of IgG1 and Celltrace violet (CTV) in transduced B cells. n = 4 for each group. C Venn diagrams highlight the overlapping gene numbers in downregulated DEGs (left) and upregulated DEGs (right) between Rag KO vs WT comparison and Ca TFEB vs vector comparison. D Summaries of the relative MTDR (left) and TMRM (right) MFIs. n = 3 for each group. E Summary of the relative MitoSox MFIs. n = 3 for each group. F Summary of the relative LAMP1 MFIs. n = 3 for each group. G Transmission electron microscope (TEM) of sorted vector or Ca TFEB transduced B cells. Arrows indicate mitochondria surrounded by double-layer structures. scale bar, 2 μm. ( H ) ELISA of pS65-Ub levels in sorted vector or Ca TFEB transduced B cells. n = 5 for each group. Flow cytometry of TMRM ( I ) or MTDR ( J ) staining of vector or TFEB TDN transduced B cells. WT ( n = 5), Cre ER Rraga fl/fl Rragb fl/fl ( n = 7). K Flow cytometry of IgG1 and CTV. B cells from indicated genotypes were activated with LPS/IL-4/BAFF for 3 days. TMRM or MTDR ( L ), MitoSox ( M ), or LAMP1 ( N ) were measured by flow cytometry. O GFP and mCherry expression in B cells with Mito-QC transduction. P B cells from the indicated mice were purified and activated with LPS/IL-4/BAFF for 72 h. Mito stress assay was performed on a Seahorse XFe96 analyzer. Right, summaries of the basal respiration and maximal respiration. Data in graphs represent mean ± SEM. ns, not significant. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001, one-way ANOVA ( A , B , D – F , K – P ), two-way ANOVA ( I and J ), two-tailed/unpaired Student’s t -test ( H ). Source data are provided as a file.

Article Snippet: Plates were washed twice with washing buffer (0.05% Tween 20 in PBS), blocked with 5% blocking protein (Bio-Rad) at 37 °C for 1 h, washed twice, and incubated with indicated sera samples at 37 °C for 1.5 h. Horseradish peroxidase (HRP)-conjugated secondary antibodies: anti-mouse IgG1, anti-mouse IgG2b, anti-mouse IgG2c, and anti-mouse IgG3 (all from SouthernBiotech), or anti-mouse IgM (Bethyl laboratories) were developed at 37 °C for 1 h after washing with a buffer for four times.

Techniques: Quantitative RT-PCR, Expressing, Plasmid Preparation, Comparison, Transmission Assay, Microscopy, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Staining, Transduction, Purification, Two Tailed Test

Journal: Nature Communications

Article Title: The nutrient-sensing Rag-GTPase complex in B cells controls humoral immunity via TFEB/TFE3-dependent mitochondrial fitness

doi: 10.1038/s41467-024-54344-5

Figure Lengend Snippet: A Flow cytometry of CD43 and B220 expression on BM cells. Below, summaries of B220 lo CD43 − , B220 hi CD43 − and B220 + CD43 + cell frequencies. B Flow cytometry of BP-1 and CD24 expression in BM B220 + CD43 + IgM – B cell precursors. Right, summary of fraction A (CD24 − BP-1 − ), fraction B (CD24 + BP-1 − ), and fraction C/C′ (CD24 + BP-1 + ) cell frequencies. C Flow cytometry of GL-7 and Fas expression the Peyer’s patches. Right, summary of the GC B cell frequencies. For A-C, WT ( n = 5), μMT:Cre ER Rraga fl/fl Rragb fl/fl ( n = 6), and μMT:Cre ER Rraga fl/fl Rragb fl/fl Tfeb fl/fl Tfe3 −/− ( n = 6). Splenic B cells were stimulated with LPS/IL-4/BAFF for 72 h. IgG1 expression ( D ), TMRM and MTDR staining ( E ), CellROX staining ( F ), and LAMP1 staining ( G ) were examined by flow cytometry. Summaries of IgG1 + B cell frequencies, the relative TMRM, MTDR, CellROX, and LAMP1 MFIs were presented. For ( D – G ), n = 4 mice for each group. H Mitostress assay was performed on a Seahorse XFe96 analyzer. Right, summaries of the basal respiration and maximal respiration. WT ( n = 6), μMT:Cre ER Rraga fl/fl Rragb fl/fl ( n = 5), μMT:Cre ER Rraga fl/fl Rragb fl/fl Tfeb fl/fl Tfe3 −/− ( n = 6). I – L Tamoxifen injected WT, μMT:Cre ER Rraga fl/fl Rragb fl/fl , and μMT:Cre ER Rraga fl/fl Rragb fl/fl Tfeb fl/fl Tfe3 −/− chimera mice were immunized intraperitoneally with NP-OVA/alum ( I , J ) or TNP-LPS ( K , L ). I Flow cytometry of CD138 and B220 expression on splenic lymphocytes. Right, summary of CD138 + plasmablast frequencies. J Flow cytometry of GL-7 and Fas expression on splenic B cells. Right, summary of GC B cell frequencies. For ( I and J ), WT ( n = 4), μMT:Cre ER Rraga fl/fl Rragb fl/fl ( n = 3), and μMT:Cre ER Rraga fl/fl Rragb fl/fl Tfeb fl/fl Tfe3 −/− ( n = 4). K Flow cytometry of CD138 and B220 expression on splenic lymphocytes. Right, summary of CD138 + plasmablast frequencies. L ELISA measurement of serum TNP-specific antibodies. For ( K and L ), WT ( n = 6), μMT:Cre ER Rraga fl/fl Rragb fl/fl ( n = 7), μMT:Cre ER Rraga fl/fl Rragb fl/fl Tfeb fl/fl Tfe3 −/− ( n = 6). Data in graphs represent mean ± SEM. ns, not significant. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001, one-way ANOVA ( A , C , D – K ), two-way ANOVA ( B and L ). Source data are provided as a file.

Article Snippet: Plates were washed twice with washing buffer (0.05% Tween 20 in PBS), blocked with 5% blocking protein (Bio-Rad) at 37 °C for 1 h, washed twice, and incubated with indicated sera samples at 37 °C for 1.5 h. Horseradish peroxidase (HRP)-conjugated secondary antibodies: anti-mouse IgG1, anti-mouse IgG2b, anti-mouse IgG2c, and anti-mouse IgG3 (all from SouthernBiotech), or anti-mouse IgM (Bethyl laboratories) were developed at 37 °C for 1 h after washing with a buffer for four times.

Techniques: Flow Cytometry, Expressing, Staining, Injection, Enzyme-linked Immunosorbent Assay