Journal: Frontiers in Immunology

Article Title: TLR9- and CD40-Targeting Vaccination Promotes Human B Cell Maturation and IgG Induction via pDC-Dependent Mechanisms in Humanized Mice

doi: 10.3389/fimmu.2021.672143

Figure Lengend Snippet: TLR9 agonist CpG-B promotes IgG responses in humanized mice vaccinated with CD40-targeting vaccine. Humanized mice were treated with PBS control (n=3) or vaccinated with αCD40-HIV5pep (n=3) alone or vaccinated with αCD40-HIV5pep plus CpG-B (n=4) at week0, week3 and week6. At week 7, mice were sacrificed. (A, B) The expression of IgM and IgG on B cells from PBMCs and spleens was detected by FACS. (C) The total level of IgM and IgG in the plasma was detected by ELISA. (D) Splenocyte from humanized mice were cultured ex vivo in the present of R848(1μg/ml) and IL-2(10 u/ml) for 48hours, the cells were used for total IgG detection by ELISpot. (E) Antigen specific IgG level in the plasma was detected by ELISA. (F) The expression of activation-induced cytidine deaminase (AID) in spleen cells was detected by RT-PCR. Each dot represents one individual mouse, bars indicate mean. *P < 0.05, **P < 0.01, by unpaired, two-tailed Student’s t-test comparing the two vaccinated groups.

Article Snippet: Total IgM and IgG were detected by ELISA kits, purchased from Bethyl Laboratories,int.(Cat.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Cell Culture, Ex Vivo, Enzyme-linked Immunospot, Activation Assay, Reverse Transcription Polymerase Chain Reaction, Two Tailed Test

Journal: The Journal of Experimental Medicine

Article Title: Repression of the B cell identity factor Pax5 is not required for plasma cell development

doi: 10.1084/jem.20200147

Figure Lengend Snippet: Normal B cell development in Igh Pax5/+ mice. (A–C) Flow-cytometric analysis of bone marrow (A), spleen (B), and lymph node (C) cells from 8–12-wk-old Igh Pax5/+ (black) and Igh +/+ (gray) littermates. The relative frequency was determined for pro-B (CD19 + Kit + CD2 – IgM – ), pre-B (CD19 + Kit – CD2 + IgM – ), immature B (CD19 + IgM + IgD – ), and recirculating B (CD19 + IgM lo IgD hi ) cells in the bone marrow (A), follicular (CD19 + CD21 lo CD23 hi ), and marginal zone (CD19 + CD21 hi CD23 lo ) B cells in the spleen (B) and mature CD4 + and CD8 + T cells in the lymph nodes (C). The flow-cytometric data were obtained in seven (A), five (B), or two (C) independent experiments. Statistical data (A–C) are shown as mean values with SD and were analyzed by the two-tailed unpaired Student’s t test; **, P < 0.01; ****, P < 0.0001. NS, not significant (P > 0.05). Each dot corresponds to one mouse. (D–F) Flow-cytometric analysis of Pax5 expression by intracellular staining of developing and recirculating B cells in the bone marrow (D), mature B cells in the spleen (E), and mature T cells in the lymph nodes of Igh Pax5/+ (black line) and Igh +/+ (gray filled) littermates. The specificity of the anti-Pax5 antibody (IgG2a) was controlled by staining of the different B cell types with a control IgG2a isotype antibody (gray dashed line).

Article Snippet: The following antibodies were used for ELISPOT and ELISA: goat anti-mouse IgM, IgG, IgG1, IgG2b, and IgA (human adsorbed; SouthernBiotech), HRP-coupled goat anti-mouse IgM, IgG, and IgG1 (human adsorbed; SouthernBiotech), and AP-coupled goat anti-mouse IgM, IgG1, IgA, and IgE (human adsorbed; SouthernBiotech).

Techniques: Two Tailed Test, Expressing, Staining

Journal: The Journal of Experimental Medicine

Article Title: Repression of the B cell identity factor Pax5 is not required for plasma cell development

doi: 10.1084/jem.20200147

Figure Lengend Snippet: Antibody secretion by Igh Pax5/+ plasma cells. (A and B) Antibody secretion by Igh Pax5/+ and Igh +/+ plasma cells under steady-state conditions. The numbers of IgM and IgG ASCs in the bone marrow of Igh Pax5/+ (black) and Igh +/+ (gray) mice at the age of 2–3 mo (A) and 6–8 mo (B) were determined by ELISPOT assay by incubating RBC-depleted bone marrow cells (2 × 10 5 ) on IgM- or IgG-coated plates for 6 h before determination of the number and size of the ELISPOTs. Images of representative ELISPOT wells (left), the mean cell number (middle), and the median ELISPOT size (right) are shown for IgM and IgG ASCs. (C and D) Antibody secretion by Igh Pax5/+ and Igh +/+ plasma cells 14 d after immunization with NP-KLH (in alum). The numbers of anti–NP-IgM or anti–NP-IgG1 ASCs in the spleen (C) and bone marrow (D) were determined by ELISPOT assay by incubating RBC-depleted cells (2 × 10 6 ) from the spleen or bone marrow on NP 20 -BSA-coated plates before the determination of the number and size of the ELISPOTs. Images of representative ELISPOT wells (left), the mean cell number (middle), and the median ELISPOT size (right) are shown for anti–NP-IgM and anti–NP-IgG1 ASCs. The data (C and D) are pooled from two independent immunization experiments. Statistical data (A–D) are shown as mean values with SD and were analyzed by the two-tailed unpaired Student’s t test; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. NS, not significant (P > 0.05). Each dot (A–D) corresponds to one mouse. (E) Electron microscope images of plasma cells isolated from the bone marrow of Igh Pax5/+ and Igh +/+ mice at the age of 6–8 mo. 1 representative image of 10 individual plasma cells analyzed per genotype is shown. The length of the indicated scale bars is 1 mm (B and D) or 0.5 µm (E).

Article Snippet: The following antibodies were used for ELISPOT and ELISA: goat anti-mouse IgM, IgG, IgG1, IgG2b, and IgA (human adsorbed; SouthernBiotech), HRP-coupled goat anti-mouse IgM, IgG, and IgG1 (human adsorbed; SouthernBiotech), and AP-coupled goat anti-mouse IgM, IgG1, IgA, and IgE (human adsorbed; SouthernBiotech).

Techniques: Enzyme-linked Immunospot, Two Tailed Test, Microscopy, Isolation

Journal: The Journal of Experimental Medicine

Article Title: Repression of the B cell identity factor Pax5 is not required for plasma cell development

doi: 10.1084/jem.20200147

Figure Lengend Snippet: Antibody titers and morphology of plasma cells in Igh Pax5/+ mice. (A and B) Serum titers of total antibody of the IgM, IgG1, IgA, and IgE isotypes in nonimmunized Igh Pax5/+ (black) and Igh +/+ (gray) mice at the age of 2–3 mo (A) or 6–8 mo (B). The results shown are representative of three independent experiments. Statistical data (A and B) are shown as mean values with SD and were analyzed by the two-tailed unpaired Student’s t test; *, P < 0.05. NS, not significant (P > 0.05). (C) Expression of the indicated immunoglobulin isotype mRNAs in Igh Pax5/+ (black) and Igh +/+ (gray) plasma cells. The expression data are shown as mean TPM values with SD and are based on two ( Igh +/+ ) and three ( Igh Pax5/+ ) independent RNA-seq experiments. Each dot represents one experiment performed with pooled plasma cells from 5–10 mice per genotype. The P values were calculated by the DESeq2 program; ***, P < 0.001; ****, P < 0.0001. NS, not significant (P > 0.05). (D) Higher magnification of the same two electron microscope images shown in . The two plasma cells were isolated from the bone marrow of 6–8-mo-old Igh Pax5/+ and Igh +/+ mice. (E) Comparison of ER content in B cells and plasma cells. Staining with the ER-Tracker Red dye (at a final concentration of 0.2 µM) was used to determine the ER content of B cells (CD19 + ) and plasma cells (Blimp1-GFP + CD138 + ) in the bone marrow of 6–8-mo-old Igh Pax5/+ and Igh +/+ mice at steady state (left). The quantification of the staining is shown as mean fluorescence intensity (MFI; right). Statistical data (A, B, and E) are shown as mean values with SD and were analyzed by the two-tailed unpaired Student’s t test; *, P < 0.05. NS, not significant (P > 0.05). Each dot (A, B, and E) corresponds to one mouse.

Article Snippet: The following antibodies were used for ELISPOT and ELISA: goat anti-mouse IgM, IgG, IgG1, IgG2b, and IgA (human adsorbed; SouthernBiotech), HRP-coupled goat anti-mouse IgM, IgG, and IgG1 (human adsorbed; SouthernBiotech), and AP-coupled goat anti-mouse IgM, IgG1, IgA, and IgE (human adsorbed; SouthernBiotech).

Techniques: Two Tailed Test, Expressing, RNA Sequencing Assay, Microscopy, Isolation, Staining, Concentration Assay, Fluorescence

Journal: The Journal of Experimental Medicine

Article Title: Repression of the B cell identity factor Pax5 is not required for plasma cell development

doi: 10.1084/jem.20200147

Figure Lengend Snippet: Rescue of IgG1 CSR in the presence of functional T cells. (A) Experimental strategy to interrogate B-lineage–intrinsic effects of ectopic Pax5 expression in response to immunization. CD43 – B cells isolated from the spleens of Igh B1-8hi/Pax5 Prdm1 Gfp/+ or Igh B1-8hi/+ Prdm1 Gfp/+ mice (CD45.1 + CD45.2 + ) were cotransferred with splenic T cells from OT-II TCR-tg Rag2 −/− mice (CD45.2 + ) into C57BL/6 recipients (CD45.1 + ). 1 d after cell transfer, the recipients were immunized with NP-OVA (in alum) and were analyzed 6 d after immunization. (B) Flow-cytometric analysis of splenic GC B cells of Igh B1-8hi donor origin. The frequencies of IgG1 + GC B cells of the Igh B1-8hi/+ Prdm1 Gfp/+ and Igh B1-8hi/Pax5 Prdm1 Gfp/+ genotypes were determined by sequential gating on donor cells (CD45.1 + CD45.2 + ), B cells (B220 + ), and GC B cells (GL7 + Fas + ) by flow cytometry (above). The quantification of the frequencies of GC B cells and IgG1 + GC B cells is shown below. The data are pooled from three independent experiments. (C) Analysis of splenic GFP + plasma cells derived from the transferred Igh B1-8hi/+ Prdm1 Gfp/+ or Igh B1-8hi/Pax5 Prdm1 Gfp/+ B cells. The flow-cytometric analysis is shown on the left, and the frequency of the GFP + plasma cells is quantified on the right. The data are pooled from two independent experiments. (D) ELISPOT assay to determine the frequency of anti–NP-IgM and anti–NP-IgG1 ASCs derived from the transferred Igh B1-8hi/+ Prdm1 Gfp/+ or Igh B1-8hi/Pax5 Prdm1 Gfp/+ cells. Six days after immunization with NP-OVA, 500 GFP + CD138 + plasma cells of donor origin were directly sorted onto NP 20 -BSA–coated plates. Cells were incubated for 6 h, before the number and size of anti–NP-IgM and anti–NP-IgG1 ELISPOTs were determined. Images of representative ELISPOT wells (left), the mean cell number (middle), and the median ELISPOT size (right) are shown for anti–NP-IgM and anti–NP-IgG1 ASCs. Statistical data (B–D) are shown as mean values with SD and were analyzed by the two-tailed unpaired Student’s t test; **, P < 0.01; ****, P < 0.0001. NS, not significant (P > 0.05). Each dot (B–D) corresponds to one mouse. A scale bar of 1 mm length is shown.

Article Snippet: The following antibodies were used for ELISPOT and ELISA: goat anti-mouse IgM, IgG, IgG1, IgG2b, and IgA (human adsorbed; SouthernBiotech), HRP-coupled goat anti-mouse IgM, IgG, and IgG1 (human adsorbed; SouthernBiotech), and AP-coupled goat anti-mouse IgM, IgG1, IgA, and IgE (human adsorbed; SouthernBiotech).

Techniques: Functional Assay, Expressing, Isolation, Flow Cytometry, Derivative Assay, Enzyme-linked Immunospot, Incubation, Two Tailed Test

Journal: Nature Communications

Article Title: A p38α-BLIMP1 signalling pathway is essential for plasma cell differentiation

doi: 10.1038/s41467-022-34969-0

Figure Lengend Snippet: a Schematic outline of in vitro plasma cell (iPC) differentiation from splenic naïve B cells. Flow cytometry analysis of the percentage of iGCB cells (CD19 + Fas hi GL7 hi ) at day 4 and iPCs (CD19 + CD138 + ) at day 8 of culture, and supernatant IgG1 and IgE of p38α KO and WT B cells at day 8 of iPC culture were measured by ELISA ( n = 6 per group). b Principal-component analysis (PCA) of RNA-seq data from sorted iGCB cells ( n = 2 per group), iPCs (CD19 + CD138 + ) and CD138 − B cells (CD19 + CD138 − ) of indicated genotypes ( n = 3 per group). Numbers of upregulated (red) and downregulated (blue) genes ( p value < 0.01) in p38α KO iGCB cells and iPCs in comparison with WT cells. c Purified splenic naïve B cells from p38α fl/fl Vav cre or p38α fl/fl mice (CD45.2 + ) were mixed and co-cultured with CD45.1 + WT splenic naïve B cells at a 1:1 ratio. Flow cytometry analysis of iPCs gated on CD45.1 + and CD45.2 + B cells in iPC differentiation system at day 8 of culture ( n = 3 per group). d Immunoblot analysis of p38α, p-p38α, IgG heavy (H)-chain and Actin in cultured p38α KO and WT B cells at indicated time points after being transferred onto IL-21 feeder cells. e Cultured p38α KO and WT B cells were transduced with retroviruses encoding p38α and p38α kinase-dead mutants (K53M or T180A/Y182F) at day 2 of iGCB cell differentiation. iPCs among retrovirus-transduced B cells (CD19 + GFP + CD138 + ) were analyzed by flow cytometry 6 days after retroviral transduction ( n = 6 per group). Protein levels of p38α, IgG heavy (H)-chain and Actin in retrovirus-transduced B cells were analyzed by immunoblot. Data were representative of at least three independent experiments ( a ), ( c – e ). Each symbol represents a representative sample. Small horizontal lines indicate the mean (±s.d.). Data were analyzed by two-tailed unpaired t -tests. Source data are provided as a Source Data file.

Article Snippet: The following antibodies were from Southern Biotech: Mouse IgE-UNLB (0114-01, 1:50-1:1,000,000), IgG1-UNLB (0102-01, 1:50-1:1,000,000), IgG3-UNLB (0105-01, 1:50-1:1,000,000), IgM-UNLB (0101-01, 1:50-1:1,000,000), goat anti-mouse IgE-BIOT (1110-08, 1:500), goat anti-mouse IgG1-BIOT (1070-08, 1:500), goat anti-mouse IgG3-BIOT (1100-08, 1:500), goat anti-mouse IgM-BIOT (1020-08, 1:500), goat anti-mouse Kappa-UNLB (1050-01, 1:200) and goat anti-mouse Kappa-UNLB (1060-01, 1:200).

Techniques: In Vitro, Clinical Proteomics, Flow Cytometry, Enzyme-linked Immunosorbent Assay, RNA Sequencing, Comparison, Purification, Cell Culture, Western Blot, Transduction, Cell Differentiation, Retroviral, Two Tailed Test

Journal: Nature Communications

Article Title: A p38α-BLIMP1 signalling pathway is essential for plasma cell differentiation

doi: 10.1038/s41467-022-34969-0

Figure Lengend Snippet: a Flow cytometry analysis of BLIMP1-expressing cells (CD19 + GFP + ), BLIMP1-expressing CD138 − cells (CD19 + GFP + CD138 − ) and BLIMP1-expressing CD138 + (CD19 + GFP + CD138 + ) cells differentiated from p38α fl/fl Blimp1 gfp/+ and p38α fl/fl Vav cre Blimp1 gfp/+ B cells in iPC culture ( n = 5 per group). b MFI of GFP in BLIMP1-expressing CD138 − B cells (CD19 + GFP + CD138 − ) and BLIMP1-expressing CD138 + B cells (CD19 + GFP + CD138 + ) from ( a ) were determined by flow cytometry ( n = 5 per group). c Immunoblot analysis of BLIMP1, IgG H-chain and Actin in cultured B cells at indicated time points after p38α KO and WT iGCB cells transferred onto IL-21 feeder cells. d Flow cytometry analysis of BLIMP1 expression by intracellular staining for BLIMP1 in splenic B cells from OVA/Alum/LPS-immunized mice described as Fig. ( n ≥ 7 per group). e Quantitative RT-PCR analysis of Blimp1 mRNA in cultured p38α KO and WT B cells at indicated time points after transferred onto IL-21 feeder cells ( n = 3 per group). f p38α KO and WT iGCB cells at day 4, CD138 − B cells (CD19 + CD138 − ) and CD138 + B cells (CD19 + CD138 + ) at day 8 were sorted, mRNA level of Blimp1 in these subsets were analyzed by quantitative RT-PCR ( n = 3 per group). g Cultured p38α KO B cells were transduced with retroviruses encoding p38α or p38α kinase-dead mutants (K53M or T180A/Y182F) and transferred onto IL-21 feeder cells. Blimp1 mRNA levels were measured by quantitative RT-PCR after 4 days of culture ( n = 3 per group). Data were representative of at least three independent experiments. Each symbol represents a representative sample or an individual mouse ( d ). Small horizontal lines indicate the mean (±s.d.). Data were analyzed by two-tailed unpaired t -tests. Source data are provided as a Source Data file.

Article Snippet: The following antibodies were from Southern Biotech: Mouse IgE-UNLB (0114-01, 1:50-1:1,000,000), IgG1-UNLB (0102-01, 1:50-1:1,000,000), IgG3-UNLB (0105-01, 1:50-1:1,000,000), IgM-UNLB (0101-01, 1:50-1:1,000,000), goat anti-mouse IgE-BIOT (1110-08, 1:500), goat anti-mouse IgG1-BIOT (1070-08, 1:500), goat anti-mouse IgG3-BIOT (1100-08, 1:500), goat anti-mouse IgM-BIOT (1020-08, 1:500), goat anti-mouse Kappa-UNLB (1050-01, 1:200) and goat anti-mouse Kappa-UNLB (1060-01, 1:200).

Techniques: Flow Cytometry, Expressing, Western Blot, Cell Culture, Staining, Quantitative RT-PCR, Transduction, Two Tailed Test

Journal: Nature Communications

Article Title: A p38α-BLIMP1 signalling pathway is essential for plasma cell differentiation

doi: 10.1038/s41467-022-34969-0

Figure Lengend Snippet: a Cultured p38α KO and WT B cells were transduced with retroviruses encoding p38α or BLIMP1 at day 2 of in vitro differentiation. Flow cytometry analysis of iPCs in retrovirally transduced B cells (CD19 + GFP + CD138 + ) 6 days after transduction ( n = 6 per group). b ELISpot analysis of IgG1 and IgE antibody-secreting cells among cultured B cells as ( a ) ( n = 3 per group). c Heatmap analysis of differential expression genes (foldchange > 2) in iPCs of indicated genotypes. Group 1: decreased in p38α KO iPCs, restored by p38α or BLIMP1 overexpression. Group 2: decreased in p38α KO iPCs, restored by p38α but not BLIMP1 overexpression. Group 3: decreased in p38α KO iPCs, restored by BLIMP1 but not p38α overexpression. Group 4: decreased in p38α KO iPCs, restored by neither p38α nor BLIMP1 overexpression. Group 5: increased in p38α KO iPCs, restored by p38α or BLIMP1 overexpression. Group 6: increased in p38α KO iPCs, restored by p38α but not BLIMP1 overexpression. Group 7: increased in p38α KO iPCs, restored by BLIMP1 but not p38α overexpression. Group 8: increased in p38α KO iPCs, restored neither p38α nor BLIMP1 overexpression. d Gene Ontology enrichment analysis of Group 1 from ( c ). e Immunoblot analysis of BLIMP1, p38α, IgG H-chain, Actin and UPR-related proteins, Bip, IRE1α, SEC61A1 and SEC61B in retrovirally transduced B cells at day 8 in iPC culture. f Cultured p38α KO and WT B cells were transduced with retroviruses encoding p38α or BLIMP1 at day 2 of in vitro differentiation. Representative transmission electron microscopy images of sorted iPCs in retrovirally transduced B cells (CD19 + GFP + CD138 + ) of indicated groups at day 8. Scale bars, 500 nm. Percentage of endoplasmic reticulum (ER) expansion and diameter of ER lumen in iPCs of indicated groups ( n ≥ 100 per group). Data were representative of at least three independent experiments ( a , b , e , f ). Each symbol represents a representative sample. Small horizontal lines indicate the mean (±s.d.). Data were analyzed by one-side hypergeometric test ( d ) and two-tailed unpaired t -tests ( a , f ). Source data are provided as a Source Data file.

Article Snippet: The following antibodies were from Southern Biotech: Mouse IgE-UNLB (0114-01, 1:50-1:1,000,000), IgG1-UNLB (0102-01, 1:50-1:1,000,000), IgG3-UNLB (0105-01, 1:50-1:1,000,000), IgM-UNLB (0101-01, 1:50-1:1,000,000), goat anti-mouse IgE-BIOT (1110-08, 1:500), goat anti-mouse IgG1-BIOT (1070-08, 1:500), goat anti-mouse IgG3-BIOT (1100-08, 1:500), goat anti-mouse IgM-BIOT (1020-08, 1:500), goat anti-mouse Kappa-UNLB (1050-01, 1:200) and goat anti-mouse Kappa-UNLB (1060-01, 1:200).

Techniques: Cell Culture, Transduction, In Vitro, Flow Cytometry, Enzyme-linked Immunospot, Quantitative Proteomics, Over Expression, Western Blot, Transmission Assay, Electron Microscopy, Two Tailed Test

Journal: Nature Communications

Article Title: A p38α-BLIMP1 signalling pathway is essential for plasma cell differentiation

doi: 10.1038/s41467-022-34969-0

Figure Lengend Snippet: a Schematic outline of CRISPR/Cas9 screen of p38α substrates in iPC differentiation. Naive B cells from Cas9 tg/+ mice (GFP + ) and WT littermates (GFP − ) were mixed at a 1:1 ratio and cultured. Cultured B cells were transduced with retroviruses encoding individual sgRNAs targeting p38α substrates, BFP, and puromycin-resistance gene at day 2. iPCs at day 8 among retrovirus-transduced Cas9-expressing B cells (CD19 + GFP + BFP + CD138 + ) were analyzed by flow cytometry. b Summary of the screen results in ( a ). X axis: The ratio between iPCs percentages of p38α substrates sgRNA-expressing and NTC sgRNA-expressing B cells (Fold change); Y axis: The variation of effect on iPCs generation of 3 sgRNAs targeting gene of interest as measured by Students’ t -tests. c Flow cytometry analysis of iPCs among cultured Cas9 tg/+ B cells transduced with retroviruses encoding p38α or Tcf3 sgRNA (CD19 + GFP + BFP + CD138 + ). Immunoblot analysis of BLIMP1, TCF3 and Actin expression in Tcf3 sgRNA expressing B cells at indicated time of culture. d Schematic diagram of a putative TCF3-binding site in the Prdm1 locus. p38α KO and WT B cells cultured on IL-21 feeder cells for 2 days were utilized for CUT&Tag assay and immunoprecipitated with anti-TCF3 antibody. The enrichment of TCF3 binding genomic fragments containing the reported TCF3 binding site in Blimp1 enhancer region was measured by quantitative RT-PCR ( n = 3 per group). e Flow cytometry analysis of iPCs among cultured p38α KO B cells transduced with retroviruses encoding the TCF3 splicing isoforms E12 and E47 (CD19 + GFP + CD138 + ) ( n = 6 per group), protein levels of TCF3, IgG H-chain, BLIMP1, p38α, and Actin were analyzed by immunoblot. f Flow cytometry analysis of iPCs among cultured Tcf3 KO B cells transduced with retroviruses encoding p38α (CD19 + GFP + CD138 + ) ( n = 7 per group). g Flow cytometry analysis of iPCs (CD19 + CD138 + ) among cultured p38α fl/fl CD19 cre , Tcf3 fl/fl CD19 cre , p38α fl/fl Tcf3 fl/fl CD19 cre , p38α fl/fl Tcf3 fl/fl B cells at day 8 of in vitro iPC differentiation ( n = 3 per group). NTC, non-targeting control. Data were representative of at least three independent experiments ( b – g ). Each symbol represents a representative sample. Small horizontal lines indicate the mean (±s.d.). Data were analyzed by two-tailed unpaired t -tests. Source data are provided as a Source Data file.

Article Snippet: The following antibodies were from Southern Biotech: Mouse IgE-UNLB (0114-01, 1:50-1:1,000,000), IgG1-UNLB (0102-01, 1:50-1:1,000,000), IgG3-UNLB (0105-01, 1:50-1:1,000,000), IgM-UNLB (0101-01, 1:50-1:1,000,000), goat anti-mouse IgE-BIOT (1110-08, 1:500), goat anti-mouse IgG1-BIOT (1070-08, 1:500), goat anti-mouse IgG3-BIOT (1100-08, 1:500), goat anti-mouse IgM-BIOT (1020-08, 1:500), goat anti-mouse Kappa-UNLB (1050-01, 1:200) and goat anti-mouse Kappa-UNLB (1060-01, 1:200).

Techniques: CRISPR, Cell Culture, Transduction, Expressing, Flow Cytometry, Western Blot, Binding Assay, Immunoprecipitation, Quantitative RT-PCR, In Vitro, Control, Two Tailed Test

Journal: Nature Communications

Article Title: A p38α-BLIMP1 signalling pathway is essential for plasma cell differentiation

doi: 10.1038/s41467-022-34969-0

Figure Lengend Snippet: a Flow cytometry analysis of iPCs differentiated from cultured Cas9 tg/+ B cells transduced with retroviruses encoding p38α or Tcf4 sgRNA (CD19 + GFP + BFP + CD138 + ). Immunoblot analysis of BLIMP1, TCF4, and Actin expression in Tcf4 sgRNA expressing B cells at indicated time points after being transferred onto IL-21 feeder cells. b Schematic diagram of a putative TCF4-binding site in the Prdm1 locus. CUT&Tag analysis of TCF4 binding genomic fragments including the reported TCF4 binding site in Blimp1 enhancer region in cultured p38α KO and WT B cells at day 2 after transferred onto IL-21 feeder cells ( n = 3 per group). c Flow cytometry analysis of iPCs among cultured p38α KO B cells transduced with retroviruses encoding TCF4 (CD19 + GFP + CD138 + ) ( n = 6 per group), protein levels of TCF4, IgG H-chain, BLIMP1, p38α and Actin were analyzed by immunoblot. d – f Same as described in ( a – c ) except that Irf4 sgRNAs were used or IRF4 was overexpressed in cultured p38α KO B cells. g Cultured p38α fl/fl Vav cre Cas9 tg/+ and p38α fl/fl Cas9 tg/+ B cells were transduced with retroviruses encoding sgRNA targeting p38α , Tcf3 , Tcf4 and Irf4 at day 2 of culture ( n = 3 per group). Percentages of iPCs in sgRNA transduced B cells (CD19 + GFP + BFP + CD138 + ) were analyzed by flow cytometry at day 8. NTC, non-targeting control. Data were representative of at least three independent experiments. Each symbol represents a representative sample. Small horizontal lines indicate the mean (±s.d.). Data were analyzed by two-tailed unpaired t -tests. Source data are provided as a Source Data file.

Article Snippet: The following antibodies were from Southern Biotech: Mouse IgE-UNLB (0114-01, 1:50-1:1,000,000), IgG1-UNLB (0102-01, 1:50-1:1,000,000), IgG3-UNLB (0105-01, 1:50-1:1,000,000), IgM-UNLB (0101-01, 1:50-1:1,000,000), goat anti-mouse IgE-BIOT (1110-08, 1:500), goat anti-mouse IgG1-BIOT (1070-08, 1:500), goat anti-mouse IgG3-BIOT (1100-08, 1:500), goat anti-mouse IgM-BIOT (1020-08, 1:500), goat anti-mouse Kappa-UNLB (1050-01, 1:200) and goat anti-mouse Kappa-UNLB (1060-01, 1:200).

Techniques: Flow Cytometry, Cell Culture, Transduction, Western Blot, Expressing, Binding Assay, Control, Two Tailed Test

Journal: Nature Communications

Article Title: A p38α-BLIMP1 signalling pathway is essential for plasma cell differentiation

doi: 10.1038/s41467-022-34969-0

Figure Lengend Snippet: a Cultured p38α − /− B cells were transduced with retroviruses encoding p38α or p38α kinase-dead mutations (K53M or T180A/Y182F) (GFP + ) and luciferase reporter harboring TCF3, TCF4 or IRF4 binding sites in Prdm1 enhance region at day 2, and luciferase activity in retrovirus-infected B cells (CD19 + GFP + ) was analyzed at day 8 in iPC culture ( n = 3 per group). b p38 −/− 293 A cells were transfected with indicated plasmids, and the luciferase activity of IRF4 mutants was analyzed 24 h after transfection. Immunoblot analysis of p38α, p-p38α, IRF4 and Actin in p38 −/− 293 A cells transfected with indicated plasmids (n = 3 per group). c Cultured Irf4 −/− B cells were transduced with retroviruses encoding IRF4 or IRF4 mutants (T267A, T268A, T269A) at day 2, iPCs in retrovirally transduced B cells (CD19 + GFP + CD138 + ) 6 days after transduction were analyzed by flow cytometry ( n = 5 per group), and protein levels of IRF4, IgG H-chain, BLIMP1 and Actin were analyzed by immunoblot. Data were representative of at least three independent experiments. Each symbol represents a representative sample. Small horizontal lines indicate the mean (±s.d.). Data were analyzed by two-tailed unpaired t-tests. Source data are provided as a Source Data file.

Article Snippet: The following antibodies were from Southern Biotech: Mouse IgE-UNLB (0114-01, 1:50-1:1,000,000), IgG1-UNLB (0102-01, 1:50-1:1,000,000), IgG3-UNLB (0105-01, 1:50-1:1,000,000), IgM-UNLB (0101-01, 1:50-1:1,000,000), goat anti-mouse IgE-BIOT (1110-08, 1:500), goat anti-mouse IgG1-BIOT (1070-08, 1:500), goat anti-mouse IgG3-BIOT (1100-08, 1:500), goat anti-mouse IgM-BIOT (1020-08, 1:500), goat anti-mouse Kappa-UNLB (1050-01, 1:200) and goat anti-mouse Kappa-UNLB (1060-01, 1:200).

Techniques: Cell Culture, Transduction, Luciferase, Binding Assay, Activity Assay, Infection, Transfection, Western Blot, Flow Cytometry, Two Tailed Test

Journal: bioRxiv

Article Title: Human effector CD8 + T cells with an exhausted-like phenotype control tumor growth in vivo

doi: 10.1101/2023.10.11.561856

Figure Lengend Snippet: Splenocytes (A-D) and tumor-infiltrating lymphocytes (TIL) (E-H) were isolated from HIS mice 16-18 days after tumor implantation and analyzed by flow cytometry. A, frequency of splenic T cells in tumor-bearing or naïve HIS mice; parent population refers to frequency (%) of CD3 + T cells within human CD45 + cells and CD4 + and CD8 + T cells within CD3 + T cells. B , CD8 + T cell differentiation defined as T naïve (CD45RA + CD62L + ), T CM (CD45RA - CD62L + ), T EM (CD45RA - CD62L - ), T EMRA (CD45RA + CD62L - ) in tumor-bearing or naïve HIS mice. C-D , expression of indicated markers on CD8 + T cells from spleen of tumor-bearing or naïve HIS mice. E, frequency of CD4 + and CD8 + T cells within TILs, gated on total CD3 + T cells. F, CD8 + T cell differentiation within TILs. G-H, expression of indicated markers on CD8 + T cells within TILs. I, expression of CD137 on splenic CD8 + and CD4 + T cells of tumor-bearing or naïve HIS mice. J, expression of PD-1 and CD137 on splenic CD8 + T cells of tumor-bearing mice. K , expression of PD-1 on splenic CD8 + T cells based on CD137 in tumor-bearing HIS mice or bulk CD8 + T cells from naïve HIS mice. L, CD8 + T cell differentiation in spleen of tumor-bearing HIS mice. M-O , expression of indicated markers on splenocyte-derived CD8 + T cell populations based on expression of CD137 and PD-1. Data are pooled from at least 3 independent experiments, n=10-23 mice per group. For each experiment, a different HPC donor was used for HIS mouse reconstitution and generation of autologous tumor. Significance by paired t-test, one-way ANOVA or 2way ANOVA, as appropriate. Data from individual experiments are indicated by different symbols.

Article Snippet: Antibodies for flow cytometry-based sorting of T cells: CD3-FITC (Biolegend, UCHT1, Cat. 300406), CD4-APC-Cy7 (Biolegend, RPA-T4, Cat. 300518), CD8-BV650 (Biolegend, SK1, Cat. 344730), CD45-Pacific Blue (Biolegend, HI30, Cat. 304029), CD137-APC (Miltenyi, REA765, Cat. 130-110-901), PD-1-PE-Dazzle (Biolegend, EH12.2H7, Cat. 329940), Zombie AquaTM Fixable Viability Kit (Biolegend, Cat. 423101).

Techniques: Isolation, Tumor Implantation, Flow Cytometry, Cell Differentiation, Expressing, Derivative Assay

Journal: bioRxiv

Article Title: Human effector CD8 + T cells with an exhausted-like phenotype control tumor growth in vivo

doi: 10.1101/2023.10.11.561856

Figure Lengend Snippet: A , schematic of generation, expansion and characterization of T cells from HIS mice bearing autologous LCL tumors. B , fold expansion of FACS sorted splenic CD8 + T cells from tumor-bearing HIS mice (CD137 + , CD137 - and CD137 - PD1 - ) or naïve HIS mice (bulk). C , CD8 + T cell differentiation after ex vivo expansion defined as T naïve (CD45RA + CD62L + ), T CM (CD45RA - CD62L + ), T EM (CD45RA - CD62L - ), T EMRA (CD45RA + CD62L - ). D, IFN-ψ ELISpot of expanded T cells in 5:1 (E:T) co-culture with autologous LCL tumor cells for 24 hours. Spot count is normalized to the spots produced by expanded CD8 + bulk T cells from naïve HIS mice. E , TNF⍺ ELISA of supernatant of expanded T cells in co-culture (5:1, E:T) with autologous LCL tumor cells for 24 hours. TNF⍺ concentration is normalized to the TNF⍺ secretion from expanded CD8 + bulk T cells derived from naïve mice. Data are pooled from at least 3 independent experiments, n=6-19 mice per group. For each experiment, a different HPC donor was used for HIS mouse reconstitution and generation of autologous tumor. Significance by mixed-effects analysis (paired), RM one-way ANOVA or 2way ANOVA, as appropriate. Data from individual experiments are indicated by different symbols.

Article Snippet: Antibodies for flow cytometry-based sorting of T cells: CD3-FITC (Biolegend, UCHT1, Cat. 300406), CD4-APC-Cy7 (Biolegend, RPA-T4, Cat. 300518), CD8-BV650 (Biolegend, SK1, Cat. 344730), CD45-Pacific Blue (Biolegend, HI30, Cat. 304029), CD137-APC (Miltenyi, REA765, Cat. 130-110-901), PD-1-PE-Dazzle (Biolegend, EH12.2H7, Cat. 329940), Zombie AquaTM Fixable Viability Kit (Biolegend, Cat. 423101).

Techniques: Cell Differentiation, Ex Vivo, Enzyme-linked Immunospot, Co-Culture Assay, Produced, Enzyme-linked Immunosorbent Assay, Concentration Assay, Derivative Assay

Journal: bioRxiv

Article Title: Human effector CD8 + T cells with an exhausted-like phenotype control tumor growth in vivo

doi: 10.1101/2023.10.11.561856

Figure Lengend Snippet: Transcriptome analysis and pathway analysis of expanded CD8 + T cells from tumor-bearing HIS mice (CD137 + , CD137 - and CD137 - PD1 - ) or naïve HIS mice (bulk). A, Upset plot (intersect) showing number of differentially expressed genes between groups in bulk RNAseq. p(FDR) < 0.05, log2 FC > 1.5. B , PCA plot of RNAseq showing PC1 and PC2. C, Top 50 upregulated and D, downregulated genes of T cell subsets based on the DEG between CD137 + vs. CD137 - PD1 - CD8 + T cells. p(FDR) < 0.05, log2 FC > 1.5. E, Volcano plot showing DEG between CD137 + and CD137 - PD1 - CD8 + T cells with genes of interest highlighted in yellow. F , differential expression of genes of interest between groups. G , Overrepresentation analysis (ORA) of upregulated pathways in CD137 + CD8 + T cells. H , Gene set enrichment analysis (GSEA) of signatures described on the y-axis. Gene ratio (# genes related to GO term / total number of sig genes) is displayed on the x-axis. Signatures with an adjusted p-value <0.05 are highlighted with a red box. Shown are data from 5 individual experiments, each experiment with different human HPC donor for reconstitution of HIS mice and autologous tumor.

Article Snippet: Antibodies for flow cytometry-based sorting of T cells: CD3-FITC (Biolegend, UCHT1, Cat. 300406), CD4-APC-Cy7 (Biolegend, RPA-T4, Cat. 300518), CD8-BV650 (Biolegend, SK1, Cat. 344730), CD45-Pacific Blue (Biolegend, HI30, Cat. 304029), CD137-APC (Miltenyi, REA765, Cat. 130-110-901), PD-1-PE-Dazzle (Biolegend, EH12.2H7, Cat. 329940), Zombie AquaTM Fixable Viability Kit (Biolegend, Cat. 423101).

Techniques: Quantitative Proteomics

Journal: bioRxiv

Article Title: Human effector CD8 + T cells with an exhausted-like phenotype control tumor growth in vivo

doi: 10.1101/2023.10.11.561856

Figure Lengend Snippet: A , total number of individual clonotypes found per population. Left: data from individual experiments, right: pooled data for group analysis. Wilcoxon test. B, TCR (CDR3 of TRA , TRB , TRG and TRD ) sequence sample diversity estimation using Hill numbers method, with Q=1 describing the Shannon diversity. C, rare clonal proportion showing the occupied repertoire space by clonotypes with defined counts (1, 2-3, 4-10, etc.). Left: data from individual experiments, right: pooled data for group analysis. Wilcoxon test. D, relative abundance of clonotypes with defined frequencies (size). Left: data from individual experiments, right: pooled data for group analysis. Wilcoxon test. E, repertoire overlap analysis cross-comparing every population from every experiment (each with different donor). F , repertoire overlap comparing the repertoire of bulk CD8 + T cells from naïve HIS mice to the populations from tumor-bearing HIS mice from individual experiments (each with different donor; data from individual experiments are indicated by different symbols). Mixed effects analysis with Tukey’s multiple comparisons test. G , tracking of clonotypes over populations. The top 10 most abundant clonotypes of the TCR repertoire of CD137 + CD8 + T cells from one representative experiment are shown. H , proportion of the top 10 most abundant clonotypes (from repertoires of CD137 + CD8 + T cells) in the repertoire of all populations, correlated with the spot count of IFN-ψ ELISpot. Shown are data from 4 individual experiments, each experiment with different human HPC donor for reconstitution of HIS mice and autologous tumor.

Article Snippet: Antibodies for flow cytometry-based sorting of T cells: CD3-FITC (Biolegend, UCHT1, Cat. 300406), CD4-APC-Cy7 (Biolegend, RPA-T4, Cat. 300518), CD8-BV650 (Biolegend, SK1, Cat. 344730), CD45-Pacific Blue (Biolegend, HI30, Cat. 304029), CD137-APC (Miltenyi, REA765, Cat. 130-110-901), PD-1-PE-Dazzle (Biolegend, EH12.2H7, Cat. 329940), Zombie AquaTM Fixable Viability Kit (Biolegend, Cat. 423101).

Techniques: Sequencing, Enzyme-linked Immunospot

Journal: bioRxiv

Article Title: Human effector CD8 + T cells with an exhausted-like phenotype control tumor growth in vivo

doi: 10.1101/2023.10.11.561856

Figure Lengend Snippet: A , schematic of generation of tumor-reactive T cells and subsequent ACT. NSG mice were injected with 2 x 10 LCL s.c. in the flank and after three days, 10 x 10 ex vivo expanded T cells were adoptively transferred intravenously. Transferred T cells and LCL tumors were autologous to each other. B , tumor volume on the day of sacrifice in NSG recipient mice after ACT of the indicated cell populations. C, waterfall plot of tumor size in NSG recipient mice of ACT on the day of sacrifice relative to the tumor volume of control mice (no ACT). Bars depict individual mice. D , frequency of CD3 + T cells (% of human CD45 + cells) in TIL from NSG mice after ACT of the indicated cell populations, measured by flow cytometry. E , frequency of CD8 + T cells (% of total cells) in tumors of NSG mice after ACT of the indicated cell populations, measured by immunohistochemistry. F , differentiation of CD8 + T cells in TIL of NSG mice after ACT of the indicated cell populations defined as T naïve (CD45RA + CD62L + ), T CM (CD45RA - CD62L + ), T EM (CD45RA - CD62L - ) and T EMRA (CD45RA + CD62L - ). G, correlation between tumor volume and infiltration of CD8 + T cells (measured by IHC) in tumors of NSG mice after adoptive transfer of CD137 + CD8 + T cells. H , schematic of ACT. CD137 + CD8 + T cells, CD137 - CD8 + T cells and CD137 - PD-1 - CD8 + T cells were isolated from spleen of tumor-bearing HIS mice or bulk CD8 + T cells from spleen of naïve HIS mice and expanded ex vivo . Recipient HIS mice were injected with 2 x 10 LCL s.c. in the flank and after three days, ex vivo expanded T cells were adoptively transferred intravenously. Tumor-bearing HIS recipient mice received 2 x 10 T cells without prior conditioning/lymphodepletion. Donor and recipient HIS mice as well as LCL were autologous to each other. I, tumor volume on the day of sacrifice in HIS recipient mice after ACT of the indicated cell populations. J, waterfall plot of tumor size on the day of sacrifice of HIS mice receiving ACT relative to the tumor volume of control HIS mice (no ACT). Bars depict individual mice. B-G: Data are pooled from 2-3 independent experiments, n=6-13 per group. I, data are pooled from 2-3 independent experiments (n=6-13 per group); J, data are from 1-3 independent experiments (n=3-13 per group). For each experiment, a with different HPC donor was used for HIS mouse reconstitution and generation of autologous tumor. Significance by one-way ANOVA. Data from individual experiments are indicated by different symbols.

Article Snippet: Antibodies for flow cytometry-based sorting of T cells: CD3-FITC (Biolegend, UCHT1, Cat. 300406), CD4-APC-Cy7 (Biolegend, RPA-T4, Cat. 300518), CD8-BV650 (Biolegend, SK1, Cat. 344730), CD45-Pacific Blue (Biolegend, HI30, Cat. 304029), CD137-APC (Miltenyi, REA765, Cat. 130-110-901), PD-1-PE-Dazzle (Biolegend, EH12.2H7, Cat. 329940), Zombie AquaTM Fixable Viability Kit (Biolegend, Cat. 423101).

Techniques: Injection, Ex Vivo, Control, Flow Cytometry, Immunohistochemistry, Adoptive Transfer Assay, Isolation

Journal: Nature Communications

Article Title: The systemic lupus erythematosus-associated NCF1 90H allele synergizes with viral infection to cause mouse lupus but also limits virus spread

doi: 10.1038/s41467-025-56857-z

Figure Lengend Snippet: a Timeline of the MNV infection experiment with male BALB/c. Ncf1 90H mice after transferring from the SPF facility to the MNV-infected facility (day 1). b The representative appearance of arthritis in the ankle of hind paws on day 49 and mean arthritis scores from days 1 to 56 ( n = 8 per group). Data were analyzed using one-way ANOVA and presented as mean ± SEM. c The levels of proteinuria ( n = 8 per group). Data were analyzed using one-way ANOVA and presented as mean ± SEM. d The level of anti-dsDNA, anti-Sm/RNP, anti-phospholipid (anti-PLs) specific antibodies, and β2-GP1 on day 56 ( n = 8 per group). Data were analyzed using one-way ANOVA and presented as mean ± SEM. e Correlation with anti-MNV antibodies (OD): maximum arthritis score ( p = 0.0138), proteinuria ( p = 0.0049), anti-dsDNA antibodies ( p = 0.0055) in Ncf1 90H mice on day 56 post of MNV infection ( n = 8 per group). Data were analyzed using the Pearson correlation test. f Enlarged spleen and spleen index in mice ( n = 7/group). The spleen index is defined by the spleen weight (mg) divided by the body weight ( g ) and then multiplied by 10 ( n = 8 per group). Data were analyzed using one-way ANOVA and presented as mean ± SEM. g Representative H&E stained joint and kidney sections (magnification x 10). Histological scoring of joint and kidney inflammation using a scale of 0-3 ( n = 8 per group). Data were analyzed using one-way ANOVA and presented as mean ± SEM. h Immunofluorescence images (magnification x 20) and histology scores of deposits of IgG and C3 in the glomerulus from wild-type (WT) R90 and 90H mice on day 56 ( n = 4 per group). Data were analyzed using one-way ANOVA and presented as mean ± SEM. i Relative expression of Ifnα and anti-viral ISGs ( Irf1 , Irf7 , Stat1 , Mx1 , Ip10 and Isg15 ) within spleens ( n = 4 per group). Data were analyzed by the Mann-Whitney test (two-tailed) and presented as mean ± SEM. j Relative expression of Ifnα and anti-viral ISGs ( Irf1 , Irf7 , Stat1 , Mx1 , Ip10 and Isg15 ) within kidneys. The expression of mRNAs was normalized to the housekeeping gene β-actin ( n = 4 per group). Data were analyzed by the Mann-Whitney test (two-tailed) and presented as mean ± SEM. k Immunoblot analysis of p-JAK1/JAK1 and p-STAT1/STAT1 proteins in the kidneys on day 56 after MNV infection ( n = 6 per group). Data were analyzed by the Mann-Whitney test (two-tailed) and presented as mean ± SEM.

Article Snippet: After washing, biotinylated goat anti-mouse IgG (1:1000 dilutions, Southern Biotech, 1030-08) or IgG2b (1:1000 dilutions, Southern Biotech, 1090-08) was added to the wells and incubated at RT for 2 h. ExtrAvidin conjugated alkaline phosphatase (Sigma-Aldrich, E2636) diluted in PBS was added and incubated for 30–45 min. 100 μL of substrate BCIP/Nitroblue Tetrazolium (Sigma-Aldrich, B3804) per well (1 tablet in 10 mL ddH 2 O and pre-filtered with 0.45 μm) was added and incubated in the dark for 10–15 min.

Techniques: Infection, Transferring, Staining, Immunofluorescence, Expressing, MANN-WHITNEY, Two Tailed Test, Western Blot

Journal: Nature Communications

Article Title: The systemic lupus erythematosus-associated NCF1 90H allele synergizes with viral infection to cause mouse lupus but also limits virus spread

doi: 10.1038/s41467-025-56857-z

Figure Lengend Snippet: a Timeline of MNV infection by gavage. b The quantification of MNV in the small intestine, colon, and spleen on day 7 by RT-qPCR ( n = 9 per group). Data were analyzed by the Mann-Whitney test (two-tailed) and presented as mean ± SEM. c Titer of anti-MNV antibodies on days 0, 14, 28, 56, 70, and 84 in females (MNV-R90: n = 6; MNV-90H: n = 13) and males (MNV-R90: n = 5; MNV-90H: n = 6). Data were analyzed by the Mann-Whitney test (two-tailed) and presented as mean ± SEM. d IgG production in femals (R90: n = 6; 90H: n = 13; MNV-R90: n = 6; MNV-90H: n = 13) and males (R90: n = 5; 90H: n = 6; MNV-R90: n = 5; MNV-90H: n = 6). Data were analyzed using one-way ANOVA and presented as mean ± SEM. e The correlation between IgG and anti-MNV antibodies in wild-type Ncf1 R90 mice ( n = 11) and Ncf1 90H mice ( n = 12). Data were analyzed using the Pearson correlation test. f Anti-ssRNA antibodies on day 56 in females (R90: n = 5; 90H: n = 5; MNV-R90: n = 5; MNV-90H: n = 13) and males (R90: n = 5; 90H: n = 5; MNV-R90: n = 5; MNV-90H: n = 13). Data were analyzed using one-way ANOVA and presented as mean ± SEM. g Anti-dsDNA antibodies on day 56 in females (R90: n = 5; 90H: n = 5; MNV-R90: n = 5; MNV-90H: n = 13) and males (R90: n = 5; 90H: n = 5; MNV-R90: n = 5; MNV-90H: n = 6). Data were analyzed using one-way ANOVA and presented as mean ± SEM. h Anti-Sm/RNP antibodies on day 56 in females (R90: n = 5; 90H: n = 5; MNV-R90: n = 5; MNV-90H: n = 13) and males (R90: n = 5; 90H: n = 5; MNV-R90: n = 5; MNV-90H: n = 6). Data were analyzed using one-way ANOVA and presented as mean ± SEM. i Proteinuria on day 56 in females (R90: n = 6; 90H: n = 13; MNV-R90: n = 6; MNV-90H: n = 13) and males (R90: n = 5; 90H: n = 6; MNV-R90: n = 5; MNV-90H: n = 6). Data were analyzed using one-way ANOVA and presented as mean ± SEM. j The ratio of Tcm or Tvm cells in CD4 + Foxp3 + in the PPs (MNV-R90: n = 11; MNV-90H: n = 19). Data were analyzed by the Mann-Whitney test (two-tailed) and presented as mean ± SEM. k, l The ratio of Tfh cells and Tfr cells in CD4 + FOXP3 + cells (MNV-R90: n = 11; MNV-90H: n = 19). Data were analyzed by the Mann-Whitney test (two-tailed) and presented as mean ± SEM. m The ratio of Trm cells in CD4 + CD44 hi cells (MNV-R90: n = 11; MNV-90H: n = 18). Data were analyzed by the Mann-Whitney test (two-tailed) and presented as mean ± SEM. n The ratio of GC-B cells in IgD lo B cells (MNV-R90: n = 11; MNV-90H: n = 19). Data were analyzed by the Mann-Whitney test (two-tailed) and presented as mean ± SEM. o The ratio of LLPCs in IgD lo CD138 + Sca-1 + cells (MNV-R90: n = 11; MNV-90H: n = 19). Data were analyzed by the Mann-Whitney test (two-tailed) and presented as mean ± SEM.

Article Snippet: After washing, biotinylated goat anti-mouse IgG (1:1000 dilutions, Southern Biotech, 1030-08) or IgG2b (1:1000 dilutions, Southern Biotech, 1090-08) was added to the wells and incubated at RT for 2 h. ExtrAvidin conjugated alkaline phosphatase (Sigma-Aldrich, E2636) diluted in PBS was added and incubated for 30–45 min. 100 μL of substrate BCIP/Nitroblue Tetrazolium (Sigma-Aldrich, B3804) per well (1 tablet in 10 mL ddH 2 O and pre-filtered with 0.45 μm) was added and incubated in the dark for 10–15 min.

Techniques: Infection, Quantitative RT-PCR, MANN-WHITNEY, Two Tailed Test

Journal: Nature Communications

Article Title: The systemic lupus erythematosus-associated NCF1 90H allele synergizes with viral infection to cause mouse lupus but also limits virus spread

doi: 10.1038/s41467-025-56857-z

Figure Lengend Snippet: a Timeline of MNV infection in male mice by i.v. and i.p. injection. b Anti-MNV antibodies on days 0, 14, 28, and 56. c Anti-ssRNA antibodies on day 21 (R90: n = 6; 90H: n = 6; MNV-R90: n = 11; MNV-90H: n = 12). Data were analyzed using one-way ANOVA and presented as mean ± SEM. d IgG production on day 21 (R90: n = 8; 90H: n = 12; MNV-R90: n = 11; MNV-90H: n = 12). Data were analyzed using one-way ANOVA and presented as mean ± SEM. e Representative paws on day 21. f Mean arthritis score (MNV-R90: n = 8; MNV-90H: n = 11). Data were analyzed by the Mann-Whitney test (two-tailed) and presented as mean ± SEM. g Arthritis incidence ( n = 9 per group). h Representative HE staining of the joints ( n = 4, magnification x 2.5 and x 10). i Anti-collagen II (COL2) IgG antibodies (R90: n = 7; 90H: n = 7; MNV-R90: n = 11; MNV-90H: n = 20). Data were analyzed using one-way ANOVA and presented as mean ± SEM. j Anti-COL2 IgG2b antibodies (R90: n = 7; 90H: n = 7; MNV-R90: n = 9; MNV-90H: n = 20). Data were analyzed using one-way ANOVA and presented as mean ± SEM. k Anti-COL2 IgM antibodies (R90: n = 8; 90H: n = 11; MNV-R90: n = 11; MNV-90H: n = 18). Data were analyzed using one-way ANOVA and presented as mean ± SEM. The level of anti-COL2 antibodies was measured in mouse sera on day 21 by ELISA. l , m Representative B cell-ELISpot and statistics of total anti-COL2 IgG and IgM ASCs in the spleen were shown (R90: n = 5; 90H: n = 5; MNV-R90: n = 8; MNV-90H: n = 12). Data were analyzed using one-way ANOVA and presented as mean ± SEM.

Article Snippet: After washing, biotinylated goat anti-mouse IgG (1:1000 dilutions, Southern Biotech, 1030-08) or IgG2b (1:1000 dilutions, Southern Biotech, 1090-08) was added to the wells and incubated at RT for 2 h. ExtrAvidin conjugated alkaline phosphatase (Sigma-Aldrich, E2636) diluted in PBS was added and incubated for 30–45 min. 100 μL of substrate BCIP/Nitroblue Tetrazolium (Sigma-Aldrich, B3804) per well (1 tablet in 10 mL ddH 2 O and pre-filtered with 0.45 μm) was added and incubated in the dark for 10–15 min.

Techniques: Infection, Injection, MANN-WHITNEY, Two Tailed Test, Staining, Enzyme-linked Immunosorbent Assay, Enzyme-linked Immunospot

Journal: Nature communications

Article Title: Antigen-selective modulation of AAV immunogenicity with tolerogenic rapamycin nanoparticles enables successful vector re-administration.

doi: 10.1038/s41467-018-06621-3

Figure Lengend Snippet: Fig. 1 SVP[Rapa] treatment prevents anti-AAV capsid antibody responses in mice. a Protocol outline. Male C57BL/6 mice (n = 5 per group) were treated intravenously with 4 × 1012 vg kg−1 of AAV8-luc together with SVP[Rapa] (200 µg) or with SVP[empty] control. Three weeks later, animals were challenged with 4 × 1012 vg kg−1 of AAV8-hF.IX vector mixed with either SVP[Rapa] (200 µg) or SVP[empty] control. One additional control group (n = 5) of naive animals received 4 × 1012 vg kg−1 of AAV8-hF.IX vector alone at day 21 (defined as “AAV8-hF.IX only”). b Analysis of anti-AAV8 IgG antibodies measured by ELISA (**p < 0.01, ****p < 0.0001, refers to the significance between SVP[Rapa] and SVP[empty] or AAV8-hF.IX groups). c Anti-AAV8 neutralizing antibodies (NAbs) titers on day 54. d hF.IX antigen levels in plasma on day 54 determined by ELISA. e AAV8 vector genome copy number (VGCN) per diploid genome performed after killing (day 53). f Protocol outline. Three groups of C57BL/6 male mice (n = 5 per group) were prime-boosted (days 0 and 93, arrows) with 5 × 1011 vg kg−1 of AAV8-SEAP alone, or AAV8-SEAP mixed with SVP[Rapa] (50 µg), or AAV8-SEAP mixed with SVP [Empty] control. g Anti-AAV8 IgG antibodies measured by ELISA (*p < 0.05, **p < 0.01, refers to significance between SVP[Rapa] and AAV-SEAP). h Expressed SEAP activity in serum. Data are shown as mean ± s.d. Statistical analyses were performed by two-way ANOVA with Tukey post hoc test in b, g, and h, and by unpaired, two-tail t-test in c and by one-way ANOVA with Tukey post hoc test in d and e (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; ns not significant)

Article Snippet: Plates were then washed, and the a secondary-antibody conjugated to HRP was added to the wells and incubated one hour at +37 °C (anti-mouse IgG-HRP, dilution 1:20,000, anti-mouse IgG1-HRP, dilution 1:10,000, anti-mouse IgG2a, dilution 1:5000, anti-mouse IgG2b, dilution 1:10,000, anti-mouse IgG3, dilution 1:10,000, all the secondary mouse antibodies conjugated to HRP were purchased from Southern Biotech).

Techniques: Control, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, Clinical Proteomics, Activity Assay

Journal: Nature communications

Article Title: Antigen-selective modulation of AAV immunogenicity with tolerogenic rapamycin nanoparticles enables successful vector re-administration.

doi: 10.1038/s41467-018-06621-3

Figure Lengend Snippet: Fig. 2 SVP[Rapa] treatment enables AAV8 re-administration in nonhuman primates. a Protocol outline. Male naive cynomolgus monkeys were treated i.v. with 2 × 1012 vg kg−1 of AAV8-Gaa vector and either SVP[Rapa] (3 mg kg−1, n = 2, SVP[Rapa]#1 and SVP[Rapa]#2) or SVP[empty] control (n = 1) and then challenged i.v. on day 30 with 2 × 1012 vg kg−1 of AAV8-hF.IX vector and either SVP[Rapa] or SVP[empty] control, as described above. b, c Analysis of b anti-AAV8 IgG antibodies and c anti-AAV8 IgM antibody responses measured by ELISA. d Analysis of anti-AAV8 neutralizing antibodies (NAb) measured with a cell-based neutralization assay. e Analysis of anti-AAV8 IgG secreting B cells in splenocytes measured by B ELISpot. Data are shown as individual replicates and the bars represent mean ± s.d. The dotted line indicates the threshold for positivity corresponding to 50 spot forming units (SFU) per million cells. f Plasma hF.IX antigen levels quantified by ELISA at the indicated time points following administration of AAV8-hF.IX vector. g AAV8-hF. IX vector genome copy number (VGCN) per diploid genome in liver. The symbols represent individual liver lobes (left, right, caudate and quadrate) and the bars represent the mean ± s.d. (4 liver lobes per monkey; one-way ANOVA with Tukey post hoc test, **p < 0.01, ****p < 0.0001) The arrows represent the timing of each AAV8 vector infusion

Article Snippet: Plates were then washed, and the a secondary-antibody conjugated to HRP was added to the wells and incubated one hour at +37 °C (anti-mouse IgG-HRP, dilution 1:20,000, anti-mouse IgG1-HRP, dilution 1:10,000, anti-mouse IgG2a, dilution 1:5000, anti-mouse IgG2b, dilution 1:10,000, anti-mouse IgG3, dilution 1:10,000, all the secondary mouse antibodies conjugated to HRP were purchased from Southern Biotech).

Techniques: Plasmid Preparation, Control, Enzyme-linked Immunosorbent Assay, Neutralization, Enzyme-linked Immunospot, Clinical Proteomics

Journal: Nature communications

Article Title: Antigen-selective modulation of AAV immunogenicity with tolerogenic rapamycin nanoparticles enables successful vector re-administration.

doi: 10.1038/s41467-018-06621-3

Figure Lengend Snippet: Fig. 4 Inhibition of anti-AAV8 capsid cellular and humoral responses with SVP[Rapa] co-administration. a CD8 T cell infiltrates in the liver. Livers from animals treated in Fig. 1a were collected after killing on day 53 and evaluated for CD8 mRNA expression by quantitative PCR using the ΔΔCt method relative to housekeeping gene and to average of untreated mice. b–d Male C57BL/6 mice were treated with 4 × 1012 vg kg−1 of AAV8-VP1 vector together with SVP[Rapa] or with SVP[empty] control. 14 days later, spleens were collected for B and T cell assays. b Analysis of T cell recall responses after overnight stimulation with an AAV8 peptide pool in splenocytes measured by IFN-γ ELISpot and c anti-AAV8 IgG and IgM secreting B cell responses in splenocytes measured by B ELISpot. d Frequency of B220+ CD19+ B cells in spleens measured by flow cytometry. e–i Male C57BL/6 mice were treated with 4 × 1012 vg kg−1 of AAV8-luc vector together with SVP[Rapa] or with SVP[empty] control. 14 days later, animals were sacrificed. e Gating of germinal center (GC) B cells (CD95+ GL7+) are shown in representative flow cytometry plot. Cells were gated on B220+IgD−cells. Shown is a mouse from the SVP [empty] control group. f Frequency of GC B cells (CD95+ GL7+) in lymph nodes of treated mice determined by flow cytometry as shown in e; g frequency of CD25+ FoxP3+ regulatory T cells in lymph nodes; h frequency of CXCR5+ PD1+ Foxp3+ follicular regulatory T (Tfr) cells and i frequency of CXCR5+PD1 +FoxP3−follicular helper T (Tfh) cells in lymph nodes. SVP[Rapa] treatment consisted of 200 µg of rapamycin. Data are shown as mean ± s.d. Statistical analyses were performed by one-way ANOVA with Tukey post hoc test in a and by unpaired, two-tail t-test in b–d, f–i (n = 5 in a–d, n = 20 in f–i. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001)

Article Snippet: Plates were then washed, and the a secondary-antibody conjugated to HRP was added to the wells and incubated one hour at +37 °C (anti-mouse IgG-HRP, dilution 1:20,000, anti-mouse IgG1-HRP, dilution 1:10,000, anti-mouse IgG2a, dilution 1:5000, anti-mouse IgG2b, dilution 1:10,000, anti-mouse IgG3, dilution 1:10,000, all the secondary mouse antibodies conjugated to HRP were purchased from Southern Biotech).

Techniques: Inhibition, Expressing, Real-time Polymerase Chain Reaction, Plasmid Preparation, Control, Enzyme-linked Immunospot, Cytometry

Journal: Nature communications

Article Title: Antigen-selective modulation of AAV immunogenicity with tolerogenic rapamycin nanoparticles enables successful vector re-administration.

doi: 10.1038/s41467-018-06621-3

Figure Lengend Snippet: Fig. 5 Control of anti-AAV8 capsid memory T cell responses with SVP[Rapa]. a Protocol outline. Male C57BL/6 mice (n = 4) were treated i.v. with AAV8- luc vector (2.5 × 1012 vg kg−1) together with SVP[Rapa] or with SVP[empty] on day 0 and with a second dose on day 77 with AAV8-luc vector (5 × 1011 vg kg−1) together with SVP[Rapa] or SVP[empty] control. Spleens were collected at day 19 and day 80 after priming and boosting, respectively. b IFN-γ ELISpot responses after 7 days of in vitro restimulation with an AAV8 peptide pool performed in splenocytes collected 19 or 80 days following initial vector injection. The symbols represent individual animals and the bars represent mean ± s.d. (n = 4, ***p < 0.05, ns not significant, two-tailed, unpaired t-test). c Protocol outline. Donor mice (n = 15) were left untreated or treated i.v with 4 × 1011 vg kg−1 of AAV8-RFP vector. 62 days later, spleens were collected and CD4+ T cells were purified. 1 × 107 of CD4+ T cells were then adoptively transferred into four groups of recipient mice (n = 6). Animals were treated with 4 × 1011 vg kg−1 of AAV8-RFP vector together with SVP[Rapa] or SVP[empty] control on day 1 post adoptive transfer and with 4 × 1011 vg kg−1 of AAV8-SEAP together with SVP[Rapa] or SVP[empty] on 21 post adoptive transfer. The tabular legend represents the summary of the treatment groups and the symbols represent the different cohorts of recipient mice. d, e Analysis of anti-AAV8 IgG antibodies in donor mice (d) and in recipient mice post adoptive transfer (e) measured by ELISA. d, e Data shown as mean ± s.d. (n = 6, *p < 0.05, ****p < 0.0001, refers to significance between cohorts 3 and 4 as determined by two-way ANOVA with Tukey post hoc test). SVP[Rapa] treatment consisted of 50 µg of rapamycin

Article Snippet: Plates were then washed, and the a secondary-antibody conjugated to HRP was added to the wells and incubated one hour at +37 °C (anti-mouse IgG-HRP, dilution 1:20,000, anti-mouse IgG1-HRP, dilution 1:10,000, anti-mouse IgG2a, dilution 1:5000, anti-mouse IgG2b, dilution 1:10,000, anti-mouse IgG3, dilution 1:10,000, all the secondary mouse antibodies conjugated to HRP were purchased from Southern Biotech).

Techniques: Control, Plasmid Preparation, Enzyme-linked Immunospot, In Vitro, Injection, Two Tailed Test, Adoptive Transfer Assay, Enzyme-linked Immunosorbent Assay

Journal: Vaccine

Article Title: Establishment of a Shigella sonnei human challenge model in Thailand ☆

doi: 10.1016/j.vaccine.2012.09.061

Figure Lengend Snippet: Demographic and baseline IgG antibody data.

Article Snippet: Statistical analysis Among the three cohorts, the mean age of subjects was compared by one-way ANOVA, the gender distribution and education levels were compared by Fisher’s exact test, and the median baseline IgG antibody titers were compared by a non-parametric equality-of-medians test using Stata 10 (Stata Corp., College Station, TX, USA).

Techniques:

Journal: Vaccine

Article Title: Establishment of a Shigella sonnei human challenge model in Thailand ☆

doi: 10.1016/j.vaccine.2012.09.061

Figure Lengend Snippet: Serum IgA, IgG, and IgM antibody titers (bars represent geometric mean (middle bar) with 95% confidence intervals) against S. sonnei lipopolysaccharide (LPS) of subjects at baseline (closed round symbol), Day 14 (open square symbols) and Day 28 (star symbols) after challenge with S. sonnei 53G at doses of 93, 440 and 1680 colony forming unit (CFU). No significant dose related linear trends were noted.

Article Snippet: Statistical analysis Among the three cohorts, the mean age of subjects was compared by one-way ANOVA, the gender distribution and education levels were compared by Fisher’s exact test, and the median baseline IgG antibody titers were compared by a non-parametric equality-of-medians test using Stata 10 (Stata Corp., College Station, TX, USA).

Techniques:

Journal: Vaccine

Article Title: Establishment of a Shigella sonnei human challenge model in Thailand ☆

doi: 10.1016/j.vaccine.2012.09.061

Figure Lengend Snippet: IgA, IgG and IgM antibody secreting cells (ASC) counts (bars represent geometric mean (middle bar) with 95% confidence intervals) to S. sonnei lipopolysaccharide (LPS) of subjects at baseline (closed round symbol), Day 7 (open square symbols) and Day 9 (star symbols) after challenge with S. sonnei 53G at doses of 93, 440 and 1680 colony forming unit (CFU). ASCs were determined by ELISPOT. Significant dose related linear trends P values (linear by linear association) are shown.

Article Snippet: Statistical analysis Among the three cohorts, the mean age of subjects was compared by one-way ANOVA, the gender distribution and education levels were compared by Fisher’s exact test, and the median baseline IgG antibody titers were compared by a non-parametric equality-of-medians test using Stata 10 (Stata Corp., College Station, TX, USA).

Techniques: Enzyme-linked Immunospot

Journal: Nature Communications

Article Title: SARS-CoV-2 virus lacking the envelope and membrane open-reading frames as a vaccine platform

doi: 10.1038/s41467-025-59533-4

Figure Lengend Snippet: a Total IgG antibody endpoint titers against the spike proteins of an ancestral isolate (spike D614G) or Omicron XBB using hamster sera after one or two vaccinations with the ΔEM virus. Each dot in the bar graph indicates an individual hamster in each group (mean with SD, n = 4 females/group). Statistical significance was determined by using a two-way ANOVA with Šídák’s multiple comparisons test (* P < 0.05, ** P < 0.01, *** P < 0.001). b Efficacy of the ΔEM vaccine virus as a booster vaccine. Virus titers of the Omicron XBB variant in lung and nasal turbinate (NT) tissues at day 3 after challenge. Each dot in the bar graph indicates an individual hamster in each group (mean with SD, n = 4 females/group). Statistical significance was determined by using the unpaired two-tailed Student’s t -test. (* P < 0.05, *** P < 0.001, **** P < 0.0001). c Induction of a spike-specific T cell population producing IFN-γ in the lungs of ΔEM virus- or mRNA-boosted hamsters that had previously received a prime mRNA vaccination as measured by use of an ELISpot assay (n = 6 females/group). Box plots show the median center line and 10/90 percentiles. Whiskers show min and max values. Statistical significance was determined by using a one-way ANOVA with Tukey’s multiple comparison test (* P < 0.05, ** P < 0.01; ns, not significant). Exact P values: ( a ) Ancestral D614G spike: mRNA x2 ( P = 0.0001), mRNA + ΔEM ( P = 0.0018), ΔEM x2 ( P = 0.0004); XBB spike: mRNA x2 ( P = 0.0011), mRNA + ΔEM ( P = 0.0003), ΔEM x2 ( P = 0.0348). ( b ) lung: control vs. mRNA x2 ( P = 0.0002), mRNA x2 vs. mRNA + ΔEM ( P < 0.0001); NT: control vs. mRNA x2 ( P = 0.0122), mRNA x2 vs. mRNA + ΔEM ( P = 0.0129). ( c ) control vs. mRNA x2 ( P = 0.2058), control vs. mRNA + ΔEM ( P = 0.0011), mRNA x2 vs. mRNA + ΔEM ( P = 0.0381). Source data are provided as a Source Data file.

Article Snippet: A hamster IgG secondary antibody conjugated with horseradish peroxidase (1:5000, Thermo Fisher Scientific), and mouse IgG1 (115-035-205), IgG2b (115-035-207), and IgG3 (115-035-209) subclass secondary antibodies conjugated with horseradish peroxidase (1:2000, Jackson ImmunoResearch) were used for detection.

Techniques: Virus, Variant Assay, Two Tailed Test, Enzyme-linked Immunospot, Comparison, Control

Journal: Oncoimmunology

Article Title: Loss of tapasin in human lung and colon cancer cells and escape from tumor-associated antigen-specific CTL recognition

doi: 10.1080/2162402X.2016.1274476

Figure Lengend Snippet: Survivin 2B-specific CTLs failed to recognize the tapasin-deficient lung cancer cells. (A) mRNA expression of survivin 2B. Expression value for YS3 is shown relative to LHK2 (with a value arbitrarily set to 1.0). (B) Western blot of survivin expression in LHK2 and YS3. Actin expression was used as a loading control. (C) Flowcytometry of the CTL clone H4 from cancer-patient PBMC, stained with PE-conjugated survivin 2B tetramers and PC5-conjugated CD8+ mAb. Numbers in plots indicate frequency of corresponding quadrants. (D) ELISPOT IFNγ assay of H4 responding to T2-A24 cells pulsed with synthetic survivin 2B peptides or HIV peptides, which are known to be bound to HLA-A24. Produced IFNγ are detected as red spots. (E) ELISPOT IFNγ assay of H4 responding to LHK2, YS3, and K562. The y-axis indicates the numbers of IFNγ-positive spots per a well. Data are shown as mean + SEM (n = 3). p-values were calculated using a two-tailed t-test (*p < 0.05). Data are representatives of three independent experiments.

Article Snippet: The anti-tapasin TO-3 mAb (sc-80647; Santa Cruz), anti-human CD8 + mAb (N1592; Daco), anti-actin mAb (clone AC-15; SIGMA), and anti-survivin rabbit polyclonal Ab (novusbiologicals) were obtained commercially.

Techniques: Expressing, Western Blot, Control, Staining, Enzyme-linked Immunospot, Produced, Two Tailed Test

Journal: Oncoimmunology

Article Title: Loss of tapasin in human lung and colon cancer cells and escape from tumor-associated antigen-specific CTL recognition

doi: 10.1080/2162402X.2016.1274476

Figure Lengend Snippet: Tapasin deficiency leads to escape from TAA-specific CTL-mediated cytotoxicity. A and B, Survivin and cep55 gene expression in SW480, 3G12 and a normal colon tissue. (B) Tumor growth rates of SW480 and 3G12. 1 × 105 SW480 or 3G12 cells were subcutaneously injected into NSG mice. The x-axis and the y-axis indicate days after injection and the sizes of tumors, respectively. Tumor volume was calculated as follows: volume = xy2/2. Data are shown as mean + SEM (n = 3). (C) Immunohistochemistry of dissected tumors 36 d after injection. Formalin-fixed and paraffin-embedded tissues were stained with tapasin mAb (magnification x200). D and E, LDH cytotoxicity assay of the CTLs specific to the survivin 2B epitope and the cep55 epitope presented by HLA-A24. LDH releases from indicated target cells cultured with survivin 2B-specific CTL clones (D) and cep55-specific CTL clones (E) were measured. The x-axis and y-axis indicate effector/target (E/T) ratios and % target cell lysis, respectively. Data are representatives of three independent experiments. F and G, The adoptive transfer of cep55-specific CTLs and tumor rejection in vivo. SW480 (F) and 3G12 (G) cells were subcutaneously injected into NSG mice. The mice received the adoptive transfer of PBS (black) or 2 × 106 cep55-specific CTLs (red) 10 and 17 d after tumor injection. The x-axis indicates days after tumor injection. Data are shown as mean + SD (n = 3). p-values were calculated using a two-tailed t-test (**p < 0.01).

Article Snippet: The anti-tapasin TO-3 mAb (sc-80647; Santa Cruz), anti-human CD8 + mAb (N1592; Daco), anti-actin mAb (clone AC-15; SIGMA), and anti-survivin rabbit polyclonal Ab (novusbiologicals) were obtained commercially.

Techniques: Gene Expression, Injection, Immunohistochemistry, Staining, LDH Cytotoxicity Assay, Cell Culture, Clone Assay, Lysis, Adoptive Transfer Assay, In Vivo, Two Tailed Test

Journal: Oncoimmunology

Article Title: Loss of tapasin in human lung and colon cancer cells and escape from tumor-associated antigen-specific CTL recognition

doi: 10.1080/2162402X.2016.1274476

Figure Lengend Snippet: The antigen processing of endogenous TAAs is limited by loss of tapasin. (A) An exogenous presentation assay using antigen-presenting cells (APC) to quantify CTL-epitopes processed in target cells. (B) IFNγ-ELISPOT assay of survivin 2B- or cepp55-CTL clones. T2-A24 cells pulsed with 60 µg of survivin 2B or cepp55 peptides were used as APC. (C) An exogenous presentation assay using T2-A24 cells as APC. IFNγ produced by indicated CTL clones were detected using ELISPOT assay. The <10 kDa cell extracts from SW480 or 3G12 were titrated and incubated with T2-A24 cells and indicated CTL clones as described in methods. The numbers indicate cell equivalents of the extracts. (D) IFNγ-ELISPOT assay of a survivin 2B-CTL clone. The CTL was incubated with 5 × 104 cells of SW480 or 3G12 pulsed with indicated amounts of survivin 2B peptides (left). A right panel shows the quantified numbers of IFNγ spots. Data are representatives of three independent experiments.

Article Snippet: The anti-tapasin TO-3 mAb (sc-80647; Santa Cruz), anti-human CD8 + mAb (N1592; Daco), anti-actin mAb (clone AC-15; SIGMA), and anti-survivin rabbit polyclonal Ab (novusbiologicals) were obtained commercially.

Techniques: Enzyme-linked Immunospot, Clone Assay, Produced, Incubation

Journal: Nature Communications

Article Title: A p38α-BLIMP1 signalling pathway is essential for plasma cell differentiation

doi: 10.1038/s41467-022-34969-0

Figure Lengend Snippet: a Schematic outline of in vitro plasma cell (iPC) differentiation from splenic naïve B cells. Flow cytometry analysis of the percentage of iGCB cells (CD19 + Fas hi GL7 hi ) at day 4 and iPCs (CD19 + CD138 + ) at day 8 of culture, and supernatant IgG1 and IgE of p38α KO and WT B cells at day 8 of iPC culture were measured by ELISA ( n = 6 per group). b Principal-component analysis (PCA) of RNA-seq data from sorted iGCB cells ( n = 2 per group), iPCs (CD19 + CD138 + ) and CD138 − B cells (CD19 + CD138 − ) of indicated genotypes ( n = 3 per group). Numbers of upregulated (red) and downregulated (blue) genes ( p value < 0.01) in p38α KO iGCB cells and iPCs in comparison with WT cells. c Purified splenic naïve B cells from p38α fl/fl Vav cre or p38α fl/fl mice (CD45.2 + ) were mixed and co-cultured with CD45.1 + WT splenic naïve B cells at a 1:1 ratio. Flow cytometry analysis of iPCs gated on CD45.1 + and CD45.2 + B cells in iPC differentiation system at day 8 of culture ( n = 3 per group). d Immunoblot analysis of p38α, p-p38α, IgG heavy (H)-chain and Actin in cultured p38α KO and WT B cells at indicated time points after being transferred onto IL-21 feeder cells. e Cultured p38α KO and WT B cells were transduced with retroviruses encoding p38α and p38α kinase-dead mutants (K53M or T180A/Y182F) at day 2 of iGCB cell differentiation. iPCs among retrovirus-transduced B cells (CD19 + GFP + CD138 + ) were analyzed by flow cytometry 6 days after retroviral transduction ( n = 6 per group). Protein levels of p38α, IgG heavy (H)-chain and Actin in retrovirus-transduced B cells were analyzed by immunoblot. Data were representative of at least three independent experiments ( a ), ( c – e ). Each symbol represents a representative sample. Small horizontal lines indicate the mean (±s.d.). Data were analyzed by two-tailed unpaired t -tests. Source data are provided as a Source Data file.

Article Snippet: The following antibodies were from Southern Biotech: Mouse IgE-UNLB (0114-01, 1:50-1:1,000,000), IgG1-UNLB (0102-01, 1:50-1:1,000,000), IgG3-UNLB (0105-01, 1:50-1:1,000,000), IgM-UNLB (0101-01, 1:50-1:1,000,000), goat anti-mouse IgE-BIOT (1110-08, 1:500), goat anti-mouse IgG1-BIOT (1070-08, 1:500), goat anti-mouse IgG3-BIOT (1100-08, 1:500), goat anti-mouse IgM-BIOT (1020-08, 1:500), goat anti-mouse Kappa-UNLB (1050-01, 1:200) and goat anti-mouse Kappa-UNLB (1060-01, 1:200).

Techniques: In Vitro, Clinical Proteomics, Flow Cytometry, Enzyme-linked Immunosorbent Assay, RNA Sequencing, Comparison, Purification, Cell Culture, Western Blot, Transduction, Cell Differentiation, Retroviral, Two Tailed Test

Journal: Nature Communications

Article Title: A p38α-BLIMP1 signalling pathway is essential for plasma cell differentiation

doi: 10.1038/s41467-022-34969-0

Figure Lengend Snippet: a Flow cytometry analysis of BLIMP1-expressing cells (CD19 + GFP + ), BLIMP1-expressing CD138 − cells (CD19 + GFP + CD138 − ) and BLIMP1-expressing CD138 + (CD19 + GFP + CD138 + ) cells differentiated from p38α fl/fl Blimp1 gfp/+ and p38α fl/fl Vav cre Blimp1 gfp/+ B cells in iPC culture ( n = 5 per group). b MFI of GFP in BLIMP1-expressing CD138 − B cells (CD19 + GFP + CD138 − ) and BLIMP1-expressing CD138 + B cells (CD19 + GFP + CD138 + ) from ( a ) were determined by flow cytometry ( n = 5 per group). c Immunoblot analysis of BLIMP1, IgG H-chain and Actin in cultured B cells at indicated time points after p38α KO and WT iGCB cells transferred onto IL-21 feeder cells. d Flow cytometry analysis of BLIMP1 expression by intracellular staining for BLIMP1 in splenic B cells from OVA/Alum/LPS-immunized mice described as Fig. ( n ≥ 7 per group). e Quantitative RT-PCR analysis of Blimp1 mRNA in cultured p38α KO and WT B cells at indicated time points after transferred onto IL-21 feeder cells ( n = 3 per group). f p38α KO and WT iGCB cells at day 4, CD138 − B cells (CD19 + CD138 − ) and CD138 + B cells (CD19 + CD138 + ) at day 8 were sorted, mRNA level of Blimp1 in these subsets were analyzed by quantitative RT-PCR ( n = 3 per group). g Cultured p38α KO B cells were transduced with retroviruses encoding p38α or p38α kinase-dead mutants (K53M or T180A/Y182F) and transferred onto IL-21 feeder cells. Blimp1 mRNA levels were measured by quantitative RT-PCR after 4 days of culture ( n = 3 per group). Data were representative of at least three independent experiments. Each symbol represents a representative sample or an individual mouse ( d ). Small horizontal lines indicate the mean (±s.d.). Data were analyzed by two-tailed unpaired t -tests. Source data are provided as a Source Data file.

Article Snippet: The following antibodies were from Southern Biotech: Mouse IgE-UNLB (0114-01, 1:50-1:1,000,000), IgG1-UNLB (0102-01, 1:50-1:1,000,000), IgG3-UNLB (0105-01, 1:50-1:1,000,000), IgM-UNLB (0101-01, 1:50-1:1,000,000), goat anti-mouse IgE-BIOT (1110-08, 1:500), goat anti-mouse IgG1-BIOT (1070-08, 1:500), goat anti-mouse IgG3-BIOT (1100-08, 1:500), goat anti-mouse IgM-BIOT (1020-08, 1:500), goat anti-mouse Kappa-UNLB (1050-01, 1:200) and goat anti-mouse Kappa-UNLB (1060-01, 1:200).

Techniques: Flow Cytometry, Expressing, Western Blot, Cell Culture, Staining, Quantitative RT-PCR, Transduction, Two Tailed Test

Journal: Nature Communications

Article Title: A p38α-BLIMP1 signalling pathway is essential for plasma cell differentiation

doi: 10.1038/s41467-022-34969-0

Figure Lengend Snippet: a Cultured p38α KO and WT B cells were transduced with retroviruses encoding p38α or BLIMP1 at day 2 of in vitro differentiation. Flow cytometry analysis of iPCs in retrovirally transduced B cells (CD19 + GFP + CD138 + ) 6 days after transduction ( n = 6 per group). b ELISpot analysis of IgG1 and IgE antibody-secreting cells among cultured B cells as ( a ) ( n = 3 per group). c Heatmap analysis of differential expression genes (foldchange > 2) in iPCs of indicated genotypes. Group 1: decreased in p38α KO iPCs, restored by p38α or BLIMP1 overexpression. Group 2: decreased in p38α KO iPCs, restored by p38α but not BLIMP1 overexpression. Group 3: decreased in p38α KO iPCs, restored by BLIMP1 but not p38α overexpression. Group 4: decreased in p38α KO iPCs, restored by neither p38α nor BLIMP1 overexpression. Group 5: increased in p38α KO iPCs, restored by p38α or BLIMP1 overexpression. Group 6: increased in p38α KO iPCs, restored by p38α but not BLIMP1 overexpression. Group 7: increased in p38α KO iPCs, restored by BLIMP1 but not p38α overexpression. Group 8: increased in p38α KO iPCs, restored neither p38α nor BLIMP1 overexpression. d Gene Ontology enrichment analysis of Group 1 from ( c ). e Immunoblot analysis of BLIMP1, p38α, IgG H-chain, Actin and UPR-related proteins, Bip, IRE1α, SEC61A1 and SEC61B in retrovirally transduced B cells at day 8 in iPC culture. f Cultured p38α KO and WT B cells were transduced with retroviruses encoding p38α or BLIMP1 at day 2 of in vitro differentiation. Representative transmission electron microscopy images of sorted iPCs in retrovirally transduced B cells (CD19 + GFP + CD138 + ) of indicated groups at day 8. Scale bars, 500 nm. Percentage of endoplasmic reticulum (ER) expansion and diameter of ER lumen in iPCs of indicated groups ( n ≥ 100 per group). Data were representative of at least three independent experiments ( a , b , e , f ). Each symbol represents a representative sample. Small horizontal lines indicate the mean (±s.d.). Data were analyzed by one-side hypergeometric test ( d ) and two-tailed unpaired t -tests ( a , f ). Source data are provided as a Source Data file.

Article Snippet: The following antibodies were from Southern Biotech: Mouse IgE-UNLB (0114-01, 1:50-1:1,000,000), IgG1-UNLB (0102-01, 1:50-1:1,000,000), IgG3-UNLB (0105-01, 1:50-1:1,000,000), IgM-UNLB (0101-01, 1:50-1:1,000,000), goat anti-mouse IgE-BIOT (1110-08, 1:500), goat anti-mouse IgG1-BIOT (1070-08, 1:500), goat anti-mouse IgG3-BIOT (1100-08, 1:500), goat anti-mouse IgM-BIOT (1020-08, 1:500), goat anti-mouse Kappa-UNLB (1050-01, 1:200) and goat anti-mouse Kappa-UNLB (1060-01, 1:200).

Techniques: Cell Culture, Transduction, In Vitro, Flow Cytometry, Enzyme-linked Immunospot, Quantitative Proteomics, Over Expression, Western Blot, Transmission Assay, Electron Microscopy, Two Tailed Test

Journal: Nature Communications

Article Title: A p38α-BLIMP1 signalling pathway is essential for plasma cell differentiation

doi: 10.1038/s41467-022-34969-0

Figure Lengend Snippet: a Schematic outline of CRISPR/Cas9 screen of p38α substrates in iPC differentiation. Naive B cells from Cas9 tg/+ mice (GFP + ) and WT littermates (GFP − ) were mixed at a 1:1 ratio and cultured. Cultured B cells were transduced with retroviruses encoding individual sgRNAs targeting p38α substrates, BFP, and puromycin-resistance gene at day 2. iPCs at day 8 among retrovirus-transduced Cas9-expressing B cells (CD19 + GFP + BFP + CD138 + ) were analyzed by flow cytometry. b Summary of the screen results in ( a ). X axis: The ratio between iPCs percentages of p38α substrates sgRNA-expressing and NTC sgRNA-expressing B cells (Fold change); Y axis: The variation of effect on iPCs generation of 3 sgRNAs targeting gene of interest as measured by Students’ t -tests. c Flow cytometry analysis of iPCs among cultured Cas9 tg/+ B cells transduced with retroviruses encoding p38α or Tcf3 sgRNA (CD19 + GFP + BFP + CD138 + ). Immunoblot analysis of BLIMP1, TCF3 and Actin expression in Tcf3 sgRNA expressing B cells at indicated time of culture. d Schematic diagram of a putative TCF3-binding site in the Prdm1 locus. p38α KO and WT B cells cultured on IL-21 feeder cells for 2 days were utilized for CUT&Tag assay and immunoprecipitated with anti-TCF3 antibody. The enrichment of TCF3 binding genomic fragments containing the reported TCF3 binding site in Blimp1 enhancer region was measured by quantitative RT-PCR ( n = 3 per group). e Flow cytometry analysis of iPCs among cultured p38α KO B cells transduced with retroviruses encoding the TCF3 splicing isoforms E12 and E47 (CD19 + GFP + CD138 + ) ( n = 6 per group), protein levels of TCF3, IgG H-chain, BLIMP1, p38α, and Actin were analyzed by immunoblot. f Flow cytometry analysis of iPCs among cultured Tcf3 KO B cells transduced with retroviruses encoding p38α (CD19 + GFP + CD138 + ) ( n = 7 per group). g Flow cytometry analysis of iPCs (CD19 + CD138 + ) among cultured p38α fl/fl CD19 cre , Tcf3 fl/fl CD19 cre , p38α fl/fl Tcf3 fl/fl CD19 cre , p38α fl/fl Tcf3 fl/fl B cells at day 8 of in vitro iPC differentiation ( n = 3 per group). NTC, non-targeting control. Data were representative of at least three independent experiments ( b – g ). Each symbol represents a representative sample. Small horizontal lines indicate the mean (±s.d.). Data were analyzed by two-tailed unpaired t -tests. Source data are provided as a Source Data file.

Article Snippet: The following antibodies were from Southern Biotech: Mouse IgE-UNLB (0114-01, 1:50-1:1,000,000), IgG1-UNLB (0102-01, 1:50-1:1,000,000), IgG3-UNLB (0105-01, 1:50-1:1,000,000), IgM-UNLB (0101-01, 1:50-1:1,000,000), goat anti-mouse IgE-BIOT (1110-08, 1:500), goat anti-mouse IgG1-BIOT (1070-08, 1:500), goat anti-mouse IgG3-BIOT (1100-08, 1:500), goat anti-mouse IgM-BIOT (1020-08, 1:500), goat anti-mouse Kappa-UNLB (1050-01, 1:200) and goat anti-mouse Kappa-UNLB (1060-01, 1:200).

Techniques: CRISPR, Cell Culture, Transduction, Expressing, Flow Cytometry, Western Blot, Binding Assay, Immunoprecipitation, Quantitative RT-PCR, In Vitro, Control, Two Tailed Test

Journal: Nature Communications

Article Title: A p38α-BLIMP1 signalling pathway is essential for plasma cell differentiation

doi: 10.1038/s41467-022-34969-0

Figure Lengend Snippet: a Flow cytometry analysis of iPCs differentiated from cultured Cas9 tg/+ B cells transduced with retroviruses encoding p38α or Tcf4 sgRNA (CD19 + GFP + BFP + CD138 + ). Immunoblot analysis of BLIMP1, TCF4, and Actin expression in Tcf4 sgRNA expressing B cells at indicated time points after being transferred onto IL-21 feeder cells. b Schematic diagram of a putative TCF4-binding site in the Prdm1 locus. CUT&Tag analysis of TCF4 binding genomic fragments including the reported TCF4 binding site in Blimp1 enhancer region in cultured p38α KO and WT B cells at day 2 after transferred onto IL-21 feeder cells ( n = 3 per group). c Flow cytometry analysis of iPCs among cultured p38α KO B cells transduced with retroviruses encoding TCF4 (CD19 + GFP + CD138 + ) ( n = 6 per group), protein levels of TCF4, IgG H-chain, BLIMP1, p38α and Actin were analyzed by immunoblot. d – f Same as described in ( a – c ) except that Irf4 sgRNAs were used or IRF4 was overexpressed in cultured p38α KO B cells. g Cultured p38α fl/fl Vav cre Cas9 tg/+ and p38α fl/fl Cas9 tg/+ B cells were transduced with retroviruses encoding sgRNA targeting p38α , Tcf3 , Tcf4 and Irf4 at day 2 of culture ( n = 3 per group). Percentages of iPCs in sgRNA transduced B cells (CD19 + GFP + BFP + CD138 + ) were analyzed by flow cytometry at day 8. NTC, non-targeting control. Data were representative of at least three independent experiments. Each symbol represents a representative sample. Small horizontal lines indicate the mean (±s.d.). Data were analyzed by two-tailed unpaired t -tests. Source data are provided as a Source Data file.

Article Snippet: The following antibodies were from Southern Biotech: Mouse IgE-UNLB (0114-01, 1:50-1:1,000,000), IgG1-UNLB (0102-01, 1:50-1:1,000,000), IgG3-UNLB (0105-01, 1:50-1:1,000,000), IgM-UNLB (0101-01, 1:50-1:1,000,000), goat anti-mouse IgE-BIOT (1110-08, 1:500), goat anti-mouse IgG1-BIOT (1070-08, 1:500), goat anti-mouse IgG3-BIOT (1100-08, 1:500), goat anti-mouse IgM-BIOT (1020-08, 1:500), goat anti-mouse Kappa-UNLB (1050-01, 1:200) and goat anti-mouse Kappa-UNLB (1060-01, 1:200).

Techniques: Flow Cytometry, Cell Culture, Transduction, Western Blot, Expressing, Binding Assay, Control, Two Tailed Test

Journal: Nature Communications

Article Title: A p38α-BLIMP1 signalling pathway is essential for plasma cell differentiation

doi: 10.1038/s41467-022-34969-0

Figure Lengend Snippet: a Cultured p38α − /− B cells were transduced with retroviruses encoding p38α or p38α kinase-dead mutations (K53M or T180A/Y182F) (GFP + ) and luciferase reporter harboring TCF3, TCF4 or IRF4 binding sites in Prdm1 enhance region at day 2, and luciferase activity in retrovirus-infected B cells (CD19 + GFP + ) was analyzed at day 8 in iPC culture ( n = 3 per group). b p38 −/− 293 A cells were transfected with indicated plasmids, and the luciferase activity of IRF4 mutants was analyzed 24 h after transfection. Immunoblot analysis of p38α, p-p38α, IRF4 and Actin in p38 −/− 293 A cells transfected with indicated plasmids (n = 3 per group). c Cultured Irf4 −/− B cells were transduced with retroviruses encoding IRF4 or IRF4 mutants (T267A, T268A, T269A) at day 2, iPCs in retrovirally transduced B cells (CD19 + GFP + CD138 + ) 6 days after transduction were analyzed by flow cytometry ( n = 5 per group), and protein levels of IRF4, IgG H-chain, BLIMP1 and Actin were analyzed by immunoblot. Data were representative of at least three independent experiments. Each symbol represents a representative sample. Small horizontal lines indicate the mean (±s.d.). Data were analyzed by two-tailed unpaired t-tests. Source data are provided as a Source Data file.

Article Snippet: The following antibodies were from Southern Biotech: Mouse IgE-UNLB (0114-01, 1:50-1:1,000,000), IgG1-UNLB (0102-01, 1:50-1:1,000,000), IgG3-UNLB (0105-01, 1:50-1:1,000,000), IgM-UNLB (0101-01, 1:50-1:1,000,000), goat anti-mouse IgE-BIOT (1110-08, 1:500), goat anti-mouse IgG1-BIOT (1070-08, 1:500), goat anti-mouse IgG3-BIOT (1100-08, 1:500), goat anti-mouse IgM-BIOT (1020-08, 1:500), goat anti-mouse Kappa-UNLB (1050-01, 1:200) and goat anti-mouse Kappa-UNLB (1060-01, 1:200).

Techniques: Cell Culture, Transduction, Luciferase, Binding Assay, Activity Assay, Infection, Transfection, Western Blot, Flow Cytometry, Two Tailed Test

Journal: Cell Reports Medicine

Article Title: IL-21/IL-21R signaling renders acute myeloid leukemia stem cells more susceptible to cytarabine treatment and CAR T cell therapy

doi: 10.1016/j.xcrm.2024.101826

Figure Lengend Snippet:

Article Snippet: Human IgG1, REA Control Antibody (S) , Miltenyi , Cat# 130-113-438; RRID: AB_2733893.

Techniques: Control, Recombinant, Virus, Enzyme-linked Immunosorbent Assay, Enzyme-linked Immunospot, Plasmid Preparation, Software, Phospho-proteomics