Journal: PLoS ONE

Article Title: Effects of subconjunctival administration of anti-high mobility group box 1 on dry eye in a mouse model of Sjӧgren’s syndrome

doi: 10.1371/journal.pone.0183678

Figure Lengend Snippet: (A) BrdU staining showing no significant proliferative changes in CD3 + T cells or B220 + B cells. (B) Percentage of Th17 (CD3 + CD4 + IL-17 hi ) cells and Tc17 (CD3 + CD8 + IL-17 hi ) cells were not altered after treatment. (C) IFNγ-secreting T cells (CD3 + CD4 + IFNγ hi or CD3 + CD8 + IFNγ hi ) were not affected by treatment. Data are presented as mean ± standard error. (Th17, T helper cells secreting IL-17; Tc17, cytotoxic T cells secreting IL-17).

Article Snippet: Color conjugation combinations and gating strategies were as follows: (1) IFNγ, and IL-17A secreting cells; CD3-PerCP cy5.5 (eBioscience, 145-2C11), CD4-APC (eBioscience, GK1.5), CD8-PE cy7 (eBioscience, 53–6.7), B220-APC cy7 (eBioscience, RA3-6B2), IL-17A-PE (BD Pharmingen ™ , TC11-18H10), IFNγ-FITC (eBioscience, XMG1.2); (2) Plasma cells; CD3-PerCP cy5.5 (eBioscience, 145-2C11), B220-APC cy7 (eBioscience, RA3-6B2), CD138-APC (BD Pharmingen ™ , 281–2); (3) IL-10 secreting B cells; CD3-PE (eBioscience, 145-2C11), CD19-PerCP cy5.5 (eBioscience, eBio 1D3), B220-APC cy7 (eBioscience, RA3-6B2), IL-10-FITC (eBioscience, JES5-16E3); (4) ILC3s; CD3-PerCP cy5.5 (eBioscience, 145-2C11), B220-FITC (eBioscience, RA3-6B2), NKp46-APC (eBioscience, 29A1.4), CD45-APC cy7 (eBioscience, 30-F11), IL-22-PE (eBioscience, 1H8PWSR).

Techniques: BrdU Staining

Journal: PLoS ONE

Article Title: Effects of subconjunctival administration of anti-high mobility group box 1 on dry eye in a mouse model of Sjӧgren’s syndrome

doi: 10.1371/journal.pone.0183678

Figure Lengend Snippet: (A) No significant change in percentage of plasma cells (CD3 - B220 + CD138 + cells). (B) No change in IL-10-secreting B regulatory cells (CD3 - CD19 + B220 + IL-10 hi ). (C) No change in the level of anti-SSA (RO 60) antibodies after treatment. (D) No significant changes in inflammatory foci scores (> 50 lymphocytes/focus) among all groups. Data are presented as mean ± standard error.

Article Snippet: Color conjugation combinations and gating strategies were as follows: (1) IFNγ, and IL-17A secreting cells; CD3-PerCP cy5.5 (eBioscience, 145-2C11), CD4-APC (eBioscience, GK1.5), CD8-PE cy7 (eBioscience, 53–6.7), B220-APC cy7 (eBioscience, RA3-6B2), IL-17A-PE (BD Pharmingen ™ , TC11-18H10), IFNγ-FITC (eBioscience, XMG1.2); (2) Plasma cells; CD3-PerCP cy5.5 (eBioscience, 145-2C11), B220-APC cy7 (eBioscience, RA3-6B2), CD138-APC (BD Pharmingen ™ , 281–2); (3) IL-10 secreting B cells; CD3-PE (eBioscience, 145-2C11), CD19-PerCP cy5.5 (eBioscience, eBio 1D3), B220-APC cy7 (eBioscience, RA3-6B2), IL-10-FITC (eBioscience, JES5-16E3); (4) ILC3s; CD3-PerCP cy5.5 (eBioscience, 145-2C11), B220-FITC (eBioscience, RA3-6B2), NKp46-APC (eBioscience, 29A1.4), CD45-APC cy7 (eBioscience, 30-F11), IL-22-PE (eBioscience, 1H8PWSR).

Techniques: Hi-C

Journal: PLoS ONE

Article Title: Effects of subconjunctival administration of anti-high mobility group box 1 on dry eye in a mouse model of Sjӧgren’s syndrome

doi: 10.1371/journal.pone.0183678

Figure Lengend Snippet: (A) Fold changes in ILC3 percentage (CD3 - B220 - CD45 + IL-22 hi cells; NCR + or NCR - ILC3) showing a significant increase following 2 μg anti-HMGB1 treatment compared with control (Kruskal-Wallis test, PBS vs. 2μg anti-HMGB1, *p = 0.025). Fold increase in NCR - ILC3 percentage (CD3 - B220 - CD45 + NKp46 - IL-22 hi cells)(Kruskal-Wallis test, PBS vs. 2 μg anti-HMGB1, **p = 0.0142). (B) Increased IL-22 levels after 2 μg anti-HMGB1 treatment (Kruskal-Wallis test, PBS vs. 2 μg anti-HMGB1, *p = 0.025). (C) No change in percentage of CD3 + IL-22 hi cells (Th22 cells or γδ T cells) in draining lymph nodes. Data are presented as mean ± standard error. (D) Representative images of NCR - ILC3s (CD3 - B220 - CD45 + NKp46 - IL-22 hi cells) in PBS- and 2 μg anti-HMGB1-treated groups. NCR, natural cytotoxicity receptor.

Article Snippet: Color conjugation combinations and gating strategies were as follows: (1) IFNγ, and IL-17A secreting cells; CD3-PerCP cy5.5 (eBioscience, 145-2C11), CD4-APC (eBioscience, GK1.5), CD8-PE cy7 (eBioscience, 53–6.7), B220-APC cy7 (eBioscience, RA3-6B2), IL-17A-PE (BD Pharmingen ™ , TC11-18H10), IFNγ-FITC (eBioscience, XMG1.2); (2) Plasma cells; CD3-PerCP cy5.5 (eBioscience, 145-2C11), B220-APC cy7 (eBioscience, RA3-6B2), CD138-APC (BD Pharmingen ™ , 281–2); (3) IL-10 secreting B cells; CD3-PE (eBioscience, 145-2C11), CD19-PerCP cy5.5 (eBioscience, eBio 1D3), B220-APC cy7 (eBioscience, RA3-6B2), IL-10-FITC (eBioscience, JES5-16E3); (4) ILC3s; CD3-PerCP cy5.5 (eBioscience, 145-2C11), B220-FITC (eBioscience, RA3-6B2), NKp46-APC (eBioscience, 29A1.4), CD45-APC cy7 (eBioscience, 30-F11), IL-22-PE (eBioscience, 1H8PWSR).

Techniques:

Journal: Scientific Reports

Article Title: Modulation of Igβ is essential for the B cell selection in germinal center

doi: 10.1038/srep10303

Figure Lengend Snippet: Down-regulated Igβ expression in the germinal center. ( a ) Frozen sections of the spleen from B6 mice, 10 days after the immunization with NP-CGG precipitated in alum, were stained with PNA (green) and antibodies to Igβ (red) and CD38 (blue). ( b ) Naïve B cells (shaded), B220 + CD38 − cells (blue line) and B220 + GL7 + cells (red line) were analyzed for the expression level of Igβ. ( c ) Quantitative RT-PCR of Igβ mRNA in sorted naïve B cells (B220 + IgM + CD38 + ) and GC B cells (B220 + CD38 − ) from B6 mice 10 days after the immunization with NP-CGG. Cells were stained with propidium iodide to exclude dead cells and apoptotic cells. Data were normalized to the expression levels of the β-actin transcript. ( d ) Surface IgM in mouse splenic B cells was cross-linked with 10 ug/ml anti-IgM F(ab’) 2 to induce endocytosis for indicated periods of time and remaining levels of cell surface IgM were analyzed ( left ). Expression levels of Igβ mRNA were analyzed by RT-qPCR in B cells whose surface IgM was down-modulated by cross-linking for the indicated periods ( right ).

Article Snippet: anti-B220-APC-Cy7 , BD Biosciences , RA3-6B2.

Techniques: Expressing, Staining, Quantitative RT-PCR

Journal: Scientific Reports

Article Title: Modulation of Igβ is essential for the B cell selection in germinal center

doi: 10.1038/srep10303

Figure Lengend Snippet: Classification of GC B cells by expression level of Igβ. ( a ) Flow cytometric analysis of splenocytes from immunized B6 mice. B220 + B cells (left) cells were further gated to B220 + CD38 + cells ( top right ) and B220 + CD38 − cells ( bottom right ) and analyzed for the expression levels of Igβ and GL7. The numbers indicate the percentage of cells in each square, and the number in the parenthesis indicate the percentage of cells in each square within the CD38 − GL7 − cells. ( b ) Suppressed IgD expression in B220 + CD38 − GL7 − and B220 + CD38 − GL7 + B cells. ( c ) Expression of cell surface Igα in GC B cells. Cell surface expression levels of Igα and GL7 were examined in B220 + CD38 + naive B cells ( top ) and B220 + CD38 − GC B cells ( bottom ) by staining with anti-GL7 mAb in combination with rabbit polyclonal antibody raised against extracellular portion of Igα. Numbers indicate the percentages of the cells within the gates. ( d) Transcription levels of Igβ, Bcl6 and AID in GC subsets ( left ) and surface Fas expression in B220 + CD38 − GL7 − and B220 + CD38 − GL7 + GC subsets ( right ). RT-qPCR were carried out in sorted B220 + CD38 − GL7 − IgβHi cells (Igβ-Hi), B220 + CD38 − GL7 − IgβInt cells (Igβ-Int), B220 + CD38 − GL7 − IgβLo cells (Igβ-Lo) and B220 + CD38 − GL7 + cells (GL7 + ) 10 days after the immunization. ( e ) Cell cycle analysis of GC B cells. Splenocytes from immunized B6 mice were stained with Hoechst 33342 dye in combination with antibodies to Igβ and indicated surface markers, followed by FACS analysis. Naive B cells ( top ), B220 + CD38 − GL7 − ( bottom left ) and B220 + CD38 − GL7 + B cells ( bottom right ) were analyzed.

Article Snippet: anti-B220-APC-Cy7 , BD Biosciences , RA3-6B2.

Techniques: Expressing, Staining, Quantitative RT-PCR, Cell Cycle Assay

Journal: Scientific Reports

Article Title: Modulation of Igβ is essential for the B cell selection in germinal center

doi: 10.1038/srep10303

Figure Lengend Snippet: Class switching events in GC B cells (a) Expression of IgG1 and Igβ in B220 + IgM + CD38 + naive B cells ( top ) and B220 + CD38 − GC B cells ( bottom ). ( b ) Expression levels of germline transcript and surface expression of IgM and IgG1 in the GC B cell subsets. Expression of IgM germline transcript (GLT-μ), IgG1 germline transcript (GLT-γ1) and IgG1 postswitch transcript were assessed by RT-qPCR in each population, respectively ( top ). Cell surface expression of IgM and IgG1 in each GC subset was examined ( bottom ). ( c ) Frequency of somatic hypermutation in VH186.2 gene of GC B cell subsets. Number of nucleic acid mutations ( left ) and amino acid mutations ( right ) are shown.

Article Snippet: anti-B220-APC-Cy7 , BD Biosciences , RA3-6B2.

Techniques: Expressing, Quantitative RT-PCR

Journal: Scientific Reports

Article Title: Modulation of Igβ is essential for the B cell selection in germinal center

doi: 10.1038/srep10303

Figure Lengend Snippet: Forced expression of Igβ in B cells impaired the antibody production and affinity maturation. ( a ) Expression of Igβ in B220 + CD38 − GL7 − splenocytes from immunized Igβ-Tg (Igβ-Tg; line) and wild-type litter mates (wild type; shaded). ( b ) Expression of CD38 and GL7 in B220 + spleen cells from immunized wild type litter mates ( top left ) and Igβ-Tg mice ( top right ). Cell cycle analysis of CD38 − GL7 − GC B cells from wild type litter mates ( bottom left ) and Igβ-Tg mice ( bottom right ). Numbers indicate the percentages of cells in each gate. ( c ) Serum anti-NP titers in each isotype after the immunization. Igβ-Tg (open circle) and wild type litter mates (closed circle) were immunized with NP-CGG on day 0 (1°) and boosted 42 d later (2°). Sera were collected at indicated day and assayed by ELISA as described in Methods section. Error bars indicate SEM for each group. Each group consists of 5 mice. Representative data from 3 repeated experiments is shown. ( d ) Affinity maturation of anti-NP IgG1 antibody in NP-CGG immunized Igβ-Tg (closed bar) and wild type litter mates (open bar). Error bars indicate SEM for each group. ( e ) Decreased frequency of somatic hypermutation in immunized Igβ-Tg mice. CD38 − GL7 + GC B cells were isolated from Igβ-Tg and wild type litter mates 21 days after the immunization. Total RNA was extracted and reverse transcribed to obtain cDNA samples. VH186.2 gene was then amplified and sequenced for the mutation analysis.

Article Snippet: anti-B220-APC-Cy7 , BD Biosciences , RA3-6B2.

Techniques: Expressing, Cell Cycle Assay, Enzyme-linked Immunosorbent Assay, Isolation, Amplification, Mutagenesis

Journal: Scientific Reports

Article Title: Modulation of Igβ is essential for the B cell selection in germinal center

doi: 10.1038/srep10303

Figure Lengend Snippet: Impaired GC formation by inhibition of IL-21-mediated Igβ down-regulation. ( a ) Expression of Igβ in activated splenic B cells supplemented with IL-21. Splenocytes depleted of T cells were cultured with IL4 (10 ng/ml), and anti-CD40 antibody (1 μg/ml) supplemented with indicated concentrations of IL-21 for 6 days. Expression levels of Igβ and GL7 were analyzed by flow cytomeric analysis. The numbers under the square gate indicate the percentages of Igβ down-regulated population in the gate. Representative data from 3 experiments is shown. ( b ) Average percentages and SEM of Igβ low cells from repeated experiments as in ( a ) were shown. ( c ) Flow cytomtric analysis of Igβ expression in B220 + CD38 − GL7 − splenocytes from immunized wild type (shaded) and IL-21R-deficient (line) mice. ( d ) Flow cytometric analysis of the splenic B cells (B220 + ) from wild type litter mates ( left ) and IL-21R-deficient mice ( right ) 10 days after the immunization with NP-CGG. The numbers indicate the percentage of CD38 − GL7 + cells within the gate. ( e ) Cell cycle analysis of B220 + CD38 − GL7 − GC B cells from immunized IL-21R-deficient mice. Splenocytes from wild type litter mates ( left ) and IL21R-deficient mice ( right ) 10 days after the immunization were stained with Hoechst 33342 dye in combination with antibodies to Igβ.

Article Snippet: anti-B220-APC-Cy7 , BD Biosciences , RA3-6B2.

Techniques: Inhibition, Expressing, Cell Culture, Cell Cycle Assay, Staining

Journal: Scientific Reports

Article Title: Modulation of Igβ is essential for the B cell selection in germinal center

doi: 10.1038/srep10303

Figure Lengend Snippet: Amelioration of auto-reactive antibody production and proteinuria by suppression of Igβ down-regulation in BXSB-Yaa autoimmune prone mice. Igβ-Tg mice were back-crossed to BXSB-Yaa for 9 generation and obtained male mice were subjected to further analyses. Serum titers in NZB and wild type C57BL/6 mice were also measured concomitantly as positive band negative controls, respectively. ( a ) Percentages of Igβ-Hi and Igβ-Int B cell population in B220 + CD38 − GL7 − GC B cell subset from immunized C57BL/6 (open bars), BXSB-Yaa background wt (dark shaded bars) and Igβ-Tg (light shaded bars) mice are shown. ( b ) Serum titers of anti-dsDNA antibodies were measured in BXSB-Yaa background wt and Igβ-Tg mice at indicated ages. Each symbol indicate the serum levels for individual mouse. Thick lines indicate the average values and thin lines indicate SEM values. ( c ) Protineuria was measured at 20 weeks of age. Each circle indicate the serum levels for individual mouse. ( d ) Survival curve of BXSB-Yaa background wt and Igβ-Tg mice.

Article Snippet: anti-B220-APC-Cy7 , BD Biosciences , RA3-6B2.

Techniques:

Journal: Scientific Reports

Article Title: Modulation of Igβ is essential for the B cell selection in germinal center

doi: 10.1038/srep10303

Figure Lengend Snippet: List of antibodies used in the experiments

Article Snippet: anti-B220-APC-Cy7 , BD Biosciences , RA3-6B2.

Techniques:

Journal: bioRxiv

Article Title: Nfkbid is required for immunity and antibody responses to Toxoplasma gondii

doi: 10.1101/2020.06.26.174151

Figure Lengend Snippet: A) Serum IgM from C57BL/6J, muMT, bumble, and A/J mice was measured by ELISA. Serum was harvested from mice either naïve, chronically infected with type III CEP strain, or on D5 post-secondary infection with type I GT1 T. gondii strain. PerC transfer (+) refers to mice adoptively transferred 5×10 6 total PerC cells as a day 2 neonate. Each dot represents the results from an individual mouse, and plotted is the average +/-SD of the O.D. obtained at 450nm; *P<0.05, unpaired two-tailed t-test. B) Bumble reconstitution of the peritoneal B-1 compartment after neonatal PerC adoptive transfer. Representative FACS plots of peritoneal B-2 cells (B220 high CD19+) and B-1 (B220 int-neg CD19+) cells from bumble mice with or without PerC adoptive transfer. Shown are mice on day 20 of primary infection with the type III CEP strain. C) B cell deficient muMT mice (n=3), muMT given WT PerC adoptive transfers as 2-day neonates then allowed to reconstitute for 6-7 weeks into adulthood (n=2), and muMT given B cell enriched splenocytes (n=3) 1 day prior to infection with the type III CEP strain were assessed for survival. D) muMT reconstitution of B-2 cell compartment, WT and muMT with B cell enriched splenocytes (EasySep™ Mouse Pan-B Cell Isolation Kit, cat# 19844) adoptively transferred 24 hrs earlier E) muMT reconstitution of peritoneal B cell compartment after neonatal adoptive transfer. Representative FACS plots of peritoneal B-2 cells (B220high CD19+) and B-1 B cells (B220int-neg CD19+) from WT, and muMT mice or muMT mice with neonatal PerC adoptive transfer. For D and E, uninfected mice are 6-8 weeks of age and numbers indicate the percent of cells that fall within the depicted gate.

Article Snippet: The following mAbs (1:100 staining dilutions) were used: anti-CD1d-BV650 (1B1, BD Bioscience), anti-CD11c-eFlour 450 (N418, eBioscience); anti-CD11c-eFlour 450 (N418, BD Bioscience); anti-CD45.2-eFlour 450 (104, eBioscience); anti-CD4-eFlour 450 (GK1.5, eBioscience), anti-CD4-PECy7 (GK1.5, eBioscience); anti-CD11b-FITC (M1/70, eBioScience), anti-CD11b-BUV395 (M1/70, BD Bioscience), anti-CD11b-BV421 (M1/70, BD Bioscience), anti-CD11b-Pacific Blue (M1/70, BioLegend); anti-IFNγ-PE (XMG1.2, BD Bioscience); anti-CD8α-APC (53-6.7, eBioscience), anti-CD8α-BV510 (53-6.7, BioLegend), anti-Ly6G-APC (1A8-Ly6g, eBioscience), anti-CD19-PerCP-Cy5.5 (ebio1D3, eBioscience), anti-CD19-PE (6D5, BioLegend), anti-CD19-BV785 (6D5, BioLegend), anti-CD3-eFlour 780 (17A2, BD Biosciences), anti-Ly6C-PECy7 (HK1.4, BioLegend), anti-CD23-Pacific Blue (B3B4, BioLegend), anti-CD23-AF700 (B3B4, BioLegend), anti-CD21/CD35-FITC (7E9, BioLegend), anti-CD21/CD35-PE (7E9, BioLegend), anti-CD5-APC (53-7.3, BioLegend), anti-CD43-BV510 (S7, BD Bioscience), anti-CD43-BUV737 (S7, BD Bioscience), anti-CD5-PerCP-Cy5.5 (53-7.3, BioLegend), anti-CD5-Cy7-APC (53-7.3, BioLegend), anti-CD45R/B220-Cy7-APC (RA3-6B2, anti-CD45R/B220-BUV-661 (RA3-6B2, BD Bioscience), anti-CD73-Cy7-PE (eBioTY/11.9, eBioscience), anti-CD80-BV711 (16-10A1, BioLegend), anti-FCRL5-af88 (polyclonal, R&D systems) anti-IgM-PECy7 (RMM-1, BioLegend), anti-IgM-BV605 (RMM-1, BioLegend), anti-IgD-FITC (11-26c.2a, BioLegend), anti-IgD-PEDazzle (11-26c.2a, BioLegend), anti-CD138/Syndecan-1-BV510 (281-2, BD Bioscience), anti-CD138/Syndecan-1-BV650 (281-2, BioLegend), anti-mouse-CD267/TACI-AlexaFlour-647 (8F10, BD Biosciences), and anti-CD268/BAFF-R-PE (7H22-E16, BioLegend).

Techniques: Enzyme-linked Immunosorbent Assay, Infection, Two Tailed Test, Adoptive Transfer Assay, Cell Isolation

Journal: bioRxiv

Article Title: Nfkbid is required for immunity and antibody responses to Toxoplasma gondii

doi: 10.1101/2020.06.26.174151

Figure Lengend Snippet: A) Serum obtained from A/J and C57BL/6J mice chronically infected (CEP) or at D5 of secondary infection (GT1) were used to probe GT1 parasite lysate separated by SDS-PAGE, western blots were detected with anti-mouse IgG. “Total Lane Signal” is the signal obtained from the entire lane of C57BL/6J compared to that of A/J (=1); western blots were developed in tandem and analyzed by Image J. Results are from 6 individual experiments; ** P < 0.01, * P < 0.05; unpaired two-tailed t-tests. B) Gating strategies for identifying memory B cells. Memory B cells are identified as CD19+ B220+ CD23+ CD21 mid CD73+. Conventional memory B cells are FCRL5-CD80-while atypical memory B cells are identified as FCRL5+, CD80+. Representative FACS plots of memory compartments in A/J and C57BL/6J mice on day 5 of secondary infection with the type I GT1 strain are shown. The frequency of class-switched (IgM-IgD-) memory cells at the indicated infection states were analyzed. Each dot represents the results from an individual mouse and the cumulative averages +SD from 2 experiments are plotted. N=3-6 mice per infection state. Significance was assessed with an unpaired two-tailed t-test; *** P<0.0001, ** P<0.01.

Article Snippet: The following mAbs (1:100 staining dilutions) were used: anti-CD1d-BV650 (1B1, BD Bioscience), anti-CD11c-eFlour 450 (N418, eBioscience); anti-CD11c-eFlour 450 (N418, BD Bioscience); anti-CD45.2-eFlour 450 (104, eBioscience); anti-CD4-eFlour 450 (GK1.5, eBioscience), anti-CD4-PECy7 (GK1.5, eBioscience); anti-CD11b-FITC (M1/70, eBioScience), anti-CD11b-BUV395 (M1/70, BD Bioscience), anti-CD11b-BV421 (M1/70, BD Bioscience), anti-CD11b-Pacific Blue (M1/70, BioLegend); anti-IFNγ-PE (XMG1.2, BD Bioscience); anti-CD8α-APC (53-6.7, eBioscience), anti-CD8α-BV510 (53-6.7, BioLegend), anti-Ly6G-APC (1A8-Ly6g, eBioscience), anti-CD19-PerCP-Cy5.5 (ebio1D3, eBioscience), anti-CD19-PE (6D5, BioLegend), anti-CD19-BV785 (6D5, BioLegend), anti-CD3-eFlour 780 (17A2, BD Biosciences), anti-Ly6C-PECy7 (HK1.4, BioLegend), anti-CD23-Pacific Blue (B3B4, BioLegend), anti-CD23-AF700 (B3B4, BioLegend), anti-CD21/CD35-FITC (7E9, BioLegend), anti-CD21/CD35-PE (7E9, BioLegend), anti-CD5-APC (53-7.3, BioLegend), anti-CD43-BV510 (S7, BD Bioscience), anti-CD43-BUV737 (S7, BD Bioscience), anti-CD5-PerCP-Cy5.5 (53-7.3, BioLegend), anti-CD5-Cy7-APC (53-7.3, BioLegend), anti-CD45R/B220-Cy7-APC (RA3-6B2, anti-CD45R/B220-BUV-661 (RA3-6B2, BD Bioscience), anti-CD73-Cy7-PE (eBioTY/11.9, eBioscience), anti-CD80-BV711 (16-10A1, BioLegend), anti-FCRL5-af88 (polyclonal, R&D systems) anti-IgM-PECy7 (RMM-1, BioLegend), anti-IgM-BV605 (RMM-1, BioLegend), anti-IgD-FITC (11-26c.2a, BioLegend), anti-IgD-PEDazzle (11-26c.2a, BioLegend), anti-CD138/Syndecan-1-BV510 (281-2, BD Bioscience), anti-CD138/Syndecan-1-BV650 (281-2, BioLegend), anti-mouse-CD267/TACI-AlexaFlour-647 (8F10, BD Biosciences), and anti-CD268/BAFF-R-PE (7H22-E16, BioLegend).

Techniques: Infection, SDS Page, Western Blot, Two Tailed Test

Journal: bioRxiv

Article Title: Nfkbid is required for immunity and antibody responses to Toxoplasma gondii

doi: 10.1101/2020.06.26.174151

Figure Lengend Snippet: A) Gating strategies for identifying B-1 (CD19+ B220 int-neg ) and B-2 (CD19+ B220 hi ) B cells. The legend applies to panels C-H. B) Representative FACS plots of the CD11b+ peritoneal B-1 B cell compartment in A/J and C57BL/6J (B6) mice at naïve, chronic (Chr), and 5 days (D5) post-secondary infection with the GT1 strain. Numbers indicate the percent of cells that fall within the indicated gate. Representative histograms of IgM surface expression and percent of cells that fall within the IgM lo gate of CD5-B-1b cells from A/J (red) and C57BL/6J (blue) at the indicated time points. C) Frequencies of splenic CD43+ B-1a (CD5+) or B-1b (CD5-) that are IgM+IgD- or IgM-IgD-in A/J and C57BL/6J mice at the indicated infection states. D) Representative FACS plots of splenic CD19+ B220 int-neg B cells stained for CD43 and CD5 in A/J and C57BL/6J mice at D5 of secondary infection. Representative CD138 expression on CD5-CD43+ B-1b cells. E) Frequencies of CD43+ splenic B-1a and B-1b cells from A/J and C57BL/6J mice that express BAFFR+, TACI+, or CD138+ at the indicated infection states. F) Heatmap depicting the relative expression of all Ighg transcripts from the indicated B cell population, mouse strain and infection state. G) Representative FACS plots of intracellular IgG (H+L) of B-1b cells, and H) frequency of both peritoneal and splenic B-1a and B-1b cells of A/J and C57BL/6J mice at day 5 of secondary infection. For C, E and H, the cumulative average +SD from 2-4 experiments is plotted and each dot represents the result from an individual mouse; P values calculated by 2-way ANOVA with Tukey correction; **** P<0.0001, *** P<0.001, ** P<0.01, * P<0.05.

Article Snippet: The following mAbs (1:100 staining dilutions) were used: anti-CD1d-BV650 (1B1, BD Bioscience), anti-CD11c-eFlour 450 (N418, eBioscience); anti-CD11c-eFlour 450 (N418, BD Bioscience); anti-CD45.2-eFlour 450 (104, eBioscience); anti-CD4-eFlour 450 (GK1.5, eBioscience), anti-CD4-PECy7 (GK1.5, eBioscience); anti-CD11b-FITC (M1/70, eBioScience), anti-CD11b-BUV395 (M1/70, BD Bioscience), anti-CD11b-BV421 (M1/70, BD Bioscience), anti-CD11b-Pacific Blue (M1/70, BioLegend); anti-IFNγ-PE (XMG1.2, BD Bioscience); anti-CD8α-APC (53-6.7, eBioscience), anti-CD8α-BV510 (53-6.7, BioLegend), anti-Ly6G-APC (1A8-Ly6g, eBioscience), anti-CD19-PerCP-Cy5.5 (ebio1D3, eBioscience), anti-CD19-PE (6D5, BioLegend), anti-CD19-BV785 (6D5, BioLegend), anti-CD3-eFlour 780 (17A2, BD Biosciences), anti-Ly6C-PECy7 (HK1.4, BioLegend), anti-CD23-Pacific Blue (B3B4, BioLegend), anti-CD23-AF700 (B3B4, BioLegend), anti-CD21/CD35-FITC (7E9, BioLegend), anti-CD21/CD35-PE (7E9, BioLegend), anti-CD5-APC (53-7.3, BioLegend), anti-CD43-BV510 (S7, BD Bioscience), anti-CD43-BUV737 (S7, BD Bioscience), anti-CD5-PerCP-Cy5.5 (53-7.3, BioLegend), anti-CD5-Cy7-APC (53-7.3, BioLegend), anti-CD45R/B220-Cy7-APC (RA3-6B2, anti-CD45R/B220-BUV-661 (RA3-6B2, BD Bioscience), anti-CD73-Cy7-PE (eBioTY/11.9, eBioscience), anti-CD80-BV711 (16-10A1, BioLegend), anti-FCRL5-af88 (polyclonal, R&D systems) anti-IgM-PECy7 (RMM-1, BioLegend), anti-IgM-BV605 (RMM-1, BioLegend), anti-IgD-FITC (11-26c.2a, BioLegend), anti-IgD-PEDazzle (11-26c.2a, BioLegend), anti-CD138/Syndecan-1-BV510 (281-2, BD Bioscience), anti-CD138/Syndecan-1-BV650 (281-2, BioLegend), anti-mouse-CD267/TACI-AlexaFlour-647 (8F10, BD Biosciences), and anti-CD268/BAFF-R-PE (7H22-E16, BioLegend).

Techniques: Infection, Expressing, Staining

Journal: bioRxiv

Article Title: Nfkbid is required for immunity and antibody responses to Toxoplasma gondii

doi: 10.1101/2020.06.26.174151

Figure Lengend Snippet: Transcriptomic analysis of peritoneal B-1a (CD19+ B220 int-neg CD11b+ CD5+), B-1b (CD19+ B220 int-neg CD11b+ CD5-) and B-2 (CD19+ B220 hi CD11b-CD5-) B cells from A/J and C57BL/6J mice was performed using 3’-Tag RNA sequencing. A) Genes that were differentially upregulated in B-1b cells on day 5 of secondary infection compared to naïve mice in the A/J genetic background. P values of differentially expressed genes were calculated using the Benjamini-Hochberg adjustment for false discovery rate, and only those genes that survived significance were included in the heatmap. For comparison, all B-1 compartments in A/J mice are shown for this gene set. A/J B-1 Pathway and GO term enrichment was assessed on the genes presented in the heatmap in A. P values for enrichment analysis were adjusted with the Holm-Bonferroni correction. B) A cluster of genes found to be differentially induced in B-1b cells in A/J compared to C57BL/6J mice on day 5 of secondary infection are plotted as a heat map. C) Gene set enrichment analysis of the rank-ordered list of differentially expressed genes between A/J and C57BL/6J B-1b cells at D5 of secondary infection. Gene set depicted was in the top 10 gene sets ranked by false discovery rate (FDR) after investigating MSigDB’s C7: immunologic signatures collection. Enrichment score is the degree of overrepresentation of a gene set at the top or bottom of a ranked list. NES is the enrichment score after normalizing for gene set size.

Article Snippet: The following mAbs (1:100 staining dilutions) were used: anti-CD1d-BV650 (1B1, BD Bioscience), anti-CD11c-eFlour 450 (N418, eBioscience); anti-CD11c-eFlour 450 (N418, BD Bioscience); anti-CD45.2-eFlour 450 (104, eBioscience); anti-CD4-eFlour 450 (GK1.5, eBioscience), anti-CD4-PECy7 (GK1.5, eBioscience); anti-CD11b-FITC (M1/70, eBioScience), anti-CD11b-BUV395 (M1/70, BD Bioscience), anti-CD11b-BV421 (M1/70, BD Bioscience), anti-CD11b-Pacific Blue (M1/70, BioLegend); anti-IFNγ-PE (XMG1.2, BD Bioscience); anti-CD8α-APC (53-6.7, eBioscience), anti-CD8α-BV510 (53-6.7, BioLegend), anti-Ly6G-APC (1A8-Ly6g, eBioscience), anti-CD19-PerCP-Cy5.5 (ebio1D3, eBioscience), anti-CD19-PE (6D5, BioLegend), anti-CD19-BV785 (6D5, BioLegend), anti-CD3-eFlour 780 (17A2, BD Biosciences), anti-Ly6C-PECy7 (HK1.4, BioLegend), anti-CD23-Pacific Blue (B3B4, BioLegend), anti-CD23-AF700 (B3B4, BioLegend), anti-CD21/CD35-FITC (7E9, BioLegend), anti-CD21/CD35-PE (7E9, BioLegend), anti-CD5-APC (53-7.3, BioLegend), anti-CD43-BV510 (S7, BD Bioscience), anti-CD43-BUV737 (S7, BD Bioscience), anti-CD5-PerCP-Cy5.5 (53-7.3, BioLegend), anti-CD5-Cy7-APC (53-7.3, BioLegend), anti-CD45R/B220-Cy7-APC (RA3-6B2, anti-CD45R/B220-BUV-661 (RA3-6B2, BD Bioscience), anti-CD73-Cy7-PE (eBioTY/11.9, eBioscience), anti-CD80-BV711 (16-10A1, BioLegend), anti-FCRL5-af88 (polyclonal, R&D systems) anti-IgM-PECy7 (RMM-1, BioLegend), anti-IgM-BV605 (RMM-1, BioLegend), anti-IgD-FITC (11-26c.2a, BioLegend), anti-IgD-PEDazzle (11-26c.2a, BioLegend), anti-CD138/Syndecan-1-BV510 (281-2, BD Bioscience), anti-CD138/Syndecan-1-BV650 (281-2, BioLegend), anti-mouse-CD267/TACI-AlexaFlour-647 (8F10, BD Biosciences), and anti-CD268/BAFF-R-PE (7H22-E16, BioLegend).

Techniques: RNA Sequencing Assay, Infection

Journal: bioRxiv

Article Title: Nfkbid is required for immunity and antibody responses to Toxoplasma gondii

doi: 10.1101/2020.06.26.174151

Figure Lengend Snippet: A) Nfkbid expression in CPM (Counts per million) of 3’-Tag RNA-seq reads of the indicated B cell populations obtained from A/J and C57BL/6J mice that were either naïve, chronically infected, or on D5 of secondary infection with the type I GT1 strain. B) Enriched B cells from the PerC and spleen were stimulated with LPS for 2 hrs and Nfkbid transcripts were quantified by qPCR; ** P< 0.01, paired t-test. C) Representative histograms display the detection of parasite-specific IgG1 bound to formaldehyde fixed GFP+ type I GT1 parasites; diluted serums (10 3 ) from C57BL/6J, Nfkbid +/-(C57BL/6J x bumble F1), and A/J mice chronically infected with the type III CEP strain were assayed. Anti-mouse IgG1 background staining in the absence of serum is shown. D) As in C, but quantification of the parasite-specific IgG1 antibody isotype binding over a range of serum concentrations is shown. Plotted is the cumulative average +/-SD of the MFI from 2-3 separate experiments (C57BL/6J n=8; Nfkbid +/-n=7; A/J n=8); significance was assessed by unpaired t-tests and Holm-Sidak corrections comparing A/J vs C57BL/6J (*) or Nfkbid+/- vs C57BL/6J (#); **** P<0.0001, # P<0.05. IgG1 staining was not significantly different between A/J and Nfkbid +/-serums. E) Representative FACS plots of the dump-CD19+ CD138+ plasmablast populations within A/J, C57BL/6J and Nfkbid +/-mice at day 5 of secondary infection with type I GT1 parasites. F) Frequency of B220-CD138+ plasmablasts of total live dump-CD19+ cells. Plotted is the cumulative average +/-SD of 2 separate experiments (n=5 per mouse strain); significance was assessed by unpaired t-tests; * P< 0.05.

Article Snippet: The following mAbs (1:100 staining dilutions) were used: anti-CD1d-BV650 (1B1, BD Bioscience), anti-CD11c-eFlour 450 (N418, eBioscience); anti-CD11c-eFlour 450 (N418, BD Bioscience); anti-CD45.2-eFlour 450 (104, eBioscience); anti-CD4-eFlour 450 (GK1.5, eBioscience), anti-CD4-PECy7 (GK1.5, eBioscience); anti-CD11b-FITC (M1/70, eBioScience), anti-CD11b-BUV395 (M1/70, BD Bioscience), anti-CD11b-BV421 (M1/70, BD Bioscience), anti-CD11b-Pacific Blue (M1/70, BioLegend); anti-IFNγ-PE (XMG1.2, BD Bioscience); anti-CD8α-APC (53-6.7, eBioscience), anti-CD8α-BV510 (53-6.7, BioLegend), anti-Ly6G-APC (1A8-Ly6g, eBioscience), anti-CD19-PerCP-Cy5.5 (ebio1D3, eBioscience), anti-CD19-PE (6D5, BioLegend), anti-CD19-BV785 (6D5, BioLegend), anti-CD3-eFlour 780 (17A2, BD Biosciences), anti-Ly6C-PECy7 (HK1.4, BioLegend), anti-CD23-Pacific Blue (B3B4, BioLegend), anti-CD23-AF700 (B3B4, BioLegend), anti-CD21/CD35-FITC (7E9, BioLegend), anti-CD21/CD35-PE (7E9, BioLegend), anti-CD5-APC (53-7.3, BioLegend), anti-CD43-BV510 (S7, BD Bioscience), anti-CD43-BUV737 (S7, BD Bioscience), anti-CD5-PerCP-Cy5.5 (53-7.3, BioLegend), anti-CD5-Cy7-APC (53-7.3, BioLegend), anti-CD45R/B220-Cy7-APC (RA3-6B2, anti-CD45R/B220-BUV-661 (RA3-6B2, BD Bioscience), anti-CD73-Cy7-PE (eBioTY/11.9, eBioscience), anti-CD80-BV711 (16-10A1, BioLegend), anti-FCRL5-af88 (polyclonal, R&D systems) anti-IgM-PECy7 (RMM-1, BioLegend), anti-IgM-BV605 (RMM-1, BioLegend), anti-IgD-FITC (11-26c.2a, BioLegend), anti-IgD-PEDazzle (11-26c.2a, BioLegend), anti-CD138/Syndecan-1-BV510 (281-2, BD Bioscience), anti-CD138/Syndecan-1-BV650 (281-2, BioLegend), anti-mouse-CD267/TACI-AlexaFlour-647 (8F10, BD Biosciences), and anti-CD268/BAFF-R-PE (7H22-E16, BioLegend).

Techniques: Expressing, RNA Sequencing Assay, Infection, Staining, Binding Assay