Journal: Nature Communications

Article Title: Influenza virus infection expands the breadth of antibody responses through IL-4 signalling in B cells

doi: 10.1038/s41467-021-24090-z

Figure Lengend Snippet: a B220 + IgM − IgD − Dump − B cells were obtained from spleen of the mice immunized with inactivated A/Narita/1/2009 (Vaccine, white bars; n = 3) and from MLN in the A/Narita/1/2009 infected mice (Infection, black bars; n = 3). Binding to A/Narita/1/2009-HA and/or A/PR/8/1934-HA was determined by flow cytometry analysis. b The data indicate binding to HA in flow cytometry analysis (left) and the number of single or dual-binding B cells (right) in spleen (white bars) or MLN (black bars) from the mice infected with A/Narita/1/2009 ( n = 5). c Narita-HA binding B cells (2000 cells each) were isolated from vaccinated mice (spleen, white bar; n = 3) and infected mice (MLN, black bar; N = 3). The V-D-J combinations are shown by connected color bands between V and D (left), V and J (middle), and D and J (right). d V h gene usage of A/Narita/1/2009-HA binding B cells in spleen from three vaccinated mice (Vaccine n = 3) or MLN (Infection n = 2) from two infected mice with A/Narita/1/2009. Total RNA isolated from sorted A/Narita/1/2009-HA binding B cells served as the template for cDNA synthesis followed by PCR to amplify IgG variable region genes. Amplified IgG gene libraries were sequenced by MiSeq. The IgG sequences were analyzed with the ImMunoGeneTics (IMGT) HighV-QUEST. e Seven hundred twenty HA probe + B cells were single sorted and cultured on NB21.2D9 feeder cells with IL-4 in individual wells. After 20 days of culture, 303 IgG-producing cells were obtained. The binding of antibodies produced by these cells to A/Narita/1/2009-HA, A/PR/8/1934-HA, A/H1N1pdm09 derived LAH (blue), or rHA2 (red) was measured by ELISA. Finally, reliable binding and V H genetic information were obtained for 101 clones. Black circles indicate clones showing no binding to LAH or rHA2. Y -axis represents the ratio of the OD 450 value in the sample vs the OD 450 value obtained with IgG [anti-Narita-HA (1 µg/ml), anti-PR8-HA (1 µg/ml), anti-LAH (1 µg/ml), and rHA-2 (3 µg/ml)]. f Amino acid sequences encoded by V h genes in single or dual-HA binding B cell clones in the region from CDR1 to CDR3 are shown with germline amino acid sequence (unhighlighted). Red boxes indicated CDR regions. The positions where amino acid substitutions occur are indicated by the red font. g Number of amino-acid mutations in the region extending from CDR1 to CDR3 of single or dual-binding B cell clones (single, blue dots; n = 18, dual, red dots; n = 31) as determined by referring to the germline sequences of the corresponding V h in the IMGT database. * p < 0.05, ** p < 0.01 by un p aired, two-tailed t -test (Vaccine group with Infection group in a and c , Spleen group with MLN group in b , Single group with Dual group in g ). All error bars represent SEM.

Article Snippet: For the ELISA assay, the viral antigens of A/Narita/1/2009 or A/PR/8/1934 propagated in MDCK cells were captured on 96-well Maxisorp ELISA plates (Thermo Fisher, Waltham, MA) by anti-California (clone: RM01; 1:10,000 dilution, SinoBiological, Beijing, China, 86001-RM01) or PR8 HA antibody (clone: R107; 1:10,000 dilution, SinoBiological, 11684-R107), respectively.

Techniques: Infection, Binding Assay, Flow Cytometry, Isolation, Amplification, Cell Culture, Produced, Derivative Assay, Enzyme-linked Immunosorbent Assay, Clone Assay, Sequencing, Two Tailed Test

Journal: Nature Communications

Article Title: Influenza virus infection expands the breadth of antibody responses through IL-4 signalling in B cells

doi: 10.1038/s41467-021-24090-z

Figure Lengend Snippet: a Top panel indicates the design of the A/Narita/1/2009 infection experiment in Tmprss2 −/− mice. Control C57BL/6 (WT) and Tmprss2 −/− mice were infected with A/Narita/1/2009 (WT: 5LD 50 n = 4 (white), 0.3LD 50 n = 4 (red), Tmprss2 −/− : 0.3LD 50 n = 7 (blue), 3000LD 50 n = 4 (black)) (left). Missing symbols mean that the mice were euthanized, as described in Fig. . The viral load (PFU) in the lungs at 3- or 10-day ( n = 4 for each) post-infection was determined by MDCK culture (right). b IgG1 and IgG2b titers against Narita-HA in BALF and serum from WT (white, n = 5) and Tmprss2 −/− mice at 14 days post-infection with A/Narita/1/2009 (3000LD 50 n = 7 (black), 300LD 50 n = 2 (yellow), 3LD 50 n = 2 (brown), 0.3LD 50 n = 3 (red)). c Top panel indicates the design of the passive transfer experiment in Tmprss2 −/− mice. Mice intranasally transferred with BALF or serum (60 µg/ml anti-Narita IgG) from WT (white) (anti-Narita n = 3, anti-PR8 n = 4) or Tmprss2 - /- (3000LD 50 anti-Narita in BALF n = 3, anti-PR8 in BALF n = 8, anti-Narita in serum n = 5, anti-PR8 in serum n = 6 (black), 0.3LD 50 anti-Narita in BALF n = 2, anti-PR8 in BALF n = 4 (red)) mice infected with A/Narita/1/2009 were challenged intranasally with a lethal dose of A/Narita/1/2009 (left) or A/PR/8/1934 (right). Missing symbols mean that the mice were euthanized, as described in Fig. . d Control WT (white bars, n = 4) and Tmprss2 −/− (black bars, n = 4) mice were infected with A/Narita/1/2009, and binding to Narita-HA or PR8-HA was analyzed at 14 days post-infection. UI represents unimmunized. Bars represent mean; * p < 0.05, ** p < 0.01 by un p aired, two-tailed t -test. WT (0.3LD 50 ) group was compared with Tmprss2 −/− (3000LD 50 ) group. All error bars represent SEM.

Article Snippet: For the ELISA assay, the viral antigens of A/Narita/1/2009 or A/PR/8/1934 propagated in MDCK cells were captured on 96-well Maxisorp ELISA plates (Thermo Fisher, Waltham, MA) by anti-California (clone: RM01; 1:10,000 dilution, SinoBiological, Beijing, China, 86001-RM01) or PR8 HA antibody (clone: R107; 1:10,000 dilution, SinoBiological, 11684-R107), respectively.

Techniques: Infection, Binding Assay, Two Tailed Test

Journal: Nature Communications

Article Title: Influenza virus infection expands the breadth of antibody responses through IL-4 signalling in B cells

doi: 10.1038/s41467-021-24090-z

Figure Lengend Snippet: a IgG1 antibody titers specific for A/PR/8/1934-HA- or A/Narita/1/2009-HA in BALF from WT (black), Il6 −/− , Il1b −/− , Il10 −/− , Il17a −/− , Il22 −/− , Il23 −/− , Ifnar −/− (white, n = 5 for each) mice were measured at day 14 post-infection with A/Narita/1/2009. b Top panel indicates the design of the BALF transfer experiment for protective antibodies in Il6 −/− mice. Mice intranasally administered with BALF from WT, Il6 −/− mice infected with A/Narita/1/2009 (WT n = 2 (white), Il6 −/− n = 2 (black)) were challenged intranasally with a lethal dose of A/PR/8/1934. The body weight change of mice was monitored until 14 days post-infection. c Top panel indicates the design of the BALF or serum transfer experiment. Mice intranasally transferred with BALF or serum (60 µg/ml anti-Narita IgG) from WT (white, n = 2–5), Ifng −/− (red, n = 4) , Il21 ΔT (red, n = 4), Il4 −/− (red) (anti-PR8 in BALF n = 9, in serum n = 6; anti-Narita in serum n = 5), Il4ra f/+ Cd79cre (blue, n = 2), or Il4ra ΔB (red, n = 4) mice infected with A/Narita/1/2009 were challenged intranasally with a lethal dose of A/Narita/1/2009 or A/PR/8/1934. Anti-A/Narita/1/2009 HA total IgG titers in serum were corrected to 60 µg/ml by dilution before the serum transfer. The body weight change of mice was monitored until 14 days post-infection. Missing symbols mean that the mice were euthanized, as described in Fig. . d T FH cells (CD4 + PD-1 + CXCR5 + ) and GC B cells (B220 + GL-7 + Fas + ) were analyzed in MLN from the A/Narita/1/2009 infected WT (white bars), Ifng −/− (black bars) , Il21 ΔT (blue bars), or Il4 −/− (red bars) ( n = 3 for each) mice. e The data show the percentage and number of single and dual-binding B cells (B220 + IgM − IgD − Dump − ) in MLN from the A/Narita/1/2009 infected WT (white bars), Il4 −/− (black bars), or Il4ra ΔB (red bars) ( n = 5 for each) mice. UI represents unimmunized. Bars represent mean; * p < 0.05, ** p < 0.01 by unpaired, two-tailed t -test (WT group with other groups respectively). All error bars represent SEM.

Article Snippet: For the ELISA assay, the viral antigens of A/Narita/1/2009 or A/PR/8/1934 propagated in MDCK cells were captured on 96-well Maxisorp ELISA plates (Thermo Fisher, Waltham, MA) by anti-California (clone: RM01; 1:10,000 dilution, SinoBiological, Beijing, China, 86001-RM01) or PR8 HA antibody (clone: R107; 1:10,000 dilution, SinoBiological, 11684-R107), respectively.

Techniques: Infection, Binding Assay, Two Tailed Test

Journal: Nature Communications

Article Title: Influenza virus infection expands the breadth of antibody responses through IL-4 signalling in B cells

doi: 10.1038/s41467-021-24090-z

Figure Lengend Snippet: a Top panel indicates the design of the BMT experiment. Irradiated CD45.1 host mice were reconstituted with a mixture of BM cells from WT (blue bars) and Il4ra ΔB CD45.2 mice (red bars) ( n = 3 for each). The host mice were infected with A/Narita/1/2009 on day 31. Fourteen days later, Binding to Narita-HA and/or PR8-HA and generation of CD38 - GL-7 + , or CD69 + GL-7 + cells in the B220 + B cells, or CD38 dull Fas + cells in the CD19 + Dump - populations, (right graph) were compared between IL-4R + and IL-4R - B220 + B cells. b Naive OT-II T cells from WT or Il4 −/− mice were transferred into Cd28 −/− mice followed by immunization with OVA + alum. Frozen sections were prepared at 6 days post-immunization and stained with antibodies against B220, pS6, and CD4 (left 4 panels). Left panels illustrate the localization of pS6 + B cells in the spleen. Dashed lines demarcate the follicle area, and these areas are divided into 0.01 mm 2 squares (white lines). Each dot in the violin plot (right top panel) represents the Number of pS6 + B cells localized in each 4 mm 2 square areas. 154 and 99 sites were counted in the WT (white dots) and the Il4 −/− (black dots) section, respectively. The bar graph in the bottom right panel indicates the mean number of pS6 + B cells per µm 2 . Scale bar indicates 100 µm. c mRNA expression levels were measured in splenic B cells from WT (blue bars, n = 3) or Il4 −/− (red bars, n = 4) T cell-transferred mice. d Ki67 expression was analyzed in splenic B220 + B cells from the transferred mice from WT ( n = 4) or Il4 −/− ( n = 4) mice. Ki67 expression is analyzed in GL-7 + (right) and GL-7 - (left) B cells. Bars represent mean; * p < 0.05, ** p < 0.01 by ratio paired, two-tailed t -test (IL-4Rα + group with IL-4Rα − in a ), or by unpaired , two-tailed t -test (WT group with Il4 −/− group in b and c ). All error bars represent SEM.

Article Snippet: For the ELISA assay, the viral antigens of A/Narita/1/2009 or A/PR/8/1934 propagated in MDCK cells were captured on 96-well Maxisorp ELISA plates (Thermo Fisher, Waltham, MA) by anti-California (clone: RM01; 1:10,000 dilution, SinoBiological, Beijing, China, 86001-RM01) or PR8 HA antibody (clone: R107; 1:10,000 dilution, SinoBiological, 11684-R107), respectively.

Techniques: Irradiation, Infection, Binding Assay, Staining, Expressing, Two Tailed Test

Journal: Vaccines

Article Title: Vaccine-Induced Adverse Effects in Cultured Neuroblastoma 2A (N2A) Cells Duplicate Toxicity of Serum from Patients with Gulf War Illness (GWI) and Are Prevented in the Presence of Specific Anti-Vaccine Antibodies

doi: 10.3390/vaccines8020232

Figure Lengend Snippet: Effect of influenza vaccine, and influenza vaccine incubated with antibodies to influenza vaccine components, H1N1, H3N2, and B strains or without the influenza vaccines on N2A cell spreading. *** p < 0.0001 (compared to medium).

Article Snippet: For this assay, the following antibodies were each incubated with GWI serum: anti-hemagglutinin (HA)–H1N1 (Cat. No. 11055-RP01), anti-HA–H3N2 (Cat. No. 11715-RP01), anti-HA–B (Cat. No.11053-RP01), strains of influenza (polyclonal, Sino Biological, Wayne, PA, USA); anti- Salmonella (Cat. No. PA1-20811, polyclonal, ThermoFisher Scientific, Rockford, IL, USA); anti-Japanese encephalitis virus (Cat. No. PA5-32237, polyclonal, ThermoFisher Scientific, Rockford, IL, USA); anti- Yersinia pestis (Cat. No. MA1-23074, monoclonal, ThermoFisher Scientific, Rockford, IL, USA); anti-hepatitis virus B (Cat. No. ab9193, polyclonal, Abcam, Cambridge, UK); anti-cholera toxin (monoclonal, Cat. No. LS-C142039, LS Bio, Seattle, WA, USA); anti-Yellow fever (Cat. No.3576, ThermoFisher Scientific, Rockford, IL, USA); anti-varicella zoster (Cat. No.LS-132860/58089, LS Bio, Seattle, WA, USA); anti-anthrax protective antigen (Cat. No. CPBT-66806RA, polyclonal, Creative Diagnostics, Shirley, NJ, USA); anti-mumps virus (Cat. No. MBS320375, monoclonal, MyBioSource, San Diego, CA, USA); anti-adenovirus (Cat. No. LS-C63691/120157, Seattle, WA, USA); anti-polio virus (Cat. No. ab22450, monoclonal, abcam, Cambridge, UK); anti-clostridium botulinum B toxoid activity (polyclonal, Cat. No. ab83064, abcam, Cambridge, UK); anti-measles (monoclonal, Cat. No. DMABT-H21849 Creative Diagnosticss, Shirley, NJ, USA).

Techniques: Incubation

Journal: Vaccines

Article Title: Vaccine-Induced Adverse Effects in Cultured Neuroblastoma 2A (N2A) Cells Duplicate Toxicity of Serum from Patients with Gulf War Illness (GWI) and Are Prevented in the Presence of Specific Anti-Vaccine Antibodies

doi: 10.3390/vaccines8020232

Figure Lengend Snippet: Ν2A cell apoptosis of cells in medium ( A ), exposed to the influenza vaccine ( B ) and ( C ) in the simultaneous presence of influenza vaccine and antibodies against H1N1. Similar effects were observed when antibodies to H3N2, and S . Typhi were used. The antibodies were added to the culture medium simultaneously with the corresponding vaccine for two days. In the presence of the influenza vaccine, most nuclei were stained red with TUNEL indicating apoptosis (B), whereas, in the combined presence of the influenza vaccine and anti-H1N1 antibodies (C), most nuclei appeared healthy and stained with DAPI, similar to control cells cultured in medium (A).

Article Snippet: For this assay, the following antibodies were each incubated with GWI serum: anti-hemagglutinin (HA)–H1N1 (Cat. No. 11055-RP01), anti-HA–H3N2 (Cat. No. 11715-RP01), anti-HA–B (Cat. No.11053-RP01), strains of influenza (polyclonal, Sino Biological, Wayne, PA, USA); anti- Salmonella (Cat. No. PA1-20811, polyclonal, ThermoFisher Scientific, Rockford, IL, USA); anti-Japanese encephalitis virus (Cat. No. PA5-32237, polyclonal, ThermoFisher Scientific, Rockford, IL, USA); anti- Yersinia pestis (Cat. No. MA1-23074, monoclonal, ThermoFisher Scientific, Rockford, IL, USA); anti-hepatitis virus B (Cat. No. ab9193, polyclonal, Abcam, Cambridge, UK); anti-cholera toxin (monoclonal, Cat. No. LS-C142039, LS Bio, Seattle, WA, USA); anti-Yellow fever (Cat. No.3576, ThermoFisher Scientific, Rockford, IL, USA); anti-varicella zoster (Cat. No.LS-132860/58089, LS Bio, Seattle, WA, USA); anti-anthrax protective antigen (Cat. No. CPBT-66806RA, polyclonal, Creative Diagnostics, Shirley, NJ, USA); anti-mumps virus (Cat. No. MBS320375, monoclonal, MyBioSource, San Diego, CA, USA); anti-adenovirus (Cat. No. LS-C63691/120157, Seattle, WA, USA); anti-polio virus (Cat. No. ab22450, monoclonal, abcam, Cambridge, UK); anti-clostridium botulinum B toxoid activity (polyclonal, Cat. No. ab83064, abcam, Cambridge, UK); anti-measles (monoclonal, Cat. No. DMABT-H21849 Creative Diagnosticss, Shirley, NJ, USA).

Techniques: Staining, TUNEL Assay, Cell Culture

Journal: Vaccines

Article Title: Vaccine-Induced Adverse Effects in Cultured Neuroblastoma 2A (N2A) Cells Duplicate Toxicity of Serum from Patients with Gulf War Illness (GWI) and Are Prevented in the Presence of Specific Anti-Vaccine Antibodies

doi: 10.3390/vaccines8020232

Figure Lengend Snippet: Ν2A cell apoptosis of cells in the presence of Gulf War illness (GWI) plus antibodies to cholera ( A ), influenza A/H1N1 ( B ), influenza A/H3N2 ( C ) influenza B ( D ), hepatitis B ( E ), measles ( F ), S . Typhi ( G ), rabies ( H ), and mumps ( I ). The antibodies were added in the culture medium simultaneously with GWI serum for two days. Most nuclei appeared healthy and stained with DAPI (blue); few nuclei were stained with TUNEL in A–G (red). In H,I, antibodies against rabies and mumps are shown to have no effect, as most nuclei appear stained red with TUNEL.

Article Snippet: For this assay, the following antibodies were each incubated with GWI serum: anti-hemagglutinin (HA)–H1N1 (Cat. No. 11055-RP01), anti-HA–H3N2 (Cat. No. 11715-RP01), anti-HA–B (Cat. No.11053-RP01), strains of influenza (polyclonal, Sino Biological, Wayne, PA, USA); anti- Salmonella (Cat. No. PA1-20811, polyclonal, ThermoFisher Scientific, Rockford, IL, USA); anti-Japanese encephalitis virus (Cat. No. PA5-32237, polyclonal, ThermoFisher Scientific, Rockford, IL, USA); anti- Yersinia pestis (Cat. No. MA1-23074, monoclonal, ThermoFisher Scientific, Rockford, IL, USA); anti-hepatitis virus B (Cat. No. ab9193, polyclonal, Abcam, Cambridge, UK); anti-cholera toxin (monoclonal, Cat. No. LS-C142039, LS Bio, Seattle, WA, USA); anti-Yellow fever (Cat. No.3576, ThermoFisher Scientific, Rockford, IL, USA); anti-varicella zoster (Cat. No.LS-132860/58089, LS Bio, Seattle, WA, USA); anti-anthrax protective antigen (Cat. No. CPBT-66806RA, polyclonal, Creative Diagnostics, Shirley, NJ, USA); anti-mumps virus (Cat. No. MBS320375, monoclonal, MyBioSource, San Diego, CA, USA); anti-adenovirus (Cat. No. LS-C63691/120157, Seattle, WA, USA); anti-polio virus (Cat. No. ab22450, monoclonal, abcam, Cambridge, UK); anti-clostridium botulinum B toxoid activity (polyclonal, Cat. No. ab83064, abcam, Cambridge, UK); anti-measles (monoclonal, Cat. No. DMABT-H21849 Creative Diagnosticss, Shirley, NJ, USA).

Techniques: Staining, TUNEL Assay