h1n1  (Sino Biological)


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    Name:
    Influenza A H1N1 Hemagglutinin HA Protein
    Description:
    A DNA sequence encoding the extracellular domain of Influenza A virus A Brisbane 59 2007 H1N1 ACA28844 1 hemagglutinin Met 1 Gln 528 Native HA1 HA2 uncleaved was expressed with a C terminal polyhistidine tag
    Catalog Number:
    11052-V08H
    Price:
    None
    Category:
    recombinant protein
    Host:
    HEK293 Cells
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    Structured Review

    Sino Biological h1n1
    Oligoclonal mAb Response Protects Mice from Group 1 or Group 2 Influenza Challenges BALB/c mice (n = 20) were pretreated with 20 U of hyaluronidase and administered 10 μg of H/L gWiz pDNA per anti-influenza mAb (C179, S139/1, and 9H10) in muscles of separate limbs followed by EP. For the control group, BALB/c mice (n = 15) were pretreated with 20 U of hyaluronidase and administered 10 μg of H/L gWiz pDNA per anti-Ebola mAb (2G4, 4G7, and 13C6) at distinct sites with EP. Blood was collected on days 12, 19, and 33 (in surviving mice) for pharmacokinetic analyses ( Figure S5 ). (A and B) On day 23, the mice were randomized and anti-influenza DNA/EP mice (n = 10) and anti-Ebola DNA/EP mice (n = 7–8) were challenged with (A) 39 MLD 50 of A/WSN/33 <t>(H1N1)</t> or (B) 21 MLD 50 of A/Aichi/2/68 (H3N2) delivered intranasally. (A and B) Mean weight loss (%) ± SEM compared with day 0 (left) and survival (%) as depicted in Kaplan-Meier plots (right) following (A) H1N1 and (B) H3N2 challenge. For percent weight loss graphs, **p = 0.0021, ***p
    A DNA sequence encoding the extracellular domain of Influenza A virus A Brisbane 59 2007 H1N1 ACA28844 1 hemagglutinin Met 1 Gln 528 Native HA1 HA2 uncleaved was expressed with a C terminal polyhistidine tag
    https://www.bioz.com/result/h1n1/product/Sino Biological
    Average 95 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    h1n1 - by Bioz Stars, 2021-09
    95/100 stars

    Images

    1) Product Images from "In Vivo Production of Monoclonal Antibodies by Gene Transfer via Electroporation Protects against Lethal Influenza and Ebola Infections"

    Article Title: In Vivo Production of Monoclonal Antibodies by Gene Transfer via Electroporation Protects against Lethal Influenza and Ebola Infections

    Journal: Molecular Therapy. Methods & Clinical Development

    doi: 10.1016/j.omtm.2017.09.003

    Oligoclonal mAb Response Protects Mice from Group 1 or Group 2 Influenza Challenges BALB/c mice (n = 20) were pretreated with 20 U of hyaluronidase and administered 10 μg of H/L gWiz pDNA per anti-influenza mAb (C179, S139/1, and 9H10) in muscles of separate limbs followed by EP. For the control group, BALB/c mice (n = 15) were pretreated with 20 U of hyaluronidase and administered 10 μg of H/L gWiz pDNA per anti-Ebola mAb (2G4, 4G7, and 13C6) at distinct sites with EP. Blood was collected on days 12, 19, and 33 (in surviving mice) for pharmacokinetic analyses ( Figure S5 ). (A and B) On day 23, the mice were randomized and anti-influenza DNA/EP mice (n = 10) and anti-Ebola DNA/EP mice (n = 7–8) were challenged with (A) 39 MLD 50 of A/WSN/33 (H1N1) or (B) 21 MLD 50 of A/Aichi/2/68 (H3N2) delivered intranasally. (A and B) Mean weight loss (%) ± SEM compared with day 0 (left) and survival (%) as depicted in Kaplan-Meier plots (right) following (A) H1N1 and (B) H3N2 challenge. For percent weight loss graphs, **p = 0.0021, ***p
    Figure Legend Snippet: Oligoclonal mAb Response Protects Mice from Group 1 or Group 2 Influenza Challenges BALB/c mice (n = 20) were pretreated with 20 U of hyaluronidase and administered 10 μg of H/L gWiz pDNA per anti-influenza mAb (C179, S139/1, and 9H10) in muscles of separate limbs followed by EP. For the control group, BALB/c mice (n = 15) were pretreated with 20 U of hyaluronidase and administered 10 μg of H/L gWiz pDNA per anti-Ebola mAb (2G4, 4G7, and 13C6) at distinct sites with EP. Blood was collected on days 12, 19, and 33 (in surviving mice) for pharmacokinetic analyses ( Figure S5 ). (A and B) On day 23, the mice were randomized and anti-influenza DNA/EP mice (n = 10) and anti-Ebola DNA/EP mice (n = 7–8) were challenged with (A) 39 MLD 50 of A/WSN/33 (H1N1) or (B) 21 MLD 50 of A/Aichi/2/68 (H3N2) delivered intranasally. (A and B) Mean weight loss (%) ± SEM compared with day 0 (left) and survival (%) as depicted in Kaplan-Meier plots (right) following (A) H1N1 and (B) H3N2 challenge. For percent weight loss graphs, **p = 0.0021, ***p

    Techniques Used: Mouse Assay

    2) Product Images from "The Effects of Aβ1-42 Binding to the SARS-CoV-2 Spike Protein S1 Subunit and Angiotensin-Converting Enzyme 2"

    Article Title: The Effects of Aβ1-42 Binding to the SARS-CoV-2 Spike Protein S1 Subunit and Angiotensin-Converting Enzyme 2

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms22158226

    Interactions between Aβ 1-42 and the S1 of SARS-CoV-2. ( a ) Aβ 1-42 differentially binds to the immobilized surface proteins of three viruses responsible for recent pandemics. The order of potency of binding based on the estimated EC 50 was the S1 of SARS-CoV-2 (200–325 ng/mL) > HA of H1N1 (HA, 372–507 ng/mL) > S1 of MERS-CoV (599–860 ng/mL). ( b ) Linear epitope mapping was performed using 17 fragment peptides of Aβ 1-42 (details in Supplementary Figure S2 ), and the binding ability is indicated by OD. Aβ 1-42 interacted with the S1 of SARS-CoV-2, mainly in the last probe containing the hydrophobic Aβ residues of 33–42. A similar binding pattern on Aβ 1-42 was observed for HA of H1N1, and to a lesser extent, for the S1 of MERS-CoV. hACE2-Fc presents three more binding sites (OD > 0.8) across the entire sequence of Aβ 1-42 in addition to a major binding site on the C-terminal end. The following binding potency was compared by OD. ( c ) Aβ 1-40 did not bind to the S1 of SARS-CoV-2. ( d ) Aβ 1-42 bound weakly to the immobilized His-tagged RBD of SARS-CoV-2 as compared to the S1 protein. ( e ) Iowa D23N and Italian E22K Aβ 1-42 mutants exerted marked reduction in the S1 binding, and amino acid sequences for normal human (Wt) and mutated Aβ 1-42 are presented. Data are presented as mean values from two independent experiments.
    Figure Legend Snippet: Interactions between Aβ 1-42 and the S1 of SARS-CoV-2. ( a ) Aβ 1-42 differentially binds to the immobilized surface proteins of three viruses responsible for recent pandemics. The order of potency of binding based on the estimated EC 50 was the S1 of SARS-CoV-2 (200–325 ng/mL) > HA of H1N1 (HA, 372–507 ng/mL) > S1 of MERS-CoV (599–860 ng/mL). ( b ) Linear epitope mapping was performed using 17 fragment peptides of Aβ 1-42 (details in Supplementary Figure S2 ), and the binding ability is indicated by OD. Aβ 1-42 interacted with the S1 of SARS-CoV-2, mainly in the last probe containing the hydrophobic Aβ residues of 33–42. A similar binding pattern on Aβ 1-42 was observed for HA of H1N1, and to a lesser extent, for the S1 of MERS-CoV. hACE2-Fc presents three more binding sites (OD > 0.8) across the entire sequence of Aβ 1-42 in addition to a major binding site on the C-terminal end. The following binding potency was compared by OD. ( c ) Aβ 1-40 did not bind to the S1 of SARS-CoV-2. ( d ) Aβ 1-42 bound weakly to the immobilized His-tagged RBD of SARS-CoV-2 as compared to the S1 protein. ( e ) Iowa D23N and Italian E22K Aβ 1-42 mutants exerted marked reduction in the S1 binding, and amino acid sequences for normal human (Wt) and mutated Aβ 1-42 are presented. Data are presented as mean values from two independent experiments.

    Techniques Used: Binding Assay, Sequencing

    3) Product Images from "Androgen supplementation improves some but not all aspects of immune senescence in aged male macaques"

    Article Title: Androgen supplementation improves some but not all aspects of immune senescence in aged male macaques

    Journal: GeroScience

    doi: 10.1007/s11357-017-9979-5

    Humoral and cellular immune responses to MVA and Flu vaccinations. a , b MVA- and H1N1-specific IgG antibody endpoint titers (means ± SEM) were measured by standard ELISA in plasma on days 0, 7, 14, and 28/35 post-vaccinations (#
    Figure Legend Snippet: Humoral and cellular immune responses to MVA and Flu vaccinations. a , b MVA- and H1N1-specific IgG antibody endpoint titers (means ± SEM) were measured by standard ELISA in plasma on days 0, 7, 14, and 28/35 post-vaccinations (#

    Techniques Used: Enzyme-linked Immunosorbent Assay

    4) Product Images from "Differences in nasal immunoglobulin A responses to influenza vaccine strains after live attenuated influenza vaccine (LAIV) immunization in children"

    Article Title: Differences in nasal immunoglobulin A responses to influenza vaccine strains after live attenuated influenza vaccine (LAIV) immunization in children

    Journal: Clinical and Experimental Immunology

    doi: 10.1111/cei.13395

    Relationship between baseline nasal immunoglobulin (Ig)A titre and fold change in nasal IgA response. Nasal IgA titre at baseline was compared to the fold change in response to H1N1 (a), H3N2 (b), B/Bris (c) and B/Phu (d) antigens.
    Figure Legend Snippet: Relationship between baseline nasal immunoglobulin (Ig)A titre and fold change in nasal IgA response. Nasal IgA titre at baseline was compared to the fold change in response to H1N1 (a), H3N2 (b), B/Bris (c) and B/Phu (d) antigens.

    Techniques Used:

    Relationship between age and baseline nasal immunoglobulin (Ig)A. Age was compared to the nasal IgA titre at baseline to H1N1 (a), H3N2 (b), B/Bris (c) and B/Phu (d) antigens for all children.
    Figure Legend Snippet: Relationship between age and baseline nasal immunoglobulin (Ig)A. Age was compared to the nasal IgA titre at baseline to H1N1 (a), H3N2 (b), B/Bris (c) and B/Phu (d) antigens for all children.

    Techniques Used:

    Relationship between baseline nasal immunoglobulin (Ig)A and fold change in haemagglutination inhibition assay (HAI) response. Nasal IgA titre at baseline was compared to the fold change in HAI response to H1N1 (a), H3N2 (b), B/Bris (c) and B/Phu (d) antigens.
    Figure Legend Snippet: Relationship between baseline nasal immunoglobulin (Ig)A and fold change in haemagglutination inhibition assay (HAI) response. Nasal IgA titre at baseline was compared to the fold change in HAI response to H1N1 (a), H3N2 (b), B/Bris (c) and B/Phu (d) antigens.

    Techniques Used: HAI Assay

    5) Product Images from "Host Cell Mimic Polymersomes for Rapid Detection of Highly Pathogenic Influenza Virus via a Viral Fusion and Cell Entry Mechanism"

    Article Title: Host Cell Mimic Polymersomes for Rapid Detection of Highly Pathogenic Influenza Virus via a Viral Fusion and Cell Entry Mechanism

    Journal: Advanced Functional Materials

    doi: 10.1002/adfm.201800960

    FluSome detection of HPAIV and LPAIV in nasal swab specimens and stool specimens, which are labeled as positive and negative samples, respectively. a) Fluorescence spectra of FluSome treated with HPAIV H5N1 and H5N6, and LPAIV H1N1, H2N1, H2N4, H2N4, H3N2, H3N8, H5N2, H5N3, H9N2, Control, and Blank. Lines: Furin at pH 5.0 (orange), furin at pH 7.4 (blue), trypsin at pH 5.0 (green), and trypsin at pH 7.4 (purple). b) Fluorescence intensity based on a). c) Fluorescence images of FluSome treated with HPAIV and LPAIV by IVIS. * indicates shed virus from experimentally infected animal by each virus.
    Figure Legend Snippet: FluSome detection of HPAIV and LPAIV in nasal swab specimens and stool specimens, which are labeled as positive and negative samples, respectively. a) Fluorescence spectra of FluSome treated with HPAIV H5N1 and H5N6, and LPAIV H1N1, H2N1, H2N4, H2N4, H3N2, H3N8, H5N2, H5N3, H9N2, Control, and Blank. Lines: Furin at pH 5.0 (orange), furin at pH 7.4 (blue), trypsin at pH 5.0 (green), and trypsin at pH 7.4 (purple). b) Fluorescence intensity based on a). c) Fluorescence images of FluSome treated with HPAIV and LPAIV by IVIS. * indicates shed virus from experimentally infected animal by each virus.

    Techniques Used: Labeling, Fluorescence, Infection

    Related Articles

    Concentration Assay:

    Article Title: Inflammation and Antiviral Immune Response Associated With Severe Progression of COVID-19
    Article Snippet: .. Indicated concentration of extra cellular domain truncation of SARS-CoV-2 Spike protein (GenScript) or Hemagglutinin/HA protein of Influenza A H1N1 (A/California/04/2009) (Sino Biological) was then added to the cultured PBMCs for 12 h, followed by RNA extraction and qRT-PCR assay. ..

    Cell Culture:

    Article Title: Inflammation and Antiviral Immune Response Associated With Severe Progression of COVID-19
    Article Snippet: .. Indicated concentration of extra cellular domain truncation of SARS-CoV-2 Spike protein (GenScript) or Hemagglutinin/HA protein of Influenza A H1N1 (A/California/04/2009) (Sino Biological) was then added to the cultured PBMCs for 12 h, followed by RNA extraction and qRT-PCR assay. ..

    RNA Extraction:

    Article Title: Inflammation and Antiviral Immune Response Associated With Severe Progression of COVID-19
    Article Snippet: .. Indicated concentration of extra cellular domain truncation of SARS-CoV-2 Spike protein (GenScript) or Hemagglutinin/HA protein of Influenza A H1N1 (A/California/04/2009) (Sino Biological) was then added to the cultured PBMCs for 12 h, followed by RNA extraction and qRT-PCR assay. ..

    Quantitative RT-PCR:

    Article Title: Inflammation and Antiviral Immune Response Associated With Severe Progression of COVID-19
    Article Snippet: .. Indicated concentration of extra cellular domain truncation of SARS-CoV-2 Spike protein (GenScript) or Hemagglutinin/HA protein of Influenza A H1N1 (A/California/04/2009) (Sino Biological) was then added to the cultured PBMCs for 12 h, followed by RNA extraction and qRT-PCR assay. ..

    Sandwich ELISA:

    Article Title: CCR5 Antagonist Maraviroc Inhibits Acute Exacerbation of Lung Inflammation Triggered by Influenza Virus in Cigarette Smoke-Exposed Mice
    Article Snippet: .. Viral load was measured indirectly, through the quantification of viral hemagglutinin (HA) protein present in the middle lobe of the right lung by solid-phase sandwich ELISA using Influenza A H1N1 (A/Puerto Rico/8/1934) HA ELISA Pair Set (Sino Biological) under manufacturer’s protocol as described by [ ]. ..

    Enzyme-linked Immunosorbent Assay:

    Article Title: CCR5 Antagonist Maraviroc Inhibits Acute Exacerbation of Lung Inflammation Triggered by Influenza Virus in Cigarette Smoke-Exposed Mice
    Article Snippet: .. Viral load was measured indirectly, through the quantification of viral hemagglutinin (HA) protein present in the middle lobe of the right lung by solid-phase sandwich ELISA using Influenza A H1N1 (A/Puerto Rico/8/1934) HA ELISA Pair Set (Sino Biological) under manufacturer’s protocol as described by [ ]. ..

    Article Title: The Effects of Aβ1-42 Binding to the SARS-CoV-2 Spike Protein S1 Subunit and Angiotensin-Converting Enzyme 2
    Article Snippet: .. For examining Aβ1-42 binding to viral proteins, His-tagged viral proteins, including the S1 of SARS-CoV-2, HA of H1N1, and S1 of MERS-CoV purchased from Sino Biological (Wayne, PA, USA), were immobilized at 125 ng (100 µL) per well in a 96-well ELISA plate (Thermo Fisher Scientific, Waltham, MA, USA) at 4 °C for overnight incubation. ..

    Binding Assay:

    Article Title: The Effects of Aβ1-42 Binding to the SARS-CoV-2 Spike Protein S1 Subunit and Angiotensin-Converting Enzyme 2
    Article Snippet: .. For examining Aβ1-42 binding to viral proteins, His-tagged viral proteins, including the S1 of SARS-CoV-2, HA of H1N1, and S1 of MERS-CoV purchased from Sino Biological (Wayne, PA, USA), were immobilized at 125 ng (100 µL) per well in a 96-well ELISA plate (Thermo Fisher Scientific, Waltham, MA, USA) at 4 °C for overnight incubation. ..

    Incubation:

    Article Title: The Effects of Aβ1-42 Binding to the SARS-CoV-2 Spike Protein S1 Subunit and Angiotensin-Converting Enzyme 2
    Article Snippet: .. For examining Aβ1-42 binding to viral proteins, His-tagged viral proteins, including the S1 of SARS-CoV-2, HA of H1N1, and S1 of MERS-CoV purchased from Sino Biological (Wayne, PA, USA), were immobilized at 125 ng (100 µL) per well in a 96-well ELISA plate (Thermo Fisher Scientific, Waltham, MA, USA) at 4 °C for overnight incubation. ..

    other:

    Article Title: Inflammation and Antiviral Immune Response Associated With Severe Progression of COVID-19
    Article Snippet: We also stimulated PBMCs from healthy subjects with either an extra-cellular domain truncation of SARS-CoV-2 Spike protein (Spike-ECD) or Hemagglutinin (HA) protein from Influenza A H1N1 (A/California/04/2009), both of which belong to the major immunity-eliciting antigens and mediate the invasion of virus to host cells.

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    Sino Biological pr8
    Heterosubtypic protection against H1 virus by anti–MHCII-MIX vaccination is independent of T cells. ( A and B ) BALB/c mice ( n = 7 per group) were immunized three times (weeks 0, 4, and 7) with 25 μg of DNA/EP of the indicated vaccines. Sera from mice of each group were harvested 2 wk after the third vaccination and transferred i.v. to naive mice ( n = 10 per group) before a lethal challenge with 5 × LD 50 <t>PR8.</t> Mice were monitored for weight loss (A) and survival (B). The results are representative of two independent experiments. ( C and D ) BALB/c mice ( n = 10) were immunized three times (weeks 0, 4, and 7) with 25 μg of DNA/EP of the indicated vaccines and subsequently were injected every other day from day 12 after the third vaccination with depleting mAbs against CD4 and CD8 T cells or isotype-matched control mAbs. A 5 × LD 50 lethal dose of PR8 was administered at day 14, and the mice were monitored for weight loss (C) and survival (D). Efficient depletion was confirmed by staining of splenocytes. * p
    Pr8, supplied by Sino Biological, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pr8/product/Sino Biological
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    pr8 - by Bioz Stars, 2021-09
    94/100 stars
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    93
    Sino Biological rabbit anti ha polyclonal antibody
    M2 interacts with TRAPPC6A and TRAPPC6AΔ in mammalian cells. (A to C) Plasmids expressing TRAPPC6A-Myc and Flag-SC09M2 (A), TRAPPC6A-Myc and Flag-AH05M2 (B), or TRAPPC6A-Myc and Flag-WSNM2 (C) were transfected individually or in combination, as indicated, in HEK293T cells. Forty-eight hours after transfection, cell lysates were immunoprecipitated with a mouse anti-Flag MAb or a mouse anti-Myc MAb and were subjected to Western blotting with a rabbit anti-Flag <t>polyclonal</t> antibody or a rabbit anti-Myc polyclonal antibody to reveal the presence of M2 and TRAPPC6A, respectively. (D) Western blotting of proteins bound to GST alone or to GST-TRAPPC6A. HEK293T cells transfected with pCAGGS-SC09M2 or with the pCAGGS vector were lysed with IP buffer, and the lysate was incubated with purified GST or GST-TRAPPC6A and then subjected to a pulldown assay. Equal volumes of proteins bound to the beads and the original cell lysates (5% of the input) were examined by Western blotting using a mouse anti-M2 MAb or a mouse anti-actin MAb, respectively. The GST-tagged proteins in the eluates were detected by Coomassie blue (CB) staining. (E) Confocal analysis of the distribution of M2 and TRAPPC6A proteins in A549 cells. pCAGGS-TRAPPC6A-myc and pCAGGS-Flag-SC09M2 were transfected individually or in combination into A549 cells and assessed by immunofluorescence staining. IAV M2 was detected with a mouse anti-Flag MAb and visualized with Alexa Fluor 488 (green). TRAPPC6A was detected with a rabbit anti-Myc polyclonal antibody and visualized with Alexa Fluor 546 (red). Yellow indicates colocalization of Alexa Fluor 546 and 488 in the merged image. (F) pCAGGS-TRAPPC6A-myc was cotransfected with pEGFP-C1 or pEGFP-C1-BM2 into HEK293T cells for 48 h before the cells were lysed. Following immunoprecipitation of the cell lysates with a mouse anti-GFP MAb, the immunoprecipitates were analyzed by Western blotting using a rabbit anti-GFP polyclonal antibody or a rabbit anti-Myc polyclonal antibody to reveal the presence of BM2 and TRAPPC6A, respectively. (G) Co-IP of M2 and TRAPPC6AΔ. pCAGGS-Flag-SC09M2 was cotransfected with pCAGGS-TRAPPC6A-myc or pCAGGS-TRAPPC6AΔ-myc into HEK293T cells. Forty-eight hours after transfection, cell lysates were immunoprecipitated with a mouse anti-Myc MAb and subjected to Western blotting with a rabbit anti-Flag polyclonal antibody or a rabbit anti-Myc polyclonal antibody to reveal the presence of M2 and TRAPPC6A or TRAPPC6AΔ, respectively.
    Rabbit Anti Ha Polyclonal Antibody, supplied by Sino Biological, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit anti ha polyclonal antibody/product/Sino Biological
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
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    95
    Sino Biological h1n1
    Oligoclonal mAb Response Protects Mice from Group 1 or Group 2 Influenza Challenges BALB/c mice (n = 20) were pretreated with 20 U of hyaluronidase and administered 10 μg of H/L gWiz pDNA per anti-influenza mAb (C179, S139/1, and 9H10) in muscles of separate limbs followed by EP. For the control group, BALB/c mice (n = 15) were pretreated with 20 U of hyaluronidase and administered 10 μg of H/L gWiz pDNA per anti-Ebola mAb (2G4, 4G7, and 13C6) at distinct sites with EP. Blood was collected on days 12, 19, and 33 (in surviving mice) for pharmacokinetic analyses ( Figure S5 ). (A and B) On day 23, the mice were randomized and anti-influenza DNA/EP mice (n = 10) and anti-Ebola DNA/EP mice (n = 7–8) were challenged with (A) 39 MLD 50 of A/WSN/33 <t>(H1N1)</t> or (B) 21 MLD 50 of A/Aichi/2/68 (H3N2) delivered intranasally. (A and B) Mean weight loss (%) ± SEM compared with day 0 (left) and survival (%) as depicted in Kaplan-Meier plots (right) following (A) H1N1 and (B) H3N2 challenge. For percent weight loss graphs, **p = 0.0021, ***p
    H1n1, supplied by Sino Biological, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Sino Biological pr8 np protein
    “Late” CD4 + T cell help shapes respiratory mucosal memory CD8 + and B cell immunity. WT mice were infected with <t>PR8</t> and treated with ctl IgG or α-CD4. a , The efficiency of CD4 + T cell depletion in the lung or spleen. b , Representative image of lung section stained with H E. Black arrows indicate tertial lymphoid structure. c , Representative dot plot and cell numbers of lung germinal center B (B GC ) cells. d , Frequencies of lung circulating (CD45 i.v. + ) or parenchymal (CD45 i.v. - ) CD8 + T or B cells in mice treated with control IgG or α-CD4. e , Influenza HA-specific B cells (HA-B) in the lungs were identified through HA antigen staining. f , Lung tissue or circulating CD8 + NP 366-374 or PA 224-233 T cells were identified through CD45 i.v. staining and analyzed by flow cytometry. g , Lung circulating CD8 + NP 366-374 or CD8 + PA 224-233 T cells were enumerated. i , Histogram of CD103 expression or frequency of CD103 + cells within CD8 + CD69 + NP 366-374 T RM cells. h, j , Gating strategies of CD8 + T RM ( h ) or B RM ( j ) cells in the lung. Representative data were from at least two independent experiments (4-5 mice per group). P values were calculated by unpaired two-tailed Student’s t-test.
    Pr8 Np Protein, supplied by Sino Biological, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Heterosubtypic protection against H1 virus by anti–MHCII-MIX vaccination is independent of T cells. ( A and B ) BALB/c mice ( n = 7 per group) were immunized three times (weeks 0, 4, and 7) with 25 μg of DNA/EP of the indicated vaccines. Sera from mice of each group were harvested 2 wk after the third vaccination and transferred i.v. to naive mice ( n = 10 per group) before a lethal challenge with 5 × LD 50 PR8. Mice were monitored for weight loss (A) and survival (B). The results are representative of two independent experiments. ( C and D ) BALB/c mice ( n = 10) were immunized three times (weeks 0, 4, and 7) with 25 μg of DNA/EP of the indicated vaccines and subsequently were injected every other day from day 12 after the third vaccination with depleting mAbs against CD4 and CD8 T cells or isotype-matched control mAbs. A 5 × LD 50 lethal dose of PR8 was administered at day 14, and the mice were monitored for weight loss (C) and survival (D). Efficient depletion was confirmed by staining of splenocytes. * p

    Journal: The Journal of Immunology Author Choice

    Article Title: Simultaneous Targeting of Multiple Hemagglutinins to APCs for Induction of Broad Immunity against Influenza

    doi: 10.4049/jimmunol.1701088

    Figure Lengend Snippet: Heterosubtypic protection against H1 virus by anti–MHCII-MIX vaccination is independent of T cells. ( A and B ) BALB/c mice ( n = 7 per group) were immunized three times (weeks 0, 4, and 7) with 25 μg of DNA/EP of the indicated vaccines. Sera from mice of each group were harvested 2 wk after the third vaccination and transferred i.v. to naive mice ( n = 10 per group) before a lethal challenge with 5 × LD 50 PR8. Mice were monitored for weight loss (A) and survival (B). The results are representative of two independent experiments. ( C and D ) BALB/c mice ( n = 10) were immunized three times (weeks 0, 4, and 7) with 25 μg of DNA/EP of the indicated vaccines and subsequently were injected every other day from day 12 after the third vaccination with depleting mAbs against CD4 and CD8 T cells or isotype-matched control mAbs. A 5 × LD 50 lethal dose of PR8 was administered at day 14, and the mice were monitored for weight loss (C) and survival (D). Efficient depletion was confirmed by staining of splenocytes. * p

    Article Snippet: ELISA 96-well plates (Costar 3590) were coated with one of the following recombinant influenza HA proteins: PR8 (11684-V08H; Sino Biological), A/Hong Kong/483/97 (H5N1) (11689-V08H; Sino Biological), A/northern shoveler/California/HKWF115/07 (H6N1) (MBS434125; MyBioSource, San Diego, CA, or 11723-V08H; Sino Biological), A/pintail duck/Alberta/114/1979 (H8N4) (11722-V08H; Sino Biological), A/Hong Kong/1073/99 (H9N2) (11229-V08H; Sino Biological), A/duck/Yangzhou/906/2002 (H11N2) (11705-V08H; Sino Biological), A/black-headed gull/Netherlands/1/00 (H13N8) (11721-V08H; Sino Biological), A/Shanghai/1/2013 (H7N9) (40104-V08B; Sino Biological), or A/Hong Kong/1/1968 (H3N2) (40116-V08B; Sino Biological).

    Techniques: Mouse Assay, Injection, Staining

    Vaccination with anti–MHCII-MIX confers heterosubtypic protection against H1 influenza viruses not included in the vaccine mixture. ( A – F ) BALB/c mice ( n = 6 per group) were immunized three times (weeks 0, 4, and 12, arrows) with 25 μg of DNA/EP of the indicated vaccines. As positive controls, anti–MHCII-H1(PR8) and anti–MHCII-H1(Cal07) were included. Serum IgG Abs were measured against PR8 (A) or Cal07 (B) in ELISAs (mean ± SEM). Microneutralization assays were performed on sera harvested 2 wk after the third vaccination with PR8 (C) and Cal07 (D). The dashed line indicates 50% threshold for positive neutralization. Two weeks after the third vaccination (above), mice were challenged with a lethal dose of Cal07 and monitored for weight loss (± SEM) (E) and survival (F). ( G and H ) In a new experiment, mice were vaccinated at weeks 0, 4, and 12 (as above) and challenged with a lethal dose of influenza PR8 2 wk after the third vaccination ( n = 10 per group [black], n = 6 per group [gray]). Mice were monitored for weight loss (± SEM) (G) and survival (H). * p

    Journal: The Journal of Immunology Author Choice

    Article Title: Simultaneous Targeting of Multiple Hemagglutinins to APCs for Induction of Broad Immunity against Influenza

    doi: 10.4049/jimmunol.1701088

    Figure Lengend Snippet: Vaccination with anti–MHCII-MIX confers heterosubtypic protection against H1 influenza viruses not included in the vaccine mixture. ( A – F ) BALB/c mice ( n = 6 per group) were immunized three times (weeks 0, 4, and 12, arrows) with 25 μg of DNA/EP of the indicated vaccines. As positive controls, anti–MHCII-H1(PR8) and anti–MHCII-H1(Cal07) were included. Serum IgG Abs were measured against PR8 (A) or Cal07 (B) in ELISAs (mean ± SEM). Microneutralization assays were performed on sera harvested 2 wk after the third vaccination with PR8 (C) and Cal07 (D). The dashed line indicates 50% threshold for positive neutralization. Two weeks after the third vaccination (above), mice were challenged with a lethal dose of Cal07 and monitored for weight loss (± SEM) (E) and survival (F). ( G and H ) In a new experiment, mice were vaccinated at weeks 0, 4, and 12 (as above) and challenged with a lethal dose of influenza PR8 2 wk after the third vaccination ( n = 10 per group [black], n = 6 per group [gray]). Mice were monitored for weight loss (± SEM) (G) and survival (H). * p

    Article Snippet: ELISA 96-well plates (Costar 3590) were coated with one of the following recombinant influenza HA proteins: PR8 (11684-V08H; Sino Biological), A/Hong Kong/483/97 (H5N1) (11689-V08H; Sino Biological), A/northern shoveler/California/HKWF115/07 (H6N1) (MBS434125; MyBioSource, San Diego, CA, or 11723-V08H; Sino Biological), A/pintail duck/Alberta/114/1979 (H8N4) (11722-V08H; Sino Biological), A/Hong Kong/1073/99 (H9N2) (11229-V08H; Sino Biological), A/duck/Yangzhou/906/2002 (H11N2) (11705-V08H; Sino Biological), A/black-headed gull/Netherlands/1/00 (H13N8) (11721-V08H; Sino Biological), A/Shanghai/1/2013 (H7N9) (40104-V08B; Sino Biological), or A/Hong Kong/1/1968 (H3N2) (40116-V08B; Sino Biological).

    Techniques: Mouse Assay, Neutralization

    Characterization of vaccine proteins. ( A ) The MHCII-targeted vaccine proteins consist of a targeting unit (scFv) that is specific for MHC class II (I), a dimerization unit containing a shortened hinge and C H 3 from human γ3 (II), and an antigenic unit encoding one of the six indicated HAs (III). Nontargeted controls have been prepared by replacement of the targeting unit with an scFv that is specific for the hapten NIP. ( B ) Supernatants from HEK 293E cells transfected with the DNA plasmids encoding the indicated vaccines were analyzed in sandwich ELISAs. An MHCII-targeted vaccine encoding HA from PR8 (anti–MHCII-H1) was included as positive control.

    Journal: The Journal of Immunology Author Choice

    Article Title: Simultaneous Targeting of Multiple Hemagglutinins to APCs for Induction of Broad Immunity against Influenza

    doi: 10.4049/jimmunol.1701088

    Figure Lengend Snippet: Characterization of vaccine proteins. ( A ) The MHCII-targeted vaccine proteins consist of a targeting unit (scFv) that is specific for MHC class II (I), a dimerization unit containing a shortened hinge and C H 3 from human γ3 (II), and an antigenic unit encoding one of the six indicated HAs (III). Nontargeted controls have been prepared by replacement of the targeting unit with an scFv that is specific for the hapten NIP. ( B ) Supernatants from HEK 293E cells transfected with the DNA plasmids encoding the indicated vaccines were analyzed in sandwich ELISAs. An MHCII-targeted vaccine encoding HA from PR8 (anti–MHCII-H1) was included as positive control.

    Article Snippet: ELISA 96-well plates (Costar 3590) were coated with one of the following recombinant influenza HA proteins: PR8 (11684-V08H; Sino Biological), A/Hong Kong/483/97 (H5N1) (11689-V08H; Sino Biological), A/northern shoveler/California/HKWF115/07 (H6N1) (MBS434125; MyBioSource, San Diego, CA, or 11723-V08H; Sino Biological), A/pintail duck/Alberta/114/1979 (H8N4) (11722-V08H; Sino Biological), A/Hong Kong/1073/99 (H9N2) (11229-V08H; Sino Biological), A/duck/Yangzhou/906/2002 (H11N2) (11705-V08H; Sino Biological), A/black-headed gull/Netherlands/1/00 (H13N8) (11721-V08H; Sino Biological), A/Shanghai/1/2013 (H7N9) (40104-V08B; Sino Biological), or A/Hong Kong/1/1968 (H3N2) (40116-V08B; Sino Biological).

    Techniques: Transfection, Positive Control

    M2 interacts with TRAPPC6A and TRAPPC6AΔ in mammalian cells. (A to C) Plasmids expressing TRAPPC6A-Myc and Flag-SC09M2 (A), TRAPPC6A-Myc and Flag-AH05M2 (B), or TRAPPC6A-Myc and Flag-WSNM2 (C) were transfected individually or in combination, as indicated, in HEK293T cells. Forty-eight hours after transfection, cell lysates were immunoprecipitated with a mouse anti-Flag MAb or a mouse anti-Myc MAb and were subjected to Western blotting with a rabbit anti-Flag polyclonal antibody or a rabbit anti-Myc polyclonal antibody to reveal the presence of M2 and TRAPPC6A, respectively. (D) Western blotting of proteins bound to GST alone or to GST-TRAPPC6A. HEK293T cells transfected with pCAGGS-SC09M2 or with the pCAGGS vector were lysed with IP buffer, and the lysate was incubated with purified GST or GST-TRAPPC6A and then subjected to a pulldown assay. Equal volumes of proteins bound to the beads and the original cell lysates (5% of the input) were examined by Western blotting using a mouse anti-M2 MAb or a mouse anti-actin MAb, respectively. The GST-tagged proteins in the eluates were detected by Coomassie blue (CB) staining. (E) Confocal analysis of the distribution of M2 and TRAPPC6A proteins in A549 cells. pCAGGS-TRAPPC6A-myc and pCAGGS-Flag-SC09M2 were transfected individually or in combination into A549 cells and assessed by immunofluorescence staining. IAV M2 was detected with a mouse anti-Flag MAb and visualized with Alexa Fluor 488 (green). TRAPPC6A was detected with a rabbit anti-Myc polyclonal antibody and visualized with Alexa Fluor 546 (red). Yellow indicates colocalization of Alexa Fluor 546 and 488 in the merged image. (F) pCAGGS-TRAPPC6A-myc was cotransfected with pEGFP-C1 or pEGFP-C1-BM2 into HEK293T cells for 48 h before the cells were lysed. Following immunoprecipitation of the cell lysates with a mouse anti-GFP MAb, the immunoprecipitates were analyzed by Western blotting using a rabbit anti-GFP polyclonal antibody or a rabbit anti-Myc polyclonal antibody to reveal the presence of BM2 and TRAPPC6A, respectively. (G) Co-IP of M2 and TRAPPC6AΔ. pCAGGS-Flag-SC09M2 was cotransfected with pCAGGS-TRAPPC6A-myc or pCAGGS-TRAPPC6AΔ-myc into HEK293T cells. Forty-eight hours after transfection, cell lysates were immunoprecipitated with a mouse anti-Myc MAb and subjected to Western blotting with a rabbit anti-Flag polyclonal antibody or a rabbit anti-Myc polyclonal antibody to reveal the presence of M2 and TRAPPC6A or TRAPPC6AΔ, respectively.

    Journal: Journal of Virology

    Article Title: Host Cellular Protein TRAPPC6AΔ Interacts with Influenza A Virus M2 Protein and Regulates Viral Propagation by Modulating M2 Trafficking

    doi: 10.1128/JVI.01757-16

    Figure Lengend Snippet: M2 interacts with TRAPPC6A and TRAPPC6AΔ in mammalian cells. (A to C) Plasmids expressing TRAPPC6A-Myc and Flag-SC09M2 (A), TRAPPC6A-Myc and Flag-AH05M2 (B), or TRAPPC6A-Myc and Flag-WSNM2 (C) were transfected individually or in combination, as indicated, in HEK293T cells. Forty-eight hours after transfection, cell lysates were immunoprecipitated with a mouse anti-Flag MAb or a mouse anti-Myc MAb and were subjected to Western blotting with a rabbit anti-Flag polyclonal antibody or a rabbit anti-Myc polyclonal antibody to reveal the presence of M2 and TRAPPC6A, respectively. (D) Western blotting of proteins bound to GST alone or to GST-TRAPPC6A. HEK293T cells transfected with pCAGGS-SC09M2 or with the pCAGGS vector were lysed with IP buffer, and the lysate was incubated with purified GST or GST-TRAPPC6A and then subjected to a pulldown assay. Equal volumes of proteins bound to the beads and the original cell lysates (5% of the input) were examined by Western blotting using a mouse anti-M2 MAb or a mouse anti-actin MAb, respectively. The GST-tagged proteins in the eluates were detected by Coomassie blue (CB) staining. (E) Confocal analysis of the distribution of M2 and TRAPPC6A proteins in A549 cells. pCAGGS-TRAPPC6A-myc and pCAGGS-Flag-SC09M2 were transfected individually or in combination into A549 cells and assessed by immunofluorescence staining. IAV M2 was detected with a mouse anti-Flag MAb and visualized with Alexa Fluor 488 (green). TRAPPC6A was detected with a rabbit anti-Myc polyclonal antibody and visualized with Alexa Fluor 546 (red). Yellow indicates colocalization of Alexa Fluor 546 and 488 in the merged image. (F) pCAGGS-TRAPPC6A-myc was cotransfected with pEGFP-C1 or pEGFP-C1-BM2 into HEK293T cells for 48 h before the cells were lysed. Following immunoprecipitation of the cell lysates with a mouse anti-GFP MAb, the immunoprecipitates were analyzed by Western blotting using a rabbit anti-GFP polyclonal antibody or a rabbit anti-Myc polyclonal antibody to reveal the presence of BM2 and TRAPPC6A, respectively. (G) Co-IP of M2 and TRAPPC6AΔ. pCAGGS-Flag-SC09M2 was cotransfected with pCAGGS-TRAPPC6A-myc or pCAGGS-TRAPPC6AΔ-myc into HEK293T cells. Forty-eight hours after transfection, cell lysates were immunoprecipitated with a mouse anti-Myc MAb and subjected to Western blotting with a rabbit anti-Flag polyclonal antibody or a rabbit anti-Myc polyclonal antibody to reveal the presence of M2 and TRAPPC6A or TRAPPC6AΔ, respectively.

    Article Snippet: The following primary antibodies were obtained from commercial sources: rabbit anti-Flag polyclonal antibody (F7425; Sigma-Aldrich), mouse anti-Flag monoclonal antibody (F3165; Sigma-Aldrich), rabbit anti-Myc polyclonal antibody (C3965; Sigma-Aldrich), mouse anti-Myc monoclonal antibody (M4439; Sigma-Aldrich), mouse anti-actin monoclonal antibody (sc-47778; Santa Cruz, Dallas, TX), rabbit anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) polyclonal antibody (10494-1-AP; Proteintech, Chicago, IL), rabbit anti-GFP polyclonal antibody (AG279; Beyotime Biotech, Shanghai, China), mouse anti-GFP monoclonal antibody (ab1218; Abcam, Cambridge, MA), mouse anti-M2 monoclonal antibody (ab5416; Abcam), rabbit anti-M2 polyclonal antibody (GTX125951; GeneTex, Irvine, CA), rabbit anti-HA polyclonal antibody (11692-T54; Sino Biological Inc., Beijing, China), rabbit anti-FGF2 monoclonal antibody (ab92337; Abcam), mouse anti-LAMP1 monoclonal antibody (ab25630; Abcam), and mouse anti-Giantin monoclonal antibody (ab37266; Abcam).

    Techniques: Expressing, Transfection, Immunoprecipitation, Western Blot, Plasmid Preparation, Incubation, Purification, Staining, Immunofluorescence, Co-Immunoprecipitation Assay

    A leucine residue at position 96 of M2 is required for the TRAPPC6A interaction. (A) pCAGGS-TRAPPC6A-myc was cotransfected with pEGFP-C1, pEGFP-C1-SC09 M2, pEGFP-C1-SC09 M2EDTM, or pEGFP-C1-SC09 M2CT into HEK293T cells for 48 h before preparation for cell lysates. Following immunoprecipitation with a mouse anti-GFP MAb, the immunoprecipitates were analyzed by Western blotting using a rabbit anti-GFP polyclonal antibody and a rabbit anti-Myc polyclonal antibody to reveal the presence of M2 and TRAPPC6A, respectively. (B and C) Plasmids expressing TRAPPC6A-myc and Flag-SC09M2 or Flag-SC09M2 with different amino acid deletions in the C terminus were cotransfected into HEK293T cells for 48 h before the preparation of cell lysates. Following immunoprecipitation with a mouse anti-Flag MAb, the immunoprecipitates were analyzed by Western blotting using a rabbit anti-Flag polyclonal antibody or a rabbit anti-Myc polyclonal antibody to reveal the presence of M2 and TRAPPC6A, respectively.

    Journal: Journal of Virology

    Article Title: Host Cellular Protein TRAPPC6AΔ Interacts with Influenza A Virus M2 Protein and Regulates Viral Propagation by Modulating M2 Trafficking

    doi: 10.1128/JVI.01757-16

    Figure Lengend Snippet: A leucine residue at position 96 of M2 is required for the TRAPPC6A interaction. (A) pCAGGS-TRAPPC6A-myc was cotransfected with pEGFP-C1, pEGFP-C1-SC09 M2, pEGFP-C1-SC09 M2EDTM, or pEGFP-C1-SC09 M2CT into HEK293T cells for 48 h before preparation for cell lysates. Following immunoprecipitation with a mouse anti-GFP MAb, the immunoprecipitates were analyzed by Western blotting using a rabbit anti-GFP polyclonal antibody and a rabbit anti-Myc polyclonal antibody to reveal the presence of M2 and TRAPPC6A, respectively. (B and C) Plasmids expressing TRAPPC6A-myc and Flag-SC09M2 or Flag-SC09M2 with different amino acid deletions in the C terminus were cotransfected into HEK293T cells for 48 h before the preparation of cell lysates. Following immunoprecipitation with a mouse anti-Flag MAb, the immunoprecipitates were analyzed by Western blotting using a rabbit anti-Flag polyclonal antibody or a rabbit anti-Myc polyclonal antibody to reveal the presence of M2 and TRAPPC6A, respectively.

    Article Snippet: The following primary antibodies were obtained from commercial sources: rabbit anti-Flag polyclonal antibody (F7425; Sigma-Aldrich), mouse anti-Flag monoclonal antibody (F3165; Sigma-Aldrich), rabbit anti-Myc polyclonal antibody (C3965; Sigma-Aldrich), mouse anti-Myc monoclonal antibody (M4439; Sigma-Aldrich), mouse anti-actin monoclonal antibody (sc-47778; Santa Cruz, Dallas, TX), rabbit anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) polyclonal antibody (10494-1-AP; Proteintech, Chicago, IL), rabbit anti-GFP polyclonal antibody (AG279; Beyotime Biotech, Shanghai, China), mouse anti-GFP monoclonal antibody (ab1218; Abcam, Cambridge, MA), mouse anti-M2 monoclonal antibody (ab5416; Abcam), rabbit anti-M2 polyclonal antibody (GTX125951; GeneTex, Irvine, CA), rabbit anti-HA polyclonal antibody (11692-T54; Sino Biological Inc., Beijing, China), rabbit anti-FGF2 monoclonal antibody (ab92337; Abcam), mouse anti-LAMP1 monoclonal antibody (ab25630; Abcam), and mouse anti-Giantin monoclonal antibody (ab37266; Abcam).

    Techniques: Immunoprecipitation, Western Blot, Expressing

    Dynamics of the interaction of M2 and TRAPPC6AΔ in wt and mutant WSN virus-infected cells. A549 cells were infected with wt influenza virus WSN (A), or one of the M2 deletion mutants WSN M2Del1 (B) and WSN M2Del2 (C), at an MOI of 5. At 4, 6, 8, 10, and 14 h p.i., the infected cells were fixed and stained with mouse anti-M2 MAb 14C2 and rabbit anti-TRAPPC6A polyclonal antibody, followed by incubation with Alexa Fluor 488 donkey anti-mouse IgG(H+L) (green) and Alexa Fluor 546 donkey anti-rabbit IgG(H+L) (red). Nuclei were stained with DAPI.

    Journal: Journal of Virology

    Article Title: Host Cellular Protein TRAPPC6AΔ Interacts with Influenza A Virus M2 Protein and Regulates Viral Propagation by Modulating M2 Trafficking

    doi: 10.1128/JVI.01757-16

    Figure Lengend Snippet: Dynamics of the interaction of M2 and TRAPPC6AΔ in wt and mutant WSN virus-infected cells. A549 cells were infected with wt influenza virus WSN (A), or one of the M2 deletion mutants WSN M2Del1 (B) and WSN M2Del2 (C), at an MOI of 5. At 4, 6, 8, 10, and 14 h p.i., the infected cells were fixed and stained with mouse anti-M2 MAb 14C2 and rabbit anti-TRAPPC6A polyclonal antibody, followed by incubation with Alexa Fluor 488 donkey anti-mouse IgG(H+L) (green) and Alexa Fluor 546 donkey anti-rabbit IgG(H+L) (red). Nuclei were stained with DAPI.

    Article Snippet: The following primary antibodies were obtained from commercial sources: rabbit anti-Flag polyclonal antibody (F7425; Sigma-Aldrich), mouse anti-Flag monoclonal antibody (F3165; Sigma-Aldrich), rabbit anti-Myc polyclonal antibody (C3965; Sigma-Aldrich), mouse anti-Myc monoclonal antibody (M4439; Sigma-Aldrich), mouse anti-actin monoclonal antibody (sc-47778; Santa Cruz, Dallas, TX), rabbit anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) polyclonal antibody (10494-1-AP; Proteintech, Chicago, IL), rabbit anti-GFP polyclonal antibody (AG279; Beyotime Biotech, Shanghai, China), mouse anti-GFP monoclonal antibody (ab1218; Abcam, Cambridge, MA), mouse anti-M2 monoclonal antibody (ab5416; Abcam), rabbit anti-M2 polyclonal antibody (GTX125951; GeneTex, Irvine, CA), rabbit anti-HA polyclonal antibody (11692-T54; Sino Biological Inc., Beijing, China), rabbit anti-FGF2 monoclonal antibody (ab92337; Abcam), mouse anti-LAMP1 monoclonal antibody (ab25630; Abcam), and mouse anti-Giantin monoclonal antibody (ab37266; Abcam).

    Techniques: Mutagenesis, Infection, Staining, Incubation

    Mutation at position 96 of M2 affects its interaction with TRAPPC6A. (A) Sequence analysis of IAV M2 at position 96. All of the IAV M2 sequences deposited in GenBank by 6 July 2014 were downloaded. The identity of the amino acids at position 96 was statistically analyzed. (B) Plasmids expressing TRAPPC6A-Myc and Flag-WSNM2 or Flag-WSNM2 with different mutations at position 96 were cotransfected into HEK293T cells for 48 h before the preparation of cell lysates. Following immunoprecipitation with a mouse anti-Flag MAb, the immunoprecipitates were analyzed by Western blotting using a rabbit anti-Flag polyclonal antibody or a rabbit anti-Myc polyclonal antibody to reveal the presence of M2 and TRAPPC6A, respectively.

    Journal: Journal of Virology

    Article Title: Host Cellular Protein TRAPPC6AΔ Interacts with Influenza A Virus M2 Protein and Regulates Viral Propagation by Modulating M2 Trafficking

    doi: 10.1128/JVI.01757-16

    Figure Lengend Snippet: Mutation at position 96 of M2 affects its interaction with TRAPPC6A. (A) Sequence analysis of IAV M2 at position 96. All of the IAV M2 sequences deposited in GenBank by 6 July 2014 were downloaded. The identity of the amino acids at position 96 was statistically analyzed. (B) Plasmids expressing TRAPPC6A-Myc and Flag-WSNM2 or Flag-WSNM2 with different mutations at position 96 were cotransfected into HEK293T cells for 48 h before the preparation of cell lysates. Following immunoprecipitation with a mouse anti-Flag MAb, the immunoprecipitates were analyzed by Western blotting using a rabbit anti-Flag polyclonal antibody or a rabbit anti-Myc polyclonal antibody to reveal the presence of M2 and TRAPPC6A, respectively.

    Article Snippet: The following primary antibodies were obtained from commercial sources: rabbit anti-Flag polyclonal antibody (F7425; Sigma-Aldrich), mouse anti-Flag monoclonal antibody (F3165; Sigma-Aldrich), rabbit anti-Myc polyclonal antibody (C3965; Sigma-Aldrich), mouse anti-Myc monoclonal antibody (M4439; Sigma-Aldrich), mouse anti-actin monoclonal antibody (sc-47778; Santa Cruz, Dallas, TX), rabbit anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) polyclonal antibody (10494-1-AP; Proteintech, Chicago, IL), rabbit anti-GFP polyclonal antibody (AG279; Beyotime Biotech, Shanghai, China), mouse anti-GFP monoclonal antibody (ab1218; Abcam, Cambridge, MA), mouse anti-M2 monoclonal antibody (ab5416; Abcam), rabbit anti-M2 polyclonal antibody (GTX125951; GeneTex, Irvine, CA), rabbit anti-HA polyclonal antibody (11692-T54; Sino Biological Inc., Beijing, China), rabbit anti-FGF2 monoclonal antibody (ab92337; Abcam), mouse anti-LAMP1 monoclonal antibody (ab25630; Abcam), and mouse anti-Giantin monoclonal antibody (ab37266; Abcam).

    Techniques: Mutagenesis, Sequencing, Expressing, Immunoprecipitation, Western Blot

    Effect of modulation of TRAPPC6AΔ expression on the cell surface expression of viral and cellular proteins. (A) A549 cells were transfected with siRNA targeting TRAPPC6AΔ or with nontargeting siRNA for 48 h and were then infected with the WSN virus at an MOI of 3. Cell lysates were processed at 8 and 10 h p.i. and subjected to Western blotting using a mouse anti-M2 MAb to detect the expression level of M2. (B) A549 cells were treated with siRNA and infected with the WSN virus as described above for panel A. Cells were fixed at 8 and 10 h p.i., left nonpermeabilized, and stained with the mouse anti-M2 MAb and Alexa Fluor 488-conjugated donkey anti-mouse IgG(H+L) for M2 surface expression analysis by flow cytometry. The graph shows the fluorescence intensity of M2 surface expression. (C) The TRAPPC6AΔ-overexpressing A549 cell line or the A549 control cell line transduced with an empty retrovirus was infected with the WSN virus at an MOI of 3. Cell lysates were processed at 8 and 10 h p.i. and subjected to Western blotting using a mouse anti-M2 MAb to detect the expression level of M2. (D) The TRAPPC6AΔ-overexpressing A549 cell line or the A549 control cell line transduced with an empty retrovirus was infected with the WSN virus at an MOI of 3. The cell surface expression of M2 was analyzed by flow cytometry at 8 and 10 h p.i. as described above for panel B. (E) A549 cells were treated with siRNA and infected with the WSN virus as described above for panel A. The cell surface expression of HA was analyzed by flow cytometry at 8 and 10 h p.i. as described above for panel B by using the rabbit anti-HA polyclonal antibody and Alexa Fluor 488-conjugated goat anti-rabbit IgG(H+L). (F) A549 cells were treated with siRNA and infected with the WSN virus as described above for panel A. The cell surface expression of FGF2 was analyzed by flow cytometry at 8 and 10 h p.i. as described above for panel B by using the rabbit anti-FGF2 MAb and Alexa Fluor 488-conjugated goat anti-rabbit IgG(H+L). (G) A549 cells were treated with siRNA and infected with the WSN virus as described above for panel A. At 2 h p.i., the culture medium was replaced with medium supplemented with 25 μM amantadine. The cell surface expression of M2 was analyzed by flow cytometry at 8 and 10 h p.i. as described above for panel B.

    Journal: Journal of Virology

    Article Title: Host Cellular Protein TRAPPC6AΔ Interacts with Influenza A Virus M2 Protein and Regulates Viral Propagation by Modulating M2 Trafficking

    doi: 10.1128/JVI.01757-16

    Figure Lengend Snippet: Effect of modulation of TRAPPC6AΔ expression on the cell surface expression of viral and cellular proteins. (A) A549 cells were transfected with siRNA targeting TRAPPC6AΔ or with nontargeting siRNA for 48 h and were then infected with the WSN virus at an MOI of 3. Cell lysates were processed at 8 and 10 h p.i. and subjected to Western blotting using a mouse anti-M2 MAb to detect the expression level of M2. (B) A549 cells were treated with siRNA and infected with the WSN virus as described above for panel A. Cells were fixed at 8 and 10 h p.i., left nonpermeabilized, and stained with the mouse anti-M2 MAb and Alexa Fluor 488-conjugated donkey anti-mouse IgG(H+L) for M2 surface expression analysis by flow cytometry. The graph shows the fluorescence intensity of M2 surface expression. (C) The TRAPPC6AΔ-overexpressing A549 cell line or the A549 control cell line transduced with an empty retrovirus was infected with the WSN virus at an MOI of 3. Cell lysates were processed at 8 and 10 h p.i. and subjected to Western blotting using a mouse anti-M2 MAb to detect the expression level of M2. (D) The TRAPPC6AΔ-overexpressing A549 cell line or the A549 control cell line transduced with an empty retrovirus was infected with the WSN virus at an MOI of 3. The cell surface expression of M2 was analyzed by flow cytometry at 8 and 10 h p.i. as described above for panel B. (E) A549 cells were treated with siRNA and infected with the WSN virus as described above for panel A. The cell surface expression of HA was analyzed by flow cytometry at 8 and 10 h p.i. as described above for panel B by using the rabbit anti-HA polyclonal antibody and Alexa Fluor 488-conjugated goat anti-rabbit IgG(H+L). (F) A549 cells were treated with siRNA and infected with the WSN virus as described above for panel A. The cell surface expression of FGF2 was analyzed by flow cytometry at 8 and 10 h p.i. as described above for panel B by using the rabbit anti-FGF2 MAb and Alexa Fluor 488-conjugated goat anti-rabbit IgG(H+L). (G) A549 cells were treated with siRNA and infected with the WSN virus as described above for panel A. At 2 h p.i., the culture medium was replaced with medium supplemented with 25 μM amantadine. The cell surface expression of M2 was analyzed by flow cytometry at 8 and 10 h p.i. as described above for panel B.

    Article Snippet: The following primary antibodies were obtained from commercial sources: rabbit anti-Flag polyclonal antibody (F7425; Sigma-Aldrich), mouse anti-Flag monoclonal antibody (F3165; Sigma-Aldrich), rabbit anti-Myc polyclonal antibody (C3965; Sigma-Aldrich), mouse anti-Myc monoclonal antibody (M4439; Sigma-Aldrich), mouse anti-actin monoclonal antibody (sc-47778; Santa Cruz, Dallas, TX), rabbit anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) polyclonal antibody (10494-1-AP; Proteintech, Chicago, IL), rabbit anti-GFP polyclonal antibody (AG279; Beyotime Biotech, Shanghai, China), mouse anti-GFP monoclonal antibody (ab1218; Abcam, Cambridge, MA), mouse anti-M2 monoclonal antibody (ab5416; Abcam), rabbit anti-M2 polyclonal antibody (GTX125951; GeneTex, Irvine, CA), rabbit anti-HA polyclonal antibody (11692-T54; Sino Biological Inc., Beijing, China), rabbit anti-FGF2 monoclonal antibody (ab92337; Abcam), mouse anti-LAMP1 monoclonal antibody (ab25630; Abcam), and mouse anti-Giantin monoclonal antibody (ab37266; Abcam).

    Techniques: Expressing, Transfection, Infection, Western Blot, Staining, Flow Cytometry, Cytometry, Fluorescence, Transduction

    TRAPPC6AΔ positively modulates influenza virus infection. (A) Endogenous expression of TRAPPC6AΔ in A549 cells. Whole lysates of A549 cells grown in 12-well plates were subjected to Western blotting with a rabbit anti-TRAPPC6A polyclonal antibody. HEK293T cell lysates transiently transfected with pCAGGS-TRAPPC6A or pCAGGS-TRAPPC6AΔ were used as a control. (B) siRNA knockdown of TRAPPC6AΔ in A549 cells. A549 cells were transfected with siRNA targeting TRAPPC6AΔ or nontargeting siRNA for 48 h. Whole-cell lysates were then collected and analyzed by Western blotting with a rabbit anti-TRAPPC6A polyclonal antibody. (C) Cell viability of siRNA-treated A549 cells measured by using the CellTiter-Glo assay. A549 cells were transfected with siRNA as described above for panel B. The data are presented as means ± standard deviations for triplicate transfections. (D) Virus replication in siRNA-treated A549 cells. Cells transfected with siRNA as described above for panel B were infected with WSN virus. At 24 and 48 h p.i., supernatants were collected and titrated for infectious virus by plaque assays in MDCK cells. Three independent experiments were performed, and data are shown as means ± standard deviations for triplicates from a representative experiment. **, P

    Journal: Journal of Virology

    Article Title: Host Cellular Protein TRAPPC6AΔ Interacts with Influenza A Virus M2 Protein and Regulates Viral Propagation by Modulating M2 Trafficking

    doi: 10.1128/JVI.01757-16

    Figure Lengend Snippet: TRAPPC6AΔ positively modulates influenza virus infection. (A) Endogenous expression of TRAPPC6AΔ in A549 cells. Whole lysates of A549 cells grown in 12-well plates were subjected to Western blotting with a rabbit anti-TRAPPC6A polyclonal antibody. HEK293T cell lysates transiently transfected with pCAGGS-TRAPPC6A or pCAGGS-TRAPPC6AΔ were used as a control. (B) siRNA knockdown of TRAPPC6AΔ in A549 cells. A549 cells were transfected with siRNA targeting TRAPPC6AΔ or nontargeting siRNA for 48 h. Whole-cell lysates were then collected and analyzed by Western blotting with a rabbit anti-TRAPPC6A polyclonal antibody. (C) Cell viability of siRNA-treated A549 cells measured by using the CellTiter-Glo assay. A549 cells were transfected with siRNA as described above for panel B. The data are presented as means ± standard deviations for triplicate transfections. (D) Virus replication in siRNA-treated A549 cells. Cells transfected with siRNA as described above for panel B were infected with WSN virus. At 24 and 48 h p.i., supernatants were collected and titrated for infectious virus by plaque assays in MDCK cells. Three independent experiments were performed, and data are shown as means ± standard deviations for triplicates from a representative experiment. **, P

    Article Snippet: The following primary antibodies were obtained from commercial sources: rabbit anti-Flag polyclonal antibody (F7425; Sigma-Aldrich), mouse anti-Flag monoclonal antibody (F3165; Sigma-Aldrich), rabbit anti-Myc polyclonal antibody (C3965; Sigma-Aldrich), mouse anti-Myc monoclonal antibody (M4439; Sigma-Aldrich), mouse anti-actin monoclonal antibody (sc-47778; Santa Cruz, Dallas, TX), rabbit anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) polyclonal antibody (10494-1-AP; Proteintech, Chicago, IL), rabbit anti-GFP polyclonal antibody (AG279; Beyotime Biotech, Shanghai, China), mouse anti-GFP monoclonal antibody (ab1218; Abcam, Cambridge, MA), mouse anti-M2 monoclonal antibody (ab5416; Abcam), rabbit anti-M2 polyclonal antibody (GTX125951; GeneTex, Irvine, CA), rabbit anti-HA polyclonal antibody (11692-T54; Sino Biological Inc., Beijing, China), rabbit anti-FGF2 monoclonal antibody (ab92337; Abcam), mouse anti-LAMP1 monoclonal antibody (ab25630; Abcam), and mouse anti-Giantin monoclonal antibody (ab37266; Abcam).

    Techniques: Infection, Expressing, Western Blot, Transfection, Glo Assay

    Confocal microscopy of WSN virus-infected cells stained for the Golgi apparatus or lysosomes. A549 cells were infected with the wt WSN virus at an MOI of 5. At the indicated time points, infected cells were fixed and stained with mouse anti-Giantin MAb and rabbit anti-M2 polyclonal antibody (A), mouse anti-LAMP1 MAb and rabbit anti-M2 polyclonal antibody (B), or mouse anti-LAMP1 MAb and rabbit anti-TRAPPC6A polyclonal antibody (C), followed by incubation with Alexa Fluor 488 donkey anti-mouse IgG(H+L) (green) and Alexa Fluor 546 donkey anti-rabbit IgG(H+L) (red). Nuclei were stained with DAPI.

    Journal: Journal of Virology

    Article Title: Host Cellular Protein TRAPPC6AΔ Interacts with Influenza A Virus M2 Protein and Regulates Viral Propagation by Modulating M2 Trafficking

    doi: 10.1128/JVI.01757-16

    Figure Lengend Snippet: Confocal microscopy of WSN virus-infected cells stained for the Golgi apparatus or lysosomes. A549 cells were infected with the wt WSN virus at an MOI of 5. At the indicated time points, infected cells were fixed and stained with mouse anti-Giantin MAb and rabbit anti-M2 polyclonal antibody (A), mouse anti-LAMP1 MAb and rabbit anti-M2 polyclonal antibody (B), or mouse anti-LAMP1 MAb and rabbit anti-TRAPPC6A polyclonal antibody (C), followed by incubation with Alexa Fluor 488 donkey anti-mouse IgG(H+L) (green) and Alexa Fluor 546 donkey anti-rabbit IgG(H+L) (red). Nuclei were stained with DAPI.

    Article Snippet: The following primary antibodies were obtained from commercial sources: rabbit anti-Flag polyclonal antibody (F7425; Sigma-Aldrich), mouse anti-Flag monoclonal antibody (F3165; Sigma-Aldrich), rabbit anti-Myc polyclonal antibody (C3965; Sigma-Aldrich), mouse anti-Myc monoclonal antibody (M4439; Sigma-Aldrich), mouse anti-actin monoclonal antibody (sc-47778; Santa Cruz, Dallas, TX), rabbit anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) polyclonal antibody (10494-1-AP; Proteintech, Chicago, IL), rabbit anti-GFP polyclonal antibody (AG279; Beyotime Biotech, Shanghai, China), mouse anti-GFP monoclonal antibody (ab1218; Abcam, Cambridge, MA), mouse anti-M2 monoclonal antibody (ab5416; Abcam), rabbit anti-M2 polyclonal antibody (GTX125951; GeneTex, Irvine, CA), rabbit anti-HA polyclonal antibody (11692-T54; Sino Biological Inc., Beijing, China), rabbit anti-FGF2 monoclonal antibody (ab92337; Abcam), mouse anti-LAMP1 monoclonal antibody (ab25630; Abcam), and mouse anti-Giantin monoclonal antibody (ab37266; Abcam).

    Techniques: Confocal Microscopy, Infection, Staining, Incubation

    Oligoclonal mAb Response Protects Mice from Group 1 or Group 2 Influenza Challenges BALB/c mice (n = 20) were pretreated with 20 U of hyaluronidase and administered 10 μg of H/L gWiz pDNA per anti-influenza mAb (C179, S139/1, and 9H10) in muscles of separate limbs followed by EP. For the control group, BALB/c mice (n = 15) were pretreated with 20 U of hyaluronidase and administered 10 μg of H/L gWiz pDNA per anti-Ebola mAb (2G4, 4G7, and 13C6) at distinct sites with EP. Blood was collected on days 12, 19, and 33 (in surviving mice) for pharmacokinetic analyses ( Figure S5 ). (A and B) On day 23, the mice were randomized and anti-influenza DNA/EP mice (n = 10) and anti-Ebola DNA/EP mice (n = 7–8) were challenged with (A) 39 MLD 50 of A/WSN/33 (H1N1) or (B) 21 MLD 50 of A/Aichi/2/68 (H3N2) delivered intranasally. (A and B) Mean weight loss (%) ± SEM compared with day 0 (left) and survival (%) as depicted in Kaplan-Meier plots (right) following (A) H1N1 and (B) H3N2 challenge. For percent weight loss graphs, **p = 0.0021, ***p

    Journal: Molecular Therapy. Methods & Clinical Development

    Article Title: In Vivo Production of Monoclonal Antibodies by Gene Transfer via Electroporation Protects against Lethal Influenza and Ebola Infections

    doi: 10.1016/j.omtm.2017.09.003

    Figure Lengend Snippet: Oligoclonal mAb Response Protects Mice from Group 1 or Group 2 Influenza Challenges BALB/c mice (n = 20) were pretreated with 20 U of hyaluronidase and administered 10 μg of H/L gWiz pDNA per anti-influenza mAb (C179, S139/1, and 9H10) in muscles of separate limbs followed by EP. For the control group, BALB/c mice (n = 15) were pretreated with 20 U of hyaluronidase and administered 10 μg of H/L gWiz pDNA per anti-Ebola mAb (2G4, 4G7, and 13C6) at distinct sites with EP. Blood was collected on days 12, 19, and 33 (in surviving mice) for pharmacokinetic analyses ( Figure S5 ). (A and B) On day 23, the mice were randomized and anti-influenza DNA/EP mice (n = 10) and anti-Ebola DNA/EP mice (n = 7–8) were challenged with (A) 39 MLD 50 of A/WSN/33 (H1N1) or (B) 21 MLD 50 of A/Aichi/2/68 (H3N2) delivered intranasally. (A and B) Mean weight loss (%) ± SEM compared with day 0 (left) and survival (%) as depicted in Kaplan-Meier plots (right) following (A) H1N1 and (B) H3N2 challenge. For percent weight loss graphs, **p = 0.0021, ***p

    Article Snippet: ELISA plates coated with 200 ng of H1N1 (A/WSN/33) HA, 50 ng of H3N2 (A/Aichi/2/1968) HA1 or HA subunit, or 200 ng of H10N8 (A/Jiangxi-Donghu/346/2013) HA (Sino Biologicals, Beijing, China) were blocked and incubated with serially diluted mouse serum.

    Techniques: Mouse Assay

    “Late” CD4 + T cell help shapes respiratory mucosal memory CD8 + and B cell immunity. WT mice were infected with PR8 and treated with ctl IgG or α-CD4. a , The efficiency of CD4 + T cell depletion in the lung or spleen. b , Representative image of lung section stained with H E. Black arrows indicate tertial lymphoid structure. c , Representative dot plot and cell numbers of lung germinal center B (B GC ) cells. d , Frequencies of lung circulating (CD45 i.v. + ) or parenchymal (CD45 i.v. - ) CD8 + T or B cells in mice treated with control IgG or α-CD4. e , Influenza HA-specific B cells (HA-B) in the lungs were identified through HA antigen staining. f , Lung tissue or circulating CD8 + NP 366-374 or PA 224-233 T cells were identified through CD45 i.v. staining and analyzed by flow cytometry. g , Lung circulating CD8 + NP 366-374 or CD8 + PA 224-233 T cells were enumerated. i , Histogram of CD103 expression or frequency of CD103 + cells within CD8 + CD69 + NP 366-374 T RM cells. h, j , Gating strategies of CD8 + T RM ( h ) or B RM ( j ) cells in the lung. Representative data were from at least two independent experiments (4-5 mice per group). P values were calculated by unpaired two-tailed Student’s t-test.

    Journal: bioRxiv

    Article Title: Tissue-resident CD4+ T helper cells assist protective respiratory mucosal B and CD8+ T cell memory responses

    doi: 10.1101/2020.02.28.970400

    Figure Lengend Snippet: “Late” CD4 + T cell help shapes respiratory mucosal memory CD8 + and B cell immunity. WT mice were infected with PR8 and treated with ctl IgG or α-CD4. a , The efficiency of CD4 + T cell depletion in the lung or spleen. b , Representative image of lung section stained with H E. Black arrows indicate tertial lymphoid structure. c , Representative dot plot and cell numbers of lung germinal center B (B GC ) cells. d , Frequencies of lung circulating (CD45 i.v. + ) or parenchymal (CD45 i.v. - ) CD8 + T or B cells in mice treated with control IgG or α-CD4. e , Influenza HA-specific B cells (HA-B) in the lungs were identified through HA antigen staining. f , Lung tissue or circulating CD8 + NP 366-374 or PA 224-233 T cells were identified through CD45 i.v. staining and analyzed by flow cytometry. g , Lung circulating CD8 + NP 366-374 or CD8 + PA 224-233 T cells were enumerated. i , Histogram of CD103 expression or frequency of CD103 + cells within CD8 + CD69 + NP 366-374 T RM cells. h, j , Gating strategies of CD8 + T RM ( h ) or B RM ( j ) cells in the lung. Representative data were from at least two independent experiments (4-5 mice per group). P values were calculated by unpaired two-tailed Student’s t-test.

    Article Snippet: PR8-NP protein was purchased from Sino Biological.

    Techniques: Mouse Assay, Infection, Staining, Flow Cytometry, Expressing, Two Tailed Test

    T RH cells help CD8 + T and B cells through IL-21 or CD40L dependent mechanisms. a , Expression of Il21 gene in sorted IL-21-VFP Hi, IL-21-VFP Low or IL-21-VFP - CD4 + T cells from the lung tissue at 21 d.p.i. (pooled from 8 mice) b , Expression of Il21 in the whole lung from Bcl6 fl/fl or Bcl6 ΔT mice at 15 or 42 d.p.i. c , Frequencies or cell numbers of lung B GC or HA-specific B cells from PR8 infected WT mice treated with ctl-IgG or α-IL-21R (Intranasal route). d , Heat map of IL-21 signaling-related genes from sorted lung CD8 + NP 366-374 or PA 224-233 T RM or splenic CD8 + NP 366-374 or PA 224-233 T MEM determined by Nanostring at 38 d.p.i. as reported previously 40 . e , Geometric M.F.I. of BATF expression in lung CD8 + NP 366-374, CD8 + PA 224-233 T RM , splenic CD8 + NP 366-374 or PA 224-233 T MEM cells from PR8 infected mice at 35 d.p.i. f , CD40L expression in the NP 311-325 T RH or non-T RH cells following PMA/Ionomycin stimulation at 28 d.p.i. g , Schematics of the summary. In b-c and e-f , representative data were from at least two independent experiments (3-5 mice per group). P values were calculated by unpaired (b and c) or paired (f) two-tailed Student’s t-test. P value in e was analyzed by one-way ANOVA.

    Journal: bioRxiv

    Article Title: Tissue-resident CD4+ T helper cells assist protective respiratory mucosal B and CD8+ T cell memory responses

    doi: 10.1101/2020.02.28.970400

    Figure Lengend Snippet: T RH cells help CD8 + T and B cells through IL-21 or CD40L dependent mechanisms. a , Expression of Il21 gene in sorted IL-21-VFP Hi, IL-21-VFP Low or IL-21-VFP - CD4 + T cells from the lung tissue at 21 d.p.i. (pooled from 8 mice) b , Expression of Il21 in the whole lung from Bcl6 fl/fl or Bcl6 ΔT mice at 15 or 42 d.p.i. c , Frequencies or cell numbers of lung B GC or HA-specific B cells from PR8 infected WT mice treated with ctl-IgG or α-IL-21R (Intranasal route). d , Heat map of IL-21 signaling-related genes from sorted lung CD8 + NP 366-374 or PA 224-233 T RM or splenic CD8 + NP 366-374 or PA 224-233 T MEM determined by Nanostring at 38 d.p.i. as reported previously 40 . e , Geometric M.F.I. of BATF expression in lung CD8 + NP 366-374, CD8 + PA 224-233 T RM , splenic CD8 + NP 366-374 or PA 224-233 T MEM cells from PR8 infected mice at 35 d.p.i. f , CD40L expression in the NP 311-325 T RH or non-T RH cells following PMA/Ionomycin stimulation at 28 d.p.i. g , Schematics of the summary. In b-c and e-f , representative data were from at least two independent experiments (3-5 mice per group). P values were calculated by unpaired (b and c) or paired (f) two-tailed Student’s t-test. P value in e was analyzed by one-way ANOVA.

    Article Snippet: PR8-NP protein was purchased from Sino Biological.

    Techniques: Expressing, Mouse Assay, Infection, Two Tailed Test

    Optimal T RH formation requires lung B cells and iBALT formation WT ( a-d ) mice were infected with PR8 and treated with ctl IgG, α-CD20 ( b ) or LTβR-Ig ( c and d ) (weekly starting at 14 d.p.i.) in the presence of daily injection of FTY-720 (13-27 d.p.i.). a , Experimental scheme. b and d , Representative dot plot and cell numbers of influenza-specific NP 311-325 lung T RH, lung non-T RH or splenic T FH cells. c , Representative image from lung tissue section stained with B220/GL-7 following ctl IgG or LTβR-Ig treatment. In c , the representative image was from at least two independent experiments (3-4 mice per group). In b , representative data were from at least two independent experiments (4-5 mice per group). In d , data were pooled from two independent experiments (3-4 mice per group). P values were calculated by unpaired two-tailed Student’s t-test.

    Journal: bioRxiv

    Article Title: Tissue-resident CD4+ T helper cells assist protective respiratory mucosal B and CD8+ T cell memory responses

    doi: 10.1101/2020.02.28.970400

    Figure Lengend Snippet: Optimal T RH formation requires lung B cells and iBALT formation WT ( a-d ) mice were infected with PR8 and treated with ctl IgG, α-CD20 ( b ) or LTβR-Ig ( c and d ) (weekly starting at 14 d.p.i.) in the presence of daily injection of FTY-720 (13-27 d.p.i.). a , Experimental scheme. b and d , Representative dot plot and cell numbers of influenza-specific NP 311-325 lung T RH, lung non-T RH or splenic T FH cells. c , Representative image from lung tissue section stained with B220/GL-7 following ctl IgG or LTβR-Ig treatment. In c , the representative image was from at least two independent experiments (3-4 mice per group). In b , representative data were from at least two independent experiments (4-5 mice per group). In d , data were pooled from two independent experiments (3-4 mice per group). P values were calculated by unpaired two-tailed Student’s t-test.

    Article Snippet: PR8-NP protein was purchased from Sino Biological.

    Techniques: Mouse Assay, Infection, Injection, Staining, Two Tailed Test

    IL-21 or CD40L-dependent T RH help to CD8 + or B cells. a-b , IL-21-VFP reporter mice were infected with PR8. a , IL-21-VFP expressing cells in the lungs or spleens were identified by flow cytometry at 28 d.p.i. b , Representative dot plot of IL-21 Hi or IL-21 Low CD4 + T cells that were PD-1 Hi FR4 Hi . c-f , WT mice were infected PR8 with or without IL-21R blockade through intraperitoneal (I.P.) route starting at 14 d.p.i. in the presence of FTY-720 administration (13-34 d.p.i.). c , Experimental scheme. Cell numbers of lung parenchymal B GC ( d ), HA-specific B RM ( e ) and CD8 + CD69 + NP 366-374 or CD8 + CD69 + PA 224-233 T RM cells ( f ). g-k , WT mice were infected with PR8 with or without IL-21R blockade through intranasal (I.N.) route at 14 d.p.i. g , Experimental scheme. h-i , Frequencies ( h ) or cell numbers ( i ) of lung tissue CD8 + CD69 + NP 366-374, CD8 + CD69 + PA 224-233 T RM , splenic CD8 + NP 366-374 or PA 224- 233 memory T cells (T MEM ) at 42 d.p.i. j , Percentage of apoptotic cells were identified by active caspase 3/7-FLICA staining within lung CD8 + NP 366-374 T RM or splenic CD8 + NP 366-374 T MEM at 28 d.p.i. k , percentages of proliferating cells were identified by Ki67 staining within lung CD8 + NP 366-374 T RM or splenic CD8 + NP 366-374 T MEM at 28 d.p.i. l , Representative histogram of BATF expression in lung CD8 + NP 366-374 or CD8 + PA 224-233 T RM of mice received with ctl IgG or α-IL21R at 35 d.p.i. m , Representative histogram of CD40L expression in influenza-specific lung T RH or non-T RH at 28 d.p.i. n-o , WT mice were infected with PR8 and received ctl IgG or α-CD40L weekly (I.P. route) starting at 14 d.p.i. in the presence of daily FTY-720 administration (13-34 d.p.i.). Representative dot plot or cell numbers B GC ( n ) and HA-specific B RM ( o ) cells at 35 d.p.i. In a-b and j-m , representative data were from at least two independent experiments (4-5 mice per group). In d, f-i and n-o , data were pooled from two independent experiments (3-5 mice per group). P values of all experiments were calculated by unpaired two-tailed Student’s t-test.

    Journal: bioRxiv

    Article Title: Tissue-resident CD4+ T helper cells assist protective respiratory mucosal B and CD8+ T cell memory responses

    doi: 10.1101/2020.02.28.970400

    Figure Lengend Snippet: IL-21 or CD40L-dependent T RH help to CD8 + or B cells. a-b , IL-21-VFP reporter mice were infected with PR8. a , IL-21-VFP expressing cells in the lungs or spleens were identified by flow cytometry at 28 d.p.i. b , Representative dot plot of IL-21 Hi or IL-21 Low CD4 + T cells that were PD-1 Hi FR4 Hi . c-f , WT mice were infected PR8 with or without IL-21R blockade through intraperitoneal (I.P.) route starting at 14 d.p.i. in the presence of FTY-720 administration (13-34 d.p.i.). c , Experimental scheme. Cell numbers of lung parenchymal B GC ( d ), HA-specific B RM ( e ) and CD8 + CD69 + NP 366-374 or CD8 + CD69 + PA 224-233 T RM cells ( f ). g-k , WT mice were infected with PR8 with or without IL-21R blockade through intranasal (I.N.) route at 14 d.p.i. g , Experimental scheme. h-i , Frequencies ( h ) or cell numbers ( i ) of lung tissue CD8 + CD69 + NP 366-374, CD8 + CD69 + PA 224-233 T RM , splenic CD8 + NP 366-374 or PA 224- 233 memory T cells (T MEM ) at 42 d.p.i. j , Percentage of apoptotic cells were identified by active caspase 3/7-FLICA staining within lung CD8 + NP 366-374 T RM or splenic CD8 + NP 366-374 T MEM at 28 d.p.i. k , percentages of proliferating cells were identified by Ki67 staining within lung CD8 + NP 366-374 T RM or splenic CD8 + NP 366-374 T MEM at 28 d.p.i. l , Representative histogram of BATF expression in lung CD8 + NP 366-374 or CD8 + PA 224-233 T RM of mice received with ctl IgG or α-IL21R at 35 d.p.i. m , Representative histogram of CD40L expression in influenza-specific lung T RH or non-T RH at 28 d.p.i. n-o , WT mice were infected with PR8 and received ctl IgG or α-CD40L weekly (I.P. route) starting at 14 d.p.i. in the presence of daily FTY-720 administration (13-34 d.p.i.). Representative dot plot or cell numbers B GC ( n ) and HA-specific B RM ( o ) cells at 35 d.p.i. In a-b and j-m , representative data were from at least two independent experiments (4-5 mice per group). In d, f-i and n-o , data were pooled from two independent experiments (3-5 mice per group). P values of all experiments were calculated by unpaired two-tailed Student’s t-test.

    Article Snippet: PR8-NP protein was purchased from Sino Biological.

    Techniques: Mouse Assay, Infection, Expressing, Flow Cytometry, Hi-C, Staining, Two Tailed Test

    T RH cells help local development of memory CD8 + and B cells. a-c , Bcl6 fl/fl (with ROSA26 LSL-YFP transgene) or Bcl6 ΔCD4ERT2 (with ROSA26 LSL-YFP transgene) mice were infected with X31. Tamoxifen was administrated daily from 12 to 16 d.p.i. a , Experimental scheme of selective deletion of Bcl6 in CD4 + T cells. b, c , YFP expression in blood CD45 + ( b ) or CD4 + T ( c ) cells following tamoxifen injection. d, e , Bcl6 fl/fl or Bcl6 ΔCD4ERT2 mice were infected with PR8. Tamoxifen was administrated daily from 12-16 d.p.i. in the presence of daily FTY720 administration (11-34 d.p.i.). d , Cell numbers of CD45 i.v. - CD4 + T, CD8 + T or B cells at 35 d.p.i. e , Representative dot plot (top) or cell numbers (bottom) of NP 311- 325 T RH or non-T RH cells at 35 d.p.i. f , Cell numbers of lung CD8 + CD69 + NP 366-374 or CD8 + CD69 + PA 224-233 T RM , B GC or HA-specific B RM cells were enumerated from X31-infected Bcl6 fl/fl and Bcl6 ΔCD4ERT2 mice in the present of FTY720 (11-34 d.p.i.) at 35 d.p.i. In a-c and f , representative data were from at least two independent experiments (2-5 mice per group). In d-e , data were pooled from two independent experiments (3 mice per group). P values of all experiments were calculated by unpaired two-tailed Student’s t-test.

    Journal: bioRxiv

    Article Title: Tissue-resident CD4+ T helper cells assist protective respiratory mucosal B and CD8+ T cell memory responses

    doi: 10.1101/2020.02.28.970400

    Figure Lengend Snippet: T RH cells help local development of memory CD8 + and B cells. a-c , Bcl6 fl/fl (with ROSA26 LSL-YFP transgene) or Bcl6 ΔCD4ERT2 (with ROSA26 LSL-YFP transgene) mice were infected with X31. Tamoxifen was administrated daily from 12 to 16 d.p.i. a , Experimental scheme of selective deletion of Bcl6 in CD4 + T cells. b, c , YFP expression in blood CD45 + ( b ) or CD4 + T ( c ) cells following tamoxifen injection. d, e , Bcl6 fl/fl or Bcl6 ΔCD4ERT2 mice were infected with PR8. Tamoxifen was administrated daily from 12-16 d.p.i. in the presence of daily FTY720 administration (11-34 d.p.i.). d , Cell numbers of CD45 i.v. - CD4 + T, CD8 + T or B cells at 35 d.p.i. e , Representative dot plot (top) or cell numbers (bottom) of NP 311- 325 T RH or non-T RH cells at 35 d.p.i. f , Cell numbers of lung CD8 + CD69 + NP 366-374 or CD8 + CD69 + PA 224-233 T RM , B GC or HA-specific B RM cells were enumerated from X31-infected Bcl6 fl/fl and Bcl6 ΔCD4ERT2 mice in the present of FTY720 (11-34 d.p.i.) at 35 d.p.i. In a-c and f , representative data were from at least two independent experiments (2-5 mice per group). In d-e , data were pooled from two independent experiments (3 mice per group). P values of all experiments were calculated by unpaired two-tailed Student’s t-test.

    Article Snippet: PR8-NP protein was purchased from Sino Biological.

    Techniques: Mouse Assay, Infection, Expressing, Injection, Two Tailed Test

    Both BCL6 and Bhlhe40 are required for optimal lung T RH responses. a-e , Bcl6 fl/fl or Bcl6 ΔT mice were infected with PR8. Representative dot plot ( a ), percentages ( b ) and cell numbers ( c ) of T RH or non-T RH in lung CD45 i.v. - total CD4 + or CD45 i.v. - influenza-specific CD4 + NP 311-325 T cells at 28 d.p.i. d-e , Representative dot plot ( d ) and percentage (top row) or cell numbers (bottom row) ( e ) of splenic total T FH or NP 311-325 T FH at 28 d.p.i. f , GSEA of the Bhlhe40 -associated genes in lung T RH (PD-1 Hi FR4 Hi ) and spleen T FH cells. g-k , Bhlhe40 fl/fl or Bhlhe40 ΔT mice were infected with PR8. g-i , Representative dot plot ( g ), percentages ( h ) or cell numbers ( i ) of influenza-specific NP 311-325 lung T RH , lung non-T RH , splenic T FH or splenic non-T FH cells at 28 d.p.i. j-k Representative dot plot ( j ) or percentages ( k ) of active caspase 3/7-FLICA + cells in lung NP 311-325 T RH or non-T RH cells at 28 d.p.i. In a-e and g-i , data were pooled from two independent experiments (3-4 mice per group). In j-k , representative data were from at least two independent experiments (4 mice per group). P values were calculated by unpaired two-tailed Student’s t-test.

    Journal: bioRxiv

    Article Title: Tissue-resident CD4+ T helper cells assist protective respiratory mucosal B and CD8+ T cell memory responses

    doi: 10.1101/2020.02.28.970400

    Figure Lengend Snippet: Both BCL6 and Bhlhe40 are required for optimal lung T RH responses. a-e , Bcl6 fl/fl or Bcl6 ΔT mice were infected with PR8. Representative dot plot ( a ), percentages ( b ) and cell numbers ( c ) of T RH or non-T RH in lung CD45 i.v. - total CD4 + or CD45 i.v. - influenza-specific CD4 + NP 311-325 T cells at 28 d.p.i. d-e , Representative dot plot ( d ) and percentage (top row) or cell numbers (bottom row) ( e ) of splenic total T FH or NP 311-325 T FH at 28 d.p.i. f , GSEA of the Bhlhe40 -associated genes in lung T RH (PD-1 Hi FR4 Hi ) and spleen T FH cells. g-k , Bhlhe40 fl/fl or Bhlhe40 ΔT mice were infected with PR8. g-i , Representative dot plot ( g ), percentages ( h ) or cell numbers ( i ) of influenza-specific NP 311-325 lung T RH , lung non-T RH , splenic T FH or splenic non-T FH cells at 28 d.p.i. j-k Representative dot plot ( j ) or percentages ( k ) of active caspase 3/7-FLICA + cells in lung NP 311-325 T RH or non-T RH cells at 28 d.p.i. In a-e and g-i , data were pooled from two independent experiments (3-4 mice per group). In j-k , representative data were from at least two independent experiments (4 mice per group). P values were calculated by unpaired two-tailed Student’s t-test.

    Article Snippet: PR8-NP protein was purchased from Sino Biological.

    Techniques: Mouse Assay, Infection, Two Tailed Test

    T cell-specific BCL6 or Bhlhe40 deficiency leads to impaired lung CD8 + memory and B cell immunity. a-d , Bcl6 fl/fl or Bcl6 ΔT mice were infected with PR8. a , Representative confocal images of lung iBALT at 28 d.p.i. Lung sections were stained with α-CD4 (red), α-B220 (green), α-GL7 (white) and DAPI (blue). Percentages of lung B GC ( b ), lung tissue HA-specific B RM or circulating HA-specific B MEM ( c ) and CD8 + CD69 + NP 366-374, or CD8 + CD69 + PA 224-233 T RM ( d ). e-g , Bhlhe40 fl/fl or Bhlhe40 ΔT mice were infected with PR8. e , Representative dot plot or cell numbers of lung tissue CD8 + T (top) or B cells (bottom) at 28 d.p.i. f , Cell numbers of NP-specific B RM or HA-specific B RM cells. g , Cell numbers of CD8 + CD69 + NP 366-374, or CD8 + CD69 + PA 224-233 T RM. . In a-d and f-g , representative data were from at least two independent experiments (4-5 mice per group). In e , data were pooled from two independent experiments (4 mice per group). P values of all experiments were calculated by unpaired two-tailed Student’s t-test.

    Journal: bioRxiv

    Article Title: Tissue-resident CD4+ T helper cells assist protective respiratory mucosal B and CD8+ T cell memory responses

    doi: 10.1101/2020.02.28.970400

    Figure Lengend Snippet: T cell-specific BCL6 or Bhlhe40 deficiency leads to impaired lung CD8 + memory and B cell immunity. a-d , Bcl6 fl/fl or Bcl6 ΔT mice were infected with PR8. a , Representative confocal images of lung iBALT at 28 d.p.i. Lung sections were stained with α-CD4 (red), α-B220 (green), α-GL7 (white) and DAPI (blue). Percentages of lung B GC ( b ), lung tissue HA-specific B RM or circulating HA-specific B MEM ( c ) and CD8 + CD69 + NP 366-374, or CD8 + CD69 + PA 224-233 T RM ( d ). e-g , Bhlhe40 fl/fl or Bhlhe40 ΔT mice were infected with PR8. e , Representative dot plot or cell numbers of lung tissue CD8 + T (top) or B cells (bottom) at 28 d.p.i. f , Cell numbers of NP-specific B RM or HA-specific B RM cells. g , Cell numbers of CD8 + CD69 + NP 366-374, or CD8 + CD69 + PA 224-233 T RM. . In a-d and f-g , representative data were from at least two independent experiments (4-5 mice per group). In e , data were pooled from two independent experiments (4 mice per group). P values of all experiments were calculated by unpaired two-tailed Student’s t-test.

    Article Snippet: PR8-NP protein was purchased from Sino Biological.

    Techniques: Mouse Assay, Infection, Staining, Two Tailed Test

    T RH cells are required for the development of lung protective CD8 + T RM and B cell immunity. a-j , Bcl6 fl/fl or Bcl6 ΔCD4ERT2 mice were infected with PR8. a-e , Tamoxifen was administrated daily from 12-16 d.p.i. in the presence of daily FTY720 administration (11-34 d.p.i.). a , Schematics of experimental design. Cell numbers of B GC ( b ), HA-specific B RM ( c ), NP-specific B RM ( d ), e , CD8 + CD69 + NP 366-374 T RM or CD8 + CD69 + PA 224-233 T RM at 35 d.p.i. f-j , Tamoxifen was administrated daily from 21-25 d.p.i. in the presence of daily FTY720 administration (20-41 d.p.i.). f , Schematics of experimental design. Cell numbers of B GC ( g ), HA-specific B RM ( h ), NP-specific B RM ( i ), j , CD8 + CD69 + NP 366-374 T RM or CD8 + CD69 + PA 224-233 T RM at 42 d.p.i. k , Bcl6 fl/fl or Bcl6 ΔCD4ERT2 mice were infected with X31 strain (H3N2) of influenza. Tamoxifen was administrated daily from 12-16 d.p.i. in the presence of daily FTY720 administration (11-34 d.p.i.). Representative dot plot (top) and cell numbers (bottom) of X31 strain-specific B RM or cross-reactive HA-specific B RM (to H3N2 A/Uruguay/716/07 strain) at 35 d.p.i. l-m , Bcl6 fl/fl (n = 11) or Bcl6 ΔCD4ERT2 (n = 15) mice were infected with X31 and administered with tamoxifen from 12 to 16 d.p.i. Mice were re-challenged with PR8 at 42 d.p.i. in the presence of FTY720 (starting from 41d). l , Schematics of experimental design. m , Host mortality following PR8 challenge was monitored. In a-h and j-k , all data were pooled from two ( c, d, k, g, h and j ) or three ( b and e ) independent experiments (2-5 mice per group). In a-k, P values were calculated by unpaired two-tailed Student’s t-test. P value of survival study in m was calculated by Logrank test.

    Journal: bioRxiv

    Article Title: Tissue-resident CD4+ T helper cells assist protective respiratory mucosal B and CD8+ T cell memory responses

    doi: 10.1101/2020.02.28.970400

    Figure Lengend Snippet: T RH cells are required for the development of lung protective CD8 + T RM and B cell immunity. a-j , Bcl6 fl/fl or Bcl6 ΔCD4ERT2 mice were infected with PR8. a-e , Tamoxifen was administrated daily from 12-16 d.p.i. in the presence of daily FTY720 administration (11-34 d.p.i.). a , Schematics of experimental design. Cell numbers of B GC ( b ), HA-specific B RM ( c ), NP-specific B RM ( d ), e , CD8 + CD69 + NP 366-374 T RM or CD8 + CD69 + PA 224-233 T RM at 35 d.p.i. f-j , Tamoxifen was administrated daily from 21-25 d.p.i. in the presence of daily FTY720 administration (20-41 d.p.i.). f , Schematics of experimental design. Cell numbers of B GC ( g ), HA-specific B RM ( h ), NP-specific B RM ( i ), j , CD8 + CD69 + NP 366-374 T RM or CD8 + CD69 + PA 224-233 T RM at 42 d.p.i. k , Bcl6 fl/fl or Bcl6 ΔCD4ERT2 mice were infected with X31 strain (H3N2) of influenza. Tamoxifen was administrated daily from 12-16 d.p.i. in the presence of daily FTY720 administration (11-34 d.p.i.). Representative dot plot (top) and cell numbers (bottom) of X31 strain-specific B RM or cross-reactive HA-specific B RM (to H3N2 A/Uruguay/716/07 strain) at 35 d.p.i. l-m , Bcl6 fl/fl (n = 11) or Bcl6 ΔCD4ERT2 (n = 15) mice were infected with X31 and administered with tamoxifen from 12 to 16 d.p.i. Mice were re-challenged with PR8 at 42 d.p.i. in the presence of FTY720 (starting from 41d). l , Schematics of experimental design. m , Host mortality following PR8 challenge was monitored. In a-h and j-k , all data were pooled from two ( c, d, k, g, h and j ) or three ( b and e ) independent experiments (2-5 mice per group). In a-k, P values were calculated by unpaired two-tailed Student’s t-test. P value of survival study in m was calculated by Logrank test.

    Article Snippet: PR8-NP protein was purchased from Sino Biological.

    Techniques: Mouse Assay, Infection, Two Tailed Test

    Lung CD4 + T cells deliver localized help for the development of tissue-resident B and CD8 + T cells. a-e , WT mice were infected with PR8 strain of influenza virus and treated with control (ctl) IgG or α-CD4 starting at 14 d.p.i. Mice were injected with α-CD45 intravenously (i.v.) 5 min before sacrifice at 42 d.p.i. a , Experimental scheme. b , Representative confocal images of iBALT in the lung. Lung sections were stained with α-CD4 (red), α-B220 (green) and DAPI (blue). c , Frequencies and cell number of influenza HA-specific B cells (HA-B) in the lung tissue (CD45 i.v. - B220 + GL7 - HA + ), lung blood vessels (CD45 i.v. + B220 + GL7 - HA + ) and spleen (B220 + GL7 - HA + ). d , Lung tissue CD8 + , CD8 + CD69 + or CD8 + CD69 + CD103 + NP 366-374 or PA 224-233 T RM cells were enumerated. e , Splenic CD8 + NP 366-374 or PA 224-233 memory cells (T MEM-SPL ) were enumerated. f-j , WT mice were infected with PR8 and treated with ctrl IgG or α-CD4 (starting at 14 d.p.i.) in the presence of daily injection of FTY-720 (starting at 13 d.p.i.). f , Schematic of experimental design. g , B GC cell numbers were enumerated by flow cytometry. h, i , Total HA-specific B cells (total HA-B) ( h ) or HA-specific tissue-resident memory B cells (HA-B RM : CD45 i.v. - B220 + GL7 - IgD - IgM - HA + CD38 + ) ( i ) were enumerated. j , Lung tissue CD8 + , CD8 + CD69 + or CD8 + CD69 + CD103 + NP 366-374 or PA 224-233 T RM cells were enumerated. k-n , WT mice were infected with PR8 and received ctl IgG, high or low dose of α-CD4. Cell number of B GC ( k ), HA-specific B RM ( l ) and NP-specific B RM cells ( m ) in the lung tissue. n , Lung tissue CD8 + , CD8 + CD69 + or CD8 + CD69 + CD103 + NP 366-374 T RM cells were enumerated. In b-e were the representative data from at least two independent experiments (4-5 mice per group). In g-h and j-n , data were pooled from two ( g, h and j ) or three ( k - n ) independent experiments (2-5 mice per group). P values were calculated by unpaired two-tailed Student’s t-test in c-j. P values in k-n were analyzed by one-way ANOVA.

    Journal: bioRxiv

    Article Title: Tissue-resident CD4+ T helper cells assist protective respiratory mucosal B and CD8+ T cell memory responses

    doi: 10.1101/2020.02.28.970400

    Figure Lengend Snippet: Lung CD4 + T cells deliver localized help for the development of tissue-resident B and CD8 + T cells. a-e , WT mice were infected with PR8 strain of influenza virus and treated with control (ctl) IgG or α-CD4 starting at 14 d.p.i. Mice were injected with α-CD45 intravenously (i.v.) 5 min before sacrifice at 42 d.p.i. a , Experimental scheme. b , Representative confocal images of iBALT in the lung. Lung sections were stained with α-CD4 (red), α-B220 (green) and DAPI (blue). c , Frequencies and cell number of influenza HA-specific B cells (HA-B) in the lung tissue (CD45 i.v. - B220 + GL7 - HA + ), lung blood vessels (CD45 i.v. + B220 + GL7 - HA + ) and spleen (B220 + GL7 - HA + ). d , Lung tissue CD8 + , CD8 + CD69 + or CD8 + CD69 + CD103 + NP 366-374 or PA 224-233 T RM cells were enumerated. e , Splenic CD8 + NP 366-374 or PA 224-233 memory cells (T MEM-SPL ) were enumerated. f-j , WT mice were infected with PR8 and treated with ctrl IgG or α-CD4 (starting at 14 d.p.i.) in the presence of daily injection of FTY-720 (starting at 13 d.p.i.). f , Schematic of experimental design. g , B GC cell numbers were enumerated by flow cytometry. h, i , Total HA-specific B cells (total HA-B) ( h ) or HA-specific tissue-resident memory B cells (HA-B RM : CD45 i.v. - B220 + GL7 - IgD - IgM - HA + CD38 + ) ( i ) were enumerated. j , Lung tissue CD8 + , CD8 + CD69 + or CD8 + CD69 + CD103 + NP 366-374 or PA 224-233 T RM cells were enumerated. k-n , WT mice were infected with PR8 and received ctl IgG, high or low dose of α-CD4. Cell number of B GC ( k ), HA-specific B RM ( l ) and NP-specific B RM cells ( m ) in the lung tissue. n , Lung tissue CD8 + , CD8 + CD69 + or CD8 + CD69 + CD103 + NP 366-374 T RM cells were enumerated. In b-e were the representative data from at least two independent experiments (4-5 mice per group). In g-h and j-n , data were pooled from two ( g, h and j ) or three ( k - n ) independent experiments (2-5 mice per group). P values were calculated by unpaired two-tailed Student’s t-test in c-j. P values in k-n were analyzed by one-way ANOVA.

    Article Snippet: PR8-NP protein was purchased from Sino Biological.

    Techniques: Mouse Assay, Infection, Injection, Staining, Flow Cytometry, Two Tailed Test

    Identification of a population of T FH -like cells in the lung tissue. WT ( a-e ) or IL-21-VFP reporter ( f ) mice were infected with PR8. a , tSNE plot of scRNAseq analysis of sorted lung CD45 i.v. - CD4 + CD44 Hi cells (pooled from 5 mice) at 28 d.p.i. b , Heat map of indicated genes in each cluster from scRNAseq data. c , Kinetics of the percentages of PD-1 Hi FR4 Hi population in lung tissue total CD4 + or influenza NP-specific (NP 311-325 ) CD4 + T cells. d , Expression of T FH cell-associated markers in lung total or influenza-specific PD-1 Hi FR4 Hi , PD-1 Low FR4 Low or splenic T FH (CD4 + CD44 Hi PD-1 + CXCR5 + ) cells at 28 d.p.i. e , Expression of IFN-γ, IL-17 or IL-4 by lung PD-1 Hi FR4 Hi , PD-1 Low FR4 Low or spleen T FH cells were identified by intracellular staining at 28 d.p.i . f , IL-21-VFP expression in lung CD4 + PD-1 Hi FR4 Hi , PD-1 Low FR4 Low or spleen T FH at 28 d.p.i. In c-f , representative plots, histograms and graphs were from at least two independent experiments (4 mice per group). P values in e and f were analyzed by one-way ANOVA.

    Journal: bioRxiv

    Article Title: Tissue-resident CD4+ T helper cells assist protective respiratory mucosal B and CD8+ T cell memory responses

    doi: 10.1101/2020.02.28.970400

    Figure Lengend Snippet: Identification of a population of T FH -like cells in the lung tissue. WT ( a-e ) or IL-21-VFP reporter ( f ) mice were infected with PR8. a , tSNE plot of scRNAseq analysis of sorted lung CD45 i.v. - CD4 + CD44 Hi cells (pooled from 5 mice) at 28 d.p.i. b , Heat map of indicated genes in each cluster from scRNAseq data. c , Kinetics of the percentages of PD-1 Hi FR4 Hi population in lung tissue total CD4 + or influenza NP-specific (NP 311-325 ) CD4 + T cells. d , Expression of T FH cell-associated markers in lung total or influenza-specific PD-1 Hi FR4 Hi , PD-1 Low FR4 Low or splenic T FH (CD4 + CD44 Hi PD-1 + CXCR5 + ) cells at 28 d.p.i. e , Expression of IFN-γ, IL-17 or IL-4 by lung PD-1 Hi FR4 Hi , PD-1 Low FR4 Low or spleen T FH cells were identified by intracellular staining at 28 d.p.i . f , IL-21-VFP expression in lung CD4 + PD-1 Hi FR4 Hi , PD-1 Low FR4 Low or spleen T FH at 28 d.p.i. In c-f , representative plots, histograms and graphs were from at least two independent experiments (4 mice per group). P values in e and f were analyzed by one-way ANOVA.

    Article Snippet: PR8-NP protein was purchased from Sino Biological.

    Techniques: Mouse Assay, Infection, Expressing, Staining

    Impaired T RH responses following B cell depletion, iBALT disruption or Bhlhe40 deficiency. a-c , WT ( a and c ) or IL-21-VFP reporter ( b ) mice were infected with PR8 and received with ctl IgG, α-CD20 or LTβR-Ig weekly in the present of FTY-720 (Experimental scheme in Fig 5 . a.). a , The efficiency of B cell depletion in the lung. b , Representative confocal images of IL-21-expressing cells in iBALT. c , Cell numbers of lung tissue B cells in mice received with ctl-IgG or LTβR-Ig. d-g , Bhlhe40 fl/fl or Bhlhe40 ΔT mice were infected with PR8. Representative dot plot ( d ), percentages (top) or cell numbers (bottom) ( e ) of total lung T RH , non-T RH , splenic T FH or splenic non-T FH at 28 d.p.i. f , Representative dot plot or cell numbers of lung tissue or splenic CD4 + T cells at 28 d.p.i. g , Cell numbers of lung or splenic influenza-NP 311-325 CD4 + T cell at 28 d.p.i. Data were pooled from two independent experiments (3-4 mice per group). P value were calculated by unpaired two-tailed Student’s t-test.

    Journal: bioRxiv

    Article Title: Tissue-resident CD4+ T helper cells assist protective respiratory mucosal B and CD8+ T cell memory responses

    doi: 10.1101/2020.02.28.970400

    Figure Lengend Snippet: Impaired T RH responses following B cell depletion, iBALT disruption or Bhlhe40 deficiency. a-c , WT ( a and c ) or IL-21-VFP reporter ( b ) mice were infected with PR8 and received with ctl IgG, α-CD20 or LTβR-Ig weekly in the present of FTY-720 (Experimental scheme in Fig 5 . a.). a , The efficiency of B cell depletion in the lung. b , Representative confocal images of IL-21-expressing cells in iBALT. c , Cell numbers of lung tissue B cells in mice received with ctl-IgG or LTβR-Ig. d-g , Bhlhe40 fl/fl or Bhlhe40 ΔT mice were infected with PR8. Representative dot plot ( d ), percentages (top) or cell numbers (bottom) ( e ) of total lung T RH , non-T RH , splenic T FH or splenic non-T FH at 28 d.p.i. f , Representative dot plot or cell numbers of lung tissue or splenic CD4 + T cells at 28 d.p.i. g , Cell numbers of lung or splenic influenza-NP 311-325 CD4 + T cell at 28 d.p.i. Data were pooled from two independent experiments (3-4 mice per group). P value were calculated by unpaired two-tailed Student’s t-test.

    Article Snippet: PR8-NP protein was purchased from Sino Biological.

    Techniques: Mouse Assay, Infection, Expressing, Two Tailed Test

    Lung “local” CD4 T cell help for the development robust memory CD8 + and B cell immunity. WT mice were infected with influenza PR8 ( a ) or X31 ( b-e ) and treated with control IgG or α-CD4 (starting at 14 d.p.i.) in the presence of daily FTY720 (starting at 13 d.p.i.). a , Blood lymphocytes in the PBS or FTY720 administrated mice. b-e , Numbers of lung B GC cells ( b ), total HA specific B cells ( c ), HA-specific B RM ( d ), and total CD8 + NP 366-374 memory T cells, CD8 + CD69 + NP 366-374 T RM or CD8 + CD69 + CD103 + NP 366-374 T RM cells ( e). f-h , WT mice were infected with PR8 and received with ctl IgG, low or high dose of α-CD4 (starting at 14 d.p.i.). Experimental scheme ( f ), representative dot plot of blood lymphocyte population ( g ) and lung lymphocyte population ( h ). Representative data were from at least two independent experiments (4-5 mice per group). P values were calculated by unpaired two-tailed Student’s t-test.

    Journal: bioRxiv

    Article Title: Tissue-resident CD4+ T helper cells assist protective respiratory mucosal B and CD8+ T cell memory responses

    doi: 10.1101/2020.02.28.970400

    Figure Lengend Snippet: Lung “local” CD4 T cell help for the development robust memory CD8 + and B cell immunity. WT mice were infected with influenza PR8 ( a ) or X31 ( b-e ) and treated with control IgG or α-CD4 (starting at 14 d.p.i.) in the presence of daily FTY720 (starting at 13 d.p.i.). a , Blood lymphocytes in the PBS or FTY720 administrated mice. b-e , Numbers of lung B GC cells ( b ), total HA specific B cells ( c ), HA-specific B RM ( d ), and total CD8 + NP 366-374 memory T cells, CD8 + CD69 + NP 366-374 T RM or CD8 + CD69 + CD103 + NP 366-374 T RM cells ( e). f-h , WT mice were infected with PR8 and received with ctl IgG, low or high dose of α-CD4 (starting at 14 d.p.i.). Experimental scheme ( f ), representative dot plot of blood lymphocyte population ( g ) and lung lymphocyte population ( h ). Representative data were from at least two independent experiments (4-5 mice per group). P values were calculated by unpaired two-tailed Student’s t-test.

    Article Snippet: PR8-NP protein was purchased from Sino Biological.

    Techniques: Mouse Assay, Infection, Two Tailed Test

    Lung PD-1 Hi FR4 Hi cells are tissue resident a , WT mice were infected with PR8. The expression of CD69, CXCR6 and Bhlhe40 in lung CD4 + PD-1 Hi FR4 Hi or CD4 + PD-1 Low FR4 Low NP 311-325 T cells or splenic T FH cells at 28 d.p.i. b-e , CD45.1 + (Host) or CD45.1 + CD45.2 + (Partner) WT mice were infected with PR8. Parabiosis surgery was performed at 21 d.p.i. Mice were sacrificed 14 days later for analysis. b , Schematics of parabiosis experiments. c , Composition of Host-derived or Partner-derived CD4 + T cells in the spleens of the parabionts. d , Frequencies of Host-derived or Partner-derived cells in the lung PD-1 Hi FR4 Hi or PD-1 Low FR4 Low total CD4 + T cell compartment. e , Frequencies of Host-derived or Partner-derived cells in influenza-specific lung CD4 + PD-1 Hi FR4 Hi or CD4 + PD-1 Low FR4 Low NP 311-325 T cell compartment. In a , the representative histograms were from at least two independent experiments (3-4 mice per group). Parabiosis data were pooled from two different experiments (two pairs per experiment). P values in d and e were analyzed by one-way ANOVA.

    Journal: bioRxiv

    Article Title: Tissue-resident CD4+ T helper cells assist protective respiratory mucosal B and CD8+ T cell memory responses

    doi: 10.1101/2020.02.28.970400

    Figure Lengend Snippet: Lung PD-1 Hi FR4 Hi cells are tissue resident a , WT mice were infected with PR8. The expression of CD69, CXCR6 and Bhlhe40 in lung CD4 + PD-1 Hi FR4 Hi or CD4 + PD-1 Low FR4 Low NP 311-325 T cells or splenic T FH cells at 28 d.p.i. b-e , CD45.1 + (Host) or CD45.1 + CD45.2 + (Partner) WT mice were infected with PR8. Parabiosis surgery was performed at 21 d.p.i. Mice were sacrificed 14 days later for analysis. b , Schematics of parabiosis experiments. c , Composition of Host-derived or Partner-derived CD4 + T cells in the spleens of the parabionts. d , Frequencies of Host-derived or Partner-derived cells in the lung PD-1 Hi FR4 Hi or PD-1 Low FR4 Low total CD4 + T cell compartment. e , Frequencies of Host-derived or Partner-derived cells in influenza-specific lung CD4 + PD-1 Hi FR4 Hi or CD4 + PD-1 Low FR4 Low NP 311-325 T cell compartment. In a , the representative histograms were from at least two independent experiments (3-4 mice per group). Parabiosis data were pooled from two different experiments (two pairs per experiment). P values in d and e were analyzed by one-way ANOVA.

    Article Snippet: PR8-NP protein was purchased from Sino Biological.

    Techniques: Mouse Assay, Infection, Expressing, Derivative Assay

    Transcriptional profiling reveals PD-1 Hi FR4 Hi cells exhibit both T FH and T RM gene signatures. a-f , WT mice were infected with PR8. Lung PD-1 Hi FR4 Hi or PD-1 Low FR4 Low CD4 + T cells and splenic T FH or non-T FH cells were sorted following exclusion of GITR Hi Treg cells and RNA-seq analysis was performed at 28 d.p.i. a , Heatmap expression of differentially expressed genes among lung PD-1 Hi FR4 Hi , PD-1 Low FR4 Low CD4 + T cells and splenic T FH or non-T FH cells. b , Principle component analysis of RNA-seq data of lung PD-1 Hi FR4 Hi , PD-1 Low FR4 Low CD4 + T cells and splenic T FH or non-T FH cells. c , Volcano plot of RNA-seq analysis of lung PD-1 Hi FR4 Hi or PD-1 Low FR4 Low CD4 + T cells. d , GSEA of the core T FH signature genes in lung CD4 + PD-1 Hi FR4 Hi and CD4 + PD-1 Low FR4 Low cells. e , Volcano plot of RNA-seq analysis on lung PD-1 Hi FR4 Hi CD4 + T and splenic T FH cells. f , GSEA of the core tissue-residency signature genes of T RM cells in lung PD-1 Hi FR4 Hi and splenic T FH cells. g-h , WT mice were infected with PR8. Lung PD-1 Hi FR4 Hi or PD-1 Low FR4 Low CD4 + T cells and splenic T FH cells were sorted at 28 d.p.i. Nanostring analysis on 560 immune-associated genes was performed. The expression of T FH -associated genes ( g ) or tissue-residency associated genes ( h ) in the three cell populations was depicted. For RNA-seq, data were from duplicates of pooled samples (n = 15). For nanostring analysis, data were from pooled samples (n = 10).

    Journal: bioRxiv

    Article Title: Tissue-resident CD4+ T helper cells assist protective respiratory mucosal B and CD8+ T cell memory responses

    doi: 10.1101/2020.02.28.970400

    Figure Lengend Snippet: Transcriptional profiling reveals PD-1 Hi FR4 Hi cells exhibit both T FH and T RM gene signatures. a-f , WT mice were infected with PR8. Lung PD-1 Hi FR4 Hi or PD-1 Low FR4 Low CD4 + T cells and splenic T FH or non-T FH cells were sorted following exclusion of GITR Hi Treg cells and RNA-seq analysis was performed at 28 d.p.i. a , Heatmap expression of differentially expressed genes among lung PD-1 Hi FR4 Hi , PD-1 Low FR4 Low CD4 + T cells and splenic T FH or non-T FH cells. b , Principle component analysis of RNA-seq data of lung PD-1 Hi FR4 Hi , PD-1 Low FR4 Low CD4 + T cells and splenic T FH or non-T FH cells. c , Volcano plot of RNA-seq analysis of lung PD-1 Hi FR4 Hi or PD-1 Low FR4 Low CD4 + T cells. d , GSEA of the core T FH signature genes in lung CD4 + PD-1 Hi FR4 Hi and CD4 + PD-1 Low FR4 Low cells. e , Volcano plot of RNA-seq analysis on lung PD-1 Hi FR4 Hi CD4 + T and splenic T FH cells. f , GSEA of the core tissue-residency signature genes of T RM cells in lung PD-1 Hi FR4 Hi and splenic T FH cells. g-h , WT mice were infected with PR8. Lung PD-1 Hi FR4 Hi or PD-1 Low FR4 Low CD4 + T cells and splenic T FH cells were sorted at 28 d.p.i. Nanostring analysis on 560 immune-associated genes was performed. The expression of T FH -associated genes ( g ) or tissue-residency associated genes ( h ) in the three cell populations was depicted. For RNA-seq, data were from duplicates of pooled samples (n = 15). For nanostring analysis, data were from pooled samples (n = 10).

    Article Snippet: PR8-NP protein was purchased from Sino Biological.

    Techniques: Mouse Assay, Infection, RNA Sequencing Assay, Expressing