pr8 np protein  (Sino Biological)


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    Name:
    Influenza A H1N1 Nucleoprotein NP Protein
    Description:
    A DNA sequence encoding the Influenza A virus A Puerto Rico 8 34 Mount Sinai H1N1 nucleoprotein AAM75159 1 Met1 Gly490 was fused with a polyhistidine tag at the C terminus
    Catalog Number:
    11675-V08B
    Price:
    None
    Category:
    recombinant protein
    Product Aliases:
    NP Protein H1N1
    Host:
    Baculovirus-Insect Cells
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    Structured Review

    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.
    A DNA sequence encoding the Influenza A virus A Puerto Rico 8 34 Mount Sinai H1N1 nucleoprotein AAM75159 1 Met1 Gly490 was fused with a polyhistidine tag at the C terminus
    https://www.bioz.com/result/pr8 np protein/product/Sino Biological
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    pr8 np protein - by Bioz Stars, 2021-09
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    Images

    1) Product Images from "Tissue-resident CD4+ T helper cells assist protective respiratory mucosal B and CD8+ T cell memory responses"

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

    Journal: bioRxiv

    doi: 10.1101/2020.02.28.970400

    “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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: 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).
    Figure Legend 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).

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

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    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.
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    Sino Biological a puerto rico 8 34 mount sinai
    Influenza A H1N1 virus T-cell epitope screen in HLA-DQ6.2 mice, and Pandemrix-vaccinated HLA-DQB1*0602 positive individuals. Spleen cells from Pandemrix-immunized HLA-DQ6.2 mice were stimulated in culture with pools of five overlapping 15-mer peptides each, covering a hemagglutinin (HA), b neuraminidase (NA) or c nucleoprotein (NP) from influenza (A/reassortant/NYMC X-179A (California/07/2009 × NYMC X-157)(H1N1)) vaccine virus used in Pandemrix (pooling cells from 2 × 2 mice, n = 2). Recombinant hemagglutinin (rHA) and nucleoprotein (rNP; A/Puerto <t>Rico/8/34</t> corresponding to the Pandemrix vaccine strain) were used as positive controls. d – f PBMC from Pandemrix-vaccinated HLA-DQB1*0602 positive individuals (sleep clinic patients without a diagnosis of NT1) were stimulated in culture with single 15-mer peptides, derived from the same vaccine virus strain ( n = 1–2). The expression of IFN-γ or IL-2 was measured by FMIA (protein) or RT-qPCR (mRNA). Results are expressed as ratios between cytokine concentrations (lines representing means) or relative gene expressions (dots representing single values, bars representing means) measured in peptide-stimulated and negative control samples (stimulation index). An asterisk (*) indicates a pool or single peptide that was selected for further testing.
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    “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

    CD4 and CD8 T cells are required for protective immunity to influenza A virus. C57BL/6 mice were vaccinated twice (at 3 weeks interval) with NP protein formulated in ADJ+GLA. At 70 days after booster vaccination, mice were challenged intranasally with H1N1/PR8 strain of influenza A virus; unvaccinated mice were challenged as controls. Cohorts of vaccinated virus-challenged mice were treated (intravenously and intranasally) with isotype control IgG, anti-CD4 or anti-CD8 antibodies at days -5, -3, -1 and 1, 3 and 5, relative to viral challenge. On the 6 th day after viral challenge, virus-specific T cells and viral titers were quantified in lungs. (A) FACS plots are gated on live lymphocytes and numbers are percentages among live lymphocytes. (B) FACS plots are gated on CD8 T cells and numbers are percentages of D b /NP366 tetramer-binding CD8 T cells among CD8 T cells. (C) FACS plots are gated on CD4 T cells and numbers are percentages of I-A b /NP311 tetramer-binding CD4 T cells among CD8 T cells. (D) Viral titers in lungs were quantified by a plaque assay. Data are from two independent experiments. *, **, and *** indicate significance at P

    Journal: bioRxiv

    Article Title: Programming Multifaceted Pulmonary T-Cell Immunity by Combination Adjuvants

    doi: 10.1101/2020.07.10.197459

    Figure Lengend Snippet: CD4 and CD8 T cells are required for protective immunity to influenza A virus. C57BL/6 mice were vaccinated twice (at 3 weeks interval) with NP protein formulated in ADJ+GLA. At 70 days after booster vaccination, mice were challenged intranasally with H1N1/PR8 strain of influenza A virus; unvaccinated mice were challenged as controls. Cohorts of vaccinated virus-challenged mice were treated (intravenously and intranasally) with isotype control IgG, anti-CD4 or anti-CD8 antibodies at days -5, -3, -1 and 1, 3 and 5, relative to viral challenge. On the 6 th day after viral challenge, virus-specific T cells and viral titers were quantified in lungs. (A) FACS plots are gated on live lymphocytes and numbers are percentages among live lymphocytes. (B) FACS plots are gated on CD8 T cells and numbers are percentages of D b /NP366 tetramer-binding CD8 T cells among CD8 T cells. (C) FACS plots are gated on CD4 T cells and numbers are percentages of I-A b /NP311 tetramer-binding CD4 T cells among CD8 T cells. (D) Viral titers in lungs were quantified by a plaque assay. Data are from two independent experiments. *, **, and *** indicate significance at P

    Article Snippet: Recombinant nucleoprotein (NP) of the PR8/H1N1 influenza virus strain was purchased from Sino Biological Inc (Beijing, China).

    Techniques: Mouse Assay, FACS, Binding Assay, Plaque Assay

    Histopathological analysis of lungs following viral challenge of vaccinated mice. Groups of C57BL/6 mice were vaccinated twice (at 3 weeks interval) with NP protein formulated in various adjuvants. At 100 days after booster vaccination, vaccinated mice were challenged intranasally with H1N1/PR8 strain of influenza A virus. On the 6 th day after viral challenge, lungs were collected in neutral-buffered formailin, and tissue sections were stained with Hematoxylin and Eosin (H E). H E stained lung sections were evaluated by a board-certified pathologist (Dr. Gasper); he was blinded to the identity of sections. In each image (40X magnification), asterisks indicate similarly sized large bronchioles, arrow heads indicate regions in which bronchial lesions extend in to the adjacent alveoli, and arrows indicate perivascular lymphoid nodules. A. Adjuplex-vaccinated mouse: there is mild necrotizing bronchitis asterisks). B. CPG-vaccinated mouse: there is obliteration of two bronchioles by inflammation that extends far into the surrounding alveoli (arrowheads). C. GLA-vaccinated mouse: there is bronchiolitis affecting 1 of the larger bronchioles, with minimal extension into the adjacent alveoli. D. ADJ+CPG-vaccinated mouse. Broncholitis is similar to that in A, but alveolar regions around the affected bronchiole (center) are infiltrated by inflammatory cells. E. ADJ+GLA vaccinated mouse: bronchiolitis is of intermediate severity between B and C, and regionally extends into the adjacent alveolar tissue (arrowhead). Each lung section was scored individually, and lesion scores from 0-3 were assigned for bronchial lesions, alveolar lesions, and specific disease patterns, with 0 = absent, 1 = mild, 2 = moderate, 3 = severe. Bronchioloar Lesions: Epithelial degeneration/necrosis; Intraepithelial neutrophils; Intraepithelial eosinophils; Intraepithelial lymphocytes; Luminal dislodged epithelial cells/debris; Luminal cellular exudate; Peribronchiolar neutrophils; Pavementing/Subendothelial leukocytes. Alveolar Lesions: Alveolar wall thickening; Interstitial macrophages; Interstitial lymphocytes; Interstitial granulocytes; Epithelial necrosis; Luminal edema; Luminal hemorrhage; Luminal cellular exudate; Luminal alveolar macrophages; Luminal neutrophils; Luminal sloughed epithelial cells.

    Journal: bioRxiv

    Article Title: Programming Multifaceted Pulmonary T-Cell Immunity by Combination Adjuvants

    doi: 10.1101/2020.07.10.197459

    Figure Lengend Snippet: Histopathological analysis of lungs following viral challenge of vaccinated mice. Groups of C57BL/6 mice were vaccinated twice (at 3 weeks interval) with NP protein formulated in various adjuvants. At 100 days after booster vaccination, vaccinated mice were challenged intranasally with H1N1/PR8 strain of influenza A virus. On the 6 th day after viral challenge, lungs were collected in neutral-buffered formailin, and tissue sections were stained with Hematoxylin and Eosin (H E). H E stained lung sections were evaluated by a board-certified pathologist (Dr. Gasper); he was blinded to the identity of sections. In each image (40X magnification), asterisks indicate similarly sized large bronchioles, arrow heads indicate regions in which bronchial lesions extend in to the adjacent alveoli, and arrows indicate perivascular lymphoid nodules. A. Adjuplex-vaccinated mouse: there is mild necrotizing bronchitis asterisks). B. CPG-vaccinated mouse: there is obliteration of two bronchioles by inflammation that extends far into the surrounding alveoli (arrowheads). C. GLA-vaccinated mouse: there is bronchiolitis affecting 1 of the larger bronchioles, with minimal extension into the adjacent alveoli. D. ADJ+CPG-vaccinated mouse. Broncholitis is similar to that in A, but alveolar regions around the affected bronchiole (center) are infiltrated by inflammatory cells. E. ADJ+GLA vaccinated mouse: bronchiolitis is of intermediate severity between B and C, and regionally extends into the adjacent alveolar tissue (arrowhead). Each lung section was scored individually, and lesion scores from 0-3 were assigned for bronchial lesions, alveolar lesions, and specific disease patterns, with 0 = absent, 1 = mild, 2 = moderate, 3 = severe. Bronchioloar Lesions: Epithelial degeneration/necrosis; Intraepithelial neutrophils; Intraepithelial eosinophils; Intraepithelial lymphocytes; Luminal dislodged epithelial cells/debris; Luminal cellular exudate; Peribronchiolar neutrophils; Pavementing/Subendothelial leukocytes. Alveolar Lesions: Alveolar wall thickening; Interstitial macrophages; Interstitial lymphocytes; Interstitial granulocytes; Epithelial necrosis; Luminal edema; Luminal hemorrhage; Luminal cellular exudate; Luminal alveolar macrophages; Luminal neutrophils; Luminal sloughed epithelial cells.

    Article Snippet: Recombinant nucleoprotein (NP) of the PR8/H1N1 influenza virus strain was purchased from Sino Biological Inc (Beijing, China).

    Techniques: Mouse Assay, Staining

    Vaccine-induced protective immunity to H1N1 and H5N1 influenza viruses. (A-C) Groups of C57BL/6 mice were vaccinated twice at 3-week intervals with NP protein formulated in various adjuvants.. (A) At 100 days after the booster vaccination, mice were challenged intranasally with H1N1/PR8 strain of influenza A virus. Viral tiers and virus-specific T cell responses in lungs were quantified on the 6 th day after virus challenge. (A) Viral titers in the lungs on the 6 th day after virus challenge. (B) Percentages of NP366-specific IFN-γ and IL-17 producing cells among CD8 T cells (bar graphs) and calculated proportions of IFN-γ and/or IL-17 producing cells among total IFN-γ+IL-7-producing peptide-stimulated NP366-specific T cells (Pie charts). (C) Percentages of NP311-specific IFN-γ and IL-17 producing cells among CD4 T cells and calculated proportions of IFN-γ and/or IL-17 producing cells among total IFN-γ+IL-7-producing peptide-stimulated NP311-specific T cells. (D) C57BL/6 mice were vaccinated with NP+ADJ+GLA twice at an interval of 3 weeks. One hundred and eighty days after the last vaccination, mice were challenged intransally with H1N1/PR8 strain of influenza A virus; unvaccinated mice were challenged with virus as controls. Cohorts of vaccinated virus-challenged mice were treated with isotype control IgG or anti-IL-17A antibodies (intravenously and intranasally) at -1, 0, 1, 3 and 5 days relative to virus challenge. On the 6 th day after viral challenge, viral titers and virus-specific T cell responses were quantified in lungs. (E) Groups of C57BL/6 mice were vaccinated twice with NP protein alone or formulated in various adjuvants. Fifty days after booster vaccination, vaccinated and unvaccinated mice were challenged intranasally with the highly pathogenic H5N1 avian influenza A virus; weight loss and survival was monitored until day 14. Data are pooled from 2 independent experiments or representative of two independent experiments. *, **, and *** indicate significance at P

    Journal: bioRxiv

    Article Title: Programming Multifaceted Pulmonary T-Cell Immunity by Combination Adjuvants

    doi: 10.1101/2020.07.10.197459

    Figure Lengend Snippet: Vaccine-induced protective immunity to H1N1 and H5N1 influenza viruses. (A-C) Groups of C57BL/6 mice were vaccinated twice at 3-week intervals with NP protein formulated in various adjuvants.. (A) At 100 days after the booster vaccination, mice were challenged intranasally with H1N1/PR8 strain of influenza A virus. Viral tiers and virus-specific T cell responses in lungs were quantified on the 6 th day after virus challenge. (A) Viral titers in the lungs on the 6 th day after virus challenge. (B) Percentages of NP366-specific IFN-γ and IL-17 producing cells among CD8 T cells (bar graphs) and calculated proportions of IFN-γ and/or IL-17 producing cells among total IFN-γ+IL-7-producing peptide-stimulated NP366-specific T cells (Pie charts). (C) Percentages of NP311-specific IFN-γ and IL-17 producing cells among CD4 T cells and calculated proportions of IFN-γ and/or IL-17 producing cells among total IFN-γ+IL-7-producing peptide-stimulated NP311-specific T cells. (D) C57BL/6 mice were vaccinated with NP+ADJ+GLA twice at an interval of 3 weeks. One hundred and eighty days after the last vaccination, mice were challenged intransally with H1N1/PR8 strain of influenza A virus; unvaccinated mice were challenged with virus as controls. Cohorts of vaccinated virus-challenged mice were treated with isotype control IgG or anti-IL-17A antibodies (intravenously and intranasally) at -1, 0, 1, 3 and 5 days relative to virus challenge. On the 6 th day after viral challenge, viral titers and virus-specific T cell responses were quantified in lungs. (E) Groups of C57BL/6 mice were vaccinated twice with NP protein alone or formulated in various adjuvants. Fifty days after booster vaccination, vaccinated and unvaccinated mice were challenged intranasally with the highly pathogenic H5N1 avian influenza A virus; weight loss and survival was monitored until day 14. Data are pooled from 2 independent experiments or representative of two independent experiments. *, **, and *** indicate significance at P

    Article Snippet: Recombinant nucleoprotein (NP) of the PR8/H1N1 influenza virus strain was purchased from Sino Biological Inc (Beijing, China).

    Techniques: Mouse Assay

    Kinetics and durability of influenza viral control in vaccinated mice. B6 mice were vaccinated twice (at 3 weeks intervals) intranasally with NP protein formulated with the indicated adjuvants. Unvaccinated mice and mice vaccinated with NP only (without adjuvants) served as controls. At 100 and 180 days after booster vaccination, mice were challenged intranasally with PR8/H1N1 influenza virus. (A) Body weight loss was assessed by calculating bodyweight at different days after challenge, relative to bodyweight before challenge at 100 days after vaccination. (B) Vaccinated mice were challenged with PR8/H1N1at 100 days after vaccination and viral titers in lungs were quantified at day 2 and 4 after challenge, using a plaque assay. (C) At day 180 after booster vaccination, mice were challenged with PR8/H1N1 virus, and viral titers in lungs were assessed at day 6 after challenge. (D) Percentages and numbers of NP366-specific CD8 T cells and NP311-specific CD4 T cells in lungs and percentage of these cells in the vascular and non-vascular compartment at day 6 after challenge (challenged at 100 days after vaccination). *, **, and *** indicate significance at P

    Journal: bioRxiv

    Article Title: Programming Multifaceted Pulmonary T-Cell Immunity by Combination Adjuvants

    doi: 10.1101/2020.07.10.197459

    Figure Lengend Snippet: Kinetics and durability of influenza viral control in vaccinated mice. B6 mice were vaccinated twice (at 3 weeks intervals) intranasally with NP protein formulated with the indicated adjuvants. Unvaccinated mice and mice vaccinated with NP only (without adjuvants) served as controls. At 100 and 180 days after booster vaccination, mice were challenged intranasally with PR8/H1N1 influenza virus. (A) Body weight loss was assessed by calculating bodyweight at different days after challenge, relative to bodyweight before challenge at 100 days after vaccination. (B) Vaccinated mice were challenged with PR8/H1N1at 100 days after vaccination and viral titers in lungs were quantified at day 2 and 4 after challenge, using a plaque assay. (C) At day 180 after booster vaccination, mice were challenged with PR8/H1N1 virus, and viral titers in lungs were assessed at day 6 after challenge. (D) Percentages and numbers of NP366-specific CD8 T cells and NP311-specific CD4 T cells in lungs and percentage of these cells in the vascular and non-vascular compartment at day 6 after challenge (challenged at 100 days after vaccination). *, **, and *** indicate significance at P

    Article Snippet: Recombinant nucleoprotein (NP) of the PR8/H1N1 influenza virus strain was purchased from Sino Biological Inc (Beijing, China).

    Techniques: Mouse Assay, Plaque Assay

    Regulation of vaccine-induced CD8 T-cell memory and protective immunity by CD4 T cells. Groups of C57BL/6 mice were vaccinated twice at 3-week intervals with NP protein formulated in ADJ+GLA. Cohorts of vaccinated mice were treated with isotype control antibodies (Non Depleted) or anti-CD4 antibodies (CD4 Depleted) intravenously and intranasally on days −1, 0, and 1 relative to prime and boost vaccination with NP+ADJ+GLA. T-cell memory in lungs (A-F) and protective immunity to influenza A virus (G-P) was determined at 80 days after booster vaccination. (A-F) T-cell memory in lungs at day 80 after booster vaccination. To stain for vascular cells, mice were injected intravenously with anti-CD45.2 antibodies, 3 minutes prior to euthanasia. Lung cells were stained directly ex vivo with D b /NP366 or I-A b /NP311 tetramers along with the indicated antibodies for cell surface markers. For cytokine analysis, lung cells were stimulated with NP366 or NP311 peptide for 5 hours before intracellular staining. (A) FACS plots are gated on total CD4 T cells and show NP311-specific tetramer-binding memory CD4 T cells only in non-depleted mice. (B) NP366-specific tetramer-binding memory CD8 T cells in lungs of non-d epleted and CD4 T cell-depleted mice. (C) Expression of tissue residency markers on NP366-specific tetramer-binding memory CD8 T cells in lungs. (D) Percentage of vascular (CD45.2 +ve ) and non-vascular (CD45.2 -ve ) cells among NP366-specific tetramer-binding memory CD8 T cells in lungs. (E) Percentages of IFN-γ- and IL-17-producing NP366-specific cells among CD8 T cells in lungs. (F) Calculated proportions of IFN-γ and/or IL-17-producing cells among cytokine-producing peptide-stimulated IFN-γ+IL-17 NP366-specific CD8 T cells. (G-P) At day 80 after booster vaccination, non-depleted and CD4 T cell-depleted mice were challenged intranasally with PR8/H1N1 influenza A virus; recall virus-specific CD8/CD4 T cell responses and viral load in lungs were assessed at day 6 after challenge. (G) Percentages of NP366-specific tetramer-binding cells among CD8 T cells in lungs. (H) Percentages of NP366-specific tetramer-binding CD8 T cells in vascular and nonvascular lung compartment. (I) Percentages of NP311-specific tetramer-binding cells among CD4 T cells in lungs. (J) Expression of tissue residency markers on NP366-specific tetramer-binding CD8 T cells. (K) Chemokine receptor and transcription factor expression in NP366-specific CD8 T cells in lungs. (L) Granzyme B expression by NP366-specific CD8 T cells directly ex vivo. (M) Percentages of IFN-γ and IL-17 producing NP366-specific CD8 T cells. (N) Relative proportions of IFN-γ and/or IL-17 producing cells among total IFN-γ plus IL-17-producing peptide-stimulated NP366-specific CD8 T cells. (O) Viral titers in lungs at day 6 after challenge. (P) Body weight, measured as a percentage of starting body weight prior to challenge. Data are pooled from two independent experiments. *, **, and *** indicate significance at P

    Journal: bioRxiv

    Article Title: Programming Multifaceted Pulmonary T-Cell Immunity by Combination Adjuvants

    doi: 10.1101/2020.07.10.197459

    Figure Lengend Snippet: Regulation of vaccine-induced CD8 T-cell memory and protective immunity by CD4 T cells. Groups of C57BL/6 mice were vaccinated twice at 3-week intervals with NP protein formulated in ADJ+GLA. Cohorts of vaccinated mice were treated with isotype control antibodies (Non Depleted) or anti-CD4 antibodies (CD4 Depleted) intravenously and intranasally on days −1, 0, and 1 relative to prime and boost vaccination with NP+ADJ+GLA. T-cell memory in lungs (A-F) and protective immunity to influenza A virus (G-P) was determined at 80 days after booster vaccination. (A-F) T-cell memory in lungs at day 80 after booster vaccination. To stain for vascular cells, mice were injected intravenously with anti-CD45.2 antibodies, 3 minutes prior to euthanasia. Lung cells were stained directly ex vivo with D b /NP366 or I-A b /NP311 tetramers along with the indicated antibodies for cell surface markers. For cytokine analysis, lung cells were stimulated with NP366 or NP311 peptide for 5 hours before intracellular staining. (A) FACS plots are gated on total CD4 T cells and show NP311-specific tetramer-binding memory CD4 T cells only in non-depleted mice. (B) NP366-specific tetramer-binding memory CD8 T cells in lungs of non-d epleted and CD4 T cell-depleted mice. (C) Expression of tissue residency markers on NP366-specific tetramer-binding memory CD8 T cells in lungs. (D) Percentage of vascular (CD45.2 +ve ) and non-vascular (CD45.2 -ve ) cells among NP366-specific tetramer-binding memory CD8 T cells in lungs. (E) Percentages of IFN-γ- and IL-17-producing NP366-specific cells among CD8 T cells in lungs. (F) Calculated proportions of IFN-γ and/or IL-17-producing cells among cytokine-producing peptide-stimulated IFN-γ+IL-17 NP366-specific CD8 T cells. (G-P) At day 80 after booster vaccination, non-depleted and CD4 T cell-depleted mice were challenged intranasally with PR8/H1N1 influenza A virus; recall virus-specific CD8/CD4 T cell responses and viral load in lungs were assessed at day 6 after challenge. (G) Percentages of NP366-specific tetramer-binding cells among CD8 T cells in lungs. (H) Percentages of NP366-specific tetramer-binding CD8 T cells in vascular and nonvascular lung compartment. (I) Percentages of NP311-specific tetramer-binding cells among CD4 T cells in lungs. (J) Expression of tissue residency markers on NP366-specific tetramer-binding CD8 T cells. (K) Chemokine receptor and transcription factor expression in NP366-specific CD8 T cells in lungs. (L) Granzyme B expression by NP366-specific CD8 T cells directly ex vivo. (M) Percentages of IFN-γ and IL-17 producing NP366-specific CD8 T cells. (N) Relative proportions of IFN-γ and/or IL-17 producing cells among total IFN-γ plus IL-17-producing peptide-stimulated NP366-specific CD8 T cells. (O) Viral titers in lungs at day 6 after challenge. (P) Body weight, measured as a percentage of starting body weight prior to challenge. Data are pooled from two independent experiments. *, **, and *** indicate significance at P

    Article Snippet: Recombinant nucleoprotein (NP) of the PR8/H1N1 influenza virus strain was purchased from Sino Biological Inc (Beijing, China).

    Techniques: Mouse Assay, Staining, Injection, Ex Vivo, FACS, Binding Assay, Expressing

    Functional polarization of recall CD8 and CD4 T cells. Cohorts of mice were vaccinated twice, as described in Figure 2 . 101 days after booster vaccination, mice were challenged with H1N1/PR8 strain of influenza A virus. Lung cells were stimulated ex vivo with NP366 or NP311 peptides for 5 h. The percentages of NP366-stimulated CD8, or NP311-stimulated CD4 T cells that produced IFN-γ, IL-17, TNF-α, and IL-2 were quantified by intracellular cytokine staining. Graphs show the percentages of cytokine-producing cells among the gated CD8 T cells or TNF-α and IL-2-producing cells among gated IFN-γ-producing CD8 T cells (A) . Graphs show the percentages of cytokine-producing cells among CD4 T cells or TNF-α and IL-2-producing cells among gated IFN-γ-producing CD4 T cells (B) . Data are representative of two independent experiments. *, **, ***, and **** indicate significance at P

    Journal: Frontiers in Immunology

    Article Title: Polymeric Pathogen-Like Particles-Based Combination Adjuvants Elicit Potent Mucosal T Cell Immunity to Influenza A Virus

    doi: 10.3389/fimmu.2020.559382

    Figure Lengend Snippet: Functional polarization of recall CD8 and CD4 T cells. Cohorts of mice were vaccinated twice, as described in Figure 2 . 101 days after booster vaccination, mice were challenged with H1N1/PR8 strain of influenza A virus. Lung cells were stimulated ex vivo with NP366 or NP311 peptides for 5 h. The percentages of NP366-stimulated CD8, or NP311-stimulated CD4 T cells that produced IFN-γ, IL-17, TNF-α, and IL-2 were quantified by intracellular cytokine staining. Graphs show the percentages of cytokine-producing cells among the gated CD8 T cells or TNF-α and IL-2-producing cells among gated IFN-γ-producing CD8 T cells (A) . Graphs show the percentages of cytokine-producing cells among CD4 T cells or TNF-α and IL-2-producing cells among gated IFN-γ-producing CD4 T cells (B) . Data are representative of two independent experiments. *, **, ***, and **** indicate significance at P

    Article Snippet: Vaccines and Vaccinations PR8 nucleoprotein (NP) was purchased from Sino Biological Inc (Beijing, China).

    Techniques: Functional Assay, Mouse Assay, Ex Vivo, Produced, Staining

    Long term memory and role of CD4 and CD8 T cells in vaccine-induced immunity to influenza virus. Three hundred sixty-two days after booster vaccination, mice were challenged with H1N1/PR8 strain of influenza A virus (A–C) . Viral titers were quantified in the lungs on D6 after challenge (A) . Numbers and frequencies of NP366-specific CD8 T cells and NP311-specific CD4 T cells in lungs (B) . The linear regression curve was plotted for data from individual mice frequencies of NP366-specific CD8 T cells or NP311-specific CD4 T cells plotted against its Log 10 viral titer value, (D, E) : Mice were vaccinated with NP formulated in ADJ+PLP-GLA, and 3 weeks later, either CD4 or CD8 T cells were depleted immediately prior to and during a lethal H1N1 PR8 influenza challenge. An additional cohort of ADJ+PLP-GLA vaccinated mice was treated with FTY720 immediately before and during viral challenge. Viral titers were quantified in the lungs on D6 after challenge (D) . Numbers and frequencies of NP366-specific CD8 T cells and NP311-specific CD4 T cells in lungs (E) . *, **, ***, and **** indicate significance at P

    Journal: Frontiers in Immunology

    Article Title: Polymeric Pathogen-Like Particles-Based Combination Adjuvants Elicit Potent Mucosal T Cell Immunity to Influenza A Virus

    doi: 10.3389/fimmu.2020.559382

    Figure Lengend Snippet: Long term memory and role of CD4 and CD8 T cells in vaccine-induced immunity to influenza virus. Three hundred sixty-two days after booster vaccination, mice were challenged with H1N1/PR8 strain of influenza A virus (A–C) . Viral titers were quantified in the lungs on D6 after challenge (A) . Numbers and frequencies of NP366-specific CD8 T cells and NP311-specific CD4 T cells in lungs (B) . The linear regression curve was plotted for data from individual mice frequencies of NP366-specific CD8 T cells or NP311-specific CD4 T cells plotted against its Log 10 viral titer value, (D, E) : Mice were vaccinated with NP formulated in ADJ+PLP-GLA, and 3 weeks later, either CD4 or CD8 T cells were depleted immediately prior to and during a lethal H1N1 PR8 influenza challenge. An additional cohort of ADJ+PLP-GLA vaccinated mice was treated with FTY720 immediately before and during viral challenge. Viral titers were quantified in the lungs on D6 after challenge (D) . Numbers and frequencies of NP366-specific CD8 T cells and NP311-specific CD4 T cells in lungs (E) . *, **, ***, and **** indicate significance at P

    Article Snippet: Vaccines and Vaccinations PR8 nucleoprotein (NP) was purchased from Sino Biological Inc (Beijing, China).

    Techniques: Mouse Assay, Plasmid Purification

    Vaccine-induced protective immunity influenza virus. Cohorts of mice were vaccinated twice, as described in Figure 2 . At 101 days after booster vaccination, mice were challenged with H1N1/PR8 strain of influenza A virus; unvaccinated mice were challenged as controls. Viral titers were quantified in the lungs on D6 after challenge (A) . Percentages or numbers of NP366-specific CD8 T cells and NP311-specific CD4 T cells in lungs (B) . Graphs show percentages of cells among the gated NP366-specific CD8 T cells or expression levels of granzyme B in the gated NP366-specific CD8 T cells (C) . Data are representative of two independent experiments. *, **, ***, and **** indicate significance at P

    Journal: Frontiers in Immunology

    Article Title: Polymeric Pathogen-Like Particles-Based Combination Adjuvants Elicit Potent Mucosal T Cell Immunity to Influenza A Virus

    doi: 10.3389/fimmu.2020.559382

    Figure Lengend Snippet: Vaccine-induced protective immunity influenza virus. Cohorts of mice were vaccinated twice, as described in Figure 2 . At 101 days after booster vaccination, mice were challenged with H1N1/PR8 strain of influenza A virus; unvaccinated mice were challenged as controls. Viral titers were quantified in the lungs on D6 after challenge (A) . Percentages or numbers of NP366-specific CD8 T cells and NP311-specific CD4 T cells in lungs (B) . Graphs show percentages of cells among the gated NP366-specific CD8 T cells or expression levels of granzyme B in the gated NP366-specific CD8 T cells (C) . Data are representative of two independent experiments. *, **, ***, and **** indicate significance at P

    Article Snippet: Vaccines and Vaccinations PR8 nucleoprotein (NP) was purchased from Sino Biological Inc (Beijing, China).

    Techniques: Mouse Assay, Expressing

    Influenza A H1N1 virus T-cell epitope screen in HLA-DQ6.2 mice, and Pandemrix-vaccinated HLA-DQB1*0602 positive individuals. Spleen cells from Pandemrix-immunized HLA-DQ6.2 mice were stimulated in culture with pools of five overlapping 15-mer peptides each, covering a hemagglutinin (HA), b neuraminidase (NA) or c nucleoprotein (NP) from influenza (A/reassortant/NYMC X-179A (California/07/2009 × NYMC X-157)(H1N1)) vaccine virus used in Pandemrix (pooling cells from 2 × 2 mice, n = 2). Recombinant hemagglutinin (rHA) and nucleoprotein (rNP; A/Puerto Rico/8/34 corresponding to the Pandemrix vaccine strain) were used as positive controls. d – f PBMC from Pandemrix-vaccinated HLA-DQB1*0602 positive individuals (sleep clinic patients without a diagnosis of NT1) were stimulated in culture with single 15-mer peptides, derived from the same vaccine virus strain ( n = 1–2). The expression of IFN-γ or IL-2 was measured by FMIA (protein) or RT-qPCR (mRNA). Results are expressed as ratios between cytokine concentrations (lines representing means) or relative gene expressions (dots representing single values, bars representing means) measured in peptide-stimulated and negative control samples (stimulation index). An asterisk (*) indicates a pool or single peptide that was selected for further testing.

    Journal: Nature Communications

    Article Title: Enhanced influenza A H1N1 T cell epitope recognition and cross-reactivity to protein-O-mannosyltransferase 1 in Pandemrix-associated narcolepsy type 1

    doi: 10.1038/s41467-021-22637-8

    Figure Lengend Snippet: Influenza A H1N1 virus T-cell epitope screen in HLA-DQ6.2 mice, and Pandemrix-vaccinated HLA-DQB1*0602 positive individuals. Spleen cells from Pandemrix-immunized HLA-DQ6.2 mice were stimulated in culture with pools of five overlapping 15-mer peptides each, covering a hemagglutinin (HA), b neuraminidase (NA) or c nucleoprotein (NP) from influenza (A/reassortant/NYMC X-179A (California/07/2009 × NYMC X-157)(H1N1)) vaccine virus used in Pandemrix (pooling cells from 2 × 2 mice, n = 2). Recombinant hemagglutinin (rHA) and nucleoprotein (rNP; A/Puerto Rico/8/34 corresponding to the Pandemrix vaccine strain) were used as positive controls. d – f PBMC from Pandemrix-vaccinated HLA-DQB1*0602 positive individuals (sleep clinic patients without a diagnosis of NT1) were stimulated in culture with single 15-mer peptides, derived from the same vaccine virus strain ( n = 1–2). The expression of IFN-γ or IL-2 was measured by FMIA (protein) or RT-qPCR (mRNA). Results are expressed as ratios between cytokine concentrations (lines representing means) or relative gene expressions (dots representing single values, bars representing means) measured in peptide-stimulated and negative control samples (stimulation index). An asterisk (*) indicates a pool or single peptide that was selected for further testing.

    Article Snippet: Recombinant influenza A H1N1 (A/California/07/2009) hemagglutinin (cat#11085-V08H), (A/California/04/2009) neuraminidase (cat#11058-VNAHC or cat#11058-V01H), and (A/California/07/2009) or (A/Puerto Rico/8/34/Mount Sinai) nucleoprotein (cat#40205-V08B and cat#11675-V08B) were purchased from Sino Biological, Beijing, China.

    Techniques: Mouse Assay, Recombinant, Derivative Assay, Expressing, Quantitative RT-PCR, Negative Control