Journal: Veterinary Research

Article Title: Characterization of NCR1+ cells residing in lymphoid tissues in the gut of lambs indicates that the majority are NK cells

doi: 10.1186/1297-9716-44-109

Figure Lengend Snippet: Primary antibodies used in this study

Article Snippet: Mouse anti-bovine CD205 (MCA1651) , CC98 , IgG2b , CD205 , Dendritic cells, some T cells, some B cells, some epithelial cells , AbD Serotec, Ltd., Oxford, UK , IF , Sheep , .

Techniques: Expressing

Journal: Nature immunology

Article Title: Regulation of T H 2 development by CXCR5 + dendritic cells and lymphotoxin-expressing B cells

doi: 10.1038/ni.2309

Figure Lengend Snippet: ( a,b ) Membrane LT expression on mesLN cells from naïve ( a ) and day 8 Hp -infected B6 mice ( b ) were were determined by flow cytometry. Control staining in the presence of anti-LTβ blocking antibody is shown. ( c ) CD19 + B cells from day 8 Hp -infected B6 and MD4μMT mice were stained with mLTβR-Fc (open histograms) or control antibody (filled histogram). ( d ) CD11c-DTR BM chimeric mice, treated with PBS (DC sufficient) or DT on days 0 and 3 (DC depletion), were infected with Hp . LT expression (open histogram) is shown on CD19 + B cells. Isotype control staining is shown in filled histogram. ( e–j ) B6 mice were treated (i.p.) with 100 μg of LTβR-Fc fusion protein or control protein at the time of Hp infection or with 250 μg of anti-CD20 or control Ab four days before Hp infection. Analyses were performed on day 8 post-infection. ( e ) Cxcl13 mRNA in mesLN cells were determined by quantitative PCR. Data show mean ± SD of three samples. nd=not detected. ( f,g ) The frequencies ( f ) and numbers ( g ) of MHCII + CD11c int mature and MHCII lo CD11c hi immature mesLN DCs in LTβR-Fc and control-treated B6 mice were determined by flow cytometry. ( h,i ) Cryosections from mesLNs of control ( h ) and LTβR-Fc treated mice ( i ) were stained with anti-B220, anti-CD11c and anti-CD4. Scale bar = 400 μm. ( j ) mesLN cells were restimulated with anti-CD3 + BFA for 4 h and the numbers of IL-4 + CD4 + T cells from LTβR-Fc-treated mice and B6 controls were determined by ICCS. Data are representative of three independent experiments (mean ± SD of five mice per group, *P<0.001 vs control, **P<0.05, ***P<0.01).

Article Snippet: Conjugated antibodies to CD205 (NLDC-145), CD11c (HL3), B220 (RA3-6B2), CD19 (1D3), CD40 (3/23), MHCII (AF6-120.1), CD4 (GK1.5), CD44 (IM7), CCR7 (4B12), CXCR5 (2G8), PD-1 (J43), ICOS (7E.17G9), CD45.1(A20) and CD45.2 (104) were purchased from from Cedarlane Laboratories (CD205) or BD-Biosciences (all others).

Techniques: Membrane, Expressing, Infection, Flow Cytometry, Control, Staining, Blocking Assay, Real-time Polymerase Chain Reaction

Journal: Nature immunology

Article Title: Regulation of T H 2 development by CXCR5 + dendritic cells and lymphotoxin-expressing B cells

doi: 10.1038/ni.2309

Figure Lengend Snippet: ( a–d ) mesLN cells from day 8 Hp -infected IL-4 (EGFP) reporter mice were analyzed by flow cytometry. ( a ) CD4 + T cells were divided into naïve (CD44 lo EGFP − ) and antigen-experienced (CD44 hi EGFP + ) populations, analyzed for expression of ICOS and PD-1 and subdivided into naïve (CD44 lo EGFP − ICOS − PD-1 − ), T FH (CD44 hi EGFP + ICOS + PD-1 hi ) and T effector (CD44 hi EGFP + ICOS + PD-1 lo ) subsets. ( b ) mesLN cells were restimulated with anti-CD3 + BFA for 4 h and the frequencies of IL-4 + naïve, ICOS + PD-1 lo and ICOS + PD-1 hi CD4 T cells were determined by ICCS. ( c,d ) Intracellular staining to detect Bcl6 was performed on non-restimulated mesLN cells. Bcl6 expression in naïve, ICOS + PD-1 lo and ICOS + PD-1 hi gated CD4 T cells is shown in ( c ) and CXCR5 and PD-1 expression on gated naïve, EGFP + Bcl6 lo and EGFP + Bcl6 hi CD4 + T cells are shown in panel d (see also gating strategy in ). ( e ) Serial cryosections of mesLNs from uninfected or day 8 Hp -infected B6 mice were stained with anti-CD8, anti-B220, anti-CD11c or anti-CD4 (Scale bar = 400 μm). ( f–h ) B6 mice were treated with anti-CXCL13 or control antibody, infected with Hp and analyzed on day 8. Cryosections ( f ) of mesLNs were stained with anti-CD4 and anti-B220 (Scale bar = 400 μm). mesLN cells were analyzed by flow cytometry immediately ex vivo ( g ) or following 4 h restimulation with anti-CD3 + BFA ( h ). The numbers of ICOS + PD-1 hi CD44 hi T FH cells ( g ) and IL-4 + ICOS + PD-1 lo T H 2 cells ( h ) are shown. ( i–l ) Chimeric mice reconstituted with 50% CD45.1 + B6 BM and 50% CD45.2 + Cxcr5 −/− BM were infected with Hp and analyzed on day 8 post-infection. ( j ) The frequencies of total CD45.1 + and CD45.2 + cells in the mesLN and within the gated naïve (CD44 lo ), ICOS + PD-1 hi T FH and ICOS + PD-1 lo effector CD4 populations were determined by flow cytometry. ( k,l ) The mesLN cells were restimulated with anti-CD3 + BFA for 4 h and the frequencies of CD45.1 + and CD45.2 + cells within the gated IL-4 + CD4 T cell population were determined in the mesLN ( k ) and PEC ( l ) by ICCS. Data are representative of three independent experiments (mean ± SD of five mice per group, *P<0.01, **P<0.001).

Article Snippet: Conjugated antibodies to CD205 (NLDC-145), CD11c (HL3), B220 (RA3-6B2), CD19 (1D3), CD40 (3/23), MHCII (AF6-120.1), CD4 (GK1.5), CD44 (IM7), CCR7 (4B12), CXCR5 (2G8), PD-1 (J43), ICOS (7E.17G9), CD45.1(A20) and CD45.2 (104) were purchased from from Cedarlane Laboratories (CD205) or BD-Biosciences (all others).

Techniques: Infection, Flow Cytometry, Expressing, Staining, Control, Ex Vivo

Journal: Nature immunology

Article Title: Regulation of T H 2 development by CXCR5 + dendritic cells and lymphotoxin-expressing B cells

doi: 10.1038/ni.2309

Figure Lengend Snippet: ( a,b ) B6 mice were infected by gavage with 200 L3 Hp or intranasally with 0.1 LD 50 of x31 influenza virus. Cryosections from the medLNs or mesLNs of uninfected or day 8 infected mice were stained anti-CD11c, anti-B220 and anti-CD21. The region within the box in panel ( a ) was enlarged in panel ( b ). Scale bar = 400 μm. All data are representative of three or more independent experiments.

Article Snippet: Conjugated antibodies to CD205 (NLDC-145), CD11c (HL3), B220 (RA3-6B2), CD19 (1D3), CD40 (3/23), MHCII (AF6-120.1), CD4 (GK1.5), CD44 (IM7), CCR7 (4B12), CXCR5 (2G8), PD-1 (J43), ICOS (7E.17G9), CD45.1(A20) and CD45.2 (104) were purchased from from Cedarlane Laboratories (CD205) or BD-Biosciences (all others).

Techniques: Infection, Virus, Staining

Journal: Nature immunology

Article Title: Regulation of T H 2 development by CXCR5 + dendritic cells and lymphotoxin-expressing B cells

doi: 10.1038/ni.2309

Figure Lengend Snippet: ( a–e ) B6 and plt/plt mice were infected with Hp and analyzed on day 8. The frequencies ( a ) and numbers ( b ) of MHCII lo CD11c hi immature DCs and MHCII + CD11c int mature DCs in the mesLNs were determined. ( c ) Cryosections of mesLNs from Hp -infected mice were stained with anti-CD11c and anti-B220. Scale bar = 400 μm. ( d–e ) mesLN cells from Hp -infected plt/plt or B6 mice were restimulated with anti-CD3 + BFA for 4 h. The frequencies ( d ) and numbers ( e ) of IL-4-producing CD4 + T cells were determined by ICCS. ( f–m ) B6 mice were treated with 200 μg anti-CXCL13 or control antibody at the time of infection with Hp ( f–k ) or influenza x31 ( l–m ). Analyses were performed 8 days post-infection. ( f ) The numbers of total cells, MHCII lo CD11c hi immature DCs and MHCII + CD11c int mature DCs in the mesLNs of Hp -infected mice were determined using flow cytometry. ( g ) Cryosections of mesLNs from Hp -infected mice were stained with anti-CD11c and anti-B220. Scale bar = 400 μm. ( h–k ) Cells from Hp -infected Cxcl13 −/− or Cxcr5 −/− mice or anti-CXCL13-treated or control-treated B6 mice were restimulated with anti-CD3 + BFA for 4 h. The frequencies ( h,j ) and numbers ( i,k ) of IL-4-producing CD4 + T cells present in the mesLN ( h–i ) and PEC ( j,k ) were determined by ICCS. ( l,m ) Cells from the medLN of influenza-infected anti-CXCL13 or control-treated B6 mice were restimulated with anti-CD3 + BFA for 4 h. The frequencies ( l ) and numbers ( m ) of IFN-γ-producing CD4 T cells were determined by ICCS. Data are representative of two or more independent experiments (mean ± SD of five mice per group, *P<0.01 vs control).

Article Snippet: Conjugated antibodies to CD205 (NLDC-145), CD11c (HL3), B220 (RA3-6B2), CD19 (1D3), CD40 (3/23), MHCII (AF6-120.1), CD4 (GK1.5), CD44 (IM7), CCR7 (4B12), CXCR5 (2G8), PD-1 (J43), ICOS (7E.17G9), CD45.1(A20) and CD45.2 (104) were purchased from from Cedarlane Laboratories (CD205) or BD-Biosciences (all others).

Techniques: Infection, Staining, Control, Flow Cytometry

Journal: Nature immunology

Article Title: Regulation of T H 2 development by CXCR5 + dendritic cells and lymphotoxin-expressing B cells

doi: 10.1038/ni.2309

Figure Lengend Snippet: ( a–g ). DC-WT and DC- Cxcr5 −/− chimeras were infected with Hp , treated with DT on days 0, 3 and 5 post-infection and analyzed on day 8. (a,b ) The frequencies of total DCs ( a ) and CXCR5-expressing MHCII + CD11c int mature DCs ( b ) are shown. ( c ) Cryosections from mesLNs of infected chimeras were stained with anti-CD11c and anti-B220. Scale bar = 400 μm ( d–g ). mesLN ( d,e ) and PEC ( f,g ) cells from infected chimeras were restimulated with plate-bound anti-CD3 + BFA for 4 h. The frequencies ( d,f ) and numbers ( e,g ) of IL-4 + donor wild-type CD45.1 + CD4 T cells in each tissue were determined by flow cytometry. Data are representative of two or more independent experiments (mean ± SD of five mice per group, *P<0.001).

Article Snippet: Conjugated antibodies to CD205 (NLDC-145), CD11c (HL3), B220 (RA3-6B2), CD19 (1D3), CD40 (3/23), MHCII (AF6-120.1), CD4 (GK1.5), CD44 (IM7), CCR7 (4B12), CXCR5 (2G8), PD-1 (J43), ICOS (7E.17G9), CD45.1(A20) and CD45.2 (104) were purchased from from Cedarlane Laboratories (CD205) or BD-Biosciences (all others).

Techniques: Infection, Expressing, Staining, Flow Cytometry

Journal: Nature immunology

Article Title: Regulation of T H 2 development by CXCR5 + dendritic cells and lymphotoxin-expressing B cells

doi: 10.1038/ni.2309

Figure Lengend Snippet: ( a–d ) mesLN cells from day 8 Hp -infected IL-4 (EGFP) reporter mice were analyzed by flow cytometry. ( a ) CD4 + T cells were divided into naïve (CD44 lo EGFP − ) and antigen-experienced (CD44 hi EGFP + ) populations, analyzed for expression of ICOS and PD-1 and subdivided into naïve (CD44 lo EGFP − ICOS − PD-1 − ), T FH (CD44 hi EGFP + ICOS + PD-1 hi ) and T effector (CD44 hi EGFP + ICOS + PD-1 lo ) subsets. ( b ) mesLN cells were restimulated with anti-CD3 + BFA for 4 h and the frequencies of IL-4 + naïve, ICOS + PD-1 lo and ICOS + PD-1 hi CD4 T cells were determined by ICCS. ( c,d ) Intracellular staining to detect Bcl6 was performed on non-restimulated mesLN cells. Bcl6 expression in naïve, ICOS + PD-1 lo and ICOS + PD-1 hi gated CD4 T cells is shown in ( c ) and CXCR5 and PD-1 expression on gated naïve, EGFP + Bcl6 lo and EGFP + Bcl6 hi CD4 + T cells are shown in panel d (see also gating strategy in ). ( e ) Serial cryosections of mesLNs from uninfected or day 8 Hp -infected B6 mice were stained with anti-CD8, anti-B220, anti-CD11c or anti-CD4 (Scale bar = 400 μm). ( f–h ) B6 mice were treated with anti-CXCL13 or control antibody, infected with Hp and analyzed on day 8. Cryosections ( f ) of mesLNs were stained with anti-CD4 and anti-B220 (Scale bar = 400 μm). mesLN cells were analyzed by flow cytometry immediately ex vivo ( g ) or following 4 h restimulation with anti-CD3 + BFA ( h ). The numbers of ICOS + PD-1 hi CD44 hi T FH cells ( g ) and IL-4 + ICOS + PD-1 lo T H 2 cells ( h ) are shown. ( i–l ) Chimeric mice reconstituted with 50% CD45.1 + B6 BM and 50% CD45.2 + Cxcr5 −/− BM were infected with Hp and analyzed on day 8 post-infection. ( j ) The frequencies of total CD45.1 + and CD45.2 + cells in the mesLN and within the gated naïve (CD44 lo ), ICOS + PD-1 hi T FH and ICOS + PD-1 lo effector CD4 populations were determined by flow cytometry. ( k,l ) The mesLN cells were restimulated with anti-CD3 + BFA for 4 h and the frequencies of CD45.1 + and CD45.2 + cells within the gated IL-4 + CD4 T cell population were determined in the mesLN ( k ) and PEC ( l ) by ICCS. Data are representative of three independent experiments (mean ± SD of five mice per group, *P<0.01, **P<0.001).

Article Snippet: Conjugated antibodies to CD205 (NLDC-145), CD11c (HL3), B220 (RA3-6B2), CD19 (1D3), CD40 (3/23), MHCII (AF6-120.1), CD4 (GK1.5), CD44 (IM7), CCR7 (4B12), CXCR5 (2G8), PD-1 (J43), ICOS (7E.17G9), CD45.1(A20) and CD45.2 (104) were purchased from from Cedarlane Laboratories (CD205) or BD-Biosciences (all others).

Techniques: Infection, Flow Cytometry, Expressing, Staining, Control, Ex Vivo

Journal: Nature immunology

Article Title: Regulation of T H 2 development by CXCR5 + dendritic cells and lymphotoxin-expressing B cells

doi: 10.1038/ni.2309

Figure Lengend Snippet: ( a–h ) B6 mice were treated with 250 μg of anti-CD20 or control antibody 4 days before infection with either Hp ( a–e ) or influenza x31 ( f–h ). Analyses were performed on day 8 post-infection. ( a,b ) Cryosections of mesLNs from Hp -infected control-treated ( a ) or anti-CD20 treated ( b ) mice were stained with anti-B220, anti-CD11c, and anti-CD4. Scale bar = 400 μm. ( c–e ) Cells from Hp -infected mesLNs were analyzed by flow cytometry either ex vivo ( c ) or following 4 h restimulation with anti-CD3 + BFA ( d,e ). The numbers of ICOS + PD-1 hi T FH cells ( c ) and the frequencies ( d ) and numbers ( e ) of IL-4 + ICOS + PD-1 lo CD4 + T cells are shown. ( f–h ) NP + (influenza-specific) CD4 + T cells from medLN of influenza-infected anti-CD20 or control treated mice were analyzed by flow cytometry either ex vivo ( f ) or following 4 h restimulation with anti-CD3 + BFA ( g,h ). The numbers of flu-specific NP + ICOS + PD-1 hi T FH cells ( f ) and the frequencies ( g ) and numbers ( h ) of IFN-γ-producing NP + ICOS + PD-1 lo CD4 + T cells are shown (mean ± SD of five mice per group, *P<0.001). Data are representative of three independent experiments.

Article Snippet: Conjugated antibodies to CD205 (NLDC-145), CD11c (HL3), B220 (RA3-6B2), CD19 (1D3), CD40 (3/23), MHCII (AF6-120.1), CD4 (GK1.5), CD44 (IM7), CCR7 (4B12), CXCR5 (2G8), PD-1 (J43), ICOS (7E.17G9), CD45.1(A20) and CD45.2 (104) were purchased from from Cedarlane Laboratories (CD205) or BD-Biosciences (all others).

Techniques: Control, Infection, Staining, Flow Cytometry, Ex Vivo

Journal: Nature immunology

Article Title: Regulation of T H 2 development by CXCR5 + dendritic cells and lymphotoxin-expressing B cells

doi: 10.1038/ni.2309

Figure Lengend Snippet: ( a,b ) Membrane LT expression on mesLN cells from naïve ( a ) and day 8 Hp -infected B6 mice ( b ) were were determined by flow cytometry. Control staining in the presence of anti-LTβ blocking antibody is shown. ( c ) CD19 + B cells from day 8 Hp -infected B6 and MD4μMT mice were stained with mLTβR-Fc (open histograms) or control antibody (filled histogram). ( d ) CD11c-DTR BM chimeric mice, treated with PBS (DC sufficient) or DT on days 0 and 3 (DC depletion), were infected with Hp . LT expression (open histogram) is shown on CD19 + B cells. Isotype control staining is shown in filled histogram. ( e–j ) B6 mice were treated (i.p.) with 100 μg of LTβR-Fc fusion protein or control protein at the time of Hp infection or with 250 μg of anti-CD20 or control Ab four days before Hp infection. Analyses were performed on day 8 post-infection. ( e ) Cxcl13 mRNA in mesLN cells were determined by quantitative PCR. Data show mean ± SD of three samples. nd=not detected. ( f,g ) The frequencies ( f ) and numbers ( g ) of MHCII + CD11c int mature and MHCII lo CD11c hi immature mesLN DCs in LTβR-Fc and control-treated B6 mice were determined by flow cytometry. ( h,i ) Cryosections from mesLNs of control ( h ) and LTβR-Fc treated mice ( i ) were stained with anti-B220, anti-CD11c and anti-CD4. Scale bar = 400 μm. ( j ) mesLN cells were restimulated with anti-CD3 + BFA for 4 h and the numbers of IL-4 + CD4 + T cells from LTβR-Fc-treated mice and B6 controls were determined by ICCS. Data are representative of three independent experiments (mean ± SD of five mice per group, *P<0.001 vs control, **P<0.05, ***P<0.01).

Article Snippet: Conjugated antibodies to CD205 (NLDC-145), CD11c (HL3), B220 (RA3-6B2), CD19 (1D3), CD40 (3/23), MHCII (AF6-120.1), CD4 (GK1.5), CD44 (IM7), CCR7 (4B12), CXCR5 (2G8), PD-1 (J43), ICOS (7E.17G9), CD45.1(A20) and CD45.2 (104) were purchased from from Cedarlane Laboratories (CD205) or BD-Biosciences (all others).

Techniques: Membrane, Expressing, Infection, Flow Cytometry, Control, Staining, Blocking Assay, Real-time Polymerase Chain Reaction

Journal: Nature immunology

Article Title: Regulation of T H 2 development by CXCR5 + dendritic cells and lymphotoxin-expressing B cells

doi: 10.1038/ni.2309

Figure Lengend Snippet: ( a–g ) B-WT and B- Lta −/− chimeras were infected with Hp and analyzed on day 8 post-infection. ( a,b ) The frequencies ( a ) and numbers ( b ) of MHCII + CD11c int mature and MHCII lo CD11c hi immature DCs in mesLNs were determined by flow cytometry. ( c,d ) Cryosections of mesLNs from infected chimeras were stained with anti-B220, anti-CD11c and anti-CD4. Scale bar = 400 μm. ( e ) Cxcl13 expression in mesLN cells were determined by quantitative PCR (mean ± SD of three samples). ( f–g ) The mesLN cells were restimulated with anti-CD3 + BFA for 4 h and the frequencies ( f ) and numbers ( g ) of IL-4 + CD4 T cells from the chimeras were determined by ICCS. Data are representative of three independent experiments (mean ± SD of five mice per group, *P<0.001, **P<0.01).

Article Snippet: Conjugated antibodies to CD205 (NLDC-145), CD11c (HL3), B220 (RA3-6B2), CD19 (1D3), CD40 (3/23), MHCII (AF6-120.1), CD4 (GK1.5), CD44 (IM7), CCR7 (4B12), CXCR5 (2G8), PD-1 (J43), ICOS (7E.17G9), CD45.1(A20) and CD45.2 (104) were purchased from from Cedarlane Laboratories (CD205) or BD-Biosciences (all others).

Techniques: Infection, Flow Cytometry, Staining, Expressing, Real-time Polymerase Chain Reaction

Journal: Nature immunology

Article Title: Regulation of T H 2 development by CXCR5 + dendritic cells and lymphotoxin-expressing B cells

doi: 10.1038/ni.2309

Figure Lengend Snippet: ( a,b ) mesLN cells from day 8 Hp -infected B6 mice were analyzed by flow cytometry. Gated mature (mat) MHCII + CD11c int and immature (imm) MHCII lo CD11c hi DCs ( a ) were analyzed for expression of DEC205 and CD40 (( b ), filled histograms). Open histograms represent non-specific control staining. ( c,d ) CD4 + T cells from mesLNs of day 5 Hp -infected IL-4 reporter (4get) mice were purified and co-cultured with total DCs (tot DCs) purified from uninfected (UI) mice or with sort-purified mature (MHCII + CD40 hi CD11c int DCs) and immature (MHCII lo CD40 lo CD11c hi ) DCs isolated from the mesLNs of day 8 Hp -infected mice. The population expansion ( c ) of IL-4 mRNA-expressing (EGFP + ) CD4 + T cells was measured at 72 h and the fold increase ( d ) in EGFP + CD4 + T cells was determined (mean ± SD n = 5). ( e–h ) MHCII lo CD40 lo CD11c hi immature ( e ) and MHCII + CD40 hi CD11c int mature ( f–h ) DCs from day 8 x31-infected mice ( e,f ), day 8 Hp -infected mice ( e,g ) and uninfected mice ( f–h ) were analyzed by flow cytometry for expression of CCR7 and CXCR5. The frequency of positive cells is shown as the Mean ± SD ( n = 4–5). ( i,j ) MHCII lo CD40 lo CD11c hi immature ( i ) and MHCII + CD40 hi CD11c int mature ( j ) DCs from x31-infected and Hp -infected mice were analyzed in transwell chemotaxis assays using CCL19 or CXCL13 as chemoattractants. Data are expressed as the mean ± SD ( n = 3) of the chemotaxis index (CI). The data are representative of at least three independent experiments. (*P<0.001)

Article Snippet: Conjugated antibodies to CD205 (NLDC-145), CD11c (HL3), B220 (RA3-6B2), CD19 (1D3), CD40 (3/23), MHCII (AF6-120.1), CD4 (GK1.5), CD44 (IM7), CCR7 (4B12), CXCR5 (2G8), PD-1 (J43), ICOS (7E.17G9), CD45.1(A20) and CD45.2 (104) were purchased from from Cedarlane Laboratories (CD205) or BD-Biosciences (all others).

Techniques: Infection, Flow Cytometry, Expressing, Control, Staining, Purification, Cell Culture, Isolation, Chemotaxis Assay