Journal: Frontiers in Immunology

Article Title: A Novel Cellular Pathway of Antigen Presentation and CD4 T Cell Activation in vivo

doi: 10.3389/fimmu.2018.02684

Figure Lengend Snippet: ISCOMATRIX does not alter the passage of soluble antigen from the injection site to the lymph node. Graphs show fluorescence intensity of (A) ISCOMATRIX-647 detected at 630 ± 17.5 nm and (B) OVA-790 detected at 830 ± 17.5 nm in the ear pinnae and (C) ISCOMATRIX-647 and (D) OVA-790 in the draining cervical lymph node over time. Representative X-ray images with pseudocoloured fluorescence images overlaid showing (E) ISCOMATRIX-647 fluorescence (630 ± 17.5 nm) and (F) OVA-790 fluorescence (830 ± 17.5 nm) over time following injection in the ear pinnae with OVA-790 alone, ISCOMATRIX-647 alone or OVA-790/ISCOMATRIX-647. Three animals were imaged at each time point. IMX = ISCOMATRIX™ adjuvant, * P < 0.05.

Article Snippet: In other experiments ISCOMATRIX labeled with the fluorescent dyes AlexaFluor 647 (CSL Limited, Australia) was used.

Techniques: Injection, Fluorescence, Adjuvant

Journal: Frontiers in Immunology

Article Title: A Novel Cellular Pathway of Antigen Presentation and CD4 T Cell Activation in vivo

doi: 10.3389/fimmu.2018.02684

Figure Lengend Snippet: Neutrophils and CD11b+ cells are recruited to both the injection site and to the draining lymph node. (A) Representative flow cytometry plots showing the recruitment of neutrophils and CD11b+ cells in the injection site at 4, 24, and 48 h following immunization with OVA/PBS (top) or OVA/ISCOMATRIX (bottom). Absolute numbers are shown in the graphs on the right. (B) Representative flow cytometry plots showing the recruitment of neutrophils and CD11b+ cells in the draining cervical lymph node at 4, 24, and 48 h following immunization with OVA/PBS (top left) or OVA/ISCOMATRIX (bottom left). Further representative plots show the identification of the majority of the CD11b cells in the OVA/ISCOMATRIX treated lymph node at 24 h were monocytes (CD64+Ly6C high) (top right). Absolute numbers of Neutrophils and monocytes recruited to the draining lymph node in response to OVA/PBS and OVA/ISCOMATRIX are shown (bottom left). Data shown is from 3 mice per group pooled and is representative of 5 independent experiments. IMX = ISCOMATRIX™ adjuvant.

Article Snippet: In other experiments ISCOMATRIX labeled with the fluorescent dyes AlexaFluor 647 (CSL Limited, Australia) was used.

Techniques: Injection, Flow Cytometry, Adjuvant

Journal: Frontiers in Immunology

Article Title: A Novel Cellular Pathway of Antigen Presentation and CD4 T Cell Activation in vivo

doi: 10.3389/fimmu.2018.02684

Figure Lengend Snippet: The injection site is not required for ISCOMATRIX adjuvant activity. (A) Representative flow cytometry plots showing Kaede red vs. Kaede green in the popliteal lymph node (top panels) draining the PBS injected (left) and the ISCOMATRIX (right) injected footpad, below plots showing the percentage of the leukocytes in the draining lymph nodes at 24 h following photoswitching immediately prior (middle left), 8 h post (middle right) 12 h post (bottom left) immunization or at 48 h from mice photoswitched at 24 h (bottom right). (B) Representative flow cytometry plots showing Kaede red vs. Kaede green in the popliteal lymph node (top panels) draining the PBS injected (left) and the ALUM/LPS (right) injected footpad, below plots showing the percentage of the leukocytes in the draining lymph nodes at 24 h following photoswitching 8 h post (bottom left) and 12 h post (bottom right) immunization. (C) Serum anti-OVA IgG1 from mice at 14 days post immunization with PBS, OVA/ISCOMATRIX with the injection site intact or OVA/ISCOMATRIX with the injection site removed shortly following immunization. Groups contained 3 animals and data is representative of two independent experiments. IMX = ISCOMATRIX™ adjuvant.

Article Snippet: In other experiments ISCOMATRIX labeled with the fluorescent dyes AlexaFluor 647 (CSL Limited, Australia) was used.

Techniques: Injection, Adjuvant, Activity Assay, Flow Cytometry

Journal: Frontiers in Immunology

Article Title: A Novel Cellular Pathway of Antigen Presentation and CD4 T Cell Activation in vivo

doi: 10.3389/fimmu.2018.02684

Figure Lengend Snippet: ISCOMATRIX localizes to the subcapsular sinus macrophages and these cells are lost from the lymph node following ISCOMATRIX treatment. (A) Mulitphoton image showing a popliteal lymph node labeled in vivo with a PE conjugated anti CD169 antibody and subsequently treated with 1 IU ISCOMATRIX-647 in the footpad. Imaging was performed 1 h after treatment with the ISCOMATRIX-647. (B) CD169 Immunohistochemical staining of cervical lymph nodes 24 h following immunization in the ear pinnae with either PBS or ISCOMATRIX. (C) Mulitphoton image showing the popliteal lymph node approximately 2 h following the co-administration of the cell impermeable DNA dye Sytox orange and ISCOMATRIX-647 in the footpad. (D) Representative plots showing CD169 and CD64 expression on Ly6G- CD11b+ cells in the draining lymph node from mice treated with PBS or ISCOMATRIX at 24 h. (E) Flow cytometry data showing the percentage and number of CD11b+ cells that are CD169+ SCS macrophages in the cervical lymph node at 24 h following immunization with PBS or ISCOMATRIX in the ear pinnae. (F) The percentage of CD11b+ cells that are CD169+ SCS macrophages in the cervical lymph node over the first 12 h following immunization with PBS or ISCOMATRIX in the ear pinnae. (G) The recovery of the of CD11b+ cells that are CD169 + SCS macrophages in the cervical lymph node over 28 days following immunization with PBS or ISCOMATRIX in the ear pinnae. Groups contained 3 animals. IMX, ISCOMATRIX™ adjuvant, ** P < 0.01, *** P < 0.001 *** P < 0.0001.

Article Snippet: In other experiments ISCOMATRIX labeled with the fluorescent dyes AlexaFluor 647 (CSL Limited, Australia) was used.

Techniques: Labeling, In Vivo, Imaging, Immunohistochemical staining, Staining, Expressing, Flow Cytometry, Adjuvant

Journal: Frontiers in Immunology

Article Title: A Novel Cellular Pathway of Antigen Presentation and CD4 T Cell Activation in vivo

doi: 10.3389/fimmu.2018.02684

Figure Lengend Snippet: SCS macrophages are taken up by monocytes following immunization with ISCOMATRIX. (A) Representative flow cytometry plots showing in vivo antibody staining with eFluor660 conjugated anti-CD169 prior to treatment with PBS (top) and ISCOMATRIX (middle) or the isotype control antibody (bottom) treatment in the ear pinnae. (B) Representative histograms and MFI (points = individual animals, lines show mean±SEM) of ex vivo PE conjugated anti-CD169 (top); anti-CD64 (middle), and anti-Ly6C (bottom) on cells positive for the in vivo CD169 label. Groups contained 3 animals and data is representative of two independent experiments ** P < 0.01. (C) Representative imaging flow cytometry images showing monocytes at 24 h post-immunization with ISCOMATRIX from the lymph nodes of mice following in vivo eFluor660 conjugated anti-CD169 (top) or the isotype control (bottom) labeling. (D) Representative imaging flow cytometry images showing macrophages (CD11b+) at 24 h post-immunization with PBS from the lymph nodes of mice following in vivo eFluor660 conjugated anti-CD169 labeling. The anti-CD169 antibody treated group contained 3 animals, while the isotype control treated group contained 2 animals. IMX = ISCOMATRIX™ adjuvant.

Article Snippet: In other experiments ISCOMATRIX labeled with the fluorescent dyes AlexaFluor 647 (CSL Limited, Australia) was used.

Techniques: Flow Cytometry, In Vivo, Staining, Control, Ex Vivo, Imaging, Labeling, Adjuvant

Journal: Frontiers in Immunology

Article Title: A Novel Cellular Pathway of Antigen Presentation and CD4 T Cell Activation in vivo

doi: 10.3389/fimmu.2018.02684

Figure Lengend Snippet: Recruited monocytes present antigen and may be observed interacting with T cells in the lymph node at 24 h post-immunization. (A) Representative plots showing flow cytometry gating strategy for the identification of CD11b+CD64+ monocytes, for CD11b+CD169+ SCS macrophages and CD11b- or CD11b+ CD11c+MHCII+ dendritic cells. Graphs showing mean (±SEM) YAe MFI on CD11b- (B) and CD11b+ (C) dendritic cells at 4, 24, and 48 h; mean (±SEM) YAe MFI SCS macrophages (D) at 4 h; mean (±SEM) YAe MFI on monocytes (E) at 4, 24, and 48 h. Groups contained 3 animals and data is representative of three independent experiments. (F) LysM-EGFP or CD11cYFP mice were immunized in the footpad (OVA or BSA/ISCOMATRIX or OVA/LPS respectively) ~20 h later the draining popliteal lymph node was surgically exposed. OTII DSRed T cells and monocytes (GFP+) or DCs (YFP+) were imaged by live real time multiphoton microscopy. Interaction between T cells and monocytes (and T cells and DCs was identified by colocalization of the DSRed signal with the GFP or YFP signals and tracked for up to 20 min, 48 such interactions were tracked in the OVA/ISCOMATRIX treated mice. IMX = ISCOMATRIX™ adjuvant, * P < 0.05, ** P < 0.01, *** P < 0.001 **** P < 0.0001.

Article Snippet: In other experiments ISCOMATRIX labeled with the fluorescent dyes AlexaFluor 647 (CSL Limited, Australia) was used.

Techniques: Flow Cytometry, Microscopy, Adjuvant

Journal: Frontiers in Immunology

Article Title: A Novel Cellular Pathway of Antigen Presentation and CD4 T Cell Activation in vivo

doi: 10.3389/fimmu.2018.02684

Figure Lengend Snippet: ISCOMATRIX stimulates Tfh and germinal center B cell responses. (A) Total number of OVA specific (OTII) CD4+ T cells in the lymph node, and (B) the number are CXCR5+PD1+ at 5 and 7 days. (C) Representative flow cytometry plots showing CXCR5 and PD1 staining on OTII CD4+ T cells from OVA-HEL and OVA-HEL/ISCOMATRIX treated mice at 5 days. (D) Total number of HEL specific (MD4) B cells and (E) the number that are GL-7+/FAS+ at 7 days. (F) Representative flow cytometry plots showing GL-7 and FAS staining on MD4 B cells from OVA-HEL and OVA-HEL/ISCOMATRIX treated mice at 7 days. (G) Graphs showing the mean ± SEM serum anti-HEL IgMa, anti-OVA IgG1, and anti-OVA IgG2c titers at 14 days post-immunization with OVA-HEL, OVA-HEL/ISCOMATRIX or PBS. Immunized groups contained 3 animals and are representative of 2 independent experiments. IMX = ISCOMATRIX™ adjuvant, * P < 0.05, *** P < 0.001, **** P < 0.0001.

Article Snippet: In other experiments ISCOMATRIX labeled with the fluorescent dyes AlexaFluor 647 (CSL Limited, Australia) was used.

Techniques: Flow Cytometry, Staining, Adjuvant