pe cy7 anti mouse cd4  (4A Biotech)

Journal: Journal of Materials Science. Materials in Medicine

Article Title: Evaluation of cellular immune response and biosafety of SV40 virus-like particle in tumor immunotherapy

doi: 10.1007/s10856-025-06986-0

Figure Lengend Snippet: ( A, B ) Flow cytometry analysis ( A ) and quantification ( B ) of CD4T cell proliferation in mesenteric lymph nodes after repeated SV40 VLP injections; ( C, D ) Flow cytometry analysis ( C ) and quantification ( D ) of NK cell activation in the spleen following repeated SV40 VLP injection; (E, F) Flow cytometry analysis ( E ) and quantification ( F ) of CD4 T cell activation in the spleen after repeated SV40 VLP injection. Gating strategies were shown in Figs. S2, S3

Article Snippet: Rosstta-PET32a-5hcVP1 strain was provided by professor Feng Li, fluorescent dye Alexa 647 was purchased from Invitrogen (OR, USA), APC/Cy7 anti-mouse CD3, PE-Cy7 anti-mouse CD4, PE anti-mouse CD8, FITC anti-mouse CD45, APC/Cy7 anti-mouse CD19, Pe-Cy7 anti-mouse CD11b, FITC anti-mouse CD11c, PE anti-mouse NK1.1, PE anti-mouse MHC2 are all purchased from 4A-BIOTECH.

Techniques: Flow Cytometry, Activation Assay, Injection

Journal: Journal of Materials Science. Materials in Medicine

Article Title: Evaluation of cellular immune response and biosafety of SV40 virus-like particle in tumor immunotherapy

doi: 10.1007/s10856-025-06986-0

Figure Lengend Snippet: ( A, C ）Flow cytometry analysis of the changes of CD4+T cells,CD8+T cells, NK cells, B cells and DCs in single-cell suspensions from the liver ( A ), lung ( B ) and kidney ( C ) after repeated SV40 VLPinjections. Statistical analysis showed no significant (ns) differences in immune cell activation among the three organs

Article Snippet: Rosstta-PET32a-5hcVP1 strain was provided by professor Feng Li, fluorescent dye Alexa 647 was purchased from Invitrogen (OR, USA), APC/Cy7 anti-mouse CD3, PE-Cy7 anti-mouse CD4, PE anti-mouse CD8, FITC anti-mouse CD45, APC/Cy7 anti-mouse CD19, Pe-Cy7 anti-mouse CD11b, FITC anti-mouse CD11c, PE anti-mouse NK1.1, PE anti-mouse MHC2 are all purchased from 4A-BIOTECH.

Techniques: Cytometry, Activation Assay

Journal: Physiological Reports

Article Title: T cell‐expressed Ift88 is required for proper thymocyte differentiation in mice

doi: 10.14814/phy2.70120

Figure Lengend Snippet: Loss of Ift88 in thymic epithelial cells does not impact T cell development. (a) Whole mount image of SSTR3:GFP cilia reporter mice co‐stained with acetylated alpha tubulin and keratin 5 using a 60× objective (b, c) Thymus (b) and spleen (c) weight in Foxn1 cre+ Ift88 f/f or control mice at 8 weeks of age. (d, e) Representative FACS plots showing DN1‐DN4 thymocytes (d), and CD4+/CD8+ DP, CD4+ SP, and CD8+ SP thymocytes (e) in 8 week old Foxn1 cre+ Ift88 f/f or control mice. (f–h) Quantification of DN1‐DN4 thymocytes (f), DP thymocytes (g), and CD4+ and CD8+ SP thymocytes (h) in Foxn1 cre+ Ift88 f/f or control mice. (i) Representative FACS plots showing CD4+ and CD8+ T cells in the spleen of Foxn1 cre+ Ift88 f/f or control mice at 8 weeks of age. (j) Quantification of CD4+ and CD8+ spleenocytes in Foxn1 cre+ Ift88 f/f or control mice. For each quantification (except spleen weight), N = 10 mice per group (Controls: 6M, 4F; Foxn1 cre+ Ift88 f/f : 7M, 3F). Two‐way ANOVA.

Article Snippet: Primary antibodies were as follows: APC rat anti‐mouse CD25 (Catalog#: 101910, clone 3C7, BioLegend), PE rat anti‐mouse CD3 (Catalog#: 12–0031‐82, clone 145–2 c11, eBioscience), PE Cy7 rat anti‐mouse CD4 (Catalog#: 25–0041‐82, clone GK1.5, Invitrogen), Percpcy5.5 rat anti‐mouse CD8 (Catalog #: 45–0081‐82, clone 53–6.7, Invitrogen), BV786 rat anti‐mouse CD44 (Catalog#: 563736, clone IM7, BD Bioscience), BV605 hamster anti‐mouse TCRβ (Catalog#: 109241, H57‐ APC 597, BioLegend), FITC rat anti‐mouse CD45 (Catalog#: 2023‐08‐08, Clone 30‐F11, ThermoFisher Scientific), and Fixable Aqua Dead Cell Stain (Catalog#: L34957, Invitrogen).

Techniques: Staining, Control

Journal: Physiological Reports

Article Title: T cell‐expressed Ift88 is required for proper thymocyte differentiation in mice

doi: 10.14814/phy2.70120

Figure Lengend Snippet: Loss of Ift88 in T cells prevents DP to SP thymocyte transition. (a, b) FACS plots showing DN1‐DN4 thymocytes (a) and CD4+/CD8+ DP, CD4+ SP, and CD8+ SP thymocytes (b) that are TdT+ (left) and GFP+ (right) 3 weeks post tamoxifen induction in CaggCre ERT2+ Ift88 f/f mTmG mice. (c) Quantification of the ratio of GFP+ to TdT+ thymocytes during thymocyte development. Data is shown as the percentage of parent cells that express GFP or TdT. (d–f) Quantification of total DN1‐DN4 thymocytes (d), DP thymocytes (e), and CD4+ and CD8+ SP thymocytes (f) in CaggCre ERT2+ Ift88 f/f mTmG or control mice. For each graph, N = 5 mice (all male). Two‐way ANOVA.

Article Snippet: Primary antibodies were as follows: APC rat anti‐mouse CD25 (Catalog#: 101910, clone 3C7, BioLegend), PE rat anti‐mouse CD3 (Catalog#: 12–0031‐82, clone 145–2 c11, eBioscience), PE Cy7 rat anti‐mouse CD4 (Catalog#: 25–0041‐82, clone GK1.5, Invitrogen), Percpcy5.5 rat anti‐mouse CD8 (Catalog #: 45–0081‐82, clone 53–6.7, Invitrogen), BV786 rat anti‐mouse CD44 (Catalog#: 563736, clone IM7, BD Bioscience), BV605 hamster anti‐mouse TCRβ (Catalog#: 109241, H57‐ APC 597, BioLegend), FITC rat anti‐mouse CD45 (Catalog#: 2023‐08‐08, Clone 30‐F11, ThermoFisher Scientific), and Fixable Aqua Dead Cell Stain (Catalog#: L34957, Invitrogen).

Techniques: Control

Journal: Physiological Reports

Article Title: T cell‐expressed Ift88 is required for proper thymocyte differentiation in mice

doi: 10.14814/phy2.70120

Figure Lengend Snippet: The ratio of GFP+ to TdT+ T cells in the spleen and kidney is substantially reduced. (a, b) FACS plots showing TdT+ and GFP+ CD4+ and CD8+ T cells in the spleen (a) and kidney (b) 3 weeks post‐tamoxifen induction in CaggCre ERT2+ Ift88 f/f mTmG mice. (c, d) Quantification of the ratio of GFP+ to TdT+ cells in the spleen (c) and kidney (d) 3 weeks post‐tamoxifen induction. (e, f) Quantification of total CD4+ and CD8+ T cells in the spleen (e) and kidney (f) 3 weeks post‐tamoxifen induction. N = 5 mice (all male). Two‐way ANOVA.

Article Snippet: Primary antibodies were as follows: APC rat anti‐mouse CD25 (Catalog#: 101910, clone 3C7, BioLegend), PE rat anti‐mouse CD3 (Catalog#: 12–0031‐82, clone 145–2 c11, eBioscience), PE Cy7 rat anti‐mouse CD4 (Catalog#: 25–0041‐82, clone GK1.5, Invitrogen), Percpcy5.5 rat anti‐mouse CD8 (Catalog #: 45–0081‐82, clone 53–6.7, Invitrogen), BV786 rat anti‐mouse CD44 (Catalog#: 563736, clone IM7, BD Bioscience), BV605 hamster anti‐mouse TCRβ (Catalog#: 109241, H57‐ APC 597, BioLegend), FITC rat anti‐mouse CD45 (Catalog#: 2023‐08‐08, Clone 30‐F11, ThermoFisher Scientific), and Fixable Aqua Dead Cell Stain (Catalog#: L34957, Invitrogen).

Techniques:

Journal: Cell Reports Medicine

Article Title: Probiotics and their metabolite spermidine enhance IFN-γ + CD4 + T cell immunity to inhibit hepatitis B virus

doi: 10.1016/j.xcrm.2024.101822

Figure Lengend Snippet: Depletion of CD4 + T cells and inactivation of IL-21 or IFN-γ abrogated the probiotics-initiated anti-HBV effects (A–C) Mice were treated i.p. with 100 mg neutralization antibody or isotype every 3 days from 0 to 6 wpi (control, n = 7; BLE, n = 7; BLE+αCD4, n = 9; BLE+αCD8, n = 5; BLE+αTCRγ/δ, n = 7; BLE+AsGM1, n = 5). Serum levels of HBsAg (A), HBV DNA (B), and liver pgRNA (C) were detected by ELISA and qPCR. HBV-carrier mice were treated with BLE or control PBS. (D and E) RT-qPCR analysis of transcription factors (D) and cytokines (E) in liver tissues. (F) FCM analysis of percentages of hepatic T-bet + Th1 cells, PD-1 + CXCR5 + Tfh cells, and RORγt + Th17 cells among CD4 + T cells. (G) IL-21 and IFN-γ in liver homogenates were analyzed by ELISA. (H and I) Percentages and MFI of IL-21 (H) and IFN-γ (I) in hepatic T-bet + Th1 cells. (J–L) Antibody-mediated blockade of IL-21R and IFN-γ abolished the effect of probiotics-induced HBV clearance. Mice were treated i.p. with 100 mg neutralization antibody (anti-IL-21R and anti-IFN-γ) or isotype every 3 days from 0 to 6 wpi (control, n = 7; BLE, n = 7; BLE+αIL-21R, n = 4; BLE+αIFN-γ, n = 7). Serum levels of HBsAg (J), HBV DNA (K), and liver pgRNA (L) were detected by ELISA and qPCR. Data were expressed as mean ± SEM. The statistical tests were performed using two-way ANOVA with Dunnett’s multiple comparison test (A, B, J, and K) or t test. ns, no significance; ∗0.01 < p value < 0.05; ∗∗0.001 < p value < 0.01; ∗∗∗0.0001 < p value < 0.001; ∗∗∗∗ p value < 0.0001.

Article Snippet: Anti-mouse CD4 (GK1.5) PE-CY7 , eBioscience , Cat# 25-0041-81; RRID: AB_469575.

Techniques: Probiotics, Neutralization, Control, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Comparison

Journal: Cell Reports Medicine

Article Title: Probiotics and their metabolite spermidine enhance IFN-γ + CD4 + T cell immunity to inhibit hepatitis B virus

doi: 10.1016/j.xcrm.2024.101822

Figure Lengend Snippet: BLE-derived spermidine promoted HBV clearance (A) Untargeted metabolomic analysis of feces from control and BLE mice by gas chromatograph system coupled with a Pegasus HT time-of-flight mass spectrometer (GC-TOF-MS). Heatmap of differentially expressed metabolites in mice fed with BLE were shown (control, n = 9; BLE, n = 9). (B) Targeted metabolomics analysis of SPD in feces, peripheral serum, and livers from GF mice. (C) Extracellular SPD concentrations in cultures of separate E . faecalis and mixed BLE in gifu anaerobe medium (GAM) medium supplemented with 2 mM arginine under different pH values. (D–F) HBV-carrier mice were fed with drinking water (control, n = 8) or 3 mM SPD-containing water (SPD, n = 8). Levels of serum HBsAg (D), HBV DNA (E), and liver pgRNA (F) were detected by ELISA or qPCR. (G and H) FCM analysis of percentages and MFI of hepatic/splenic IL-21 + CD4 + T cells (G) and IFN-γ + CD4 + T cells (H). (I) IL-21 and IFN-γ in liver homogenates were analyzed by ELISA. Data were expressed as mean ± SEM. The statistical tests were performed using t test and two-way ANOVA (D and E). ∗0.01 < p value < 0.05; ∗∗0.001 < p value < 0.01; ∗∗∗∗ p value < 0.0001.

Article Snippet: Anti-mouse CD4 (GK1.5) PE-CY7 , eBioscience , Cat# 25-0041-81; RRID: AB_469575.

Techniques: Derivative Assay, Control, Mass Spectrometry, Enzyme-linked Immunosorbent Assay

Journal: Cell Reports Medicine

Article Title: Probiotics and their metabolite spermidine enhance IFN-γ + CD4 + T cell immunity to inhibit hepatitis B virus

doi: 10.1016/j.xcrm.2024.101822

Figure Lengend Snippet: SPD enhanced CD4 + T cell function through autophagy in vivo and in vitro (A–D) OT-II CD4 + T cells were prepared by culturing splenic mononuclear cells isolated from OT-II mice with OVA 323-339 peptide for 3 days. OT-II CD4 + T cells were then treated for 24 h with increasing concentrations of SPD (A and B). 3-MA (1 mM) and GC7 (10 μM) were used to inhibit autophagy (C and D). IFN-γ levels (A and D) and autophagosome levels (B and C) in CD4 + T cells were measured by FCM. (E) Hepatic mononuclear cells were isolated from HBV mice, and levels of autophagosome in CD4 + T cells were measured by FCM. (F and G) PBMCs isolated from HBV patients were stimulated with SPD and/or GC7 for 24 h. Autophagosome levels (F) and IFN-γ levels (G) in CD4 + T cells were measured by FCM. Data were expressed as mean ± SEM. The statistical tests were performed using t test. ns, no significance; ∗0.01 < p value < 0.05; ∗∗0.001 < p value < 0.01; ∗∗∗ 0.0001 < p value < 0.001; ∗∗∗∗ p value < 0.0001.

Article Snippet: Anti-mouse CD4 (GK1.5) PE-CY7 , eBioscience , Cat# 25-0041-81; RRID: AB_469575.

Techniques: Cell Function Assay, In Vivo, In Vitro, Isolation

Journal: Cell Reports Medicine

Article Title: Probiotics and their metabolite spermidine enhance IFN-γ + CD4 + T cell immunity to inhibit hepatitis B virus

doi: 10.1016/j.xcrm.2024.101822

Figure Lengend Snippet: Probiotics accelerated HBsAg clearance in HFA mice and HBV patients (A) Study design: male three-week-old C57BL/6J GF mice were fed with fecal microbiota from HBV patients daily for 3 weeks and then hydrodynamically injected with AAV/HBV1.2 plasmid and fed with BLE or SPD for 6 weeks (control, n = 4; BLE, n = 4; SPD, n = 4). (B–D) Levels of serum HBsAg (B), HBV DNA (C), and hepatic pgRNA (D) were detected by ELISA and qPCR. (E and F) Levels of autophagosome (E) and IFN-γ (F) in CD4 + T cells were measured by FCM. (G) MHC II-HBcAg 128-140 -Tetramer was used to evaluate HBV-specific CD4 + T cells. Percentages of IFN-γ in hepatic HBV-specific CD4 + T cells were measured by FCM. (H) A total of 20 eligible patients who remained persistently positive for HBsAg following >6 months of ongoing nucleos(t)ide analogs- or PegIFNα2b-based antiviral therapy were enrolled; among them, 10 patients received BLE therapy (control, n = 10; BLE, n = 10). (I) HBsAg levels in each patient before and after BLE treatment were analyzed and compared with HBV patients without BLE treatment. (J and K) Levels of autophagosome (J) and IFN-γ (K) in CD4 + T cells from PBMCs were measured by FCM. Data were expressed as mean ± SEM. The statistical tests were performed using two-way ANOVA with Dunnett’s multiple comparison test (B and C) or t test. ∗0.01 < p value < 0.05; ∗∗0.001 < p value < 0.01; ∗∗∗ 0.0001 < p value < 0.001.

Article Snippet: Anti-mouse CD4 (GK1.5) PE-CY7 , eBioscience , Cat# 25-0041-81; RRID: AB_469575.

Techniques: Probiotics, Injection, Plasmid Preparation, Control, Enzyme-linked Immunosorbent Assay, Comparison

Journal: Cell Reports Medicine

Article Title: Probiotics and their metabolite spermidine enhance IFN-γ + CD4 + T cell immunity to inhibit hepatitis B virus

doi: 10.1016/j.xcrm.2024.101822

Figure Lengend Snippet: Combination of SPD promoted the HBV inhibition in HBV-carrier mice (A) Male five-week-old HBV-carrier mice were administered with entecavir (ETV) or co-administered with 3 mM SPD water (ETV+SPD) combined with ETV (control, n = 5; SPD, n = 5; ETV, n = 5; ETV+SPD, n = 5). (B–D) Levels of serum HBsAg (B), HBV DNA (C), and liver pgRNA (D) were detected by ELISA or qPCR. (E) RT-qPCR analysis of transcription factors and cytokines in liver tissues. (F and G) FCM analysis of percentages and MFI of splenic (F) and hepatic (G) IFN-γ + CD4 + T cells. Data were expressed as mean ± SEM. The statistical tests were performed using t test. ns, no significance; ∗0.01 < p value < 0.05; ∗∗0.001 < p value < 0.01; ∗∗∗ 0.0001 < p value < 0.001.

Article Snippet: Anti-mouse CD4 (GK1.5) PE-CY7 , eBioscience , Cat# 25-0041-81; RRID: AB_469575.

Techniques: Inhibition, Control, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR

Journal: Cell Reports Medicine

Article Title: Probiotics and their metabolite spermidine enhance IFN-γ + CD4 + T cell immunity to inhibit hepatitis B virus

doi: 10.1016/j.xcrm.2024.101822

Figure Lengend Snippet:

Article Snippet: Anti-mouse CD4 (GK1.5) PE-CY7 , eBioscience , Cat# 25-0041-81; RRID: AB_469575.

Techniques: Staining, Virus, Capsules, Recombinant, Control, Enzyme-linked Immunosorbent Assay, cDNA Synthesis, AST Assay, Northern Blot, Sequencing, Transgenic Assay, Software

Journal: bioRxiv

Article Title: Amphiphile-engineered DNA adjuvants stimulate strong type I IFN production in lymph nodes via cytosolic danger-sensing to induce potent cellular and humoral immunity in mice and non-human primates

doi: 10.1101/2024.11.01.621501

Figure Lengend Snippet: a) Schematic drawings depicting the chemical structure of the AMP-DNA adjuvants. The ssDNA variant, dT 50 consists of 50 phosphorothioate (PS)-linked deoxythymidines. When annealed to a 50-mer deoxyadenosine DNA the resulting dsDNA forms dA:dT 50 . The diacyl lipid (AMP-tail) is conjugated to the 5’-end of dT 50 in both ssDNA and dsDNA forms. b) Vaccine formulation consists of the protein antigen (OVA or RBD) admixed with the candidate DNA-adjuvants depicted in a) and ), and ISD or HSV 60 . c) Schema showing animal dosing and experimental schedule. C57BL/6J mice (n = 5) were immunized twice with 5 μg OVA protein and 5 nmol SOL or AMP-DNA adjuvants. Cells were assayed 7 days post booster dose. d) ELISpot analysis of splenocytes restimulated with OVA OLPs overnight and assayed for IFNγ production. Shown is the frequency of IFNγ SFCs per 10 6 splenocytes. e) FluoroSpot analysis of splenocytes restimulated with OVA OLPs overnight and assayed for IFNγ, TNFα and IL2 production. Shown is the frequency of SFCs per 10 6 splenocytes that produce at least one cytokine. Donut charts represent percentages of polyfunctional versus single cytokine secreting cells. f) Multiplexed proteomics analysis (Luminex) of splenocytes restimulated with OVA OLPs overnight and assayed for IFNγ, TNFα, IL2, GM-CSF and GzmB production. Depicted are quantities of secreted cytokines in the culture supernatant. g) Peripheral blood CD8 + T cells were assayed ex vivo for OVA reactive TCRs using a SIINFEKL-specific tetramer. h-i) Flow cytometric analysis of cytokine production by CD8 + (g) and CD4 + (h) T cells in peripheral blood. Shown are percentages of cytokine + cells among CD8 + or CD4 + T cells and representative flow cytometry scatter plots of IFNγ and TNFα positive CD8 + T cells. j) Serum IgG titers were determined against OVA protein. Mock vaccines contained vehicle only. Values depicted are means±SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by one-way ANOVA followed by Tukey’s post-hoc analysis. n.d., not detected

Article Snippet: Cells were surface-stained with antibodies against anti-mouse CD4 (PE-Cy7, clone: GK1.5, Invitrogen, cat# 25-0041-82, 1:200 dilution), and anti-mouse CD8a (APC, clone: 53-6.7, eBioscience, cat# 17-0081-83, 1:200) and subsequently fixed and permeabilized with BD CytoFix/CytoPerm (BD, cat# 554714).

Techniques: Variant Assay, Formulation, Enzyme-linked Immunospot, Luminex, Ex Vivo, Flow Cytometry, Vaccines

Journal: bioRxiv

Article Title: Amphiphile-engineered DNA adjuvants stimulate strong type I IFN production in lymph nodes via cytosolic danger-sensing to induce potent cellular and humoral immunity in mice and non-human primates

doi: 10.1101/2024.11.01.621501

Figure Lengend Snippet: a) Schematic drawings depicting the chemical structure of the AMP-DNA adjuvants. The ssDNA variants, dT 50 and dA 50 , consist of 50 phosphorothioate (PS)-linked deoxythymidines and deoxyadenosines, respectively. When annealed to each other the resulting dsDNA forms dA:dT 50 . The diacyl lipid (AMP-tail) is conjugated to the 5’-end of each ssDNA variant, or to the dT strand in dA:dT 50 . The dT:dA 50 molecule is AMP-conjugated on the dA strand. The dAdT:dAdT 50 molecule consists of alternating deoxyadenosine and deoxythymidine residues. b-d) C57BL/6J mice (n = 5) were immunized twice with 5 μg OVA protein and 5 nmol SOL or AMP-DNA adjuvants as in . Cells were assayed 7 days post booster dose. b) Example flow cytometric scatter plots of IFNγ and TNFα production by CD4 + T cells in peripheral blood (see ). b,c) flow cytometric analysis of IFNγ and TNFα production in CD8 + and CD4 + T cells from perfused lung tissue. e-f) Gating strategy for intracellular cytokine staining of T cells re-stimulated with peptides overnight (e), and gating strategy for tetramer + CD8 + T cells and memory phenotypes of freshly processed PBMCs (f).

Article Snippet: Cells were surface-stained with antibodies against anti-mouse CD4 (PE-Cy7, clone: GK1.5, Invitrogen, cat# 25-0041-82, 1:200 dilution), and anti-mouse CD8a (APC, clone: 53-6.7, eBioscience, cat# 17-0081-83, 1:200) and subsequently fixed and permeabilized with BD CytoFix/CytoPerm (BD, cat# 554714).

Techniques: Variant Assay, Staining

Journal: bioRxiv

Article Title: Amphiphile-engineered DNA adjuvants stimulate strong type I IFN production in lymph nodes via cytosolic danger-sensing to induce potent cellular and humoral immunity in mice and non-human primates

doi: 10.1101/2024.11.01.621501

Figure Lengend Snippet: a) Schema showing animal dosing and experimental schedule. C57BL/6J mice (n = 5) were immunized twice with 5 μg WH-01 RBD protein and 5 nmol SOL or AMP-DNA adjuvants. Cells were assayed 7 days post booster dose. b) Splenocytes were restimulated with RBD OLPs overnight and assayed for IFNγ production by ELISpot. Shown is the frequency of IFNγ SFCs per 10 6 splenocytes. c) Serum anti-RBD IgG titers were determined against WH-01 RBD protein. d-g) Flow cytometric analysis of cytokine production by CD8 + (d, f) and CD4 + T cells (e, g) in peripheral blood and perfused lung tissue. Shown are percentages of cytokine + cells among CD8 + or CD4 + T cells and representative flow cytometry dot plots of TNFα and IFNγ positive T cells. Mock vaccines contained vehicle only. Values depicted are means±SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by one-way ANOVA followed by Tukey’s post-hoc analysis. n.d., not detected.

Article Snippet: Cells were surface-stained with antibodies against anti-mouse CD4 (PE-Cy7, clone: GK1.5, Invitrogen, cat# 25-0041-82, 1:200 dilution), and anti-mouse CD8a (APC, clone: 53-6.7, eBioscience, cat# 17-0081-83, 1:200) and subsequently fixed and permeabilized with BD CytoFix/CytoPerm (BD, cat# 554714).

Techniques: Enzyme-linked Immunospot, Flow Cytometry, Vaccines

Journal: bioRxiv

Article Title: Amphiphile-engineered DNA adjuvants stimulate strong type I IFN production in lymph nodes via cytosolic danger-sensing to induce potent cellular and humoral immunity in mice and non-human primates

doi: 10.1101/2024.11.01.621501

Figure Lengend Snippet: C57BL/6J mice (n = 5) were immunized as described in . Immunizations contained 5 μg OVA protein and either 5 nmol AMP-DNA, 1 nmol AMP-CpG-7909, or the indicated comparator adjuvants (100 μg alum; 10 μg MPLA; 1:2 emulsions of IFA, AddaVax, AddaS03). Cells were assayed 7 days post booster dose. a) Peripheral blood CD8 + T cells were assayed ex vivo for OVA-reactive TCRs using a SIINFEKL-specific tetramer. b-c) Flow cytometric analysis of cytokine production by CD8 + (b) and CD4 + T cells (c) in peripheral blood. Shown are percentages of cytokine + cells among CD8 + or CD4 + T cells and representative flow cytometry dot plots of TNFα and IFNγ positive CD8 + T cells. d) ELISpot analysis of splenocytes restimulated with OVA OLPs overnight. Shown is the frequency of IFNγ SFCs per 10 6 splenocytes. e-f) Flow cytometric analysis of cytokine production by CD8 + (e) and CD4 + T cells (f) in perfused lung tissue. Representation same as b-c. g-i) Serum anti-OVA pan-IgG (g), IgG1 (h), and IgG2c (j) titers were determined against OVA protein. j) Ratio of IgG2c:IgG1 antibody response from h) and i). Mock vaccines contained antigen only. Values depicted are means±SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by one-way ANOVA followed by Tukey’s post-hoc analysis. n.d., not detected.

Article Snippet: Cells were surface-stained with antibodies against anti-mouse CD4 (PE-Cy7, clone: GK1.5, Invitrogen, cat# 25-0041-82, 1:200 dilution), and anti-mouse CD8a (APC, clone: 53-6.7, eBioscience, cat# 17-0081-83, 1:200) and subsequently fixed and permeabilized with BD CytoFix/CytoPerm (BD, cat# 554714).

Techniques: Ex Vivo, Flow Cytometry, Enzyme-linked Immunospot, Vaccines

Journal: bioRxiv

Article Title: Amphiphile-engineered DNA adjuvants stimulate strong type I IFN production in lymph nodes via cytosolic danger-sensing to induce potent cellular and humoral immunity in mice and non-human primates

doi: 10.1101/2024.11.01.621501

Figure Lengend Snippet: Cellular and humoral responses to WH-01 RBD and AMP-DNA adjuvant were assessed in Rhesus macaques. a) 3 animals were immunized at week 0 (Baseline, BL) and 4 with 140 µg WH-01 RBD protein admixed with 5 mg of AMP-dT 50 . b-d) PBMCs were collected at BL and week 6 and stimulated with WH-01 RBD OLPs overnight. b) IFNγ ELISpot analysis. Shown are IFNγ SFCs per 1 x 10 6 PBMCs. c, d) Flow cytometric analysis of cytokine production by CD4 + and CD8 + T cells. Shown are frequencies of combined IFNγ, TNFα and IL2 cytokine production. e) For antibody response assessment serum was collected at BL and weeks 2, 4, 6, and 8. RBD-specific serum IgG binding antibody units (BAU) were assessed for WH-01 at each time point. f) For neutralizing antibody (nAb) response assessment through pseudovirus inhibition serum was collected at BL and week 6. Shown are ID 50 values for WH-01 in comparison to convalescent human plasma (CHP). The dashed lines in e-f) represent the lower limit of detection discriminating between samples positive or negative for seroconversion. The dotted line in f) represents the mean value observed for the human plasma comparators. g) For GC B cell assessment LN fine needle aspirates (FNA) were collected at BL and weeks 2 and 6. Shown are frequencies of RBD-specific GC B cells (CD20 + BcL6 + Ki-67 + ) in individual animals over time with corresponding representative flow cytometry scatter plots of RBD-specific B cells. Values depicted are mean±SD. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001 by paired t test.

Article Snippet: Cells were surface-stained with antibodies against anti-mouse CD4 (PE-Cy7, clone: GK1.5, Invitrogen, cat# 25-0041-82, 1:200 dilution), and anti-mouse CD8a (APC, clone: 53-6.7, eBioscience, cat# 17-0081-83, 1:200) and subsequently fixed and permeabilized with BD CytoFix/CytoPerm (BD, cat# 554714).

Techniques: Adjuvant, Enzyme-linked Immunospot, Binding Assay, Inhibition, Comparison, Flow Cytometry

Journal: bioRxiv

Article Title: Amphiphile-engineered DNA adjuvants stimulate strong type I IFN production in lymph nodes via cytosolic danger-sensing to induce potent cellular and humoral immunity in mice and non-human primates

doi: 10.1101/2024.11.01.621501

Figure Lengend Snippet: Cellular and humoral responses to RBD and AMP-DNA adjuvant were assessed in rhesus macaques as in . Three animals were immunized at week 0 and 4 with 140 µg WH-01 RBD protein admixed with 5 mg of AMP-dT 50 . a-c) T cell cross-reactive responses were compared upon overnight restimulation with RBD OLPs to variants of concern (VOC) at week 6 by a) IFNγ ELISpot analysis [shown are IFNγ SFCs per 1 x 10 6 PBMCs], and b-c) flow cytometric analysis of cytokine production by CD4 + and CD8 + T cells [shown are frequencies of IFNγ, TNFα and IL2 cytokine production]. d) RBD-specific serum IgG binding antibody units (BAU) were assessed for VOCs at each sample collection time point and e) compared among VOCs at week 6. f, g) Neutralizing antibody responses were assessed through pseudovirus inhibition at week 6. Shown are ID 50 values for Delta and Omicron variants in comparison to convalescent human plasma (CHP). The dashed lines represent the lower limit of detection discriminating between samples positive or negative for seroconversion and dotted lines represent the mean value observed for the human plasma comparators where appropriate. Values depicted are mean±SD. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001 by paired t test.

Article Snippet: Cells were surface-stained with antibodies against anti-mouse CD4 (PE-Cy7, clone: GK1.5, Invitrogen, cat# 25-0041-82, 1:200 dilution), and anti-mouse CD8a (APC, clone: 53-6.7, eBioscience, cat# 17-0081-83, 1:200) and subsequently fixed and permeabilized with BD CytoFix/CytoPerm (BD, cat# 554714).

Techniques: Adjuvant, Enzyme-linked Immunospot, Binding Assay, Inhibition, Comparison

Journal: bioRxiv

Article Title: Amphiphile-engineered DNA adjuvants stimulate strong type I IFN production in lymph nodes via cytosolic danger-sensing to induce potent cellular and humoral immunity in mice and non-human primates

doi: 10.1101/2024.11.01.621501

Figure Lengend Snippet: a-d) As in , C57BL/6J mice (n = 5) were dosed intraperitoneally with IFNAR1 blocking antibody or isotype control one day prior to immunization with one dose of 5 μg WH-01 RBD protein and 5 nmol AMP-dT 50 . Protein from lymph nodes was extracted 24 hours post immunization and analyzed by Luminex. Shown are concentrations of measured analytes (from ) separated by functional groups: (1) growth factors, (2) inflammatory cytokines, (3) chemokines, (4) inflammasome, and (5) IFN-I. e-h) As in , C57BL/6J mice (n = 5) were immunized twice with 5 μg WH-01 RBD protein and 5 nmol AMP-dT 50 . Mice received IFNAR1 blocking antibody or isotype control intraperitoneally one day prior to each dose. Cells were assayed 7 days post booster dose. e-g) Shown are flow cytometric analyses of cytokine production by CD4 + T cells in peripheral blood (e), and CD8 + (f) and CD4 + (g) lung-resident T cells. h) Serum anti-RBD IgG titers were determined against WH-01 RBD protein. Mock immunized animals received vehicle only. Values depicted are mean±SD. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 by one-way ANOVA followed by Tukey’s post-hoc analysis. n.d., not detected.

Article Snippet: Cells were surface-stained with antibodies against anti-mouse CD4 (PE-Cy7, clone: GK1.5, Invitrogen, cat# 25-0041-82, 1:200 dilution), and anti-mouse CD8a (APC, clone: 53-6.7, eBioscience, cat# 17-0081-83, 1:200) and subsequently fixed and permeabilized with BD CytoFix/CytoPerm (BD, cat# 554714).

Techniques: Blocking Assay, Control, Luminex, Functional Assay

Journal: bioRxiv

Article Title: Amphiphile-engineered DNA adjuvants stimulate strong type I IFN production in lymph nodes via cytosolic danger-sensing to induce potent cellular and humoral immunity in mice and non-human primates

doi: 10.1101/2024.11.01.621501

Figure Lengend Snippet: a-e) C57BL/6J mice (n = 5) were dosed intraperitoneally with IFNAR1 blocking antibody or isotype control one day prior to immunization with one dose of 5 μg WH-01 RBD protein and 5 nmol AMP-dAdT 50 . Protein from lymph nodes was extracted 24 hours post immunization. a) Luminex assessed analyte concentrations were expressed in a heat map showing mock-subtracted Z-scores of analyte concentration (pg/ml). Functional group annotations as in . b-e) Concentrations of measured analytes from panel a) for functional groups 1 to 5 as indicated. f-k) C57BL/6J mice (n = 5) were immunized twice with 5 μg WH-01 RBD protein and 5 nmol AMP-dAdT 50 . Mice received IFNAR1 blocking antibody or isotype control intraperitoneally one day prior to each dose. Cells were assayed 7 days post booster dose. f) Splenocytes were restimulated with RBD OLPs overnight and assayed for IFNγ production by ELISpot. Shown is the frequency of IFNγ SFCs per 10 6 splenocytes. g, h) Flow cytometric analysis of cytokine production by CD8 + (g) and CD4 + T cells (h) in peripheral blood. k) Serum anti-RBD IgG titers were determined against WH-01 RBD protein. Mock immunized animals received vehicle only. Values depicted are mean±SD. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 by one-way ANOVA followed by Tukey’s post-hoc analysis. n.d., not detected.

Article Snippet: Cells were surface-stained with antibodies against anti-mouse CD4 (PE-Cy7, clone: GK1.5, Invitrogen, cat# 25-0041-82, 1:200 dilution), and anti-mouse CD8a (APC, clone: 53-6.7, eBioscience, cat# 17-0081-83, 1:200) and subsequently fixed and permeabilized with BD CytoFix/CytoPerm (BD, cat# 554714).

Techniques: Blocking Assay, Control, Luminex, Concentration Assay, Functional Assay, Enzyme-linked Immunospot