Journal: The Journal of Experimental Medicine

Article Title: Subspecialization of Cxcr5 + T Cells

doi:

Figure Lengend Snippet: Effector machinery of CD57 +/ −CD45RA +/ − or CD57 +/ − CXCR5 +/ − CD4 T cells. (A) Intracellular cytokine analyses of TNF-α, IL-2, IL-10, IFN-γ, and IL-4. (B) ELISA of IL-10 and IL-4. Isolated T cells were activated with 50 ng/ml PMA and 1 μg/ml ionomycin for 4 h for intracellular cytokine analyses, or 24 h for ELISA of IL-10 and IL-4. Representatives of at least four independent experiments are shown.

Article Snippet: In brief, acetone-fixed sections were stained with the primary monoclonal antibody to CXCR5 (R&D Systems), followed by incubation in biotinylated goat anti–mouse IgG (Vector Laboratories).

Techniques: Enzyme-linked Immunosorbent Assay, Isolation

Journal: The Journal of Experimental Medicine

Article Title: Subspecialization of Cxcr5 + T Cells

doi:

Figure Lengend Snippet: Identification of CD57 + CXCR5 + T cells and their localization in GCs. (A) Flow analyses of CD57 and CXCR5 expression on tonsil CD4 T cells. (B) Specific localization of CD57 + CD4 T cells in GCs. (C) Localization of CD57 + CXCR5 + CD4 T cells in GCs. Antibodies to CD57 (green), CD4 (blue), and IgD (red; B) or CXCR5 (red; C) were used for in situ immunohistochemistry.

Article Snippet: In brief, acetone-fixed sections were stained with the primary monoclonal antibody to CXCR5 (R&D Systems), followed by incubation in biotinylated goat anti–mouse IgG (Vector Laboratories).

Techniques: Expressing, In Situ, Immunohistochemistry

Journal: The Journal of Experimental Medicine

Article Title: Subspecialization of Cxcr5 + T Cells

doi:

Figure Lengend Snippet: Chemotactic responses (A) and chemokine receptor expression (B) by CD57 +/2 CXCR5 +/ − CD4 T cells. Optimal concentrations of 5 μg/ml BLC, 1 μg/ml ELC, and 100 ng/ml SDF-1 were used for chemotaxis experiments. Freshly isolated tonsil cells were used for chemotaxis and flow analyses of tonsil CD4 T cell subsets. Representatives of three independent experiments are shown.

Article Snippet: In brief, acetone-fixed sections were stained with the primary monoclonal antibody to CXCR5 (R&D Systems), followed by incubation in biotinylated goat anti–mouse IgG (Vector Laboratories).

Techniques: Expressing, Chemotaxis Assay, Isolation

Journal: The Journal of Experimental Medicine

Article Title: Subspecialization of Cxcr5 + T Cells

doi:

Figure Lengend Snippet: Spontaneous B cell helper activity of CD57 +/ − CXCR5 +/ − CD4 T cells. Sorted CD57 + CXCR5 + , CD57 − CXCR5 + , and CXCR5 − CD4 T cells were cocultured with B cells from the same tonsil for 11–13 d in the absence of any stimulatory agents followed by analyses of secreted IgG, IgA, and IgM. Representatives of at least three independent experiments are shown.

Article Snippet: In brief, acetone-fixed sections were stained with the primary monoclonal antibody to CXCR5 (R&D Systems), followed by incubation in biotinylated goat anti–mouse IgG (Vector Laboratories).

Techniques: Activity Assay

Journal: The Journal of Experimental Medicine

Article Title: Subspecialization of Cxcr5 + T Cells

doi:

Figure Lengend Snippet: Phenotype and effector function of circulating CXCR5 + T cells. (A) Surface phenotype of circulating naive or CXCR5 +/ − memory T cells. **Significant differences between CXCR5 + and CXCR5 − memory cells. (B) IL-4/IFN-γ production capabilities of CXCR5 +/ − T cells during repeated T cell receptor activation (each cycle is composed of 4-d activation with anti-CD3 and anti-CD28 followed by 3-d resting in the presence of IL-2). (C) B cell help activity of circulating CXCR5 + T cells after T cell receptor activation. Naive (IgD + ) or memory (IgD − ) B cells (2 × 10 4 ) from peripheral blood were cultured for 14 d in the presence or absence of various numbers (10 3 , 5 × 10 3 , 10 4 , 2.5 × 10 4 , 5 × 10 4 , and 10 5 ) of autologous T cells (CXCR5 − CD45RA + , CXCR5 + CD45RA − , or CXCR5 − CD45RA − ). Concentrations of IgG, IgA, and IgM in the culture supernatants were measured by ELISA. T cell numbers required for peak levels of antibody production varied among donors or experiments. One peak value with the best antibody production in each T cell group is shown. (D) Loss of CXCR5 expression during T cell receptor activation with anti-CD3 and anti-CD28. Error bars indicate SD of results from at least five different experiments (A). Representatives of three independent experiments are shown (B and D), and results from seven different donors (C) are shown.

Article Snippet: In brief, acetone-fixed sections were stained with the primary monoclonal antibody to CXCR5 (R&D Systems), followed by incubation in biotinylated goat anti–mouse IgG (Vector Laboratories).

Techniques: Activation Assay, Activity Assay, Cell Culture, Enzyme-linked Immunosorbent Assay, Expressing

Journal: NPJ Vaccines

Article Title: A candidate subunit vaccine induces protective immunity against Mycobacterium avium subspecies paratuberculosis in mice

doi: 10.1038/s41541-023-00675-1

Figure Lengend Snippet: a Schematic diagram of the vaccination timeline. C57BL/6 mice were subcutaneously injected two times (three weeks apart) with 50 μg 66NC fusion protein formulated in Montanide ISA 61 VG adjuvant. Control groups include mice immunized with Montanide ISA 61 VG adjuvant alone as a negative control or 74 F mixed MPL adjuvant as subunit vaccine control. Three weeks after the booster immunization, the mice were bled and sacrificed. b Antigen-specific IgG1 and IgG2a isotypes were measured by ELISA. c Pictures of the IFN-γ and IL-4 ELISpot assay (representative of one experiment with at least five independent replicates). d , e Quantitation of the IFN-γ and IL-4 ELISpot assay. f The percentage of cytokine-positive (%Cyt + ) antigen-specific T-lymphocytes was quantified by intracellular cytokine staining (ICS) in CD4 + and CD8 + following stimulation of splenic T lymphocytes with the indicated antigen. g Serum cytokines levels of IFN-γ, TNF-α, and IL-17A were measured by ELISA. d – g Error bars indicate the Standard Error of Mean (SEM). One-way ANOVA was used to analyze the statistical significance, followed by Tukey’s multiple comparisons tests. ** P < 0.01, *** P < 0.001.

Article Snippet: Antibody PE/Cy5 Rat IgG1 (1:50, RG1, NBP1-43076, Novus Biologicals, Littleton, Co, USA), Alexa Fluor 647 Rat IgG1 (1:50, KLH/G1-2-2, 0116-31, SouthernBiotech, Birmingham, AL, USA), and eFluor 450 Rat IgG1 (1:50, eBRG1, 48-4301-82, ThermoFisher) were used according to the manufacturer’s instructions for isotype control.

Techniques: Injection, Adjuvant, Control, Negative Control, Enzyme-linked Immunosorbent Assay, Enzyme-linked Immunospot, Quantitation Assay, Staining

Journal: NPJ Vaccines

Article Title: A candidate subunit vaccine induces protective immunity against Mycobacterium avium subspecies paratuberculosis in mice

doi: 10.1038/s41541-023-00675-1

Figure Lengend Snippet: a Schematic diagram of the vaccination and MAP challenge timeline. C57BL/6 mice were subcutaneously injected two times (three weeks apart) with 50 μg 66NC fusion protein formulated in Montanide ISA 61 VG (61 VG) adjuvant. Control groups include mice immunized with Montanide ISA 61 VG adjuvant alone as a negative control or 74 F mixed MPL adjuvant as subunit vaccine control. Three weeks after the booster immunization, the mice were challenged with MAP K-10 by intraperitoneal injection. b Body weight was measured for 2, 4, 8, and 12 weeks after the MAP challenge ( n = 6 per group). Monitor IgG ( c ) and IgM ( d ) antibody levels. Sera were collected and measured for specific anti-66NC or 74 F antibody response of both IgG and IgM at 2, 4, 6, 8, 10, 12, 14, 16, and 18 weeks after prime vaccination by ELISA. Bacterial load quantification was evaluated in the liver ( e ) and intestine ( f ) from mice challenged by intraperitoneal injection with MAP K-10 for 2, 4, and 8 weeks after vaccination. g Representative images of Ziehl-Neelsen acid-fast stain in the liver of vaccinated mice, two weeks after being challenged with MAP, are presented (scale bars, 100 μm (top) and 10 μm (bottom)). The bottom images are enlarged from the outlined areas of the top images. b , e , f Data indicate cumulative results from at least three to six independent replicates. Mean ± SEM and Two-way ANOVA were used to analyze the statistical significance, followed by Tukey’s multiple comparisons tests. ns, non-significant; * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: Antibody PE/Cy5 Rat IgG1 (1:50, RG1, NBP1-43076, Novus Biologicals, Littleton, Co, USA), Alexa Fluor 647 Rat IgG1 (1:50, KLH/G1-2-2, 0116-31, SouthernBiotech, Birmingham, AL, USA), and eFluor 450 Rat IgG1 (1:50, eBRG1, 48-4301-82, ThermoFisher) were used according to the manufacturer’s instructions for isotype control.

Techniques: Injection, Adjuvant, Control, Negative Control, Enzyme-linked Immunosorbent Assay, Ziehl-Neelsen Stain

Journal: Advanced healthcare materials

Article Title: Architectural Modification of Conformal PEG-Bottlebrush Coatings Minimizes Anti-PEG Antigenicity While Preserving Stealth Properties

doi: 10.1002/adhm.201801177

Figure Lengend Snippet: Direct comparison of reactivity of APAs toward POEGMA brushes with different EG sidechain lengths versus linear PEG (MW = 20 K). a, Schematic of printing microspots of PEG20K-BSA Ag onto a background of POEGMA brush surfaces. Surfaces were incubated with a dilution series of rabbit-derived pAPA1 in serum, labeled with Cy5-anti-rabbit dAb, and then read by a fluorescence scanner. b, Spatial intensity plots of Cy5 fluorescence from EG2-OMe polymer brush surfaces functionalized by PEG20K-BSA Ag microspots (outlined by white dashes). Shown are 330 × 330 μm regions corresponding to surfaces (containing a single Ag microspot) exposed to serum alone (left) versus serum spiked with 2 μg/mL pAPA1 (right). c, Concentration curves of pAPA1 binding measured by fluorescence intensity from PEG20K-BSA Ag microspots (black squares) versus that from EG2-OMe POEGMA background (open circles). Similar spatial intensity plots and concentration curves as shown for EG2-OMe in (b, c) are shown for EG3-OMe in (d, e) and EG5-OMe in (f, g). Data plotted in (c, e, g) represent mean ± s.d. (n = 3). LODs determined from PEG20K-BSA microspots versus polymer background (LODAg vs. LODbkg, respectively) are displayed adjacent to each curve.

Article Snippet: Next, the surfaces were exposed to a 1 μg/mL solution of Cy5-goat-anti-human IgG detection Ab (R & D Systems, Inc.) for 30 min.

Techniques: Comparison, Incubation, Derivative Assay, Labeling, Fluorescence, Polymer, Concentration Assay, Binding Assay

Journal: Advanced healthcare materials

Article Title: Architectural Modification of Conformal PEG-Bottlebrush Coatings Minimizes Anti-PEG Antigenicity While Preserving Stealth Properties

doi: 10.1002/adhm.201801177

Figure Lengend Snippet: Screening POEGMA bottlebrush surfaces for immune reactivity toward a polyclonal APA (pAPA1). a, Schematic of pAPA1 fluoroimmunoassay. Surfaces were incubated with a solution of rabbit-derived pAPA1 spiked into undiluted calf serum, rinsed, and then labeled with Cy5-donkey-α-rabbit dAbs, and then imaged with a fluorescence scanner. b, c, Representative Cy5 channel fluorescence images (b) and quantitation of mean fluorescence intensities (c). Results in (c) are plotted as mean ± 95% CI for n ≥ 4 replicates. Vehicle-only and rabbit-IgG (not specifically reactive to PEG) controls are also included to show baseline values. Incubating surfaces with pAPA1 leads to significant fluorescence, indicating APA binding, from EG5-OMe, EG6-OH, and EG9-OMe surfaces, but not from bare, EG1-OMe, EG2-OM2, or EG3-OMe surfaces. Bars marked with different letters indicate significant differences within the pAPA1-treated groups by multiple comparison testing in one-way ANOVA (Tukey post hoc test, p ≤ 0.05).

Article Snippet: Next, the surfaces were exposed to a 1 μg/mL solution of Cy5-goat-anti-human IgG detection Ab (R & D Systems, Inc.) for 30 min.

Techniques: Incubation, Derivative Assay, Labeling, Fluorescence, Quantitation Assay, Binding Assay, Comparison

Journal: Advanced healthcare materials

Article Title: Architectural Modification of Conformal PEG-Bottlebrush Coatings Minimizes Anti-PEG Antigenicity While Preserving Stealth Properties

doi: 10.1002/adhm.201801177

Figure Lengend Snippet: Screening POEGMA brush surfaces for protein adsorption and cell adhesion. a, Schematic of surface fluorescence assay used to evaluate protein fouling. Cy5-labeled BSA was incubated on surfaces, rinsed and then read with a scanner for residual fluorescence. b, c, Representative Cy5 channel fluorescence images (b) and quantitation of mean ± 95% CI fluorescence intensities (c) (n = 3 for bare glass, and n ≥ 6 for others). Vehicle groups are plotted for comparison. Bars marked with different letters indicate significant differences within the Cy5-BSA-treated groups by multiple comparison testing in one-way ANOVA (Tukey post hoc test, p ≤ 0.05). d, Schematic of in vitro cell adhesion assay. NIH 3T3 cells expressing GFP (3T3-GFP) were incubated on surfaces in complete medium, washed, and then imaged for residual fluorescence on GFP channel by epifluorescence imaging. e, f, Representative epifluorescence images of cells (e) and quantitation of cell adhesion to surfaces (f) expressed as mean %FOV ± 95% CI (n = 6). Bars marked with different letters indicate significantly different groups by multiple comparison testing in one-way ANOVA (Tukey post hoc test, p ≤ 0.05)

Article Snippet: Next, the surfaces were exposed to a 1 μg/mL solution of Cy5-goat-anti-human IgG detection Ab (R & D Systems, Inc.) for 30 min.

Techniques: Adsorption, Fluorescence, Labeling, Incubation, Quantitation Assay, Comparison, In Vitro, Cell Adhesion Assay, Expressing, Imaging

Journal: Advanced healthcare materials

Article Title: Architectural Modification of Conformal PEG-Bottlebrush Coatings Minimizes Anti-PEG Antigenicity While Preserving Stealth Properties

doi: 10.1002/adhm.201801177

Figure Lengend Snippet: Evaluating interference from APA reactivity in indirect sandwich immunoassays (ISIAs) for antibody detection (“serology”) fabricated on polymer bottlebrushes. a, Schematic of serological antibody ISIA. ISIAs comprised of p24 Ag spotted onto bottlebrush overlayers were incubated with a dilution series of rabbit anti-HIV p24 polyclonal Ab, either with (bottom pathway) or without (top pathway) the presence of APA interferent (pAPA1). Surfaces were labeled with Cy5-donkey-anti-rabbit dAb, and then read by a scanner. b, c, d, e, f, Concentration binding curves for detecting polyclonal anti-p24 Ab (analyte) on polymer brush-based ISIAs, either with or without 100 ng/mL of APA interferent (black squares and open circles, respectively). LODs for each curve are provided in ng/mL, except for the EG5-OMe curve run with APA interferent, which was not calculated due to high background noise. Each data point represents mean ± s.d. from duplicate runs.

Article Snippet: Next, the surfaces were exposed to a 1 μg/mL solution of Cy5-goat-anti-human IgG detection Ab (R & D Systems, Inc.) for 30 min.

Techniques: Polymer, Incubation, Labeling, Concentration Assay, Binding Assay

Journal: Advanced healthcare materials

Article Title: Architectural Modification of Conformal PEG-Bottlebrush Coatings Minimizes Anti-PEG Antigenicity While Preserving Stealth Properties

doi: 10.1002/adhm.201801177

Figure Lengend Snippet: Reactivity of backbone-selective versus endgroup selective APAs toward EG2-OMe, EG3-OMe, and EG5-OMe POEGMA brushes. a, Schematic of backbone-selective (blue) versus endgroup-selective (tan) APA binding to PEG backbone and methoxy terminus of a bottlebrush, respectively. b, Schematic of surface fluoroimmunoassay for APA binding. Surfaces were incubated with a solution of APA-spiked calf serum, then labeled with Cy5-conjugated dAbs, and then read with a scanner. c–f, Reactivity of polyclonal APAs toward bottlebrush surfaces with known selectivity for PEG endgroups (pAPA1) versus backbone (pAPA2) (c, d), and similar plots shown for endgroup-selective (e-mAPA) versus backbone-selective (b-mAPA) monoclonal APAs (e, f) as assessed by surface fluoroimmunoassays. Data are plotted as mean fluorescence intensities ± s.d. (n = 9). Bars marked with different letters indicate significant differences by multiple comparison testing in one-way ANOVA (Tukey post hoc test, p ≤ 0.05).

Article Snippet: Next, the surfaces were exposed to a 1 μg/mL solution of Cy5-goat-anti-human IgG detection Ab (R & D Systems, Inc.) for 30 min.

Techniques: Binding Assay, Incubation, Labeling, Fluorescence, Comparison

Journal: Journal of Virology

Article Title: CD8+ T Cell Immunodominance in Lymphocytic Choriomeningitis Virus Infection Is Modified in the Presence of Toll-Like Receptor Agonists

doi: 10.1128/jvi.05996-11

Figure Lengend Snippet: FIG. 1. Immunodominance hierarchies of LCMV-specific CD8 T cells are altered by TLR23-L administration. Mice were injected with 500 PFU LCMV s.c. with pIC (100 g) and pam3cysk4 (20 g) individually or in combination before quantifying T cell responses in the spleen at days 8 (A) and 12 (B). LCMV-specific CD8 T cell responses were estimated for the NP396, NP205, GP33, or GP276 epitope (A and B) using ICS and measuring IFN- production after in vitro restimulation. Dot plots are representative of immunodominance profiles (i), and representative data of one experiment out of three independent trials standard deviations from triplicate animals in each condition are shown (ii). The controls

Article Snippet: T lymphocytes were stained with phycoerythrin (PE)Cy5-conjugated, rat anti-mouse CD8 clone 53-6.7 (Cedarlane) at 4°C and then fixed with 1% paraformaldehyde before adding fluorescein isothiocyanate (FITC)-conjugated anti-IFN- antibody (0.1% saponin) clone XMG1.2 (Cedarlane) overnight at 4°C.

Techniques: Injection, In Vitro

Journal: Journal of Virology

Article Title: CD8+ T Cell Immunodominance in Lymphocytic Choriomeningitis Virus Infection Is Modified in the Presence of Toll-Like Receptor Agonists

doi: 10.1128/jvi.05996-11

Figure Lengend Snippet: FIG. 2. Tetramer analysis of altered immunodominance between NP396 and GP276. Mice were injected with 500 PFU LCMV s.c. as described in Materials and Methods, with or without pIC and pam3cysk4 individually or in combination. (A to C) LCMV-specific CD8 T cell responses in the spleen were estimated at day 12 with tetramer staining for the NP396 or GP276 epitopes. (A) Dot plots show tetramer-positive CD8 cells from a representative mouse. (B) Graphs summarize the data from three experiments (n 3 mice in each trial). (C) Number of CD8 T cells that are tetramer positive for NP396 or GP276 were estimated. Controls (C) represent infected spleens with CD8 labeling to adjust for the compensation. Naïve splenocytes stained with the same tetramers gave similar background data (data not shown). For immunodominance analyses between TLR-L-treated and untreated mice, the condition where NP396 becomes subdominant is depicted by an asterisk.

Article Snippet: T lymphocytes were stained with phycoerythrin (PE)Cy5-conjugated, rat anti-mouse CD8 clone 53-6.7 (Cedarlane) at 4°C and then fixed with 1% paraformaldehyde before adding fluorescein isothiocyanate (FITC)-conjugated anti-IFN- antibody (0.1% saponin) clone XMG1.2 (Cedarlane) overnight at 4°C.

Techniques: Injection, Staining, Infection, Labeling

Journal: Journal of Virology

Article Title: CD8+ T Cell Immunodominance in Lymphocytic Choriomeningitis Virus Infection Is Modified in the Presence of Toll-Like Receptor Agonists

doi: 10.1128/jvi.05996-11

Figure Lengend Snippet: FIG. 7. Analyses of the conditions that favor changing immunodominance hierarchies during viral infection. Mice were injected with 500 PFU LCMV-WE and TLR23L with TLR4-L (10 g) simultaneously or TLR23L 3 days prior to virus infection. For different flank conditions, mice were injected with virus in one flank and TLR23L in another flank. Eight days p.i., LCMV-specific CD8 T cell responses were estimated using ICS. The data are representative of three experiments. For immunodominance analyses of TLR-L-treated and untreated mice, the change in the profile where NP396 becomes subdominant was depicted by an asterisk.

Article Snippet: T lymphocytes were stained with phycoerythrin (PE)Cy5-conjugated, rat anti-mouse CD8 clone 53-6.7 (Cedarlane) at 4°C and then fixed with 1% paraformaldehyde before adding fluorescein isothiocyanate (FITC)-conjugated anti-IFN- antibody (0.1% saponin) clone XMG1.2 (Cedarlane) overnight at 4°C.

Techniques: Infection, Injection, Virus