Journal: Nature Communications

Article Title: Loss of HIV candidate vaccine efficacy in male macaques by mucosal nanoparticle immunization rescued by V2-specific response

doi: 10.1038/s41467-024-53359-2

Figure Lengend Snippet: a , b Comparison of mucosal B cells in rectal mucosa producing a IgG (V2-TTB NP, n = 12; TTB NP, n = 9; empty NP, n = 9) and b IgA (V2-TTB NP, n = 12; TTB NP, n = 9; empty NP, n = 9) antibodies that bind to ∆V1 gp120. c Comparison of plasma and mucosal IgG binding titer (AUC) against different SIV immunogens in all groups of animals (V2-TTB NP, n = 12; TTB NP, n = 9; empty NP, n = 9). d , e Comparison of d ADCC titers (the endpoint reciprocal dilution at which the ADCC killing was greater than control killing + 3 SD) and e ADCC activity among different groups of animals (V2-TTB NP, n = 12; TTB NP, n = 9; empty NP, n = 9; systemic vaccine, n = 14). f–h Correlation of f ADCC titer in the V2-TTB NP group ( n = 12, p = 0.02), g ADCC activity in the V2-TTB NP group ( n = 12, p = 0.02), and h ADCC titer in all animal groups combined ( n = 44, p < 0.0001) with number of intrarectal challenges. In h , red dots represent V2-TTB NP, black dots represent systemic vaccine, and blue squares represent TTB NP. Enlarged shapes represent multiple animals, as indicated. i , j Comparison of i V2 (NCI05)-specific ADCC activity and j frequency of V2 (NCI05)-specific ADCC activity in total ADCC among different groups of animals (V2-TTB NP, n = 12; TTB NP, n = 9; empty NP, n = 9; systemic vaccine, n = 14). k , l Correlation of V2 (NCI05)-specific ADCC activity in k the V2-TTB NP group ( n = 12, p = 0.003) and l in all animal groups combined ( n = 44, p = 0.003) with number of intrarectal challenges. m Correlation of V2 (NCI05)-specific ADCC activity at week 17 with VL at 15 wpi in the systemic vaccine group ( n = 9, p = 0.03). n Comparison of plasma antibody avidity score against ∆V1gp120 protein in different groups of animals (V2-TTB NP, n = 10; TTB NP, n = 7; empty NP, n = 8; systemic vaccine, n = 14). o Correlation of antibody response units against ∆V1gp120 (higher RU values indicated stronger binding of antibody to antigen) with number of intrarectal challenges in the V2-TTB NP+vaccine group ( n = 12, p = 0.02). Data shown in a – c , e , i , j , n were analyzed with the two-sided Mann-Whitney test. Data shown in d were analyzed with two-sided Cochran-Armitage tests for the comparisons between V2-TTB NP and TTB NP and Empty NP, separately. The two-sided Mann-Whitney test was used for the comparison between V2-TTB NP and systemic vaccine. Data shown in f – h , k – m , o were analyzed with the two-sided Spearman correlation test. Horizontal and vertical bars denote mean and SD. Source data are provided as a Source Data file. Here, the black, red, blue, and purple symbols represent male macaques immunized with a systemic vaccine, vaccine combined with V2-TTB NP, vaccine combined with TTB NP, and vaccine combined with empty NP, respectively.

Article Snippet: The primary antibodies were mouse (IgG2a) monoclonal anti-CD4 (clone OTI5D9, Novus Biologicals) and rabbit anti-Ki67 (Abcam).

Techniques: Comparison, Clinical Proteomics, Binding Assay, Control, Activity Assay, MANN-WHITNEY

Journal:

Article Title: Induction of Specific Cytotoxic Lymphocytes in Mice Vaccinated with Brucella abortus RB51

doi: 10.1128/IAI.69.9.5502-5508.2001

Figure Lengend Snippet: Different roles of CD4+ and CD8+ T cells in the cytotoxic lysis and release of IFN-γ. The two T-cell populations were isolated by the MACS magnetic kit and showed more than 92% purity by flow cytometry. The individual T-cell populations were cocultured at various concentrations with either strain RB51-infected or normal J774.A1 cells. The cytotoxic activity (A) and the amount of IFN-γ released into the supernatants (B) were measured. The data are means for triplicate estimations, and standard deviations did not exceed 20% of the means. Cocultures of CD4+ T cells with noninfected target macrophages (solid squares) and RB51-pulsed target macrophages (solid triangles) and CD8+ T cells with noninfected target cells (solid diamonds) and RB51-pulsed target cells (solid circles) were tested.

Article Snippet: Briefly, live T cells isolated by Histopaque column purification were incubated with MACS magnetic MicroBeads to which monoclonal antibodies against CD4 molecule (clone GK1.5; isotype, rat IgG2b) or CD8 molecule (clone 53-6.7; isotype, rat IgG2a) had been coupled (Miltenyi Biotec, Auburn, Calif.) at the concentration of 10 μl of MicroBeads per 10 7 total cells for 15 min in a refrigerator at 4°C (see Fig. ).

Techniques: Lysis, Isolation, Flow Cytometry, Infection, Activity Assay

Journal:

Article Title: Induction of Specific Cytotoxic Lymphocytes in Mice Vaccinated with Brucella abortus RB51

doi: 10.1128/IAI.69.9.5502-5508.2001

Figure Lengend Snippet: Specific phenotype analysis of effector cells by flow cytometry a

Article Snippet: Briefly, live T cells isolated by Histopaque column purification were incubated with MACS magnetic MicroBeads to which monoclonal antibodies against CD4 molecule (clone GK1.5; isotype, rat IgG2b) or CD8 molecule (clone 53-6.7; isotype, rat IgG2a) had been coupled (Miltenyi Biotec, Auburn, Calif.) at the concentration of 10 μl of MicroBeads per 10 7 total cells for 15 min in a refrigerator at 4°C (see Fig. ).

Techniques: Flow Cytometry

Journal: Frontiers in immunology

Article Title: IRF4 downregulation improves sensitivity and endurance of CAR T cell functional capacities.

doi: 10.3389/fimmu.2023.1185618

Figure Lengend Snippet: FIGURE 1 Downregulation of IRF4 expression in CAR T cells. (A) Schematic of CAR constructs. (B) CAR expression in T cells detected by staining with a phycoerythrin (PE)-labeled goat anti-human IgG antibody before (upper panels) and after (lower panels) magnetic cell separation (MACS). One representative donor out of four is shown. (C) Intracellular staining of IRF4 after stimulation with CEA+ BxPC-3 cells at the indicated time points in CD8+ (left panel) and CD4+ CAR T cells (right panel). CAR T cells were generated as described in the materials and methods section by activation of PBMCs followed by retroviral transduction. Untransduced cells were generated by activation of PBMCs and subsequent expansion with IL-2, but without retroviral transduction. Data represent means ± SEM of five donors, p values were calculated by Student´s t test, *indicates p ≤0.05, ***indicates p ≤0.001.

Article Snippet: Four hours later, T cells were stained with the viability dye eFluor 780, the goat F(ab’)2 antihuman IgG-PE antibody, a BV421-conjugated anti-CD8 antibody (BD), and an APC-conjugated anti-CD4 antibody (Miltenyi Biotech).

Techniques: Expressing, Construct, Staining, Labeling, Magnetic Cell Separation, Generated, Activation Assay, Retroviral, Transduction

Journal: Frontiers in immunology

Article Title: IRF4 downregulation improves sensitivity and endurance of CAR T cell functional capacities.

doi: 10.3389/fimmu.2023.1185618

Figure Lengend Snippet: FIGURE 3 Downregulation of IRF4 enhances CAR T cell functionality. (A) CAR T cells (starting with 1 × 105 CAR T cells) underwent four rounds (R1-R4) of stimulation with GFP-labeled CEA+ BxPC-3 cells (1 × 105 tumor cells at the beginning of each round). At the end of each round, CAR T cells (live CD3+ CAR+) (left panel) and BxPC-3 cells (right panel) were quantified by flow cytometry. Data represent means ± SEM of six donors, p values were calculated by Student´s t test, ns indicates not significant, and * indicates p ≤0.05. (B-G) Phenotypic analysis of CD8+ CAR T cells during repetitive antigen stimulation. CAR T cells underwent three rounds (R1-R3) of antigen-stimulation with unlabeled BxPC-3 cells. At the end of each round, the CD8/CD4 T cell ratio was determined (B). CAR T cells were stained for CD8 and further characterized regarding TIM-3 (C), PD-1 (D), TIGIT (E) expression and effector-memory cell differentiation: SCM = T stem-cell-memory (CD45RO+ CD62L+), EM = effector-memory (CD45RO+ CD62L-), CM = central-memory (CD45RO+ CD62L+), E = effector (CD45RO- CD62L-) (F), and CD27 expression (G). Data represent geometric means of ± SEM of at least four donors, p values were calculated by paired t test, ns indicates not significant, *indicates p ≤0.05, **indicates p ≤0.01.

Article Snippet: Four hours later, T cells were stained with the viability dye eFluor 780, the goat F(ab’)2 antihuman IgG-PE antibody, a BV421-conjugated anti-CD8 antibody (BD), and an APC-conjugated anti-CD4 antibody (Miltenyi Biotech).

Techniques: Labeling, Cytometry, Staining, Expressing, Cell Differentiation

Journal: Nature Communications

Article Title: Fasting mimicking diet in mice delays cancer growth and reduces immunotherapy-associated cardiovascular and systemic side effects

doi: 10.1038/s41467-023-41066-3

Figure Lengend Snippet:

Article Snippet: Anti-mouse CD4, VioBright FITC (REA604) , Miltenyi Biotec , 130-118-692.

Techniques: In Vivo

Journal: Frontiers in Microbiology

Article Title: A Functional Slow Recycling Pathway of Transferrin is Required for Growth of Chlamydia

doi: 10.3389/fmicb.2010.00112

Figure Lengend Snippet: Analysis of host cell trafficking pathways during treatment with FR179254 . HEp-2 cells were treated as indicated, and trafficking pathways were analyzed as described in Section (A) Lipid droplet (LD) visualization with the neutral lipid stain Bodipy 493. (B) Fluid-phase uptake as measured by fluorescent dextran. (C) Live cell visualization of multivesicular bodies/late endosomes using CD63-GFP expressing HEp-2 cells. (D) Analysis of late endosomes/lysosomes in LAMP1-YFP transfected cells. (E) Exocytic trafficking as measured by incorporation of fluorescent sphingomyelin (SM) in Golgi and chlamydiae. (F) Exocytic trafficking of glycoproteins as measured by localization of CD4 on the surface of transfected cells. (G,H) Analysis of retrograde trafficking using fluorescently tagged cholera toxin subunit B (CTxB) after pulse-chase labeling. Arrows within (C) and (E) indicate inclusions. Note the lack of significant staining in (C) (inset) and the presence of staining in (E) , which is contrast to a published study (Beatty, ).

Article Snippet: For CD4, non-permeabilized cells were incubated with primary Ab mouse anti-human CD4 (R&D Systems, Minneapolis, MN, USA) followed by secondary goat anti-mouse Alexa488 (Invitrogen).

Techniques: Staining, Expressing, Transfection, Pulse Chase, Labeling

Journal: Journal of Respiration

Article Title: Immune Correlates of Non-Necrotic and Necrotic Granulomas in Pulmonary Tuberculosis: A Pilot Study

doi: 10.3390/jor1040023

Figure Lengend Snippet: Figure 2. Distribution of CD15, S100A8/A9, CD4 and IBA1 positive immune cells in human lung TB granulomas. (A) Representative images singleplex (CD15 and S100A8/A9; red spots) and multiplex (CD4; green spots and IBA1; red spots) immunostaining of control, solid/non-necrotic and necrotic granulomas. Host cell nucleus is stained blue with DAPI. Scale bar in S100A8/A9 panel (50 microns) is common for CD15 and S100A8/A9 images. Scale bar in the IBA1 panel (50 microns) is common for CD4 and IBA1 images. (B) Distribution of CD15 positive cells (including MDSCs) relative to the total number of cells in the field in control, solid/non-necrotic granulomas and necrotic granulomas. (C) Distribution of S100A8/A9 positive cells (mainly neutrophils) relative to the total number of cells in the field in control, solid/non-necrotic granulomas and necrotic granulomas. (D) Distribution of CD4 positive T-cells relative to the total number of cells in the field in control, solid/non-necrotic granulomas and necrotic granulomas. (E) Distribution of IBA1 positive cells (activated macrophages) relative to the total number of cells in the field in control, solid/non-necrotic granulomas and necrotic granulomas. Data plotted in (B–E) are mean and standard error mean (SEM) of n = 3 (control); n = 5 (solid lesion) and n = 18 (necrotic lesion). Data were analyzed by One-way ANOVA with Tukey’s post-hoc correction for multiple group comparison; * p < 0.05; ** p < 0.01; *** p < 0.005.

Article Snippet: S100A8/A9) (cat no. ab22506) were purchased from (Abcam, Waltham, MA, USA), CD33 (cat no. sc19660), CD15 (cat no. sc-19649), ARG1 and NOS2 (cat no. sc-7271) (Santa Cruz Biotechnology, Dallas, TX, USA), HIF-1α (cat no. MA5160048) Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA), IL-6 (cat no. MAB206) and IL-10 (cat no. AF-217-NA) (R& D Systems Inc, Minneapolis, MN, USA) and CD4 from (cat no. NBP1-19371AF647) Novus Biologicals, Centennial, CO, USA).

Techniques: Multiplex Assay, Immunostaining, Control, Staining, Comparison