Journal: Molecular medicine reports

Article Title: BCG stimulation promotes dendritic cell proliferation and expression of VDR and CYP27B1 in vitamin D‑deficient mice.

doi: 10.3892/mmr.2019.10780

Figure Lengend Snippet: Figure 2. Effect of BCG on BMDC differentiation and maturation. (A) Cell morphology during BMDC induction from days 1 to 6, as observed by light micros- copy. (B) BCG stimulated BMDC maturation. (C) Phenotypic changes of the BMDCs induced by BCG stimulation and (D) the levels of the surface molecules of DCs. All images were captured under identical magnification and microscopy conditions. **P<0.01 vs. 0 mg/ml in the normal control or vitamin D‑deficiency groups. ##P<0.01, vitamin D‑deficiency group vs. normal control group. Scale bar, 20 µm. BCG, Bacillus Calmette‑Guérin; BMDC, bone marrow‑derived dendritic cell; CD11c, integrin alpha‑X; CD80, T‑lymphocyte activation antigen CD80; MHC‑I, major histocompatibility complex class I; MHC‑II, major histocompatibility complex class II; CD86, T‑lymphocyte activation antigen CD86.

Article Snippet: The cell concentration was adjusted to 1x106 cells/ml and the cells were fixed with 0.1 ml polyformaldehyde for 30 min at 25 ̊C Subsequently, fluorescein isothiocyanate‐labeled integrin alpha‐X (CD11c; 1:50; 130‐110‐700; Miltenyi Biotech), T‐lymphocyte activation antigen CD86 (CD86; 1:50; 130‐102‐506; Miltenyi Biotech), T-lymphocyte activation antigen cd80 (cd80; 1:50; 130‐116‐459; Miltenyi Biotech), major histocompatibility complex class i (MHc-i; 1:50; ab95572; abcam) and MHC‐II (1:50; 130‐112‐386; Miltenyi Biotech) were added, respectively.

Techniques: Microscopy, Control, Activation Assay, Immunopeptidomics

Journal: iScience

Article Title: Microglial transcription profiles in mouse and human are driven by APOE4 and sex.

doi: 10.1016/j.isci.2021.103238

Figure Lengend Snippet: Figure 2. Sex and APOE genotype drive amyloid pathology and microglial gene expression profiles in APOE-FAD mice (A) Representative images of immunohistochemical staining for amyloid-b (red) and the microglial marker IBA1 (green) in male and female APOE3- and APOE4-FAD mice. Scale bar, 50 mm. (B) Samples separate by APOE and sex along PC1, with APOE3-FAD males clustering separately from mice of both sexes and APOE3-FAD females. A large proportion of the genes driving this separation (76.4%) are DAM-APOE genes. APOE3-FAD males are shown in yellow, APOE3-FAD females in gray, APOE4-FAD males in magenta, and APOE4-FAD females in teal. (C) Heatmap of the DAM-APOE genes associated with sample clustering along PC1. Gene expression levels were normalized, scaled, and centered and are displayed as Z scores. (D and E) Levels of the DAM-APOE marker, CD11c, are increased in APOE3-FAD females and in APOE4-FAD mice of both sexes, both at the (D) transcript and (E) protein levels. (D) Gene expression of Itgax in normalized counts; data are presented as mean (GSEM) values; n = 5–7/group. *p < 0.05, **p < 0.01; one-way ANOVA with Tukey’s multiple comparisons test correction. (E) Representative immunofluorescence images of microglia (IBA1, red) and CD11c (green) in the hippocampal subiculum of male and female APOE3- and APOE4-FAD mice. Scale bar, 25 mm.

Article Snippet: For IBA1/CD11c double labeling, antigen retrieval was performed using boiling EDTA and sections were incubated for 2 days at 4 C in primary antibodies directed against IBA1 (1:500) and CD11c (#NB110-97871, 1:500, Novus Biologicals).

Techniques: Gene Expression, Immunohistochemical staining, Staining, Marker

Journal: Journal of Nanobiotechnology

Article Title: Lymph node-targeted neoantigen nanovaccines potentiate anti-tumor immune responses of post-surgical melanoma

doi: 10.1186/s12951-022-01397-7

Figure Lengend Snippet: Characterization and in vitro DC-activation of neoantigen nanovaccines. a 1 H NMR of DSPE-PEG 2000 -NHS (vehicle), DSPE-PEG 2000 -peptide and peptide (peptide: M27). b MALDI-TOF–MS of DSPE-PEG 2000 -peptide (peptide: M27). c Size, PDI, encapsulation efficiency, drug loading content and zeta-potential of nanovaccines. d Size of nanovaccines. e The transmission electron microscopy (TEM) image of nanovaccines. f Curves of peptide (M27) release from nanovaccines in different solutions. g Proportion of mature DC (CD11c + CD80 + CD86 + ) after incubation with normal saline (NS), peptide (Tyrp1, M20 or M27) or nanovaccines (Tyrp1-NP, M20-NP or M27-NP) for 48 h. P -values were determined by one-way ANOVA with Tukey’s multiple comparisons test. ** P = 0.0026 (M20 vs M20-NP), ** P = 0.0029 (NS vs M27), * P = 0.0429 (M27 vs M27-NP), *** P < 0.001

Article Snippet: Inguinal lymph nodes were obtained 48 h after immunization, made into frozen sections, and incubated with anti-CD3 rat monoclonal antibody (1:200) (Abcam, UK), anti-CD11c rabbit monoclonal antibody (1:200) (Cell Signaling Technology, USA) overnight at 4 °C.

Techniques: In Vitro, Activation Assay, Encapsulation, Zeta Potential Analyzer, Transmission Assay, Electron Microscopy, Incubation, Saline

Journal: Journal of Nanobiotechnology

Article Title: Lymph node-targeted neoantigen nanovaccines potentiate anti-tumor immune responses of post-surgical melanoma

doi: 10.1186/s12951-022-01397-7

Figure Lengend Snippet: Lymph node-targeting characteristic of neoantigen nanovaccines. a Equivalent peptide-NIR797 (Free vaccine group) and DSPE-PEG 2000 -peptide-NIR797 (Nanovaccine group) were separately mixed with Montanide™ ISA 51 and injected subcutaneously at the tail base of mice. The fluorescence distribution in mice at different time points was photographed by small animal in vivo imaging (n = 3). b Fluorescence image of inguinal lymph nodes 48 h after injection. c The average radiant efficiency of inguinal lymph nodes, spleens and kidneys 48 h after injection. P -values were determined by one-way ANOVA with Tukey’s multiple comparisons test. ** P = 0.0062. d – e A FITC labeled model antigen ovalbumin (OVA) was used to evaluate the distribution of nanovaccines. d Localization of nanovaccines and CD3 + T cells in inguinal lymph nodes 48 h after subcutaneous injection of DSPE-PEG 2000 -OVA-FITC, was shown by immunofluorescence staining. Nanovaccine: green (FITC); T cells (CD3): gray (Cy5); Scale: 500 μm. e Localization of nanovaccines and DCs in lymph nodes 48 h after subcutaneous injection of DSPE-PEG 2000 -OVA-FITC. Nanovaccine: green (FITC); DCs (CD11c): red (Cy3); Scale: 25 μm

Article Snippet: Inguinal lymph nodes were obtained 48 h after immunization, made into frozen sections, and incubated with anti-CD3 rat monoclonal antibody (1:200) (Abcam, UK), anti-CD11c rabbit monoclonal antibody (1:200) (Cell Signaling Technology, USA) overnight at 4 °C.

Techniques: Injection, Fluorescence, In Vivo Imaging, Labeling, Immunofluorescence, Staining

Journal: Journal of Nanobiotechnology

Article Title: Lymph node-targeted neoantigen nanovaccines potentiate anti-tumor immune responses of post-surgical melanoma

doi: 10.1186/s12951-022-01397-7

Figure Lengend Snippet: T cell responses activated by neoantigen nanovaccines. One week after last treatment, Proportions of mature DCs (CD11c + CD80 + CD86 + ) in lymph nodes ( a ), proportions of neoantigen specific T cells (CD3 + CD8 + M27-H 2 K b+ ) in spleens ( b ) and tumors ( c ), and proportions of effector memory T cells (CD3 + CD8 + CD44 + CD62L − ) in spleens ( d ) were analyzed by flow cytometry. P -values were determined by one-way ANOVA with Tukey’s multiple comparisons test. ** P = 0.0034 ( a ), ** P = 0.0026 (d, NS vs Nanovaccine), ** P = 0.0056 (d, Free vaccine vs Nanovaccine), *** P < 0.001. e Lymphocytes in spleens were incubated with CFSE labeled B16F10 melanoma cells and MFC forestomach cancer cells at effector-to-target ratio (E: T) of 10:1. PI was added 4 h after incubation and the percentage of dead tumor cells (CFSE + PI + / CFSE + ) was analyzed by flow cytometry. P -values were determined by one-way ANOVA with Tukey’s multiple comparisons test. * P = 0.0114 (NS vs Free vaccine), ** P = 0.0046 (Free vaccine vs Nanovaccine). f Cytokines in the supernatant after co-incubation of lymphocytes and tumor cells. P -values were determined by two-way ANOVA with Tukey’s HSD multiple comparison post hoc test. *** P < 0.001. The level of TNF-α ( g ) and IL-6 ( h ) in the tumor microenvironment. i Proportions of regulatory T cells (CD3 + CD4 + Foxp3 + ) in the tumor microenvironment. j The expression of PD-L1 in tumors. P -values were determined by one-way ANOVA with Tukey’s multiple comparisons test. ** P = 0.0014 ( g ), *** P < 0.001

Article Snippet: Inguinal lymph nodes were obtained 48 h after immunization, made into frozen sections, and incubated with anti-CD3 rat monoclonal antibody (1:200) (Abcam, UK), anti-CD11c rabbit monoclonal antibody (1:200) (Cell Signaling Technology, USA) overnight at 4 °C.

Techniques: Flow Cytometry, Incubation, Labeling, Comparison, Expressing

Journal: Nuclear medicine and biology

Article Title: Initial insights into the interaction of antibodies radiolabeled with Lutetium-177 and Actinium-225 with tumor microenvironment in experimental human and canine osteosarcoma.

doi: 10.1016/j.nucmedbio.2024.108917

Figure Lengend Snippet: Fig. 5. Quantitative analysis and representative IHC images of M1 macrophages in human OS-33 tumors and canine Gracie tumors obtained from the OS tumors bearing mice treated with [177Lu]Lu-IF3 or [225Ac]Ac-IF3 radioconjugates. Tumor slides were stained with Anti-CD11c (D1V9Y) (97585) rabbit IgG mAb. (A-D) M1 macrophages in human OS-33 tumors and canine Gracie tumors obtained from the OS tumors bearing mice treated with [177Lu]Lu-IF3 or [225Ac]Ac-IF3 and har vested at different time points (control: black; 24 h: pink; 72 h: green; 168 h: purple) (A) canine Gracie tumors treated with [177Lu]Lu-IF3. (B) human OS-33 tumors treated with [177Lu]Lu-IF3. (C) canine Gracie tumors treated with [225Ac]Ac-IF3. (D) human OS-33 tumors treated with [225Ac]Ac-IF3. * p < 0.05.

Article Snippet: Rabbit IgG anti-CD11c (D1V9Y) (#97585) was purchased from Cell Signaling (Danvers, Massachusetts, USA).

Techniques: Staining, Control

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Fusion proteins for versatile antigen targeting to cell surface receptors reveal differential capacity to prime immune responses.

doi: 10.4049/jimmunol.0902555

Figure Lengend Snippet: FIGURE 4. Superior CD8+ T cell priming by a fusion protein containing three protein G domains and a Ub mutation. A, Proliferation of adoptively transferred, CFSE-labeled OT-I T cells recovered 3 d after Ag injection from the draining lymph nodes of injected mice. Animals were injected with CD11c-targeted fusion protein containing 50 ng OVA. Fusion proteins containing the physiological residues (KG, Lys-Gly) or substituted (RV, Arg-Val) at Ub positions 48 and 76 were tested. Experiments were per- formed twice, using seven mice per group total. Fusion protein P3UOrv primed significantly better than P2UOrv (p = 0.001), whereas the differ- ence was not statistically significant for P3UOkg versus P2UOkg (p = 0.10). B, Number of divisions per OT-I precursor induced upon priming with P3UO fusion proteins carrying the amino acids shown to the left in positions 48 and 76 (e.g., RV, Arg48Val76; KG, Lys48Gly76). Experiment was performed once, using four mice per group. Priming by 50 ng P3UOrv was significantly better than priming by all of the other proteins (p , 0.03), whereas the other differences shown were not statis- tically significant.

Article Snippet: The following Abs were used: rat anti-mouse DEC-205 (clone NLDC-145), hamster anti-mouse CD11c (clone N418), and control mAb mouse anti–HLA-A2 (clone BB7.2) were purchased from American Type Culture Collection and purified from hybridoma supernatant; rat antimouse TLR2 and rat anti-mouse CD206 (MR1) were from AbD Serotec (Cergy Saint-Christophe, France); control mAb mouse anti-human TAP1 (clone 148.3) was purified from ascites.

Techniques: Mutagenesis, Labeling, Injection

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Fusion proteins for versatile antigen targeting to cell surface receptors reveal differential capacity to prime immune responses.

doi: 10.4049/jimmunol.0902555

Figure Lengend Snippet: FIGURE 5. Localization of fusion protein in draining lymph nodes. Mice were injected with fusion protein in the hind footpads. Draining lymph node cryosections were stained for OVA fusion protein (P2EUO or P3UO, green) and cell markers (red). In the top row, mice were injected with 11 mg anti-CD11c targeted (left panel) or hamster Ig complexed (right panel) P2EUO 24 h before lymph node analysis. All of the other images are from mice injected with 2.5 mg CD11c-targeted fusion protein. The times between injection and lymph node collection were 1 h (CD169), 4 h (CD3, DEC205), and 24 h (CD19, CD35). Experiments were per- formed four times. Anti-CD169–stained section, original magnification 3100; anti-DEC205–stained section, original magnification 340; all others, original magnification 310.

Article Snippet: The following Abs were used: rat anti-mouse DEC-205 (clone NLDC-145), hamster anti-mouse CD11c (clone N418), and control mAb mouse anti–HLA-A2 (clone BB7.2) were purchased from American Type Culture Collection and purified from hybridoma supernatant; rat antimouse TLR2 and rat anti-mouse CD206 (MR1) were from AbD Serotec (Cergy Saint-Christophe, France); control mAb mouse anti-human TAP1 (clone 148.3) was purified from ascites.

Techniques: Injection, Staining

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Fusion proteins for versatile antigen targeting to cell surface receptors reveal differential capacity to prime immune responses.

doi: 10.4049/jimmunol.0902555

Figure Lengend Snippet: FIGURE 6. Effector functions of fusion protein-primed T cells. A, A total of 2 3 106 naive OT-I cells expressing the congeneic marker CD45.2 were injected i.v. in C57BL/6 mice expressing CD45.1. Twenty-four hours later, the mice were injected in the footpads with PBS, OVA, or CD11c/P3UOrv with or without adjuvants LPS, poly(I:C), and/or anti-CD40. Two weeks later, the mice were boosted s.c. with the same Ag without adjuvant. Three days later, the draining lymph nodes were taken and analyzed by flow cytometry. B, C57BL/6 mice were immunized s.c. with the indicated amounts of CD11c-targeted P3UOrv together with poly(I:C) and anti-CD40, which were injected s.c. or i.p., as indicated. One week later, the draining lymph node and spleen lymphocytes were stimulated in vitro with peptide S8L for 6 h before staining for intracellular cytokines. The insets show the aggregate percentages of cytokine-producing cells among all of the CD8+ T lymphocytes. The percentages of CD8+ cells producing one (white), two (gray), or three (black) cytokines are indicated in the pie charts. Cells stimulated with control peptide T8V did not produce cytokines (data not shown). C, C57BL/6 mice, injected 24 h earlier with 1 3 106 OT-I cells, were immunized s.c. with CD11c-targeted P3UOrv equivalent containing 130 ng OVA with or without adjuvant poly (I:C) and anti-CD40. Twelve days later, 3 3 106 CFSE-labeled syngeneic splenocytes pulsed with control peptide T8V (CFSE low) or cognate peptide S8L (CFSE high) were injected, followed 16 h later by flow cytometric analysis of draining lymph nodes and spleen. D, C57BL/6 mice were immunized s.c. with different amounts of Ab/fusion protein complexes (DEC205-P3UOrv, 1.3 mg; MR-P3UOrv, 1.3 mg; TLR2-P3UOrv, 0.225 mg; CD11c-P3UOrv, 0.13 mg; P3UOrv, 1 mg) and adjuvant poly(I:C) s.c. and anti-CD40 i.p. and boosted 5 wk later s.c. with the same Ags without adjuvant. One week later, the presence of OVA-specific serum Abs with different isotypes was tested by ELISA. Experiments were performed twice.

Article Snippet: The following Abs were used: rat anti-mouse DEC-205 (clone NLDC-145), hamster anti-mouse CD11c (clone N418), and control mAb mouse anti–HLA-A2 (clone BB7.2) were purchased from American Type Culture Collection and purified from hybridoma supernatant; rat antimouse TLR2 and rat anti-mouse CD206 (MR1) were from AbD Serotec (Cergy Saint-Christophe, France); control mAb mouse anti-human TAP1 (clone 148.3) was purified from ascites.

Techniques: Expressing, Marker, Injection, Adjuvant, Cytometry, In Vitro, Staining, Control, Labeling, Enzyme-linked Immunosorbent Assay

Journal: Pharmacological research

Article Title: C4BP(β-)-mediated immunomodulation attenuates inflammation in DSS-induced murine colitis and in myeloid cells from IBD patients.

doi: 10.1016/j.phrs.2023.106948

Figure Lengend Snippet: Fig. 5. Immunohistochemistry of the intestinal epithelium from DSS-induced murine colitis. (A) Assessment of immune cell infiltrates in distal colon cross sections from uninduced control mice (Blank) and DSS-induced mice either untreated (DSS), C4BP(β-)-treated (C4BP(β-)), or minocycline-treated (Minocycline) at the end of the study. Hyperplasic villi and immune cell infiltration of crypts were apparent in DSS-induced, untreated mice surveyed for dendritic cells (CD11c), monocytes/ macrophages (F4/80), and activated B cells (BAFFR). Particularly, colonic tissue from C4BP(β-)-treated mice was found scarcely stained for the above-referred inflammatory markers. Bars = 50 µm. (B) Staining was quantitatively assessed by digital image analysis through Image J and the IHC Profiler plugin. The results shown are the mean ± SD of 3,3’-diaminobenzidine positive pixels (%) stained for each of the tested markers on the colonic sections (n = 6 mice/group) (*, p < 0.05, **, p < 0.01; ***, p < 0.001 compared with DSS colitic mice).

Article Snippet: For the immunohistochemical analysis, sample slides were incubated overnight at 4 oC with the primary antibodies: rabbit polyclonal antimouse CD11c antibody (1:200) (Biorbyt, Cambridge, UK), rabbit polyclonal anti-mouse BAFFR antibody (1:200) (ThermoFisher), rat monoclonal antibody anti-mouse F4/80 (1:50) (ThermoFisher).

Techniques: Immunohistochemistry, Control, Staining