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