Figure - PMC

Skip to main content

An official website of the United States government

Here's how you know

Here's how you know

Official websites use .gov
A .gov website belongs to an official government organization in the United States.

Secure .gov websites use HTTPS
A lock ( ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites.

View full-text article in PMC

. 2017 Sep 4;214(9):2795–2810. doi: 10.1084/jem.20161955

Search in PMC
Search in PubMed
View in NLM Catalog
Add to search

This is a work of the U.S. Government and is not subject to copyright protection in the United States. Foreign copyrights may apply.

This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms/). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 4.0 International license, as described at https://creativecommons.org/licenses/by-nc-sa/4.0/).

PMC Copyright notice

Figure 2. — B7 on DCs but not B cells is critical for TD antigen–specific IgG1 production. (A) Generation of B7.1^flox mice. LoxP sites were inserted to flox exon 2 of the B7.1 gene on the BAC. B7.1^flox BAC Tg mice were backcrossed to B7.1^−/− B7.2^−/− DKO mice to eliminate endogenous B7.1 and B7.2 expression. B7.1^flox Tg-B7 DKO mice expressed B7.1 on splenic DCs at a level similar to that of WT B6 mice before and after LPS stimulation in vitro. Data are representative of at least three independent experiments. (B) B7.1 expression on DCs and B cells of DC-specific and B cell–specific B7 cKO mice. Splenocytes were stimulated with ConA/LPS/IL-2 for 24 h, and B7.1 expression on DCs (CD11c^high) and B cells (B220⁺) was analyzed. Shaded histograms indicate anti-B7.1 antibody staining of B7 cKO strains, and dashed lines indicate anti-B7.1 staining of B7.1/B7.2 DKO mice. Numbers indicate percentages of B7.1⁺ cells. Data are representative of three independent experiments. (C) Antigen-specific IgG1 production by B7 cKO strains. B7 cKO mice were immunized with NP-KLH/Alum. At 3 wk after immunization, serum anti-NP IgG1 titer was analyzed by ELISA. Dashed line indicates background OD value in empty wells. Each strain, n = 3. Data are representative of three independent experiments. ns, not significant. (D) Antibody affinity maturation was determined by ratio of high-affinity NP-specific IgG1 (measured by binding to NP₄-BSA) to total NP-specific IgG1 (measured by binding to NP₂₅-BSA) in serum at 7, 21, and 63 d after immunization. B7.1^flox, n = 5; BC-B7cKO, n = 6 for day 7 and day 21 and n = 3 at day 63. Data are representative of three independent experiments. (E) NP-specific GC B cell development in B7 cKO strains. B7 cKO strains were immunized with NP-KLH/Alum. 1 wk after immunization, NP-binding GC B cells (B220⁺ GL7⁺ Fas⁺ NP-PE⁺) in the spleen were determined by flow cytometry. Data presented are the combined result of three independent experiments. The total numbers of mice in the three combined experiments are B7.1^flox, n = 8; B7 DKO, n = 8; DC-B7 cKO, n = 6; and BC-B7 cKO, n = 8. (F) 1 wk after NP-KLH/Alum immunization, B7.1 expression on GC B cells was analyzed. Shaded histograms indicate anti-B7.1 staining, and dashed lines indicate isotype control staining. Data are representative of three independent experiments. (G) NP-specific GC B cell development in B cell–specific B7-deficient BM chimeras. BM chimeras were made that were completely and specifically deficient in expression of B7 on B cells by reconstitution of B cell KO hosts with a mixture of B7 DKO BM and B cell KO (μMT) BM (B7.1^−/− B7.2^−/− + μMT). Control chimeras were reconstituted with a mixture of B7 WT BM and B cell KO (μMT) BM (B7.1^+/+ B7.2^+/+ + μMT). Chimeras were immunized with NP-KLH/Alum. 1 wk after immunization, NP-binding GC B cells (B220⁺ GL7⁺ CD38^dull NP-PE⁺ IgG1⁺) in the spleen were determined by flow cytometry. Each group, n = 3. Data are representative of two independent experiments. Statistical significance was determined by Student’s t test for single comparison or one-way ANOVA followed by Dunnett’s method for multiple comparisons. All error bars represent the mean ± SEM.