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

. 2018 Mar 5;215(3):801–813. doi: 10.1084/jem.20171067

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

© 2018 Kara et al.

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 4. — ACKR4 limits early proliferation of antigen-engaged B cells and limits early PB responses. (A and B) WT (CD45.1/2) and Ackr4^−/− (CD45.2) SW_HEL B cells were cotransferred into B6.Ly5.1 (CD45.1) mice and immunized with HEL^2X-SRBC. (A) Representative flow-cytometric CFSE profiles and analysis of CFSE GMFI among responding SW_HEL B cells (B220⁺Igκ⁺HEL⁺CD138⁻) on day 2 (n = 6). Undivided SW_HEL B cells were analyzed from mock-SRBC–immunized recipients (n = 2). (B) Contribution of WT and Ackr4^−/− among responding SW_HEL B cells (B220⁺Igκ⁺HEL⁺CD138⁻) over the first 3 d of the HEL^2X-SRBC response (n = 4 mice/time point). (C–H) WT or Ackr4^−/− (both CD45.2) SW_HEL B cells were transferred into B6.Ly5.1 (CD45.1) mice and immunized with HEL^2X-SRBC (n = 4 mice/genotype per time point). (C) Frequency of total responding SW_HEL B cells (CD45.2⁺Igκ⁺HEL⁺) per 10⁶ splenocytes (means ± SEM). (D) Representative plots of early PB and GC B cell and B220⁺HEL^hiGL7⁻ cell responses as a frequency of total responding SW_HEL B cells (plots are pregated CD45.2⁺Igκ⁺HEL⁺) on day 5.5. Numbers in plots represent the means ± SEM frequency of early PBs, GC B, or B220⁺HEL^hiGL7⁻ cells as the frequency of the total responding SW_HEL B cells. (E) Frequency of SW_HEL B220⁺HEL^hiGL7⁻ cells, GC B cells and early PBs (gated as shown in D) per 10⁶ splenocytes and total number per spleen on day 5.5 (means ± SEM). (F) Representative histology of responding SW_HEL B cells on day 5.5 (n = 4 mice/genotype). Bar, 200 µm. HEL, green; IgD, blue; and CD4, red. (G) Frequency of SW_HEL early PBs (gated as in E) per 10⁶ splenocytes (means ± SEM). (H) Anti-HEL IgM and IgG1 serum concentration as determined by ELISA (see Materials and methods; means ± SEM). (I–K) WT (CD45.1/2) and Ackr4^−/− (CD45.2) were cotransferred into B6.Ly5.1 (CD45.1) recipients and immunized with HEL^2X (intermediate affinity) or HEL^3X (low affinity) conjugated to SRBCs at intermediate or low epitope densities (n = 4 mice/genotype per condition). (I) HEL^2X or HEL^3X were conjugated to SRBCs at intermediate (black histogram; 100 µg/ml; SRBC) and low (gray histogram; 5 µg/ml; SRBC) epitope densities and detected using HyHEL9 mAb. Mock-conjugated SRBCs (open histogram) are shown for comparison. (J) Representative plots of concurrent WT and Ackr4^−/− SW_HEL early PBs on day 5 (pregated Igκ⁺HEL⁺ and CD45.1⁺CD45.2⁺ [WT] or CD45.1⁻CD45.2⁺ [Ackr4^−/−]). Numbers in plots represent the mean (± SEM) frequency of early PB as the frequency of total responding SW_HEL B cells. (K) SW_HEL.Ackr4^−/− competitive ratio is plotted as the ratio of Ackr4^−/− to WT among SW_HEL GC B cell (left) or B220⁺HEL^hiGL7⁻ cell (right) compartments (gated as in D), normalized to the input ratio, as determined from mock-SRBC–immunized mice. n = 4 mice (means ± SEM) on day 5. (A–K) *, P < 0.05; **, P < 0.01; ****, P < 0.0001; data are representative of two independent experiments. (A and J) Two-tailed, paired Student’s t test. (B and C–H) Two-tailed, unpaired Student’s t test. (C–K) Means ± SEM.