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Science 336 (6080): 485-489

Copyright © 2012 by the American Association for the Advancement of Science

The Inhibitory Receptor PD-1 Regulates IgA Selection and Bacterial Composition in the Gut

Shimpei Kawamoto1,* , Thinh H. Tran1,2,* , Mikako Maruya1,* , Keiichiro Suzuki1,3, Yasuko Doi1, Yumi Tsutsui1, Lucia M. Kato1,4, and Sidonia Fagarasan1,{dagger}

1 Laboratory for Mucosal Immunity, Research Center for Allergy and Immunology, RIKEN Yokohama 1-7-22, Tsurumi, Yokohama 230-0045, Japan.
2 Department of Biochemistry, Hanoi Medical University, 1st Ton That Tung, Hanoi, Vietnam.
3 AK project, Graduate School of Medicine, Kyoto University, Yoshida Sakyo-ku, Kyoto 606-8501, Japan.
4 Department of Immunology and Genomic Medicine, Graduate School of Medicine, Kyoto University, Yoshida Sakyo-ku, Kyoto 606-8501, Japan.


Figure 1
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Fig. 1. The gut microbial community composition and the repertoires of LP-resident IgA-producing cells are altered in Pdcd1–/– mice. (A) Small intestine microbiota composition in 3-month-old, specific pathogen–free (SPF) Pdcd1–/–and WT mice. Contents of the entire small intestine were pooled, and bacteria were identified with standard microbiological methods. Data from two experiments, n = 3 mice per group, are shown. ND, not detected. (B) The percentages of fecal bacteria coated with IgA as determined by flow cytometry and (C) free IgA concentrations in the feces as determined by enzyme-linked immunosorbent assay (ELISA). Data points represent individual mice. (D) IgH V family usage and (E) the affinity maturation of IgA-producing cells from the LP of Pdcd1–/–and WT mice. RCDR, replacement in CDR1 and CDR2; Stotal, silent mutations in both CDRs and in framework regions 1 to 3 (FWR1-3) (12). The number of sequences analyzed (pooled from three mice per group) is indicated. (F) Frequency of BrdU+ and (G) Caspasehi IgA PBs and PCs from the LP of Pdcd1–/– and WT mice as determined by flow cytometry. Note that some of the BrdU+ PCs may be also derived from peritoneal B1 cells. Data shown are combined from two independent sets of experiments. Mean ± SEM (n = 3 to 5 mice per group). Two-tailed unpaired Student’s t test was used for all statistical analyses; ***P < 0.001; **P < 0.01, *P < 0.05.

 

Figure 2
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Fig. 2. Increased numbers and enhanced dynamics of GC and IgA+ B cells in PPs of Pdcd1–/– mice. (A) Representative sections of the PPs and (B) flow cytometric profiles of PP cells from WT and Pdcd1–/– mice stained as indicated to reveal the structure and characteristics of GCs. Scale bars, 100 μm. LZ (light-zone AIDlow) and DZ (dark-zone AIDhi) are shown. (C) Absolute numbers of indicated B cell populations isolated from PPs of WT and Pdcd1–/– mice. Means ± SEM (n = 16 mice per group). (D) Frequency of BrdU+ GC B cells (PNA+Fas+) and B220+IgA+ B cells from PPs of Pdcd1–/– and WT mice as determined by flow cytometry. Data are combined from two independent sets of experiments. Means ± SEM (n = 3 to 5 mice per group). (E) Representative charts of clonal diversity, calculated as frequency of transcripts of a given in-frame VH-(N)-D-(N)-JH rearrangement of GC IgA+ B cells in PPs of Pdcd1–/– and WT mice. The number of productive sequences compared is indicated. Two-tailed unpaired Student’s t test was used for all statistical analyses; **P < 0.01; *P < 0.05.

 

Figure 3
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Fig. 3. Expansion of activated T cells and skewed cytokine profiles of TFH cells in PP GCs of Pdcd1–/– mice. (A) Representative flow cytometric profiles and plots of PP cells from WT and Pdcd1–/– mice stained for the indicated markers. Numbers in the graphs indicate the geometric mean fluorescence intensity of BCL6 and IRF4 in the corresponding color-coded T cell subset gates. (B) Absolute numbers of major T cell populations and (C) the ratio of TFH cells to GC B cells in the PPs of Pdcd1–/– and WT mice. Means ± SEM (n = at least 5 mice per group). (D) Flow cytometric profiles and (E) mRNA expression of indicated cytokines in PP T cell subsets. Numbers represent percentage of cells in the quadrants or gates. Relative amounts of mRNA normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) are shown. Means ± SEM of at least two independent experiments. Two-tailed unpaired Student’s t test was used for all statistical analyses; ***P < 0.001; **P < 0.01, *P < 0.05.

 

Figure 4
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Fig. 4. TFH cells from Pdcd1–/– mice are impaired in their ability to support the generation of IgA plasma cells in gut. (A) Representative flow cytometric profiles of cells isolated from PPs and LP stained for the indicated markers. Numbers represent percentage of cells in the indicated gates. (B) Sections of the PPs and small intestine stained as indicated and (C) fecal IgA levels as determined by ELISA from the Cd3e–/– mice 3 months after reconstitution with TFH cells and Foxp3+ T cells from WT and Pdcd1–/– mice. Scale bars, 100 μm. (D) Absolute number of somatic mutations in VH genes and (E) the affinity maturation of the IgA-producing cells from the LP of Cd3e–/– mice at 3 months after the reconstitution with the T cells indicated. The number of sequences analyzed: WT, TFH = 171; Pdcd1–/–, TFH = 175; WT Foxp3+, T = 153; Pdcd1–/– Foxp3+, T = 177. (F) Total numbers of indicated cells from the PPs of Cd3e–/– mice at 3 months after the reconstitution with the T cells indicated. Means ± SEM (n = 2 to 3 mice per group). Two-tailed unpaired Student’s t test was used for all statistical analyses; ***P < 0.001; **P < 0.01.

 


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