The immunoglobulin heavy chain 3′ regulatory region superenhancer controls mouse B1 B-cell fate and late VDJ repertoire diversity by Nour Ghazzaui, Hussein Issaoui, Alexis Saintamand, Christelle Oblet, Claire Carrion, and Yves Denizot BloodAdv Volume 2(3):252-262 February 13, 2018 © 2018 by The American Society of Hematology

Nour Ghazzaui et al. Blood Adv 2018;2:252-262 © 2018 by The American Society of Hematology

Backcross for aΔ3′RR /bwt and awt /bwt mice, and membrane expression of IgMa or IgMb allele by B cells. Backcross for aΔ3′RR/bwtand awt/bwtmice, and membrane expression of IgMaor IgMballele by B cells. The equilibrium between IgMa- or IgMb-expressing B cells will be disrupted if the expression of the 3′RR-deleted allele impedes B-cell development. Lowered number of IgMa-expressing B cells in aΔ3′RR/bwt mice thus will demonstrate that deletion of the 3′RR alters B-cell development or recruitment. Nour Ghazzaui et al. Blood Adv 2018;2:252-262 © 2018 by The American Society of Hematology

Flow cytometry for mouse B1 and B2 B cells. Flow cytometry for mouse B1 and B2 B cells. B1 and B2 B cells are distinguished on the basis of membrane cell surface markers. B1 B cells are B220lowIgMhighIgDlowCD23−CD11b+/low, whereas B2 B cells are B220highIgMhighIgDhighCD23+CD11b−.2,4,20 Nour Ghazzaui et al. Blood Adv 2018;2:252-262 © 2018 by The American Society of Hematology

Expression of a 3′RR-deleted allele in B1 B cells of the spleen, peritoneal cavity, and fetal liver. Expression of a 3′RR-deleted allele in B1 B cells of the spleen, peritoneal cavity, and fetal liver. (A) Flow cytometry analysis of IgMa and IgMb on spleen B1 B cells of aΔ3′RR/bwt and awt/bwt mice. One representative experiment is shown. (B) IgMa/IgMb ratios for spleen B1 B cells. Six aΔ3′RR/bwt and nine awt/bwt mice were investigated. Results are reported as means ± standard error of the mean (SEM), Mann-Whitney U test for significance. (C) IgMa and IgMb on spleen B2 B cells of aΔ3′RR/bwt and awt/bwt mice. One representative experiment is shown. (D) IgMa/IgMb ratios for spleen B2 B cells. Same mice as in panel B. (E) IgMa and IgMb on peritoneal cavity B1 B cells of aΔ3′RR/bwt and awt/bwt mice. One representative experiment is shown. (F) IgMa/IgMb ratios for peritoneal cavity B1 B cells, 7 mice per groups. (G) IgMa and IgMb on peritoneal cavity B2 B cells of aΔ3′RR/bwt and awt/bwt mice. One representative experiment is shown. (H) IgMa/IgMb ratio for peritoneal cavity B2 B cells, 7 mice per groups. Same mice as in panel E. (I) IgMa and IgMb on B1 B cells in fetal liver (12 hours before birth) of aΔ3′RR/bwt and awt/bwt mice. One representative experiment is shown. (J) IgMa/IgMb ratio for B1 B cells in the liver 12 hours before and 12 hours after birth. Eleven aΔ3′RR/bwt and nine awt/bwt mice were investigated before birth. Twelve aΔ3′RR/bwt and seven awt/bwt mice were investigated after birth. Nour Ghazzaui et al. Blood Adv 2018;2:252-262 © 2018 by The American Society of Hematology

B1 and B2 B-cell repertoire in 3′RR-deficient and wt mice. B1 and B2 B-cell repertoire in 3′RR-deficient and wt mice. B1 and B2 B cells of wt and 3′RR-deficient mice were cell sorted from pooled cells of 12 and 13 mice, respectively. 2.1 × 106 and 2.8 × 106 B1 B cells were sorted for wt and 3′RR-deficient mice, respectively. 0.8 × 106 and 1.3 × 106 B2 B cells were sorted for wt and 3′RR-deficient mice, respectively. (A-B) V usage frequency in (A) B2 and (B) B1 B cells of 3′RR and wt mice. (C) Gini index for B1 and B2 B cells of 3′RR and wt mice. The Gini index measures the inequality of clone size distribution and is bound between zero and 1. An index of zero represents a clone set of equally distributed clones, all having the same size, whereas a Gini index of 1 would point to a set including only 1 clone. Nour Ghazzaui et al. Blood Adv 2018;2:252-262 © 2018 by The American Society of Hematology

Effect of the 3′RR on BCR intensity and μ transcription in B1 B cells. Effect of the 3′RR on BCR intensity and μ transcription in B1 B cells. (A) IgMa and IgMb intensities on the membrane of fetal liver B1 B cells. Results are reported as means ± SEM. Mann-Whitney U test for significance. Four aΔ3′RR/bwt and five awt/bwt mice were investigated. NS: not significant. (B) IgMa and IgMb intensities on the membrane of peritoneal cavity B1 B cells. Seven aΔ3′RR/bwt and seven awt/bwt mice were investigated. (C) μ transcription in sorted peritoneal cavity B1 B cells of 3′RR-deficient mice and wt mice. Four separate experiments with pooled peritoneal cells from 4 to 6 mice for each genotype. Values were normalized to GAPDH transcripts. Locations of PCR primers are indicated in the scheme. (D) κ transcription in sorted peritoneal cavity B1 cells of 3′RR-deficient mice and wt mice. Same mice as in panel C. Nour Ghazzaui et al. Blood Adv 2018;2:252-262 © 2018 by The American Society of Hematology

NIgM in sera of 3′RR-deficient mice and wt mice. NIgM in sera of 3′RR-deficient mice and wt mice. (A) Indirect immunofluorescence assay for detection of NIgM. Sera from wt and 3′RR-deficient mice were investigated on Hep-2 cell-coated slides. Because reduced circulating IgM levels were found in 3′RR-deficient mice, sera were adjusted at 20-μg/mL IgM. MLR/lpr and RAGγC sera were used as positive and negative controls, respectively (1/10 dilution). Representative experiments from 1 to 5 sera per genotypes. Original magnification ×20. (B) Data, expressed as means ± SEM of the indicated number (n) of values, were analyzed using Prism software (GraphPad Software, La Jolla, CA). Significance was calculated with a nonparametric Mann-Whitney U test. PBS signal was arbitrary quoted to 1. (C) ELISA for detection of NIgM against kidney cell lysates. Sera from 3′RR-deficient and wt mice were adjusted at 20-μg/mL IgM. Data are reported as means ± SEM of 4 sera. *P = .02, Mann-Whitney U test for significance. A RAGγC serum was used as a negative control (1/10 dilution) to define baseline value. Nour Ghazzaui et al. Blood Adv 2018;2:252-262 © 2018 by The American Society of Hematology