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Human blood IgM “memory” B cells are circulating splenic marginal zone B cells harboring a prediversified immunoglobulin repertoire

Sandra Weller, Moritz C. Braun, Bruce K. Tan, Andreas Rosenwald, Corinne Cordier, Mary Ellen Conley, Alessandro Plebani, Dinakhanta S. Kumararatne, Damien Bonnet, Olivier Tournilhac, Gil Tchernia, Birte Steiniger, Louis M. Staudt, Jean-Laurent Casanova, Claude-Agnès Reynaud and Jean-Claude Weill

Data supplements

  • Supplemental materials for: Weller et al, Vol 104, Issue 12, 3647-3654

    Files in this Data Supplement:

    • Figure 1. CDR3 spectratyping of V3-15-C� transcripts (JPG, 68 KB) -

      CDR3 size distribution analysis (spectratyping) of V3-15-Cµ transcripts expressed in peripheral and splenic IgM+IgD+CD27+ B cells of a 9-year-old child before and after immunization with a polysaccharidic vaccine. Polymerase chain reaction (PCR) amplification of the CDR3 region of V3-15-Cµ transcripts was achieved by a 2-step strategy. First, cDNA from sorted IgM+IgD+CD27+ B cells was amplified using a sense V3-15-leader-specific primer with an antisense primer specific for the constant region Cµ The resulting PCR products were then amplified using a sense V3-15-FR3 specific primer with the same antisense Cµ primer. After electrophoresis on a 6% denaturing polyacrylamide gel, PCR products were revealed by silver staining. The bands indicated by arrows corresponding to one particular CDR3 length were excised, reamplified, cloned, and sequenced.

Article Figures & Data

Figures

  • Figure 1.

    Markers specific of marginal zone B cells shared by blood and spleen IgM+IgD+CD27+ subsets. Purified B cells from blood and spleen are analyzed separately for IgM, CD21, CD23, and CD1c surface expression after gating of the 3 different CD19+ lymphocyte subsets distinguished by IgD and CD27 labeling. These data correspond to 1 representative case out of 4 different individuals. Naive B cells (IgD+CD27-) are indicated by the bold line; IgD+CD27+ B cells, the gray shadow; IgD-CD27+ cells, the thin line. Percentages of cells in the 2 CD27+ quadrants are indicated. The absence of IgM+ cells among the IgD-CD27+ subset is noticeable.

  • Figure 2.

    CD1c as a strong marker of splenic marginal zone B cells in humans. Serial cryosections of an adult human spleen are stained with anti-CD20, anti-IgD, anti-CD1c, and anti-CD27 antibodies (ABC technique; original magnification, × 25) marginal zone B cells are IgDlowCD27+CD1chigh. Note the more intense staining for IgD of the corona (Co) compared with the MZ B cells while the reverse is true for CD1c. The intense IgD staining of outer marginal zone B cells has been described previously.16 GC indicates germinal center.

  • Figure 3.

    Presence of an IgM+IgD+CD27+ subset in hyper-IgM patients. Patients AID.1 and 40.1 have been reported previously.20,21 Control is an 11-year-old child, age-matched with patient 40.1. Since IgD+CD27+cells coexpress IgM, the IgM+IgD+CD27+ subset is analyzed after IgD, CD27, and CD19 labeling of purified B cells. IgD and CD27 expression is shown after gating on CD19+ cells. Percentages of cells in the naive and the 2 CD27+ quadrants are indicated.

  • Figure 4.

    A common gene-expression signature for IgM+IgD+CD27+ B cells from blood and spleen. Each column represents microarray data from a sample of the indicated cell subtype, and each row represents the expression of a single gene. The spleen IgD+CD27+ and IgD-CD27+ populations are obtained from 2 separate donors, with 1 of the 2 samples prepared in duplicate. Red squares indicate increased expression and green squares indicate decreased expression relative to the median expression of the gene according to the color bar shown. Gray squares indicate missing or excluded data. (A) The array dendrogram obtained by clustering the 49 genes that differentiated (P < .005, 2-fold higher expression) the splenic IgD+CD27+ samples from the splenic IgD-CD27+ samples. The red branches indicate the coclustering of the blood IgD+CD27+ samples, with the splenic IgD+CD27+ samples, and the blue branches indicate the coclustering of the blood IgD-CD27+ samples with their respective splenic IgD-CD27+ samples. (B) The 37 genes that achieved statistical significance with a 2-fold higher mean expression when the IgD+CD27+ cell populations are compared with the memory IgD-CD27+ cell populations.

  • Figure 5.

    B-cell clones with an identical V3-15 CDR3 shared by IgM+IgD+CD27+ B cells from blood and spleen during a T-independent response. Amplification of V3-15–Cμ mRNA sequences was performed from naive and IgM+IgD+CD27+ B cells of a 9-year-old child undergoing splenectomy and immunized against S pneumoniae and N meningitidis (plain polysaccharidic vaccines). The following samples were analyzed: blood before immunization, blood at the time of splenectomy (ie, 8 days after immunization), spleen, and blood 5 weeks after immunization. The first V3-15–specific PCR products were further amplified with V3-15–specific FR3 and Cμ primers, and the resulting products fractionated by denaturing gel electrophoresis. A specific CDR3 size was excised from the gel after silver staining and reamplified with the same FR3 and Cμ primers, and sequences were determined after cloning. Several PCR amplifications were performed from 2 independent cDNAs for each cell sample. The recurrent CDR3s encompassing the V3-15 and JH3 junctions observed in the various IgM+IgD+CD27+ fractions are shown, with the most frequently occurring sequence taken as reference (CDR3 is defined as amino acids included between the conserved Cys residue of FR3 and Trp residue of JH segments). *Clones found repeatedly in independent PCR amplifications.

  • Figure 6.

    Development and diversification of IgM+IgD+CD27+ peripheral B cells from healthy and asplenic children. (A) (B) (C) (D) Percentage of IgM+IgD+CD27+ peripheral B cells from healthy (A) and asplenic children (C) younger than 5 years. Mutation frequency of rearranged V3-23 genes from IgM+IgD+CD27+ peripheral B cells of healthy (B) and asplenic children (D). In panels B and D, each bar represents the mutation frequency of one individual, and the values marked above the bars represent the mutation range over the 288 bp V3-23 sequence analyzed; healthy adult values are pooled from 5 individuals. (E) IgD/CD27 staining profiles of peripheral CD19+ lymphocytes of asplenic individuals from 14 months to 71 years. The percentage of cells in each CD27+ quadrant is indicated. A complete description of asplenic patients is given in Table 3.

Tables

  • Table 1.

    Proportion and V3-23 gene mutation frequency of CD27+ B-cell subsets from blood and spleen of healthy individuals

    IgD+CD27+IgD-CD27+
    B cells, %Mutation frequency per 100 bp (total no. mutations; range of mutation per V sequence)B cells, %Mutation frequency per 100 bp (total no. mutations; range of mutation per V sequence)
    DonorsAgeSpleenBloodSpleenBloodSpleenBloodSpleenBlood
    C1 23 mo 20 7.5 2.2 (58 mut; 0-17) 3.1 (144 mut; 0-28) 14 7 4.6 (225 mut; 0-37) 4.7 (162 mut; 0-29)
    C2 25 mo 20 9 2.0 (82 mut; 0-12) 3.8 (222 mut; 0-27) 25 13 5.3 (216 mut; 8-29) 4.9 (269 mut; 0-33)
    C3 46 mo 28 22 1.9 (95 mut; 0-25) 2.8 (124 mut; 0-21) 22 17 7.0 (182 mut; 6-31) 6.6 (267 mut; 4-33)
    C4 8 y 31 35 2.4 (120 mut; 0-16) 3.9 (183 mut; 2-26) 10 16 4.8 (221 mut; 0-26) 4.5 (90 mut; 8-20)
    C5 9 y 17 18 3.4 (109 mut; 1-17) 5.2 (226 mut; 5-22) 18 14 7.5 (196 mut; 6-32) 5.2 (166 mut; 5-25)
    2.4 ± 0.6* 3.8 ± 1* 5.7 ± 1* 5.2 ± 0.8*
    • Mut indicates mutation.

    • * Average mutation frequency. Mutation frequencies were significantly higher in IgD- compared with IgD+ cells, both in the overall sample (P < 10-9) and after stratification by tissue type (blood, P = .03; spleen, P < 10-9; nonparametric Wilcoxon score test)

  • Table 2.

    Somatic mutations in IgM+IgD+CD27+ peripheral blood B cells of CD40- and AID-deficient patients

    Sequences, no.Mutations
    Patient characteristicsDonorAge, yTotalMutatedRangeNo.Frequency per 100 bp
    CD40-deficient 40.1 11 22 21 0-29 243 3.8
    AID-deficient AID.1* 41 10 0 0 0 0
    AID-deficient AID.2* 25 11 1 0-1 1 0.02
    • * Rearranged intronic JH4-JH5 sequences were amplified and sequenced for these 2 patients, as previously.22 The sequences obtained were compared over 334 bp with the germline sequences, from His 102 in the JH4 gene to 298 bp in the JH4-JH5 intron

  • Table 3.

    Characteristics of 10 asplenic patients

    % of B cellsMutation frequency, %
    Patient, ageDiagnosisUltrasound and/or computerized tomographyHowell-Jolly bodiesSevere infectionsIgD+CD27+IgD-CD27+IgD+CD27+IgD-CD27+
    AS1
       4 mo Asplenia syndrome No spleen + None 5 2 ND ND
       14 mo 7 5 3.2 2.5
    AS2, 18 mo Congenital asplenia No spleen + Purpura fulminans 7 4.5 3.2 4
    AS3
       23 mo Functional asplenia Small size§ + Meningitis, purpura fulminans, septicemia (S. pneumoniae) 4 6 2.2 4
       30 mo 8 8.5 ND ND
    AS4, 35 mo Congenital asplenia No spleen + Purpura fulminans 13 17 ND ND
    AS5, 50 mo* Congenital asplenia No spleen + None 3 8 3.7 4.7
    AS6, 5 y Congenital asplenia No spleen + None 9 11 ND ND
    AS7, 18 y Congenital asplenia No spleen + Septic shock with otitis 8 11 ND ND
    AS8, 43 y Congenital asplenia No spleen + Septicemia (S. pneumoniae) 5 14 ND ND
    AS9, 52 y Congenital asplenia No spleen + None 3 5 ND ND
    AS10, 71 y Congenital asplenia No spleen + None 11 3 ND ND
    • ND indicates not determined.

    • * One asplenic brother died from invasive infection (Streptococcus spp)

    • One asplenic brother died from invasive infection (S. pneumoniae)

    • Or ivemark syndrome (visceral heterotaxia: right atrial isomerism, cardiac malformation and asplenia)

    • § Not visualized by 99mTc sulfur colloid scintigraphy

    • Confirmed by 99mTc scintigraphy

    • Germ not identified