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

Abstract

The human peripheral B-cell compartment displays a large population of immunoglobulin M-positive, immunoglobulin D-positive CD27(+) (IgM(+)IgD(+)CD27(+)) "memory" B cells carrying a mutated immunoglobulin receptor. By means of phenotypic analysis, complementarity-determining region 3 (CDR3) spectratyping during a T-independent response, and gene-expression profiling of the different blood and splenic B-cell subsets, we show here that blood IgM(+)IgD(+)CD27(+) cells correspond to circulating splenic marginal zone B cells. Furthermore, analysis of this peripheral subset in healthy children younger than 2 years shows that these B cells develop and mutate their immunoglobulin receptor during ontogeny, prior to their differentiation into T-independent antigen-responsive cells. It is therefore proposed that these IgM(+)IgD(+)CD27(+) B cells provide the splenic marginal zone with a diversified and protective preimmune repertoire in charge of the responses against encapsulated bacteria.

Figure 1. Blood and spleen IgM⁺IgD⁺CD27⁺ subsets share markers specific of marginal zone B cells

Purified B cells from blood and spleen are analyzed separately for IgM, CD21, CD23 and CDlc surface expression after gating of the three different CD19-positive lymphocyte subsets distinguished by IgD and CD27 labelling. These data correspond to one representative case out of 4 different individuals. Naive B cells (IgD⁺CD27⁻), bold line; IgD⁺CD27⁺ B cells, grey shadow; IgD⁻CD27⁺, thin line. Percentages of cells in the two CD27⁺ quadrants are indicated. The absence of IgM-positive cells among the IgD⁻CD27⁺ subset is noticeable.

Figure 2. CDlc marks strongly splenic marginal zone B cells in humans

Serial cryosections of an adult human spleen are stained with anti-CD20, anti-IgD, anti-CD 1c and anti-CD27 antibodies (ABC technique; Original magnification: 25X) Marginal zone B cells are IgD^low CD27⁺CDlc^high. Note the more intense staining for IgD of the corona (Co) compared to the marginal zone B cells (MZ) while the reverse is true for CDlc. The intense IgD staining of outer marginal zone B cells has been described previousl(16). GC, germinal center.

Figure 3. Presence of an IgM⁺IgD⁺CD27⁺ subset in hyper-IgM patients

Patients L.D. and C.A. (patient one) have been reported previously (20,21). Control is a eleven-year-old child, age-matched with patient C.A. Since IgD⁺CD27⁺cells co-express 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-positive cells. Percentages of cells in the naive and the two 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 two separate donors, with one of the two 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<0.005, two-fold higher expression) the splenic IgD⁺CD27⁺ samples from the splenic IgD⁻CD27⁺ samples. The red branches indicate the co-clustering of the blood IgD⁺CD27⁺ samples with the splenic IgD⁺CD27⁺ samples and the blue branches indicate the co-clustering of the blood IgD⁻CD27⁺ samples with their respective splenic IgD⁻CD27⁺ samples, b) The 37 genes that achieved statistical significance with a two-fold higher mean expression when comparing the IgD⁺CD27⁺ cell populations with the memory IgD⁻CD27⁺ cell populations.

Figure 5. IgM⁺IgD⁺CD27⁺ B cells from blood and spleen share B cell clones with an identical V3-15 CDR3 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 Streptococcus pneumoniae and Neisseria meningitidis (plain polysaccharidic vaccines). The following samples were analyzed: blood before immunization, blood at the time of splenectomy (i.e. 8 days after immunization), spleen, blood 5 weeks after immunization. The first V3-15-specific PCR products were further amplified with V3-15-specific FR3 and C7μ 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 determined after cloning. Several PCR amplifications were performed from two independent cDNAs for each cell sample. The recurrent CDR3s encompassing the V3-15 and J_H3 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 J_H segments). The asterisk (*) marks clones found repeatedly in independent PCR amplifications.

Figure 6. Development and diversification of IgM⁺IgD⁺CD27⁺ peripheral B cells from normal and asplenic children

A, C. Percentage of IgM⁺IgD⁺CD27⁺ peripheral B cells from normal (A) and asplenic children (C) below five years. B, D. Mutation frequency of rearranged V3-23 genes from IgM⁺IgD⁺CD27⁺ peripheral B cells of normal (B) and asplenic children (D). Each bar represents the mutation frequency of one individual and the values marked above represent the mutation range over the 288 bp V3-23 sequence analyzed. Normal adult values are pooled from five individuals. E. IgD/CD27 staining profiles of peripheral CD19⁺ lymphocytes of asplenic individuals from 14 months to 71 years. Percentages of cells in each CD27⁺ quadrants are indicated. A complete description of asplenic patients is given in Supplementary Table.