Somatic diversification in the absence of antigen-driven responses is the hallmark of the IgM+ IgD+ CD27+ B cell repertoire in infants

Abstract

T cell-dependent immune responses develop soon after birth, whereas it takes 2 yr for humans to develop T cell-independent responses. We used this dissociation to analyze the repertoire diversification of IgM(+)IgD(+)CD27(+) B cells (also known as "IgM memory" B cells), comparing these cells with switched B cells in children <2 yr of age, with the aim of determining whether these two subsets are developmentally related. We show that the repertoire of IgM(+)IgD(+)CD27(+) B cells in the spleen and blood displays no sign of antigen-driven activation and expansion on H-CDR3 spectratyping, despite the many antigenic challenges provided by childhood vaccinations. This repertoire differed markedly from those of switched B cells and splenic germinal center B cells, even at the early stage of differentiation associated with mu heavy chain expression. These data provide evidence for the developmental diversification of IgM(+)IgD(+)CD27(+) B cells, at least in very young children, outside of T cell-dependent and -independent immune responses.

Figure 1.

H-CDR3 spectratypes of the V_H6 or V_H3.15 transcripts expressed by the blood B cell subsets of two 11-mo-old children. Naive IgD⁺CD27⁻, IgD⁺CD27⁺, and switched IgD⁻CD27⁺ B cells were sorted from the blood samples of donors 1 and 2. Total RNA from each cell fraction was reverse transcribed and V_H6 or V_H3.15 μ or γ transcripts were amplified by PCR, using a seminested strategy (see Materials and methods). The PCR products were labeled by a run-off reaction with specific fluorescent V_H-FR3 primers, and subjected to electrophoresis on an automated sequencer. The resulting size distribution of the peaks directly reflects the size distribution of H-CDR3 for the given transcripts. Peaks identified by an asterisk were further sequenced to evaluate intrapeak clonal diversity.

Figure 2.

H-CDR3 spectratypes of the V_H3.15 and V_H6 transcripts expressed by the splenic B cell subsets of an 8-mo-old child. (A) Two different marker combinations were used to isolate splenic GC (CD19⁺CD24⁻CD38⁺) and transitional B cells (CD19⁺CD38⁺⁺CD24⁺⁺) or naive IgD⁺CD27⁺, IgD⁺CD27⁺ and switched IgD⁻CD27⁺ B cells. (B) Spectratyping was performed on V_H3.15 and V_H6 μ and/or γ transcripts from each cell fraction, as described in the legend of Fig. 1. Peaks identified by an asterisk were further sequenced to evaluate intrapeak clonal diversity.

Figure 3.

Detectable AID expression in splenic IgD⁺CD27⁺ cells from very young children. Relative AID and Bcl-6 expression levels were determined for various splenic B cell subsets from different donors. AID and Bcl-6 sequences were amplified by real-time quantitative PCR from cDNA from IgD⁺CD27⁺, IgD⁺CD27⁻ (naive), and IgD⁻CD27⁺ cells purified by two consecutive cell sortings. The results shown were obtained from two independent PCRs (except for D6), each performed in triplicate. The relative expression of AID or Bcl-6 in each subset was calculated by the comparative method, normalizing to 1 the expression of AID in the IgD⁺CD27⁺ fraction and of Bcl-6 in the naive subset of the 8-mo-old donor (D3). PCRs with a threshold cycle (Ct) >35 were considered NS.

Figure 4.

IgD⁺CD27⁺CD1c^high cells are already present in the SMZ of an 8-mo-old child. Serial splenic cryosections were double-labeled either with anti-IgD (green) and anti-CD27 (red) antibodies (A–D) or with anti-IgD (green) and anti-CD1c (red) antibodies (E–H), and then examined under a confocal microscope. CD27^low cells (B and D) were present in the MZ corresponding to the outer zone of the IgD-positive ring surrounding the GC (A and C). Boxes with dotted lines in A and B indicate the zone magnified in C and D. A higher level of CD1c expression was observed in the MZ (F), resulting in a yellow appearance in the merged images (G and H), caused by the coexpression of IgD and CD1c at similar intensities. H shows higher magnification of the zone delimited by the box with dotted lines in G. Co, corona. Bars, 50 μm.