T cell-independent development and induction of somatic hypermutation in human IgM+ IgD+ CD27+ B cells

Abstract

IgM(+)IgD(+)CD27(+) B cells from peripheral blood have been described as circulating marginal zone B cells. It is still unknown when and where these cells develop. These IgM(+)IgD(+)CD27(+) B cells exhibit somatic hypermutations (SHMs) in their B cell receptors, but the exact nature of the signals leading to induction of these SHMs remains elusive. Here, we show that IgM(+)IgD(+)CD27(+) B cells carrying SHMs are observed during human fetal development. To examine the role of T cells in human IgM(+)IgD(+)CD27(+) B cell development we used an in vivo model in which Rag2(-/-)gamma(C)(-/-) mice were repopulated with human hematopoietic stem cells. Using Rag2(-/-)gamma(C)(-/-) mice on a Nude background, we demonstrated that development and induction of SHMs of human IgM(+)IgD(+)CD27(+) B cells can occur in a T cell-independent manner.

Figure 1.

Mature B cells with an MZ B cell phenotype are present in the human fetus. (A) Flow cytometry analysis for CD27 and sIgD of neonatal cord blood (CB), fetal cord blood, liver, MLNs, spleen, and BM. Dot plots presented are gated on CD19⁺ cells. (B) Percentages of CD27⁺ cells within the CD19⁺sIgD⁺ gate (fetal cord blood, n = 16; fetal liver, n = 18; fetal MLNs, n = 25; fetal spleen, n = 47; fetal BM, n = 45). (C) Flow cytometry analysis for sIgM, CD12, and CD1c of the CD19⁺sIgD⁺CD27⁻ cells (thin line) and CD19⁺sIgD⁺CD27⁺ cells (bold line) using gates indicated in A. Dotted lines represent the staining with the matched isotype control. (D) RT-PCR for AID expression in fetal MLNs, liver, spleen, and BM. Adult tonsil was used as a positive control. Fetal liver B cells were enriched for B cells by CD19 MACS isolation because after Ficoll isolation of mononuclear cells, still <1% of the cells were B cells. The other samples had ≥20% CD19⁺ cells and thus were not further purified. Data are from one donor representative of three.

Figure 2.

Human B cells with MZ B cell phenotype are present in HIS (BALB-Rag/γ) mice. (A) Flow cytometry analysis of blood and spleen lymphocytes of the human HSC-reconstituted Rag2^–/– γc^–/– mice. The sIgD-CD27 dot plots were obtained after gating on CD45⁺ and CD19⁺CD3⁻ cells, as indicated by the outlined areas. (B) Expression of sIgM, CD21, and CD1c by CD19⁺sIgD⁺CD27⁻ cells (thin line) and CD19⁺sIgD⁺CD27⁺ cells (bold line) from the spleens of HIS (BALB-Rag/γ) mice. Dotted lines represent staining with the matched isotype control. (C) Percentage of CD27⁺ cells within the CD19⁺sIgD⁺ population in the spleen and the blood of HIS (BALB-Rag/γ) mice. Data were obtained from 12 reconstituted Rag2^–/– γc^–/– mice with human HSC s isolated from five independent donors.

Figure 3.

NOTCH2 is essential for development of CD19⁺sIgD⁺CD27⁻ cells. (A) The relative percentage of CD19⁺sIgD⁺CD27⁺ cells in the spleen of HIS (BALB-Rag/γ) mice reconstituted with human HSCs transduced either with the NOTCH2 shRNA-mediated knockdown construct (GFP⁺ cells) or a control knockdown construct (YFP⁺ cells). (B) Relative percentage of the CD3⁺ cells with the NOTCH2 shRNA-mediated knockdown construct (GFP⁺ cells) compared with the CD3⁺ cells with a control knockdown construct (YFP⁺ cells). Three HIS (BALB-Rag/γ) mice from three different experiments using different donors for HSC transductions were analyzed. The shRNA-mediated NOTCH2 knockdown is indicated as siNOTCH2.

Figure 4.

MZ B cells develop in the absence of T cells. (A) Rag2^−/− γc^−/− mice on Nude background were reconstituted with human HSCs, and flow cytometry analysis was performed 9 wk later to detect T cells (CD4, CD8, and CD3) and B cells (CD19). (B) Flow cytometry analysis of blood and spleen lymphocytes of HIS (BALB-nude-Rag/γ). sIgD-CD27 dot plots were obtained after gating on CD45⁺ and CD19⁺CD3⁻ cells, as indicated by the outlined areas. (C) Expression of sIgM, CD21, and CD1c of CD19⁺sIgD⁺CD27⁻ cells (thin line) and CD19⁺sIgD⁺CD27⁺ cells (bold line) on splenocytes of HIS (BALB-nude-Rag/γ) mice. Dotted lines represent the staining with a matched isotype control. (D) Percentage of CD27⁺ cells within the CD19⁺sIgD⁺ population in the spleen and the blood lymphocytes of HIS (BALB-nude-Rag/γ) mice. In total, nine HIS (BALB-nude-Rag/γ) mice, which were reconstituted with HCS from three different donors, were analyzed.