Microbial colonization influences early B-lineage development in the gut lamina propria

Abstract

The RAG1/RAG2 endonuclease (RAG) initiates the V(D)J recombination reaction that assembles immunoglobulin heavy (IgH) and light (IgL) chain variable region exons from germline gene segments to generate primary antibody repertoires. IgH V(D)J assembly occurs in progenitor (pro-) B cells followed by that of IgL in precursor (pre-) B cells. Expression of IgH μ and IgL (Igκ or Igλ) chains generates IgM, which is expressed on immature B cells as the B-cell antigen-binding receptor (BCR). Rag expression can continue in immature B cells, allowing continued Igκ V(D)J recombination that replaces the initial VκJκ exon with one that generates a new specificity. This 'receptor editing' process, which can also lead to Igλ V(D)J recombination and expression, provides a mechanism whereby antigen encounter at the Rag-expressing immature B-cell stage helps shape pre-immune BCR repertoires. As the major site of postnatal B-cell development, the bone marrow is the principal location of primary immunoglobulin repertoire diversification in mice. Here we report that early B-cell development also occurs within the mouse intestinal lamina propria (LP), where the associated V(D)J recombination/receptor editing processes modulate primary LP immunoglobulin repertoires. At weanling age in normally housed mice, the LP contains a population of Rag-expressing B-lineage cells that harbour intermediates indicative of ongoing V(D)J recombination and which contain cells with pro-B, pre-B and editing phenotypes. Consistent with LP-specific receptor editing, Rag-expressing LP B-lineage cells have similar VH repertoires, but significantly different Vκ repertoires, compared to those of Rag2-expressing bone marrow counterparts. Moreover, colonization of germ-free mice leads to an increased ratio of Igλ-expressing versus Igκ-expressing B cells specifically in the LP. We conclude that B-cell development occurs in the intestinal mucosa, where it is regulated by extracellular signals from commensal microbes that influence gut immunoglobulin repertoires.

Figure 1. Gut LP RAG2⁺ B Lineage Cells in Weanling Age Mice

a, FACS plots of CD19⁺ cells from the indicated tissues taken from wild type (WT) (top) or homozygous Rag2-Gfp knock-in (bottom) mice. B220 expression is plotted against GFP fluorescence. Numbers denote percentage of CD19⁺ B220^low RAG2-GFP⁺ cells. b–d, dot plots showing percentage of RAG2-GFP⁺ cells in listed tissues from indicated post-natal ages. Each point represents one mouse. Horizontal bars indicate mean values ± s.e.m. e, Immunohistochemistry of paraffin-embedded sections from bone marrow (BM) and small intestine (SI) stained with an anti-TdT antibody. Dark brown indicates TdT-reactivity.

Figure 2. RAG2-GFP⁺ LP B lineage developmental subsets

a, Plots show the relative percentage Rag2-expressing pro-B cells, pre-B cells, editing B cells and surface IgM⁺ B cells (see text for definition of each) in the bone marrow (blue bars) and lamina propria (red bars). Plotted are mean values ± s.e.m. and each are derived from experiments of 4 independent mice at post-natal day 17–24 (see Supplementary Fig. 7 for more details). b, Plots show quantitative ligation-mediated PCR of RAG2⁺ BM B cells and RAG2⁺ LP B cells normalized to genomic DNA. BM cells from RAG2^−/− mice were a negative control. Values on the y-axis are units relative to the signal obtained from a RAG2⁺ BM B cell samples.

Figure 3. Distinct Vκ segment usage in RAG2⁺ cells from BM versus LP

a,b, Dot plots show contributions (in order of highest to lowest BM utilization) of different V_Hs (a) and Vκs (b) to in-frame rearrangements in RAG2-GFP⁺ BM (black dots) and LP (red dots) cells. The two most highly utilized V_Hs are omitted to increase plot resolution. Each point shows mean ± s.e.m. of at least 4 experiments. The χ² calculated P values for overall differences between BM and LP are indicated. Significant V segment utilization differences between BM and LP are indicated on heat map (P values scale indicated in inset). Full data set at increased resolution is in Supplementary Fig. 12.

Figure 4. Effects of Gut colonization on Development of LP B Lineage Cells

a, Plots of percentage of pro-B cells versus total CD19⁺ B lineage cells from bone marrow (BM), spleen (SpL) and lamina propria (LP) of 4 wk-old germfree (GF) mice and littermates colonized (Col) by co-housing with serum pathogen free mice for 7 days. b, Bar graphs show ratios of Igλ⁺ versus Igκ⁺ B cells within mesenteric lymph nodes (mLN), inter-epithelial lymphocyes (IEL) and tissues indicated in (a) from GF and Col mice. Mean values and s.e.m are shown. The P values are indicated as: *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. ns = not significant. (Details in Supplementary Fig. 16 and 17)