Surface mu heavy chain signals down-regulation of the V(D)J-recombinase machinery in the absence of surrogate light chain components

Abstract

Early B cell development is characterized by stepwise, ordered rearrangement of the immunoglobulin (Ig) heavy (HC) and light (LC) chain genes. Only one of the two alleles of these genes is used to produce a receptor, a phenomenon referred to as allelic exclusion. It has been suggested that pre-B cell receptor (pre-BCR) signals are responsible for down-regulation of the VDJH-recombinase machinery (Rag1, Rag2, and terminal deoxynucleotidyl transferase [TdT]), thereby preventing further rearrangement on the second HC allele. Using a mouse model, we show that expression of an inducible muHC transgene in Rag2-/- pro-B cells induces down-regulation of the following: (a) TdT protein, (b) a transgenic green fluorescent protein reporter reflecting endogenous Rag2 expression, and (c) Rag1 primary transcripts. Similar effects were also observed in the absence of surrogate LC (SLC) components, but not in the absence of the signaling subunit Ig-alpha. Furthermore, in wild-type mice and in mice lacking either lambda5, VpreB1/2, or the entire SLC, the TdT protein is down-regulated in muHC+LC- pre-B cells. Surprisingly, muHC without LC is expressed on the surface of pro-/pre-B cells from lambda5-/-, VpreB1-/-VpreB2-/-, and SLC-/- mice. Thus, SLC or LC is not required for muHC cell surface expression and signaling in these cells. Therefore, these findings offer an explanation for the occurrence of HC allelic exclusion in mice lacking SLC components.

Figure 1.

TdT is down-regulated in pre–B cells after de novo synthesis of transgenic μHC in the absence of λ5 or VpreB1/2. (A) CD19⁺ BM cells were isolated by MACS from tet-μHC, tet-μHC λ5^−/−, and tet-μHC VpreB1^−/−VpreB2^−/− mice that had received Tet in the drinking water for 7 d and cultured on stromal cells in medium containing IL-7 in the presence (top) or absence (bottom) of Tet. After 24 h, cells were fixed, permeabilized, and stained for cytoplasmatic μHC expression in combination with TdT, Ku70, or βGal (isotype control). Fluorescence was determined by flow cytometry. Numbers within the gates represent mean fluorescence intensities for βGal, TdT, and Ku70, respectively. (B) CD19⁺ BM cells were isolated by MACS from tet-μHC λ5^−/− Ig-α^−/− mice that had received Tet in the drinking water for 7 d and cultured on stromal cells in medium containing IL-7 in the absence of Tet. After 48 h, cells were fixed, permeabilized, and stained for cytoplasmatic μHC expression in combination with TdT or βGal (isotype control). Numbers within the gates represent mean fluorescence intensities for βGal and TdT, respectively.

Figure 2.

TdT is down-regulated in μHC-expressing BM pre–B cells in the absence of SLC components and conventional LC. CD19⁺ BM cells isolated from C57BL/6, VpreB1/2^−/−, or VpreB1/2^−/− λ5^−/− (SLC^−/−) mice were stained for surface c-kit or CD25, followed by fixation and permeabilization to detect intracellular κ/λLC and μHC in combination with TdT or βGal (isotype control). Cells positive for conventional LC were excluded from the analysis. (A) Cells positive for c-kit (i.e., pro–B cells) and negative (G1) or positive (G2) for cytoplasmic μHC were analyzed separately for TdT expression (unshaded). Staining for βGal was used as isotype control (shaded). (B) Cells positive for surface CD25 and cytoplasmic μHC (i.e., pre–B cells) were analyzed as in A. Numbers in the dot plots indicate the percentage of cells within the gates. Numbers in the histograms represent mean fluorescence intensities for TdT or βGal (shaded). The purity of CD19⁺ enriched cells was 96 (C57BL/6), 91 (VpreB1/2^−/−), and 89% (SLC^−/−) in this experiment. Representative results of two FACS^® analyses are shown.

Figure 3.

Expression of μHC in pro–B cells mediates down-regulation of a transgenic GFP reporter reflecting endogenous Rag2 expression in the absence of λ5. GFP⁺ BM cells were isolated from tet-μHC and tet-μHC λ5^−/− mice carrying a bacterial artificial chromosome that encodes a green fluorescent protein (GFP) reporter instead of Rag2 (NG-BAC) and cultured on stromal cells in medium containing IL-7 without Tet. At 24, 48, and 72 h, cells were fixed and stained for cytoplasmic μHC expression. Fluorescence intensities for μHC versus GFP were analyzed by FACS^®. Numbers printed within the gates indicate mean fluorescence intensity for GFP.

Figure 4.

Induction of μHC in pro–B cells induces down-regulation of Rag1 primary transcripts in the absence of λ5. CD19⁺ BM cells from tet-μHC and tet-μHC λ5^−/− mice were isolated by MACS and cultured on stromal cells with or without Tet for 12 h. Cells were fixed onto slides and subjected to RNA FISH to detect primary transcripts on individual alleles in single nuclei using ssDNA probes specific for Rag1 and CD45 (control). Hybridized probes were detected and visualized by fluorochrome-labeled Abs, and cells displaying signals were counted (top, >150 nuclei per slide). Induction of cytoplasmic μHC expression was verified by intracellular FACS^® (middle, unshaded). Nuclei were counterstained with 4,6-diamino-2-phenylindole. Microscopical images (bottom) show representative nuclei hybridized for Rag1 or CD45, respectively. The experiment was repeated with comparable results.

Figure 5.

μH chain is detectable on the surface of λ5^−/−, VpreB1^−/−VpreB2^−/−, and SLC^−/− pre–B cells. (A) CD19⁺ BM cells were isolated by MACS from tet-μHC, tet-μHC λ5^−/−, and tet-μHC VpreB1^−/−VpreB2^−/− mice that had received Tet in drinking water for 7 d. Cells were expanded on stromal cells in medium containing IL-7 and Tet for 48 h, washed, and recultured for an additional 18 h in the presence of IL-7 with (shaded) or without Tet (unshaded). Cells were harvested and stained with biotinylated Abs against μH chain (goat polyclonal), pre-BCR (SL156), or SLC components (LM34, VP245, and R5). Surface-bound biotin was amplified and revealed by EAS and streptavidin-PE. Propidium iodide negative cells were analyzed by flow cytometry. (B) CD19⁺ BM cells were isolated by MACS from SLC^−/− or Rag1^−/− (control) mice and expanded on stromal cells in medium containing IL-7 for 4 d. Cells were harvested and stained with FITC-labeled Abs against κ/λLC and biotinylated Abs against μHC (goat polyclonal) or λ5 (LM34). Surface-bound biotin was amplified and revealed aforementioned. Numbers represent the percentage of cells within the quadrants.