Antigen receptor engagement turns off the V(D)J recombination machinery in human tonsil B cells

Abstract

The germinal center (GC) is an anatomic compartment found in peripheral lymphoid organs, wherein B cells undergo clonal expansion, somatic mutation, switch recombination, and reactivate immunoglobulin gene V(D)J recombination. As a result of somatic mutation, some GC B cells develop higher affinity antibodies, whereas others suffer mutations that decrease affinity, and still others may become self-reactive. It has been proposed that secondary V(D)J rearrangements in GCs might rescue B cells whose receptors are damaged by somatic mutations. Here we present evidence that mature human tonsil B cells coexpress conventional light chains and recombination associated genes, and that they extinguish recombination activating gene and terminal deoxynucleotidyl transferase expression when their receptors are cross-linked. Thus, the response of the recombinase to receptor engagement in peripheral B cells is the opposite of the response in developing B cells to the same stimulus. These observations suggest that receptor revision is a mechanism for receptor diversification that is turned off when antigen receptors are cross-linked by the cognate antigen.

Figure 1

Ligation-mediated (LM)-PCR assay for detecting signal breaks in human GC B cells. (a) Scheme for LM-PCR strategy to detect RSS DNA breaks within the human Ig κ light chain locus. A primary V_κJ_κ1 rearrangement is represented at the κ locus, and after V(D)J recombinase activation, downstream J_κ segments may be chosen for a secondary rearrangement; J_κ5 is depicted. Blunt signal ends at the nonamer–heptamer sequences (triangles) are ligated to BW linkers. Linker-ligated DNA molecules are then amplified by PCR using two sets of specific primers (see Materials and Methods). (b) J_κ signal breaks in human GC B cells. LM-PCR for J_H6 (top) and J_κ5 (middle) signal breaks in human mononuclear bone marrow (BM) cells, naive CD38⁻IgD⁺ FM tonsil B cells, and CD38⁺IgD⁻ GC B cells (left) and in centroblasts CD38⁺CD77⁺ and centrocytes CD38⁺CD77⁻ (right). PCR products were visualized with locus-specific probes. PCR from a region overlapping the germline J_κ5 segment was used as a control for DNA loading (bottom).

Figure 1

Ligation-mediated (LM)-PCR assay for detecting signal breaks in human GC B cells. (a) Scheme for LM-PCR strategy to detect RSS DNA breaks within the human Ig κ light chain locus. A primary V_κJ_κ1 rearrangement is represented at the κ locus, and after V(D)J recombinase activation, downstream J_κ segments may be chosen for a secondary rearrangement; J_κ5 is depicted. Blunt signal ends at the nonamer–heptamer sequences (triangles) are ligated to BW linkers. Linker-ligated DNA molecules are then amplified by PCR using two sets of specific primers (see Materials and Methods). (b) J_κ signal breaks in human GC B cells. LM-PCR for J_H6 (top) and J_κ5 (middle) signal breaks in human mononuclear bone marrow (BM) cells, naive CD38⁻IgD⁺ FM tonsil B cells, and CD38⁺IgD⁻ GC B cells (left) and in centroblasts CD38⁺CD77⁺ and centrocytes CD38⁺CD77⁻ (right). PCR products were visualized with locus-specific probes. PCR from a region overlapping the germline J_κ5 segment was used as a control for DNA loading (bottom).

Figure 2

RAG, TdT, λ-like, and V-preB gene expression in human tonsil B cell subsets. RNA from FACS^®-sorted CD38⁻IgD⁺ FM B lymphocytes, CD38⁻CD77⁻ FM and memory B cells, CD38⁺CD77⁺ GC centroblasts, CD38⁺CD77⁻ GC centrocytes, CD38⁺IgD⁻ total GC B cells, and mononuclear bone marrow (BM) cells was analyzed by semiquantitative RT-PCR and visualized with labeled oligonucleotide probes. Igβ RT-PCR was used as a B cell–specific mRNA loading control. PCR assays were designed to distinguish between genomic DNA and mRNA using primers on different exons. PhosphorImager analysis showed that there is a fivefold difference in the amount of RAG1 mRNA amplified between centroblasts and centrocytes.

Figure 3

ΨL cell surface expression on GC B cells. (a) Flow cytometric analysis were performed on human tonsil B cells using anti-CD38, anti–V-preB, anti-Igκ, or anti-Igλ. Two different patient samples are shown. (b) Flow cytometric analysis of immunized wild-type and λ5-targeted (λ5^−/−) mouse spleen cells using anti-B220, anti-GL7, anti-κ, and anti-λ, and either LM34 or SL156 anti-λ5 antibodies (42).

Figure 3

ΨL cell surface expression on GC B cells. (a) Flow cytometric analysis were performed on human tonsil B cells using anti-CD38, anti–V-preB, anti-Igκ, or anti-Igλ. Two different patient samples are shown. (b) Flow cytometric analysis of immunized wild-type and λ5-targeted (λ5^−/−) mouse spleen cells using anti-B220, anti-GL7, anti-κ, and anti-λ, and either LM34 or SL156 anti-λ5 antibodies (42).

Figure 4

ΨL expression in human tonsil B cells. Immunohistochemical staining of human tonsil sections was performed using anti–V-preB (left), anti-CD21 (middle), or both (right). GCs, FM, and the T cell zones (T zone) are indicated.

Figure 5

Regulated expression of RAG, TdT, λ-like, and V-preB genes. (a) Human tonsil CD38⁻IgD⁺ FM or CD38⁺IgD⁻ GC B cells were sorted (d0) and cultured on CD40L-transfected fibroblasts with or without anti-κ+λ (α Ig) at 10 μg/ml, or IL-2, IL-4, or IL-10 for 3 d (d3). Gene expression was analyzed by RT-PCR as described above. (b) CD38⁺IgD⁻ GC B cells were cultured with CD40L alone or with CD40L and an irrelevant IgG control (Control Ig), or with intact anti-κ (Ig α–κ) or anti-λ (Ig α–λ) or both (Ig α–κ + α–λ) or with Fab′2 anti-κ or anti-λ (Fab′2 α–κ + α–λ). Antibody concentrations are indicated in micrograms per milliliter. (c) GC cells reanalyzed by flow cytometry using anti-CD38 and anti–V-preB mAbs after 3 d of culture with CD40L and the indicated antibodies. GC B cells cultured with Ig α–κ + α–λ or Fab′2 α–κ + α–λ antibodies contained 1.5–2-fold more B cells than those with cultured with control antibody, and the percentage of dead cells was 40–50% in all of cultures as determined by trypan blue exclusion.

Figure 5

Regulated expression of RAG, TdT, λ-like, and V-preB genes. (a) Human tonsil CD38⁻IgD⁺ FM or CD38⁺IgD⁻ GC B cells were sorted (d0) and cultured on CD40L-transfected fibroblasts with or without anti-κ+λ (α Ig) at 10 μg/ml, or IL-2, IL-4, or IL-10 for 3 d (d3). Gene expression was analyzed by RT-PCR as described above. (b) CD38⁺IgD⁻ GC B cells were cultured with CD40L alone or with CD40L and an irrelevant IgG control (Control Ig), or with intact anti-κ (Ig α–κ) or anti-λ (Ig α–λ) or both (Ig α–κ + α–λ) or with Fab′2 anti-κ or anti-λ (Fab′2 α–κ + α–λ). Antibody concentrations are indicated in micrograms per milliliter. (c) GC cells reanalyzed by flow cytometry using anti-CD38 and anti–V-preB mAbs after 3 d of culture with CD40L and the indicated antibodies. GC B cells cultured with Ig α–κ + α–λ or Fab′2 α–κ + α–λ antibodies contained 1.5–2-fold more B cells than those with cultured with control antibody, and the percentage of dead cells was 40–50% in all of cultures as determined by trypan blue exclusion.

Figure 5

Regulated expression of RAG, TdT, λ-like, and V-preB genes. (a) Human tonsil CD38⁻IgD⁺ FM or CD38⁺IgD⁻ GC B cells were sorted (d0) and cultured on CD40L-transfected fibroblasts with or without anti-κ+λ (α Ig) at 10 μg/ml, or IL-2, IL-4, or IL-10 for 3 d (d3). Gene expression was analyzed by RT-PCR as described above. (b) CD38⁺IgD⁻ GC B cells were cultured with CD40L alone or with CD40L and an irrelevant IgG control (Control Ig), or with intact anti-κ (Ig α–κ) or anti-λ (Ig α–λ) or both (Ig α–κ + α–λ) or with Fab′2 anti-κ or anti-λ (Fab′2 α–κ + α–λ). Antibody concentrations are indicated in micrograms per milliliter. (c) GC cells reanalyzed by flow cytometry using anti-CD38 and anti–V-preB mAbs after 3 d of culture with CD40L and the indicated antibodies. GC B cells cultured with Ig α–κ + α–λ or Fab′2 α–κ + α–λ antibodies contained 1.5–2-fold more B cells than those with cultured with control antibody, and the percentage of dead cells was 40–50% in all of cultures as determined by trypan blue exclusion.