Ongoing in vivo immunoglobulin class switch DNA recombination in chronic lymphocytic leukemia B cells

Abstract

Chronic lymphocytic leukemia (CLL) results from the expansion of malignant CD5(+) B cells that usually express IgD and IgM. These leukemic cells can give rise in vivo to clonally related IgG(+) or IgA(+) elements. The requirements and modalities of this process remain elusive. Here we show that leukemic B cells from 14 of 20 CLLs contain the hallmarks of ongoing Ig class switch DNA recombination (CSR), including extrachromosomal switch circular DNAs and circle transcripts generated by direct S micro -->Sgamma, S micro -->Salpha, and S micro -->Sepsilon as well as sequential Sgamma-->Salpha and Sgamma-->Sepsilon CSR. Similar CLL B cells express transcripts for activation-induced cytidine deaminase, a critical component of the CSR machinery, and contain germline I(H)-C(H) and mature V(H)DJ(H)-C(H) transcripts encoded by multiple Cgamma, Calpha, and Cepsilon genes. Ongoing CSR occurs in only a fraction of the CLL clone, as only small proportions of CD5(+)CD19(+) cells express surface IgG or IgA and lack IgM and IgD. In vivo class-switching CLL B cells down-regulate switch circles and circle transcripts in vitro unless exposed to exogenous CD40 ligand and IL-4. In addition, CLL B cells that do not class switch in vivo activate the CSR machinery and secrete IgG, IgA, or IgE upon in vitro exposure to CD40 ligand and IL-4. These findings indicate that in CLL at least some members of the malignant clone actively differentiate in vivo along a pathway that induces CSR. They also suggest that this process is elicited by external stimuli, including CD40 ligand and IL-4, provided by bystander immune cells.

FIGURE 1

Generation and composition of SCs and CTs. The human IgH chain locus and the molecular events involved in switching from Cμ to Cε are shown schematically. Ovals indicate S regions; rectangles before and after the S regions are I_H exons and C_H gene exons, respectively; iEμ is the IgH chain intronic enhancer; triangles are the 3′ IgH chain enhancers; V-shaped lines indicate splicing; arrowheads indicate the positions and directions of the primers used to amplify SCs, CTs, germline transcripts, and mature transcripts.

FIGURE 2

Normal GC B cells, but not naive and memory B cells, contain SCs and express CTs and AID. Sγ-Sμ, Sα-Sμ, Sε-Sμ, Sα-Sγ, and Sε-Sμ SCs (all ranging from 500–4000 bp; A) as well as Iγ-Cμ (557 bp), Iα-Cμ (666 bp), Iε-Cμ (408 bp), Iα-Cγ (604 bp), and Iε-Cγ (346 bp) CTs (B), and AID (382 bp; B) were amplified from total peripheral blood (PB) CD19⁺ B cells from four healthy subjects, and from normal tonsillar IgD⁺CD38⁻ naive (N), IgD⁺CD38⁺ founder GC (FGC), IgD⁻CD38⁺ GC (GC), and IgD⁻CD38⁻ memory (M) B cells. Sγ-Sμ, Sα-Sμ, and Sε-Sμ were hybridized with an Sγ probe; Sγ-Sμ and Sα-Sμ were hybridized with an Sγ probe; Iγ-Cμ, Iα-Cμ, and Iε-Cμ were hybridized with a Cμ probe; Iα-Cγ and Iε-Cγ were hybridized with a Cγ probe. β-Actin (593 bp) was amplified to control the loading of genomic DNA or cDNA. The results depicted are from one of three experiments yielding similar results.

FIGURE 3

Malignant CLL B cells contain SCs and express CTs and AID. Sγ-Sμ, Sα-Sμ, Sε-Sμ, Sα-Sγ, and Sε-Sμ SCs (A); and Iγ-Cμ, Iα-Cμ, Iε-Cμ, Iα-Cγ, and Iε-Cγ CTs and AID (B) were PCR amplified from peripheral blood malignant B cells from 12 CLLs. Two Iγ-Cμ and Iα-Cμ bands represent alternatively spliced transcripts. β-Actin genomic DNA was PCR amplified to control DNA loading. Sγ-Sμ, Sα-Sμ, and Sε-Sμ were hybridized with an Sμ probe; Sα-Sγ and Sε-Sγ were hybridized with an Sγ probe; Iγ-Cμ, Iα-Cμ, and Iε-Cμ were hybridized with a Cμ probe; Iα-Cγ and Iε-Cγ were hybridized with a Cγ probe.

FIGURE 4

Malignant CLL B cells down-regulate SCs and CTs after transferring in vitro. Total Sγ-Sμ SCs and Iγ-Cμ were PCR amplified from purified CLL E-137 B cells incubated in medium alone (control) or in medium containing htCD40L and IL-4 for 1, 2, 3, or 4 days. β-Actin was PCR amplified to control DNA loading. Data shown are from one representative experiment of three performed yielding similar results.

FIGURE 5

Actively class-switching malignant CLL B cells comprise clonally related, but phenotypically distinct, subsets. A, CD19, CD5, IgM, IgD, IgG, IgA, CD38, CD77, CD44, CD71, and CD138 (syndecan-1) on purified CLL E-123 B cells. B, SSCP of 48 V_HDJ_H transcripts from purified CLL E-123 B cells. The electrophoretic migration profiles of dsDNA and ssDNA are indicated. C, Nucleotide V_H sequences of V_HDJ_H-Cμ, -Cδ, -Cγ, and -Cα cDNAs from purified CLL E-123 B cells. The top sequence corresponds to the germline V_H3 gene DP77/VHG16 to which leukemic V_H sequences were compared. Dashes indicate identities. Solid lines depict complementary-determining regions (CDRs).

FIGURE 6

The transcriptional status of the Ig H chain locus in actively class-switching malignant CLL B cells is similar to that of nonmalignant GC B cells. Iγ1-Cγ1 (603 bp), Iγ2-Cγ2 (597 bp), Iγ3-Cγ3 (670 bp), Iγ4-Cγ4 (411 bp), Iα1-Cα1 (1194 bp), Iα2-Cα2 (1181 bp), Iε-Cε (409 bp), V_HDJ_H-Cμ (~150 bp), V_HDJ_H-Cδ (~350 bp), V_HDJ_H-Cγ 1 (~415 bp), V_HDJ_H-Cγ2 (~415 bp), V_HDJ_H-Cγ 3 (~415 bp), V_HDJ_H-Cγ4 (~415 bp), V_HDJ_H-Cα1 (~900 bp), V_HDJ_H-Cα2 (~890 bp), and V_HDJ_H-Cε (~380 bp) were PCR amplified from normal IgD⁺CD38⁻ naive (A), IgD⁺CD38⁺ founder GC (B), IgD⁻CD38⁺ GC (C), and IgD⁻CD38⁻ memory (D) tonsillar B cells. Similar cDNAs were also amplified from actively class-switching CLL E-137 (E) and nonactively class-switching CLL E-69 (F) B cells. Data shown are from a representative experiment of four performed yielding similar results.

FIGURE 7

Malignant CLL B cells that are not actively class switching in vivo undergo CSR upon exposure to CD40L and IL-4 in vitro. A, CLL E-69 B cells were incubated with medium only or with htCD40L and/or IL-4 for two hours. Left panels, Total proteins were pulled down with GST-CD40IC and immunoblotted for TRAF-2 and TRAF-6; the IKKα activity was assessed using IκBα-GST as substrate. Right panels, Binding of nuclear proteins to radiolabeled oligonucleotides encompassing the NF-κB and STAT-6 binding sites within the human Cγ3 promoter. B–D, Germline and mature transcripts, AID, β-actin, and SCs before and after CLL E-69 B cell incubation with medium only (control) or htCD40L and IL-4 for 4 days. AID and β-actin cDNAs were diluted with water (1/1, 1/2, and 1/4) before amplification. E, IgM, IgG, IgA, and IgE secretion after CLL E-69 B cell incubation with medium only (control), htCD40L and IL-4, or htCD40L, IL-4, and IL-10 for 7 days. Data shown are from a representative experiment of four performed yielding similar results.