Early generated B1 B cells with restricted BCRs become chronic lymphocytic leukemia with continued c-Myc and low Bmf expression

Abstract

In mice, generation of autoreactive CD5⁺ B cells occurs as a consequence of BCR signaling induced by (self)-ligand exposure from fetal/neonatal B-1 B cell development. A fraction of these cells self-renew and persist as a minor B1 B cell subset throughout life. Here, we show that transfer of early generated B1 B cells from Eμ-TCL1 transgenic mice resulted in chronic lymphocytic leukemia (CLL) with a biased repertoire, including stereotyped BCRs. Thus, B1 B cells bearing restricted BCRs can become CLL during aging. Increased anti-thymocyte/Thy-1 autoreactive (ATA) BCR cells in the B1 B cell subset by transgenic expression yielded spontaneous ATA B-CLL/lymphoma incidence, enhanced by TCL1 transgenesis. In contrast, ATA B-CLL did not develop from other B cell subsets, even when the identical ATA BCR was expressed on a Thy-1 low/null background. Thus, both a specific BCR and B1 B cell context were important for CLL progression. Neonatal B1 B cells and their CLL progeny in aged mice continued to express moderately up-regulated c-Myc and down-regulated proapoptotic Bmf, unlike most mature B cells in the adult. Thus, there is a genetic predisposition inherent in B-1 development generating restricted BCRs and self-renewal capacity, with both features contributing to potential for progression to CLL.

Figure 1.

T cell–independent CLL in Eμ-TCL1 Tg mice. (A) Increase in CD5⁺B220^lo B cell frequency in PBL of both CD40^+/− and CD40^−/− TC^+/− littermates (C.B17 background) and CD3γδ^−/− TC^+/− mice (C.B17 and C57BL/6 mixed background), compared with WT (C.B17), at 6–7 mo. Mean is indicated by a bar. (right) B220^loCD5⁺ B CLL in a 7-mo-old TC^+/−CD3γδ^−/− mouse (marked) compared with a TC^−/− littermate. (B) Incidence of CLL over time. No strong reduction of CLL incidence occurred in CD40^−/− background.

Figure 2.

CLL development from early generated B-1–derived B cells. (A) Comparison of CLL incidence in C.B17.scid recipients transferred with BM HSC, B1a, and non-B1a B cells purified from 2 mo TC⁺ C.B17 mice. pB1a and sB1a (each 5 × 10⁵/recipient), and non-B1a spleen B cells (sB; 3 × 10⁶/recipient) were cotransferred with TC^– BALB/c BM. 2 mo after transfer, PBL showed similar numbers of B and T cells (top, using TC⁺ HSC alone were all IgM^b, others were predominantly IgM^a). Increase in B220^loCD5⁺ B cells of IgM^b B1a and CLL development are marked. PerC, peritoneal cavity. Representative of six transfer cases using 2–3-mo-old mice. TC⁺ mice together with TC^– mice. (B) Cotransfer of purified B1 B cells from ≤2-mo-old TC⁺C.B17 mice with TC^– BALB/c BM into C.B17.scid mice, monitoring PBL after transfer. (C) Transfer of B1a cells from 10-d-old TC^+/− mice and development of CLL. 10⁶ purified sB1a B cells (5 × 10⁵ TC⁺) from 12 10-d-old (TC^+/− x CB17)F₁ mice were transferred together with 5 × 10⁶ TC^– BM cells into CB.17. mice. (left) Surface IgM expression of sB1a cells. (right) Frequency of B1a cells in PBL of recipient after adoptive transfer (middle). Characteristic mouse CLL CD5⁺B220⁺ phenotype and lymphomatous spleen B cells (thick black line) compared with original transferred B1a cells (thin black line and CD5⁻B220⁺ B cells (gray). Expression of hTCL1 Tg in this CLL is visualized by cytoplasmic staining (gray, second step control). Total leukemia B cell number indicated as a sum of IgM^b+ B cells in PBL (2 × 10⁸), spleen (4.6 × 10⁸), and peritoneal cavity (1.8 × 10⁸), as compared with the size of the initial TC⁺ inoculum. Representative of 15 cases with CLL/lymphoma generation by ≤2-mo-old mice. TC⁺ B1 B cell transfer. (D) H&E staining of spleen section with CLL/lymphoma of day 10 B1a cell origin. (E) Splenomegaly 8 mo after TC⁺ pB1a transferred together with TC^– BM cells, compared with a recipient of TC^– BM alone. (F) Summary of CLL/lymphoma incidence by purified B1 B cell transfer from ≤2-mo-old TC⁺ mice.

Figure 3.

Increased incidence of MBL/CLL/lymphoma in B1 B cells with ATA BCR. (A) Self-Thy-1 antigen-dependent CD5 induction by ATA B cells through B-1 development does not involve CD40 signal. Comparative CD5 level by mature (AA4^–) ATA B cells (ATAid⁺) in PerC in 2-mo-old ATAμκTg mice in Thy-1^−/− versus WT (Thy-1^+/+) and CD40^−/− (n = 5; mean ± SE). (B) Increased ATA B cell frequency in PBL B cells in aging ATAμTg mice. (left) ATA B increased case (marked) as MBL at 12 mo, with further B220 reduction. CD5 reduction also occurred in around half the cases of increased ATA B cells. (C) ATA B-CLL/lymphoma incidence in ATAμTg and ATAμκTg mice with or without TCL1 Tg (left). Incidence over time by ATA μκTg mice (middle). (right) Splenomegaly by TC⁺ATAμκTg mice (>90% cases) together with CLL (top), and representative total spleen cell data analysis (bottom).

Figure 4.

hTCL1 expression by all B cell subsets. (left) Summary of generation of different B cell subsets in spleen through B-2 development from BM; ATA B cell subset generation in ATAμκTg mice by different levels of self-Thy-1 antigen exposure, and AGcA B cell subsets (dominantly autoreactive to mucin 2) in AGcAμκTg mice, expressing different light chain from ATA BCR. (right) 2-mo-old TC⁺ ATAμκTg (with different Thy-1 levels) and AGcAμκTg mice. CD21/CD24 staining of ATA B or AGcA B cells in spleen (both constituting the dominant B cells among total B cells) to show arrested, MZ B, or FO B dominance, and B220/CD5 staining of total (lymphocyte) spleen and PerC. AA4 (CD93) and CD23 staining was also included to confirm each subset. Marked cell subsets for cytoplasmic hTCL1 staining analysis. CD21^hiCD24^med; MZ B, CD21^medCD24^lo; FO B. Thin dotted lines in hTCL1 staining are the data from TC^– μκTg⁺ littermates as controls. Representative data of two to three samples each.

Figure 5.

CLL/lymphoma incidence predominantly arises from B1 B cells. (A) Summary of IgM⁺ B cell tumor (CLL/lymphoma) incidence and frequency of tumor IgM with either original μκTg BCR, endogenous IgM (IgM^b), or IgM^b coexpressed with original μκTg BCR. In ATAμκTg.J_H^– Thy-1^–, also in AGcA μκTg mice, IgL edited B cell tumor occurred together with original V_H3609μ IgH expression (*). (B) Representative case of endogenous IgM (IgM^b) increase as B220^loCD5⁺ B cells in PBL of TC⁺ATAμκTg.Thy-1^– mice (Gr. 4). As shown on right, original B220⁺CD5^– cells at 3 mo in PBL were circulating FO B cells with IgM^a+ ATA BCR, which did not develop B cell tumor. (C) TC⁺ATAμκTg.J_H^–Thy-1^– mouse PBL at the same 14 mo, without or with B cell tumor development. The edited IgL by this tumor B cells (*) was V_k19-23, together with V_H3609μ IgH. (D) Edited IgL in V_H3609μ⁺ B tumors resemble normal mouse V_H3609μ⁺ B1a Igκ. (left) Igκ single-cell sequence data by V_H3609μ⁺ B cells (V_H3609id)⁺kappa⁺ in 2–3-mo-old ATAμTg mouse pB1a. Summary of data from four mice (107 shown of the total 117 κ listed). (right) Igκ list of IgL-edited V_H3609μ⁺ B1a (CD5⁺) tumors. 9/11 of Gr5 and 5/6 of Gr 6 edited Igκ were similar to normal mouse V_H3609μ⁺ B1a, including identical CDR3 use by Vκ21/Jκ2 and Vκ32/Jκ1, the most frequently expressed by V_H3609μ⁺ B1a (CDR3 amino acids are shown).

Figure 6.

ATA B-CLL generation from mature B1 B cells than arrested B cells. (A) CD5 down-regulation occurrance at CLL stage in TC⁺ATAμκTg mice. CD5⁺ and CD5^– CLL (marked) found in two littermates, both with splenomegaly. CD5 down-regulation also occurred in TC^– ATA B cells as shown in Fig. 3 B. (B) 3 d after stimulation of ATA B cells in peritoneal cavity (pB1) and arrested B cells in spleen of ATAμκTg mice. Cell proliferation by LPS and CpG with CD5 down-regulation. Increased viability and proliferation by anti-IgM + anti-CD40 + IL-4 stimulation by arrested ATA B cells. Similar outcome by two experiments. (C) Data from one representative mouse with spontaneous CLL generation in ATAμκTg.CD40^−/− mice (6/12 cases). (D) Cotransfer of HSC enriched BM, arrested ATA B in spleen (arr. B), or ATA B in PerC (pB1) from TC⁺ATAμκTg (IgM^a) mice, together with C.B17 mouse BM. Percentage of C.B17-derived B cells (IgM^b) and ATA B in PBL are shown. ATA B-CLL generated by pB1 transfer is marked. Representative of five recipients each.

Figure 7.

Continued up-regulated c-Myc and down-regulated Bmf by B1 B cells to become CLL. (A) CXCR5 up-regulated B1a cell migration, interaction, and renewal/maintenance from young to aged, with cyclin D2 dependence. (B) Myc mRNA levels by B1 B cells at normal and tumor stage, including TC^– ATA B cell tumors, relative to FO B cells. Mean percentages indicated in lines. S, spleen; p, peritoneal cavity. (C) Reduction of Myc mRNA by normal mouse (C.B17) B1a during 24-h culture. After plating in a 96-well U-plate for culture (with 10% FCS), the immediately harvested samples before 37°C incubation was defined as 0 h. FO B Myc mRNA levels were similar before and after culture (as 1.0). n = 4; mean ± SE; *, P = 0.0007. (D) Myc, Mcl-1, Bcl-2, Bim, and Bmf mRNA levels by B1 B cells at TC^– nontumor and TC⁺ (and TC^–) tumor stage relative to 2-mo-old FO B cells. (left) WT (C.B17 mice). (right) ATA B cells in ATAμκTg mice, including arrested ATA B cells in spleen. Values are mean ± SE. (E) Western blot of samples of FO B and pB1a cells in 2-mo-old WT mice together with TC⁺ CLL/lymphomas stained for Myc, Mcl-1, Bcl-2, Bim, Bmf, and β-actin. Another set of WT/ATA B cell tumors was assembled to compare Myc, Bim, Bmf, and β-actin. WT FO B and pB1a sample data were similar to WT data on left (not depicted). Representative data of two to three experiments. (F) Western blot using rabbit anti-Bmf, in comparison with Myc. (G) Myc, Bim, and Bmf mRNA levels in immature B cells (CD19⁺B220⁺AA4⁺IgM⁺IgD^–) in day 1 liver and 2 mo BM. 2-mo-old spleen FO B cells in 1.0. C.B17 mouse. n = 4; mean ± SE; *, P = 0.016; **, P = 0.001.