Characterization of a late transitional B cell population highly sensitive to BAFF-mediated homeostatic proliferation - PubMed (original) (raw)

Characterization of a late transitional B cell population highly sensitive to BAFF-mediated homeostatic proliferation

Almut Meyer-Bahlburg et al. J Exp Med. 2008.

Abstract

We have characterized a distinct, late transitional B cell subset, CD21(int) transitional 2 (T2) B cells. In contrast to early transitional B cells, CD21(int) T2 B cells exhibit augmented responses to a range of potential microenvironmental stimuli. Adoptive transfer studies demonstrate that this subset is an immediate precursor of both follicular mature and marginal zone (MZ) B cells. In vivo, a large percentage of CD21(int) T2 B cells has entered the cell cycle, and the cycling subpopulation exhibits further augmentation in mitogenic responses and B cell-activating factor of the TNF family (BAFF) receptor expression. Consistent with these features, CD21(int) T2 cells exhibit preferential responses to BAFF-facilitated homeostatic signals in vivo. In addition, we demonstrate that M167 B cell receptor (BCR) idiotypic-specific B cells are first selected within the cycling CD21(int) T2 population, ultimately leading to preferential enrichment of these cells within the MZ B cell compartment. These data, in association with the coordinate role for BAFF and microenvironmental cues in determining the mature BCR repertoire, imply that this subset functions as a unique selection point in peripheral B cell development.

PubMed Disclaimer

Figures

Figure 1.

Figure 1.

Characterization of CD21int T2 B cells. (A–C) Expression of surface markers on splenic B cell subsets. (A) B220+/CD19+ splenic B cell subsets gated based on CD21 and CD24. (B) Percentage and absolute number of CD21intT2 cells within total splenic lymphocytes in 8–12-wk-old C57BL/6 mice (mean of 3 independent experiments). (C) Expression of IgM, IgD, CD62L, AA4.1, CD23, and CD1d in splenic B cell subsets. Representative of more than five independent experiments. (D) GFP expression in splenic B cell subsets from an 8-wk-old Rag2p-GFP transgenic mouse. (E) Expression of Notch target genes. Relative Hes1 and Deltex1 expression are shown as fold change of each subset relative to FM B cells set as 1. Data represented as the mean with SD from four independent experiments.

Figure 2.

Figure 2.

CD21int T2 B cells are functionally distinct. (A) Proliferative responses of purified B cell subsets after incubation for 48 h in media alone, with anti-IgM or LPS. (B) CFSE-labeled B cell subsets were cultured in media alone (dashed lines) or with anti-IgM (solid lines) for 48 h, and proliferation was analyzed by dilution of CFSE. Numbers indicate the percentage of live cells in unstimulated versus anti-IgM–stimulated (bold) cultures. T1 cells did not survive anti-IgM stimulation, so no trace is shown. (C) Sorted cells were cultured with anti-IgM and apoptosis measured by caspase 3 activity at indicated time points. Numbers indicate the percentage of active caspase 3+ cells within the live lymphocyte gate. (D) Kinetic analysis of caspase 3 activity and cell survival. (top) The percentage of active caspase 3+ cells from the experiment shown in C. (bottom) The percentage of live cells, based on 7-AAD staining over time with or without BCR engagement. Data are representative of at least three independent experiments.

Figure 3.

Figure 3.

CD21int T2 B cells develop after T1 B cells. Kinetic analysis of splenic B cell reconstitution after sublethal irradiation. C57BL/6 mice were irradiated with 500 cGy, and splenocytes were analyzed at the indicated time points. Shown is the mean of absolute numbers of each B cell subset with SD from three animals. Representative of three independent experiments.

Figure 4.

Figure 4.

CD21int T2 B cells contain precursors for both FM and T2-MZP/MZ B cells. CD21int T2 B cells were purified from sublethally irradiated mice, labeled with CFSE, and 1–2 × 106 cells/recipient were transferred into Ly5.2+ C57BL/6 mice. Recipient animals were killed at day 1 or 2 after transfer, and splenocytes were analyzed by staining with the indicated markers. (A) After initial gating of host B220+CD19+ B cells, splenic B cell subsets were identified as in Fig. 1 (left); and T2-MZP/MZ were additionally characterized as IgMhiCD21hi cells (right). (B, left) Donor cells identified as Ly5.1+CFSE+. (middle and right) Donor B cell subsets determined based on gating shown in A. (C) Donor and recipient FM and T2-MZP/MZ B cells were compared for relative expression of CD1d. Data shown are representative of three experiments with two mice per day for each experiment.

Figure 5.

Figure 5.

CD21int T2 B cells cycle in vivo. (A) Cell cycle analysis. Total splenocytes from BALB/c mice were stained for B220, IgM, CD21, and CD24, and then fixed and stained with DAPI and Pyronin Y. After doublet exclusion, B220+ cells were identified as T1, CD21int T2, or FM B cells, and the percentage of cells within each phase of cell cycle was determined. Data are shown as the mean with SD of five independent experiments. (B–D) BrdU labeling of splenic B cell subsets. (B) Representative FACS histogram of BrdU labeling in splenic B cells 1 d after continuous BrdU administration. (C and D) Percentage of BrdU+ cells in splenic B cell subsets (C) and CD23lo versus CD23hi T1 and CD21int T2 B cells (D) over time after continuous BrdU administration. *, P < 0.05; **, P < 0.01 for CD21int T2 compared with T1 or CD23lo T1 cells. Data are representative of two independent experiments, and the mean with SD of three mice per time point is shown.

Figure 6.

Figure 6.

Cycling CD21int T2 B cells are GFP−. (A) Cell cycle status of GFP+ versus GFP− CD21int T2 B cells. Data are the mean with SD from three independent experiments showing the percentage of CD21int T2 B cells within each phase of the cell cycle. (B) GFP expression in CD21int T2 B cells at distinct phases of cell cycle. CD21int T2 B cells from Rag2p-GFP Tg mice were identified, and relative GFP expression was determined for each phase of the cell cycle. (C) CFSE dilution of GFP+ versus GFP− CD21int T2 cells in response to BCR engagement. CFSE-labeled CD21int T2 B cells were cultured in media alone (dashed lines) or anti-IgM (black lines) for 48 h, and proliferation was determined by dilution of CFSE. Numbers shown indicate the percentage of live cells under each condition. (D) Mitogenic responses of GFP+ versus GFP− CD21int T2 B cells. Sorted splenic B cell subsets were cultured in media alone, with anti-IgM, or with LPS, and proliferation was determined. (E) Expression of Notch target genes. Splenic B cell subsets were sorted from Rag2p-GFP Tg mice, and the transcript levels of Deltex1 and Hes1 was determined. Displayed as fold change in expression relative to FM B cells set as 1. All data shown are representative of at least three separate experiments.

Figure 7.

Figure 7.

GFP− CD21intT2 have undergone several cell divisions. B cell subsets were sorted from Rag2p-GFP transgenic mice. The number of cell divisions, based on the ΔCT between the coding joint of rearranged genomic DNA and signal joint of KRECs formed during IRS-to-RS rearrangement in the κ-chain locus was determined by real-time PCR. Mean with SD from two independent experiments.

Figure 8.

Figure 8.

CD21int T2 B cells cycle preferentially in response to homeostatic signals. (A–C) Transfer of T1, CD21int T2, or FM B cells into μMT mice. 1–2 × 106 purified Ly5.1+ T1 B cells (A), CD21int T2 B cells from irradiated mice (B), or GFP− FM B cells (C) were labeled with CFSE and transferred into μMT mice together with feeder B cells (13–14 × 106 unlabeled CD43−Ly5.2+ splenic B cells/recipient). Recipients were killed on indicated days after transfer, and splenocytes were stained as indicated. Gates were set according to cells from a control C57BL/6 mouse. (first column) Gated analysis of B220+CD19+ B showing Ly5.1+CFSE+ donor cells. (second column) Analysis of Ly5.1+CFSE+ B cell subsets. (third column) CFSE dilution in transferred Ly5.1+ B cells. (fourth column; A and B only) CFSE dilution within each Ly5.1+ B cell subset. Data from one of two (A and C) or three (B) independent experiments (with two mice per time point) are shown.

Figure 9.

Figure 9.

BAFF signals contribute to CD21int T2 B cell HP. (A) Effect of BAFF inhibition on HP. μMT mice were treated with TACI- or control GFP-adenovirus 7 d before transfer of CFSE-labeled splenic B cells (20 × 106 CD43− Ly5.1+ cells/recipient). Mice were killed 5 d after transfer, and splenocytes were surface stained. Ly5.1+ transferred cells (left) were analyzed for dilution of CFSE (right). (B) BAFF-R expression. Total splenic B cells from Rag2p-GFP Tg mice were stained for B220, IgM, CD24, and CD21 and BAFF-R, and BAFF-R expression was compared between B cell subsets (left) or in CD21int T2 cells at each phase of cell cycle (right). Representative of more than five experiments.

Figure 10.

Figure 10.

CD21int T2 cells show evidence of antigen-mediated selection. (A) BCR κ+/λ+ ratio. The percentage of κ+ versus λ+ B cells within each B cell subset in wt mice was determined by FACS and displayed as the ratio of κ+/λ+ B cells. Mean of four mice with SD is shown. (B and C) Determination of percentage of Id-specific cells within each B cell subset in M167 transgenic mice. (B) Representative histograms from one M167 transgenic and wt animal. (C) Average of Id-specific cells with SD from four mice in two independent experiments. (D) Cell cycle status of Id-specific cells. Total splenocytes were stained as in Fig. 5 A, with the addition of the anti-Id antibody. Shown is the percentage of Id-specific cells in G0 or G1 within the CD21int T2 population.

Similar articles

Cited by

References

    1. Hardy, R.R., and K. Hayakawa. 2001. B cell development pathways. Annu. Rev. Immunol. 19:595–621. - PubMed
    1. Goodnow, C.C. 1996. Balancing immunity and tolerance: deleting and tuning lymphocyte repertoires. Proc. Natl. Acad. Sci. USA. 93:2264–2271. - PMC - PubMed
    1. Melchers, F. 2006. Anergic B cells caught in the act. Immunity. 25:864–867. - PubMed
    1. Wardemann, H., S. Yurasov, A. Schaefer, J.W. Young, E. Meffre, and M.C. Nussenzweig. 2003. Predominant autoantibody production by early human B cell precursors. Science. 301:1374–1377. - PubMed
    1. Samuels, J., Y.S. Ng, C. Coupillaud, D. Paget, and E. Meffre. 2005. Impaired early B cell tolerance in patients with rheumatoid arthritis. J. Exp. Med. 201:1659–1667. - PMC - PubMed

Publication types

MeSH terms

Substances

Grants and funding

LinkOut - more resources