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BCR-dependent lineage plasticity in mature B cells

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Science  15 Feb 2019:
Vol. 363, Issue 6428, pp. 748-753
DOI: 10.1126/science.aau8475

B1 or B2? The BCR decides

Immunological B cells are generally divided into two major subsets. B2 cells generate specific antibodies against foreign antigens in secondary lymphoid organs. B1 cells, found predominantly in the peritoneal and pleural cavities, instead produce “natural” antibodies as part of the innate immune system. Two models to explain this split exist: the “lineage model,” wherein both subsets have distinct progenitors, and the “selection model,” in which fates are directed by different B cell antigen receptors (BCRs). Graf et al. provide support for the selection model using a transgenic system in which BCR specificities can be changed. Mature B2 cells differentiated into functional B1 cells when a self-reactive B1 BCR was swapped in, in the absence of B1 lineage precommitment.

Science, this issue p. 748

Abstract

B2 cells engage in classical antibody responses, whereas B1 cells are considered carriers of innate immunity, biased toward recognizing epitopes present on the surfaces of common pathogens and self antigens. To explore the role of B cell antigen receptor (BCR) specificity in driving B1 cell differentiation, we developed a transgenic system allowing us to change BCR specificity in B cells in an inducible and programmed manner. Mature B2 cells differentiated into bona fide B1 cells upon acquisition of a B1 cell–typical self-reactive BCR through a phase of proliferative expansion. Thus, B2 cells have B1 cell differentiation potential in addition to their classical capacity to differentiate into memory and plasma cells, and B1 differentiation can be instructed by BCR-mediated self-reactivity, in the absence of B1-lineage precommitment.

B cells are the carriers of humoral immunity. On the basis of their phenotype and functional properties, mature B cells can be subdivided into B1 and B2 cells, with the former further divided into the CD5+ B1a and CD5 B1b subsets. B2 cells form follicles in the spleen and lymph nodes and are responsible for generating specific antibody responses against foreign antigens, typically involving T cell–dependent affinity maturation and somatic hypermutation of the B cell antigen receptor (BCR) (1). In contrast, B1 cells are predominantly found in peritoneal and pleural cavities and produce natural antibodies, providing a “first line of defense” against common bacterial pathogens and contributing to the clearance of apoptotic cells and oxidized lipids (2, 3). Therefore, B1 cells are thought to perform innate-like functions in the immune system, predominantly expressing evolutionarily selected BCRs with low levels of somatic mutation and junctional diversity (46).

Despite major advances in the field, the origin of B1 cells remains controversial. The “lineage model” posits that B1 and B2 cells represent separate lineages that arise from different progenitor populations, committed to either lineage prior to BCR expression. In the case of B1 cells, this possibly even occurs prior to hematopoietic stem cell differentiation (710). As recently reviewed in detail, definitive evidence for the lineage model is still lacking, with both putative BCR-negative B1 progenitors and exclusive progenitor commitment to B2 differentiation being controversial (11, 12). In the “selection model,” B cell progenitors are instructed by BCR specificity to differentiate into B1 cells, depending on BCR-mediated recognition of common bacterial and certain self antigens (2, 13). Although strong evidence indeed indicates that certain BCR specificities can exclusively drive B1 cell differentiation, this does not rule out a possible commitment of progenitor cells to either lineage prior to BCR expression (14). However, as demonstrated below, the existence of B1 and B2 cell–typical BCRs can be exploited to test lineage commitment and its control by BCR specificity in mature B cells.

To address this issue, we generated a genetic system that would allow a BCR-dependent interconversion of mature B1 and B2 cells. For this purpose, we targeted two prearranged immunoglobulin heavy (IgH) chain variable (V) region gene segments—VH12 and B1-8, respectively—in a head-to-head orientation and flanked by inverted loxP sites into the JH region of the IgH locus. Cre-mediated recombination results in inversion of this VH12B1-8fli cassette, leading to a situation in which some cells express B1-8 and others VH12 IgH chains. In combination with Vκ4 Ig light chains, the VH12 and B1-8 IgH chains form B1 and B2 cell–typical BCRs, respectively (14, 15). VH12B1-8fli mice (hereafter termed B2 mice) and B1-8VH12fli mice (hereafter termed B1 mice) should predominantly develop B cells with either B1-8/Vκ4 or VH12/Vκ4 BCRs (Fig. 1A). In agreement with previous work, the knocked-in BCRs allowed the developing cells to circumvent the pro/pre–B cell stage (fig. S1, A to E) (15). When the B cells in these animals were stained with the BCR-specific anti-idiotypic antibodies Ac146 and 5C5, respectively (14, 16), essentially all splenic B cells of B2 mice were Ac146+ and of the B2 cell phenotype, characterized by their small size and a CD19+, B220+, IgDhi, CD5, CD23+/lo, and CD43 surface phenotype. In contrast, B cells of B1 mice were 5C5+ and acquired the B1 cell phenotype, characterized by large cell size and a CD19hi, B220lo, IgDlo, CD5+, CD23, and CD43+ surface phenotype (Fig. 1B). This is consistent with the natural expression of this BCR that is specific for phosphatidylcholine (PtC) and exclusively expressed on B1 cells of wild-type mice (13) (fig. S1D). PtC is a common membrane phospholipid and is exposed on senescent red blood cells, which suggests that VH12/Vκ4 antibodies are involved in the clearance of these cells (17, 18). When splenic B cells of B1 and B2 mice were isolated and transduced with TAT-Cre in vitro, approximately 10% of these cells inverted the IgH insertion cassette (fig. S1G). This led to detectable populations of cells that switched from the expression of the original BCR to that of the new BCR, thereby confirming the functionality of the transgenic system (Fig. 1C and fig. S1F). For simplicity, we refer to B1-8VH12fli– and VH12B1-8fli–derived switched B cells as B1B2 and B2B1 cells, respectively.

Fig. 1 Genetic system to switch BCR specificity.

(A) Schematic of Cre-mediated switch of BCR specificity. Mice with the IgH cassette in the VH12B1-8fli orientation (B2 mice) develop B2 cells with the B1-8/Vκ4 BCR (left), whereas mice with the B1-8VH12fli orientation (B1 mice) develop B1 cells with the VH12/Vκ4 BCR (right). The flanking inverted loxP sites (bottom) allow the orientation of the cassette, and thus BCR specificity, to be changed by application of Cre. (B) Cell surface phenotype of splenic Ac146+ B cells from B2 mice (blue) and peritoneal 5C5+ B cells isolated from B1 mice (red) measured by flow cytometry. FSC, forward scatter (area). (C) BCR expression via flow cytometry on B cells isolated from spleens of B2 mice (top) or B1 mice (bottom), 4 days after treatment with TAT-Cre in vitro. B1-8 and VH12 were detected by the anti-idiotypic antibodies Ac146 and 5C5, respectively. Data are representative of 10 to 20 independent experiments.

To test whether a change of BCR specificity would change the phenotype of fully mature B cells, we treated B1 and B2 mice for 2 weeks with antibodies against the interleukin-7 receptor (IL-7R) to block the influx of immature B cells into the spleen (fig. S2). Purified splenic B cells from these mice were then transduced with TAT-Cre in vitro. Only very low numbers of B1B2 cells developed. In contrast, B2B1 cells developed into a dominant population (Fig. 1C and fig. S1F). However, although these cells acquired some B1-typical markers, their phenotypic change was only partial in vitro (fig. S3). We therefore transferred IgMa experimental (TAT-Cre transduced) mature B cells together with IgMb carrier B cells into immunodeficient recipients to study the consequences of the BCR switch in a more physiological environment (Fig. 2A) (19). Consistent with the in vitro data, only low numbers of B1B2 cells were recovered from the recipients. These were located predominantly in the peritoneal cavity and had acquired a phenotype intermediate between B1 and B2 (fig. S4). In contrast, B2B1 cells expressing the 5C5 idiotype were readily detectable in the spleen and peritoneal cavity of the recipients 4, 8, and 30 days after transfer and became the dominant population among the IgMa experimental cells (Fig. 2B). Contributing to this dominance was an initial, transient phase of rapid proliferation, similar to the clonal expansion of PtC-specific B1 cells observed in wild-type mice over the first 3 to 4 weeks of life (Fig. 2C and fig. S5A) (4, 20). Strikingly, after completion of this expansion phase, the B2B1 cells became resting cells that phenotypically were essentially indistinguishable from bona fide B1 cells. B2B1 cells reproducibly increased in size, down-regulated B220 and IgD, lost CD23 and CD21 expression, and increased the expression of CD19, CD43, and CD5 (Fig. 2, D and E, and fig. S5B). In the case of the B1a-specific marker CD5, the up-regulation was modest in splenic and not significant in peritoneal B2B1 cells. This may relate to the observation that adult bone marrow–derived B1a cells express lower levels of CD5 than fetal liver–derived B1a cells (21, 22).

Fig. 2 B2B1 cells acquire the surface phenotype of B1 cells and persist in vivo after a phase of proliferative expansion.

(A) Experimental scheme: B1 and B2 mice were treated for 2 weeks with antibodies to IL-7R to block B cell development. Splenic B cells were then treated with TAT-Cre in vitro and transferred intravenously to immunodeficient recipients the next day. FACS, fluorescence-activated cell sorting. (B) BCR expression via flow cytometry on experimental IgMa B cells isolated from the indicated anatomical sites at the indicated time points after transfer. (C) Proliferation analysis based on bromodeoxyuridine (BrdU). Animals were fed with BrdU for the indicated time windows. The BrdU content of the indicated cells was analyzed on day 8 (left and center) or day 30 (right) by flow cytometry. (D) Flow cytometric analysis of cell surface phenotype of peritoneal B2B1 cells (red) isolated from the recipients 30 days after transfer, and of control splenic B2 (gray) and peritoneal (5C5+) B1a cells (blue) isolated from wild-type animals. Splenic B2B1 cells (dashed red curves) are only shown for the markers in which they differ relative to peritoneal B2B1 cells [see (E)]. (E) Summary of the B2B1 cell surface phenotype from five independent experiments. Each dot represents the median fluorescence intensity (mfi) of the indicated surface marker and cell population of one recipient mouse or control mouse, normalized to the median mfi in PeC B2B1 cells (set to 1000) per experiment. Tamoxifen/CreERT2-derived B2B1 cells are colored turquoise (see fig. S7 for details). ****P < 0.0001, ***P < 0.001, **P < 0.01 (ordinary one-way analysis of variance and Bonferroni multiple-comparisons test); n.s., not significant.

The B2B1 conversion was not dependent on B2 cell subtype or donor organ, as marginal zone and spleen- or lymph node–derived follicular B2 cells were all able to acquire the B1 cell phenotype (fig. S6). Consistent with this, we calculated that at least ~40% of the splenic B cells that successfully inverted the VH12B1-8fli allele assumed a B1 cell phenotype, excluding the possibility that the B2→B1 cells developed from some small cellular subset (fig. S7 and table S1). To control for potential biases caused by (unlikely) endogenous IgL gene rearrangements, all experiments on B2→B1 conversion were also performed using Rag2-deficient donor B2 mice, with identical results (see supplementary materials). Furthermore, to exclude that the B2B1 conversion was peculiar to the in vitro treatment of these cells with bacterial-derived TAT-Cre protein (with its possible endotoxin contamination), we crossed the tamoxifen-inducible conditional R26CreERT2 allele into the donor B2 mice (23). The recipients of B cells from these mice were then administered tamoxifen 1 day after transfer. As in the case of TAT-Cre, B2B1 cells developed and adopted the B1 cell phenotype (Fig. 2E and fig. S8). In the course of these experiments, we noticed that the donor B2 mice contained minute numbers of spontaneously arising IgMa 5C5+ cells in the spleen and a dominant population of these cells in the peritoneal cavity (fig. S9). This was likely a consequence of rare spontaneous recombination events between the inverted loxP sites, as has been previously reported, followed by positive selection and clonal expansion (24). The frequency of this population was increased in the presence of the R26CreERT2 allele (fig. S9). To exclude the possibility that these cellular contaminants contributed to B2B1 cell development in the recipients (even though they should have been eliminated by the B cell purification procedure), we performed cell transfer experiments without the delivery or induction of Cre. In this situation, IgMa 5C5+ cells were undetectable in the recipients, validating our experimental approach (fig. S9). Thus, upon expression of a B1-typical BCR, mature B2 cells acquire the phenotype of B1 cells and persist as such in vivo.

The phenotypic change of B2B1 cells suggested that these cells might also adopt B1-typical functions. B1 cells home to the peritoneal cavity and secrete natural antibodies into the blood. In the B2B1 recipient animals, the percentages of the experimental IgMa B cells were markedly increased in the peritoneal cavity relative to the spleen. Moreover, essentially all of these peritoneal IgMa B cells were 5C5+ B2B1 cells, indicating that these cells homed more efficiently to this anatomical site than did the IgMb carrier B cells or the nonswitched Ac146+ B cells, similar to natural B1 cells (Fig. 3, A and B). B2B1 cells were also present in the pleural cavity, the second natural niche of B1 cells (Fig. 3C). Consistent with the spontaneous production of antibodies by B1 cells, 5C5-binding IgMa-positive antibodies were detectable in the circulation of the recipients (Fig. 3D). B1 and B2 cells are also known to have different functional properties when cultured in vitro. Relative to B2 cells, B1 cells show prolonged survival in vitro in the absence of stimulation and faster differentiation into CD138+ plasma cells upon lipopolysaccharide (LPS) stimulation (25, 26). In addition, unlike B2 cells, they are refractory to anti-IgM F(ab)2–induced BCR engagement and show efficient class switching to IgA upon stimulation with B cell–activating factor (BAFF), LPS, and transforming growth factor–β (TGF-β) (2729). Without exception, the response of peritoneal B2B1 cells to these stimuli was similar to that of B1 cells (Fig. 3E and fig. S10). Thus, B2B1 cells not only acquired the B1 cell surface phenotype, but also adopted B1-typical functional properties in vitro and in vivo.

Fig. 3 B2B1 cells acquire the functional characteristics of B1 cells.

(A) Flow cytometric analysis of the experimental IgMa B cells (by TAT-Cre) within all B cells in recipients 30 days after transfer in the spleen (SP) and peritoneal cavity (PeC) (top) and the 5C5+ B2B1 cells within these cells (bottom). (B) Quantification of the data shown in (A). Each dot represents one recipient mouse analyzed in one of five independent experiments. ****P < 0.0001 (paired t test). (C) Flow cytometry showing the B2B1 cells within the B cells in the pleural cavity. (D) Enzyme-linked immunosorbent assay measurements of 5C5-specific antibodies with IgMa allotype in the serum of three B2B1 recipients (30 days after transfer) and the indicated controls (P < 0.001 between B2B1 recipients and negative controls). OD 405, optical density at wavelength 405 nm. (E) Stimulation of wild-type splenic B2 cells, control peritoneal cavity 5C5+ B1 cells (from B1 mice), and peritoneal B2B1 cells isolated 30 days after transfer. The BCR switch was induced by TAT-Cre or CreERT2/tamoxifen, as indicated. Cells were stimulated by anti-IgM for 4 days (first row), LPS for 2 days (second row), or BAFF, LPS, and TGF-β for 4 days (last two rows) and analyzed by flow cytometry. Gates show survival and activation of cells based on propidium iodide (PI) and size (first row), plasma cell differentiation based on CD138 (second row), and isotype switching from IgM to IgA or IgG2a/2b (last two rows, gated on IgM-negative cells). Data are representative of two independent experiments.

To expand the analysis of the identity of B2B1 cells beyond the limited number of surface markers, we performed RNA sequencing (RNA-seq) of peritoneal B2B1 cells and compared their transcriptome to that of peritoneal B1 and splenic B2 control cells. In terms of a previously defined B1 signature consisting of 14 B1-specific and 14 B2-specific genes (30), B2B1 cells showed an expression profile almost identical to that of B1 control cells (Fig. 4A). This result was further confirmed by a transcriptome-wide analysis, where the Spearman correlations between B2B1 and B1 control samples were as high as those between biological replicates and significantly higher than those between B2B1 and B2 control samples, closely clustering B1 and B2B1 samples together (Fig. 4B). Thus, the B2B1 conversion entails profound genetic alterations, leading to a B1-typical transcriptome profile in B2B1 cells.

Fig. 4 B2B1 cells acquire the transcriptome profile of B1 cells.

(A) Heat map with sample clustering of gene expression levels of 14 genes specifically expressed in B1 cells (red) and 14 genes specifically expressed in B2 cells (blue) previously defined as B1 signature (30). RNA was isolated from splenic control B2 cells from B2 mice (B2 Ctrl), peritoneal 5C5+ cells from B1 mice (B1 Ctrl), and B2B1 cells isolated from the peritoneal cavity of recipients 30 days after transfer (B2B1). (B) Heat map of Spearman correlations between each sample, based on the expression levels of 7629 expressed genes. Each B2B1 sample is based on one donor animal and was pooled from three or four recipients.

Taken together, mature B2 cells of the various subsets exhibit plasticity toward B1 cell differentiation. The cells undergo B1 differentiation upon substitution of their BCR by an autoreactive BCR that is known to drive B1 cell development in early postnatal life. In both instances, differentiation is initially linked to extensive cell proliferation, likely reflecting a special mode of B cell activation by autoantigen (4, 20). The present experimental system offers a unique opportunity to study this process, which is key to the understanding of BCR repertoire selection in the B1 compartment. In contrast, only low numbers of B1 cells switching to the expression of the B2-typical BCR were recovered from the animals, with a surface phenotype intermediate between B1 and B2. This is likely a consequence of the fact that B1 cells represent B cells that have undergone antigen-driven activation, proliferation, and differentiation. The profound genetic and morphological changes that the cells undergo during these processes may well be irreversible.

Jerne’s notion that autoreactive receptors initially drive lymphocyte development, with the subsequent selection of escape somatic mutants, was accommodated in the classical model of B cell development through ordered V(D)J gene rearrangements, in first the IgH and then the IgL loci of progenitor B cells (1, 31, 32). In this model, IgH chain–containing pre-BCRs constitutively drive clonal expansion sustained by the surrogate light chain, followed by sequential IgL chain gene rearrangements and IgL chain editing. Although these ordered gene rearrangements clearly dominate postnatal B cell development, recent evidence indicates that during fetal development, very much in agreement with Jerne, IgH and IgL chains are often initially coexpressed (33). In this situation, autoreactive BCRs instead of pre-BCRs drive the expansion and selection of B cells, which at that developmental stage predominantly develop into the B1 variety (11, 34).

Although the control of these different pathways of differentiation remains to be fully elucidated, the present results establish that mature B2 cells are not irreversibly committed to a B2 “lineage” but maintain the potential to develop into B1 cells. That these cells do so upon engraftment of an autoreactive, B1 cell–typical BCR demonstrates the instructive role of BCR specificity and mode of signaling in driving B1 cell differentiation, in the absence of B1-lineage precommitment.

Supplementary Materials

www.sciencemag.org/content/363/6428/748/suppl/DC1

Materials and Methods

Figs. S1 to S10

Table S1

References (3540)

References and Notes

Acknowledgments: We thank S. Clarke for reagents and the Vκ4 mice; J. di Santo for providing the Rag2−/−−/− mice; J. Wang, K. Jenssen, V. Blinder, E. Derudder, T. Chakraborty, J. Xia, D. Ghitza, C. Aristoff, A. Monti, A. Tetreault, J. Grundy, and A. Pellerin for technical help in Boston; K. Petsch, C. Grosse, J. Pempe, S. Mackowiak, and the MDC animal caretakers for their help; and H. P. Rahn for excellent FACS-related support. We apologize to colleagues whose work we could not cite because of space restrictions. Funding: Supported by NIH grant AI054636 and the European Research Council (ERC Advanced Grant 268921 to K.R.). Author contributions: J.S. and K.L.O. started the experimental work at the Immune Disease Institute, Harvard Medical School; R.G. substantially broadened and completed the study at the MDC; K.-P.L. generated the VH12B1-8fli mouse strain; S.A. helped to generate the RNA-seq library; B.Z. helped with cell transfer experiments; S.S. provided reagents; V.T.C. provided scientific and experimental advice; R.G. and K.R. wrote the manuscript; and K.R. supervised the project. Competing interests: The authors declare no competing financial interest. Data and materials availability: The RNA-seq data are available in the Gene Expression Omnibus (GEO) database (www.ncbi.nlm.nih.gov/geo) under accession number GSE124827. The materials required to use the transgenic system described here will be available through K.R. under a material transfer agreement from the MDC.
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