Cutting Edge: An In Vivo Reporter Reveals Active B Cell Receptor Signaling in the Germinal Center

Affinity maturation of BCRs is vital for optimizing Ag-specific protective Abs. Upon Ag recognition in the context of cognate T cell help, a subset of activated B cells is recruited into the germinal center (GC) (1–5). GC B cells undergo random somatic hypermutation (SHM) to diversify their BCR repertoires and subsequently undergo selection and expansion that are regulated by resident follicular dendritic cells and recruited T follicular helper (Tfh) cells. GC B cells with highest affinity BCRs survive to populate the memory and long-lived plasma cell (LLPC) pools, thereby conferring potent and lasting humoral immunity. Ag capture by the BCR and recruitment of Tfh help regulate GC B cell selection. However, it is not known whether BCR signaling contributes to affinity maturation. These two models are not mutually exclusive, and data in favor of both have been reported (2–4, 6, 7).

The GC has been histologically divided into light and dark zones (LZ and DZ), both populated by GC B cells. Importantly, Tfh and follicular dendritic cells are enriched in the LZ, suggesting that this may be the site of Ag-driven selection (1, 4). GC B cells shuttle rapidly (on the order of hours) between these two zones in a chemokine-dependent manner (8, 9). This is thought to be the dynamic spatiotemporal correlate of iterative rounds of SHM and selection in the GC reaction. LZ and DZ GC B cells can be identified reliably according to expression of the chemokine receptor CXCR4 and the costimulatory ligand CD86 (7, 10). Gene expression profiling of these subsets has revealed a signature of both BCR and CD40-dependent signaling in LZ GC B cells (7, 10). However, recent work by Shlomchik and colleagues (6) has demonstrated that BCR signaling in GC B cells is markedly dampened due to phosphatase activity.

Because only a small fraction of GC B cells ultimately undergo selection, there must be hidden heterogeneity among GC B cells with respect to signaling and gene expression that is obscured by bulk analysis. In this study we use an in vivo sensor of BCR signaling, Nur77-eGFP BAC transgenic line (Nur77-GFP), to unmask such heterogeneity (11). In contrast to prior reports that BCR signaling is largely undetectable in GC B cells, our data demonstrate directly that BCR signaling, although markedly reduced relative to activated B cells, nevertheless occurs in vivo among a subpopulation of LZ GC B cells.

Nur77-GFP mice were previously described (11). C57BL/6, BoyJ, and B1-8i mice were obtained from The Jackson Laboratory (12). All mice were housed in a specific pathogen-free facility at the University of California San Francisco according to University and National Institutes of Health guidelines.

Abs to B220, CD4, CD45.2, CD69, CD83, CD86, CXCR4, GL-7, Fas, IgD, IgM, IgG, and λ1 L chain conjugated to biotin, PE, PE-Cy7, PerCP-Cy5.5, allophycocyanin, Pacific Blue, and QDot605 (eBioscience or BD Biosciences); 4-hydroxy-3-nitrophenylacetyl (NP) conjugated to PE or keyhole limpet hemocyanin (KLH; Biosearch Technologies); Goat anti-mouse IgM F(ab′)₂ (Jackson ImmunoResearch Laboratories); anti-CD3ε (2C11 clone; Harlan Laboratories); anti-CD40 (hm40-3 clone; BD Pharmingen); and ibrutinib (J. Taunton, University of California San Francisco) were used.

Mice were either immunized with 100 μg NP-KLH (BioSearch Technologies) mixed 1:1 with alum adjuvant (Alhydrogel; Accurate Chemical and Scientific) injected i.p. or infected i.p. with 2 × 10⁵ PFU lymphocytic choriomeningitis virus (LCMV) Armstrong.

Splenocytes from CD45.2 B1-8i reporter mice were loaded with CellTrace Violet (Invitrogen) per the manufacturer’s protocol. Cells (2 × 10⁶) were adoptively transferred into CD45.1 BoyJ hosts, which were then immunized with NP-KLH as above. Three days later splenocytes were surface stained and analyzed by FACS.

Lymphocyte activation assays were performed as previously described (13).

Cells were collected on a BD LSRFortessa and analyzed on FlowJo (v9.7.6; Tree Star). Graphs were generated with Prism v6 (GraphPad Software). Bulk cells were sorted on a MoFlo and single cells on a FACSAria. BCR sequence data analyzed with IMGT/V-QUEST (http://www.imgt.org).

Ten days after after NP-KLH immunization, B6 reporter splenocytes were stained with Fas, GL7, CXCR4, and CD86, fixed, and single cell sorted (gating in Supplemental Fig. 1B) into 96-well plates with catch media. Dump gating and prepurification were not used. Plates were frozen and subjected to nested PCR and Sanger sequencing as described (14), except that secondary nested PCR was run with AmpliTaq DNA polymerase (Applied Biosystems) and 1× PCR buffer (Roche).

Nine days after LCMV infection, B6 reporter splenocytes were negatively selected with Abs to IgD, CD4, and CD8 to enrich for GC B cells. Cells were then stained for IgD, CXCR4, CD86, Gl7, Fas, CD19, and DAPI and sorted (gating in Supplemental Fig. 2E) into TRIzol (Invitrogen) and stored at −80°C. cDNA was prepared with a SuperScript III kit (Invitrogen). Quantitative PCR reactions were run on a QuantStudio 12K Flex thermal cycler (Applied Biosystems) using either TaqMan assays (Bcl2A1, Pax5, Bcl6, and GAPDH) with TaqMan Universal PCR Master Mix (Applied Biosystems) or 250 nM (each) primer pairs [Aicda, Irf4 (15), Cxcr4, Ccnd2, Ccnb2, and cMyc (7)] with FastStart Universal SYBR Green Master Mix (Roche).

Mice were infected with LCMV as above, and on day 10 postinfection they were injected i.p. twice each day for 3 d with either vehicle or ibrutinib at 12.5 mg/kg/dose dissolved in Captex355.

The B cell response of C57BL/6 mice to immunization with the hapten NP is characterized by predominant use of H chain Vh186.2 (16). The B1-8i mouse strain was created by targeted insertion of an unmutated V_H186.2(DFL16.2)J_H2 gene into the H chain locus (12). B1-8i transgenic B cells expressing endogenous λ1 L chain are capable of binding NP with a precursor frequency of 2–3% in the preimmune B cell repertoire (17). To track Ag-specific B cells both before and after immunization, we generated B1-8i Nur77-GFP reporter mice. We adoptively transferred B1-8i reporter splenocytes loaded with CellTrace Violet dilutional dye into congenically marked hosts and immunized recipients with NP coupled to the protein Ag KLH. As expected, after 3 d, we observed expanded cell number, GFP upregulation, and concurrent dye dilution in transferred NP-specific λ1⁺ B cells (Fig. 1A, 1B, Supplemental Fig. 1A).

To further characterize BCR signaling in GC B cells, we immunized unmanipulated B1-8i reporter mice with NP-KLH and assessed GFP expression in λ1⁺NP⁺ B cells 9 d later. Although GFP was upregulated in λ1⁺NP⁺IgD⁺ B cells, GFP expression in λ1⁺NP⁺ GC B cells was markedly reduced (Fig. 1C, 1D). It is possible that GFP protein dilution upon cell division contributes to reduced GFP expression; however, GFP was markedly upregulated in dividing NP-specific adoptively transferred B cells despite such dilution (Fig. 1A, 1B). Although BCR expression was reduced among GC B cells by ∼50%, reduction in GFP expression in GC B cells was disproportionate and did not correlate with surface BCR (Fig. 1D and data not shown). Therefore, we hypothesize that GC B cells “see” less Ag and/or signaling is suppressed.

Although GFP expression is very low in GC B cells relative to naive or activated B cells, we nevertheless observe a “shoulder” of high GFP-expressing cells, suggesting that a subset of GC B cells is undergoing active signaling (Fig. 1D). We further subdivided GC B cells into LZ and DZ phenotype cells on the basis of CD86 and CXCR4 expression (Fig. 1E) (7). High GFP expression is enriched among LZ phenotype GC B cells (CD86^hiCXCR4^lo), consistent with previously identified gene expression suggestive of active BCR and CD40 signaling in this compartment (7). Moreover, GFP expression was correlated with CD86 and CD83 surface expression but not CXCR4 (Fig. 1F). To reduce the high precursor frequency of NP-specific clones in the B1-8i model system, we immunized unrestricted repertoire B6 reporter mice with NP-KLH. We observed that GFP expression was again markedly reduced in GC B cells relative to naive B cells (Fig. 2A), similarly enriched among LZ phenotype cells, and correlated with CD86 expression in GC B cells (Fig. 2B, 2C).

To determine whether heterogeneity of GFP expression in the GC was due to timing of GC entry such that “recent recruits” might retain higher GFP expression, we assessed the extent of SHM among GFP-high and GFP-low GC subsets. In the first 2 wk after immunization with NP coupled to a protein Ag, mutation of a single residue in the Vh186.2 H chain, W33L, arises in immunized mice with very high frequency (90%) and increases Ab affinity for NP by a factor of 10 (16). Analysis of the ratio of coding to silent mutations in the CDR sequences of the Vh186.2 H chain has also been used as a proxy measure for affinity maturation (17). We assessed Vh186.2 sequences from GFP-high and GFP-low LZ and DZ phenotype GC B cells sorted 10 d after NP-KLH immunization of B6 reporter mice (see gating in Supplemental Fig. 1B). We observed comparable levels of missense mutations in Vh186.2 germline sequence among all sorted subsets, suggesting that neither high nor low GFP-expressing GC B cell compartments were preferentially enriched with unmutated recent arrivals (Fig. 2D, Supplemental Fig. 1C) (14).

We next infected reporter mice with LCMV to determine whether GFP expression is similarly regulated in the context of a complex, physiologic stimulus. GC B cell number was markedly expanded at 8 and 15 d following infection (data not shown). At both of these time points, we observed a marked reduction in overall GFP expression among GC B cells relative to IgD⁺ B cells, as well as a unique shoulder of GFP-high GC B cells among LZ but not DZ phenotype B cells (Fig. 3A, 3B, Supplemental Fig. 2A–C). We confirmed that these GC B cells are not only Fas^hi and GL-7^hi, but also CD38^lo and peanut agglutinin (PNA^hi) (data not shown) (3, 4). Importantly, we found no correlation between either isotype or extent of surface BCR expression and GFP levels within LZ GC B cells (data not shown).

The Nur77-GFP reporter marks a subset of LZ GC B cells that may represent the actively signaling subpopulation of cells within the LZ compartment. To test this hypothesis, we assessed expression of LZ and DZ “signature” genes in bulk LZ and DZ cells sorted with or without further subdivision on the basis of GFP expression from reporter mice infected with LCMV (see gating in Supplemental Fig. 2D). We observed predicted enrichment of relevant gene expression in bulk-sorted LZ and DZ populations (Fig. 3C, black bars). Importantly, by enriching LZ GC B cells for active signaling by collecting high GFP-expressing cells, and, conversely, depleting the DZ of “contaminating” GFP-high cells, we see further enrichment of the putative LZ and DZ gene expression signatures relative to bulk sort (Fig. 3C, 3D, green bars).

Critical cell fate decisions in the GC control which clones become quiescent memory B cells, which populate the LLPC pool, and which continue to undergo further rounds of SHM (2–4). The transcriptional regulators Blimp1, Irf4, and Xbp-1 favor plasma cell fate and are counteracted by Bcl6 and Pax5, which maintain undifferentiated GC B cells (2, 4, 18, 19). LZ phenotype genes Irf4 and Blimp1 were enriched in high GFP GC B cells whereas Pax5 and Bcl6 expression were reduced (Fig. 3E). Importantly, GFP-high GC B cells do not express CD138, and conversely fully differentiated plasma cells that do express CD138 as well as intracellular IgG do not express GFP (data not shown). Therefore, cells falling into the high GFP-expressing GC B cell gate do not merely represent contaminating plasma cells or plasmablasts (18, 19). Rather, we propose that this high GFP–expressing population may contain plasma cell precursors undergoing selection and differentiation in the GC.

GFP expression in naive Nur77-GFP reporter B cells is dependent on Ag and BCR signaling (11). However, we have previously shown that signals other than BCR, including ligation of CD40, are capable of inducing GFP expression in vitro, albeit much less efficiently (11). We therefore wanted to determine whether GFP expression among GC B cells specifically requires BCR-dependent signaling, or may be driven by indirect consequences of Ag capture such as Tfh engagement and CD40-dependent signaling. To distinguish between these possibilities, we used the well-characterized irreversible Btk inhibitor ibrutinib (20). We found ∼60-fold selectivity of ibrutinib for inhibition of BCR signaling over TCR signaling in vitro (Fig. 4A, Supplemental Fig. 2E). CD40-induced GFP upregulation was insensitive to ibrutinib at all doses used (Fig. 4B). We next treated reporter mice that had been infected 10 d earlier with LCMV, a time point when GCs had already been well established, for 3 additional days with either ibrutinib or vehicle. GC cell number was modestly reduced by about half among inhibitor-treated mice, but relative LZ and DZ composition remained unaffected (Supplemental Fig. 2F, 2G). Importantly, we found that LZ GFP expression was markedly reduced relative to vehicle-treated infected animals, implying that a Btk-dependent, BCR-specific signal was driving GFP expression in reporter GC B cells in vivo (Fig. 4C, Supplemental Fig. 2H–J). We restimulated lymphocytes taken from ibrutinib- or vehicle-treated mice immediately ex vivo (without further supplementation of inhibitor) through AgR for 4 h. We observed persistent inhibition of AgR-mediated GFP and CD69 upregulation among B, but not T, cells (Fig. 4D, E, Supplemental Fig. 2K, 2L). These data confirm in vivo specificity of ibrutinib treatment for Btk over Itk at doses used in our assay, and they thereby suggest that GFP expression in reporter GC B cells is indeed specifically driven by BCR signaling in vivo.

Although a variety of signals are integrated by GC B cells in vivo to make decisions about selection and differentiation, the contribution of active BCR signaling to this process has been controversial (2–4, 6, 7). The Nur77-eGFP reporter reveals a marked reduction in BCR signaling among GC B cells relative to activated B cells in vivo. This may render GC B cells especially dependent on Ag and/or T cell help for survival and selection, enhancing competition and selection pressure within the GC. Furthermore, dampened yet active BCR signaling could resolve subtle affinity differences among competing clones.

BCR-dependent GFP expression is detected among a small subset of LZ phenotype cells and correlates with gene expression characteristic of GC B cells undergoing selection as well as differentiation into the plasma cell compartment. These high GFP–expressing GC B cells exhibit high rates of SHM, confirming their status as bona fide GC-resident B cells rather than recent immigrants or contaminating activated B cells. These cells may correspond partly to those GC B cells identified in recent work with a c-Myc reporter (15, 21). Whether heterogeneity in BCR signaling as revealed by GFP expression among GC B cells is in turn controlled by limiting Ag, BCR affinity, and/or cell-intrinsic differences in signaling machinery remains to be determined as does the contribution of BCR signaling to affinity maturation. The Nur77-eGFP reporter marks cells undergoing BCR signaling and possibly selection in the GC and has great potential as a new tool to further refine our understanding of this complex and dynamic process.

We thank Chris Allen for critical reading of the manuscript and SHM protocol, Jack Taunton for providing ibrutinib, Wan-Lin Lo for help with FACSAria sorting, and Al Roque for help with animal husbandry.

The authors have no financial conflicts of interest.