Nat. Immunol. 13, 396–404 (2012); published online 26 February 2012; addendum published after print 15 November 2013
It has come to our attention that in our studies of the differentiation and memory of IgE+ B cells in mice, we did not appropriately distinguish IgG1+ and IgE+ B cells because of technical flaws in our original flow cytometry1,2. After changing the fluorochrome of the antibody to IgG1 from phycoerythrin to allophycocyanin to overcome problems in the compensation between the green fluorescent protein (GFP) and IgG1 channels of our original nine-color analysis of in vivo IgG1+ and IgE+ B cell populations, we identified the following three distinct IgG1+ and IgE+ B cell populations in our IgE-GFP reporter mice infected with Nippostrongylus brasiliensis: IgG1+GFP− IgG1-expressing cells, IgG1+GFPhi IgG1-expressing cells and IgG1−GFPhi IgE-expressing cells (Fig. 1a). The mean fluorescence intensity for GFP in the population of IgG1+GFPhi cells was slightly lower than that of the IgG1−GFPhi IgE+ cells, but the range of GFP fluorescence of these two cell populations overlapped significantly, in contrast to the results we obtained before (Supplementary Fig. 3 in ref. 2) and also in contrast the results we obtained for in vitro–derived IgG1+ and IgE+ cells, which exhibited a more distinct separation in GFP fluorescence (Fig. 1 in ref. 1). The IgG1+GFPhi cell population was present in both germinal center (GC) B cell populations and memory B cell populations and, although it constituted only ∼10% of all IgG1+ B cells, it ranged from approximately equal to the number of IgG1−GFPhi IgE+ B cells (for memory B cells) to approximately threefold that number (for GC B cells) (Fig. 1b). We further confirmed that for cells in GCs, both IgG1+GFP− and IgG1+GFPhi cells were IgG1-expressing B cells and IgG1−GFPhi cells were IgE-expressing B cells, as assessed by quantitative real-time PCR analysis of transcripts encoding membrane IgG1 and membrane IgE (Supplementary Fig. 1), as well as by flow cytometry staining of each cell population for membrane IgE with antibody to IgE after treatment of the cells with acid to remove serum IgE bound to the low-affinity CD23 receptor for IgE (Supplementary Fig. 2). Thus, our original studies, which defined in vivo IgE+ B cells as B220+IgD−GFPhi cells1, probably included contributions from both IgG1+GFPhi B cells and IgG1−GFPhi IgE+ B cells, since the fluorescence intensity of GFP in the IgG1+GFPhi cells was similar to that of the IgG1−GFPhi IgE+ cells.
We were concerned that in our previous study we may not have clearly separated IgG1+ and IgE+ B cells in our studies of IgE memory, which used the fluorescence intensity of GFP to separate IgG1+ and IgE+ memory B cell populations for adoptive transfer1. If the GFPhi B cells used in those studies included both IgG1+GFPhi and IgG1−GFPhi cells, then both IgG1+ memory B cells and IgE+ memory B cells could have contributed to IgE memory responses. To address this, we repeated the cell-transfer studies as follows: we sorted equal numbers of memory B cells from each of the three different populations (IgG1+GFP−, IgG1+GFPhi and IgG1−GFPhi) obtained from mice 33–35 d after infection with N. brasiliensis and transferred those cells into B cell–deficient mMT mice along with memory T cells and carrier splenocytes from mice deficient in recombination-activating gene 2. Subsequently, we challenged the recipient mice with N. brasiliensis and measured the concentration of IgE and IgG1 in serum. Similar to results obtained in our earlier study (Fig. 8 in ref. 1), IgG1−GFPhi IgE+ memory B cells gave rise to serum IgE responses but not serum IgG1 responses, while IgG1+GFP− cells memory B cells gave rise to serum IgG1 responses but not serum IgE responses (Fig. 2). IgG1+GFPhi memory B cells produced both serum IgE responses and serum IgG1 responses after transfer and challenge with N. brasiliensis. Given the approximately equal frequency of IgG1+GFPhi and IgG1−GFPhi memory B cells in N. brasiliensis–infected mice (Fig. 1b), we calculate that the IgG1+GFPhi memory B cells contributed approximately 25% of the total serum IgE response in one study (Fig. 2a) and significantly less in a second study (Fig. 2b). Thus, we have identified a small population of IgG1+ memory B cells that contributed to IgE memory responses in N. brasiliensis–infected IgE-GFP reporter mice. However, IgE+ memory B cells (i.e., IgG1−GFPhi memory B cells) were still the main contributors to IgE memory responses in our new studies, consistent with our earlier work in which we concluded that IgE-switched memory B cells were the main source of cellular IgE memory1.
In summary, after changing the conditions of our flow cytometry to rectify the analysis of IgG1+ and GFP+ B cells, we have resolved three distinct GC and memory B cell populations in our IgE-GFP reporter mice: a large IgG1+GFP− IgG1-expressing B cell population, a small IgG1+GFPhi IgG1-expressing B cell population, and a small IgG1−GFPhi IgE-expressing B cell population. Since the GFP fluorescence of the IgG1+GFPhi B cells and the IgG1−GFPhi IgE+ B cells was similar, our analyses of GFPhi B cells in earlier publications included contributions from both IgG1+GFPhi B cells and IgG1−GFPhi IgE+ B cells. Notably, in our new studies, we still observed IgE+ GC B cells (i.e., IgG1−GFPhi GC B cells), and the in vivo kinetics of those cells were still consistent with our original observations1,2 and other published studies3. In addition, we still found that IgE+ memory B cells were the main source of IgE memory responses and that the majority of IgG1+ memory B cells did not contribute significantly to IgE memory. However, we have now identified a small subset of IgG1+ memory B cells that made minor contributions to cellular IgE memory. With this Addendum, we seek to correct the mistake in our earlier flow cytometry and to clarify the different populations of IgG1+ and IgE+ B cells that we found in our IgE-GFP reporter mice. We apologize for any confusion that this error may have caused.
N. brasiliensis infection.
Larvae of N. brasiliensis (R. Locksley) were isolated from the feces of infected Lewis rats (Charles River). Mice were infected subcutaneously with 500 L3 larvae in 200 mL PBS.
Antibodies and flow cytometry.
The following monoclonal antibodies and reagents were used for flow cytometry: Pacific Blue–conjugated antibody to anti-mouse B220 (RA3-6B2; BD Bioscience), allophycocyanin-indotricarbocyanine–conjugated antibody to mouse IgD (11-26c.2a; BioLegend), biotin-conjugated antibody to mouse GL7 (GL7; eBioscience), phycoerythrin-indotricarbocyanine–conjugated anti-mouse CD95 (Jo2; BD Bioscience), Alexa Fluor 700–conjugated antibody to mouse CD38 (90; eBioscience), allophycocyanin-conjugated antibody to mouse IgG1 (A85-1; BD Bioscience), phycoerythrin-conjugated antibody to mouse IgE (R35-72; BD Bioscience), streptavidin-V500 (BD Bioscience) and 7-amino-actinomycin D (eBioscience). Cells were analyzed on an LSR II (Becton Dickinson) or were sorted on a FACSAria (Becton Dickinson). Data were processed with FlowJo software (TreeStar).
Quantitative real-time PCR analysis of transcripts encoding membrane IgG1 and membrane IgE.
Total RNA was prepared by an RNeasy protocol (Qiagen) with DNase treatment to remove genomic DNA contamination from cells sorted from lymph nodes of IgE-GFP reporter mice (n = 39) at 15 d after infection with N. brasiliensis. Cells were first gated as B220+IgD−GL7+CD95+CD38− GC B cells and were then sorted into IgG1+GFP−, IgG1+GFPhi and IgG1−GFPhi cell populations. Sequences for real-time PCR analysis of membrane IgG1 were as follows: forward primer, 5′-CAAGGCCACATTGACTGTAGA-3′; probe, 5′-FAM-AAGTCCTCCAGCACAGCCTACATGG-TAMRA-3′; and reverse primer, 5′-GAGTGTGACAGCAGCGCTGTAG-3′. Sequences for real-time PCR analysis of membrane IgE were as follows: forward primer, 5′-CAAGGCCACATTGACTGGTAGA-3′; probe, 5′-FAM-TCTGAATACCAGGTCACAGT-TAMRA-3′; and reverse primer, 5′-AGTTCACAGTGCTCATGTTCAG-3′. The approximate cell input for each assay was as follows: 4 × 104 IgG1+GFP− cells, 1 × 104 IgG1+GFPhi cells, and 1 × 104 IgG1−GFPhi cells. For each assay, results were normalized to those of control transcription of the gene encoding ribosomal protein L19, and relative transcription was determined by calculation of the change in cycling threshold (ΔCt) as 2−ΔCt, with reference to an absolute baseline cycling threshold of 40 cycles.
B220+IgD−GL7−CD38+ memory B cells were sorted from the pooled lymph nodes and spleens of IgE-GFP mice (n = 30–40) at day 35 after infection with N. brasiliensis. Memory T cells were sorted from the pooled lymph nodes and spleens of C57BL/6 mice (n = 20) at day 35 after infection with N. brasiliensis. Approximately 1.4 × 104 memory B cells, along with 1 × 105 memory T cells and 1 × 106 splenocytes from mice deficient in recombination-activating gene 2 (as carrier cells), were injected intravenously (in 100 ml sterile PBS) into mMT recipient mice (n = 2 mice per group for IgG1+GFPhi and IgG1−GFPhi memory B cells; n = 2–3 mice per group for IgG1+GFP− memory B cells). Recipient mMT mice were infected with N. brasiliensis 1 d after cell transfer.
Enzyme-linked immunosorbent assay of IgE and IgG1.
Mouse serum IgE was measured with a mouse IgE ELISA set according to the manufacturer's instructions and the manufacturer's IgE standard (BD Biosciences Pharmingen). Mouse serum IgG1 was measured with purified antibody to mouse IgG1 (A85-3; BD Bioscience), biotin-conjugated antibody to mouse IgG1 (A85-1; BD Bioscience) and streptavidin–horseradish peroxidase (BD Bioscience Pharmingen) with a purified mouse IgG1 standard (BD Bioscience).
P values were calculated with JMP statistical software (SAS).
Talay, O. et al. IgE+ memory B cells and plasma cells generated through a germinal-center pathway. Nat. Immunol. 13, 396–404 (2012).
Talay, O. et al. On the differentiation of mouse IgE+ cells. Nat. Immunol. 13, 623–624 (2012).
Yang, Z., Sullivan, B.M. & Allen, C.D. Fluorescent in vivo detection reveals that IgE+ B cells are restrained by an intrinsic cell fate predisposition. Immunity 36, 857–872 (2012).
We thank R. Locksley (University of California, San Francisco) for larvae of N. brasiliensis; and J. Jackman for technical assistance.
All authors are employed by Genentech, and D.Y., H.D.B., E.E.M.S., M.Z., E.L., W.P.L., J.G.E., C.D.A., M.X. and L.C.W. hold equity in the Roche Group.
Integrated supplementary information
Supplementary Figure 1 Quantitative real-time PCR analysis of membrane IgG1 and membrane IgE transcripts in IgG1+ GFP− (IgG1), IgG1+ GFPhi (DP), and IgG1− GFPhi (IgE) cells sorted from pooled mesenteric and mediastinal lymph nodes of IgE-GFP reporter mice 15 days after infection with N. brasiliensis.
(a) Membrane IgG1 transcript. (b) Membrane IgE transcript. Cells were first gated as B220+ IgD− B cells, then as GL7+ CD95+ CD38− germinal center B cells, and then separated based on IgG1 and GFP expression. The location and sequences of primers and probes for the membrane IgG1 and membrane IgE assays are as shown in the figure. Approximate cell input for each assay was as follows: 4×104 IgG1 cells, 1×104 DP cells, and 1×104 IgE cells. Data are plotted as relative expression, with the normalized cycling threshold (CT) value denoted above each bar graph. For each assay, results were normalized to those of control transcription of the gene encoding ribosomal protein L19, and relative transcription was determined by the calculation 2−ΔCT with reference to an absolute baseline CT of 40 cycles. The graph depicts the mean ± s.e.m. of two technical replicates. Source data
Supplementary Figure 2 Membrane IgE staining of IgG1+ GFP− (red, IgG1), IgG1+ GFPhi (blue, DP), and IgG1− GFPhi (black, IgE) cells from mesenteric lymph nodes of IgE-GFP reporter mice at 12 days after infection with N. brasiliensis.
At this time point, almost all B220+ IgD− cells are germinal center B cells. Membrane IgE staining with an anti-IgE antibody was performed after treatment of cells with an acid buffer to first remove serum IgE bound to the B cells through the low affinity IgE receptor CD23. The graph shows the results of three measurements of the mean fluorescence intensity (MFI) of each cell population, with a line drawn at the mean value of the three measurements. * p=0.01 using a Student's t-test for each pair-wise comparison. Source data
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Talay, O., Yan, D., Brightbill, H. et al. Addendum: IgE+ memory B cells and plasma cells generated through a germinal-center pathway. Nat Immunol 14, 1302–1304 (2013). https://doi.org/10.1038/ni.2770
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