Research ArticleALLERGY

Chronic allergen exposure drives accumulation of long-lived IgE plasma cells in the bone marrow, giving rise to serological memory

See allHide authors and affiliations

Science Immunology  10 Jan 2020:
Vol. 5, Issue 43, eaav8402
DOI: 10.1126/sciimmunol.aav8402
  • Fig. 1 IgE+ PCs accumulate in the BM during chronic allergen exposure in IgEVenus and Blimp-1mCherry reporter mice.

    (A) Representative plots of BMPCs in mice exposed to either saline or HDM for 4 or 15 weeks (wks). Numbers on each plot indicate the percentage of CD138+ PCs within a dump population (left) and quantification as the percentage of live cells (right). (B) Ig heavy-chain RNA expression in sorted CD138+ BMPCs exposed to HDM for 15 weeks. (C) Quantification of total Blimp-1mCherry+ PCs in the BM of saline or HDM-exposed mice (percentage of live cells). (D) Comparison of serum IgE in WT and IgEVenus homozygous/Blimp-1mCherry heterozygous double reporter mice exposed to saline or HDM for 15 weeks. (E) Membrane IgEVenus/Blimp-1mCherry single and double reporter mice were intranasally exposed to either saline or HDM for 4 and 15 weeks. Representative dot plots of IgEVenus+ cells within dump/IgD population in the BM (left) (refer to fig. S2D for gating). Quantification (shown as the percentage of live cells) of IgEVenus+ single reporters (center graph) and IgEVenus/Blimp-1mCherry double reporters (right graph). FSC, forward scatter height. (F) The frequency of PCs (Blimp-1mCherry+) and B cells (B220+ Blimp-1mCherry−) was analyzed within the IgEVenus+ gate in the BM of 15-week HDM-exposed double reporter mice. (G) CD138 expression assessed within the IgEVenus/Blimp-1mCherry IgE PC population in the BM (left) and quantified (right). (H) Representative dot plots of IgEVenus+ cells within dump/IgD population in the spleen of saline- or HDM-exposed mice (left) and (I) the distribution of IgE PCs (Blimp-1mCherry+B220) and IgE B cells/plasmablasts (B220+ Blimp-1mCherry±). *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001.

  • Fig. 2 IgE+ PCs generated during chronic allergic inflammation contribute to IgE serological memory during rest.

    (A) Experimental setup for HDM exposure and rest. (B) Serum IgE ELISA in mice exposed to HDM for four weeks or (C) 15 weeks of HDM, with or without subsequent 9 weeks of rest. ns, not significant. (D) Representative plots of Venus+ cells in the BM after saline or HDM exposure ± 9 weeks of rest (left) and quantification of membrane IgEVenus+ cells in the BM (right). (E) CXCR4 expression on membrane IgEVenus+ PCs (B220 Venus+) compared with IgG1 PCs (IgG1+, B220) in mice treated with chronic HDM ± rest. Experiments were performed using IgEVenus heterozygous and Blimp-1mCherry heterozygous mice. MFI, mean fluorescence intensity. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001.

  • Fig. 3 IgE+ BMPCs generated during chronic HDM exposure are maintained in the BM after 8 months of rest.

    (A) Serum IgE ELISA in mice exposed to HDM for 15 weeks and rested for 29 weeks. (B) Experimental setup for HDM exposure and rest ± anti-CD20 and IgG2a isotype (Iso) control monoclonal antibody (mAb) treatments. (C) Circulating B cell frequency before antibody treatment (left graph) and 7 days after antibody treatment (right graph) in mice exposed to HDM for 15 weeks and treated with anti-CD20 or IgG2a control monoclonal antibody. (D) Serum IgE levels at various time points over the course of a 32-week (8-month) rest period after 15 weeks of HDM exposure. (E) Representative plots of Venus+ cells in the BM after HDM exposure and 32-week rest ± anti-CD20 or IgG2a control monoclonal antibody treatment (top left) or intracellular IgE+ cells in the BM (bottom left) and quantification of membrane IgEVenus+ cells in the BM (top right) or intracellular IgE+ cells in the BM (bottom right). (F) Intracellular IgE staining within BM Venus+ cells. ****P ≤ 0.0001.

  • Fig. 4 IgE+ PCs that migrate to the BM are primarily derived from sequential class switching of IgG1+ cells.

    (A) Quantification of the percentage of sequential class switching (presence of IgG1 remnants) within IgE-switched cells in the spleen of mice exposed to HDM for 4 weeks or the BM of mice exposed to HDM for 15 weeks (refer to table S1). VDJ, variable-diversity-joining. (B) The presence of Sγ1 remnant sequence within IgE clones in the BM of 15-week HDM-exposed mice was confirmed by PCR using primers specific to Sγ1 repeat region and Sε. Image shows representative gel from one experiment. The presence of ≥1 band indicates that the IgE-switched clone contains IgG1 remnants and was derived from sequential class switching of an IgG1+ cell.

  • Fig. 5 IgE+ PCs secrete Der p 1–specific IgE that can induce mast cell degranulation and systemic anaphylaxis.

    (A) Groups of naïve mice received an intravenous (I.V.) injection of serum from mice that were exposed to saline, 4 weeks, or 15 weeks HDM ± rest allowing allergen-specific IgE to bind FcεRI-expressing cells systemically. After 24 hours, basal core temperature measurements were taken for all mice, followed by intravenous challenge with Der p 1 allergen. Core temperature change relative to basal temperature is shown as a readout for systemic anaphylaxis. (B) Histamine levels in the plasma of mice 30 min after Der p 1 challenge. (C) PCA (mast cell degranulation) was assayed by intradermal (I.D.) injection of the same sera as (A) from saline- or HDM-exposed mice. After 24 hours, the mice were challenged intravenously with Der p 1 diluted in 0.5% Evans blue dye. Evans blue dye was extracted from ear tissue and measured spectrophotometrically. Plot shows Evans blue dye extravasation in the tissue quantified as nanograms of Evans blue per milligram of tissue as a measure of local mast cell degranulation. **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001.

  • Fig. 6 IgE+ PCs are present in the BM of allergic patients and secrete IgE when cultured in vitro.

    (A) Intracellular IgE staining of BM mononuclear cells from nonallergic and allergic individuals. Plots show Dump CD27+ CD38+ BMPCs (gating strategy: fig. S6B). Quantification of IgE BMPCs shown as percentage of live (middle graph) and percentage of BMPCs (right graph). (B) Comparison of expression levels of surface proteins (MFI) in allergic and nonallergic BMPCs and naïve B cells. (C) BM mononuclear cells from three cat-allergic and two nonallergic donors were cultured in stromal cell–conditioned media for 8 days, and supernatants (sup) were collected. IgE and IgG levels in the supernatants were measured by ELISA. ND, not detectable. (D) Serum IgE and total IgG were measured by ELISA in the same donors. (E) ImmunoCAP scores for cat dander–specific IgE within cultured BM supernatants from allergic and nonallergic donors. **P ≤ 0.01.

  • Fig. 7 IgE PCs in the BM of allergic patients secrete allergen-specific IgE that can drive mast cell degranulation in FcεRIα humanized mice.

    (A) Humanization strategy for the full coding sequence of FcεRIα. (B) Spleens were harvested from WT or Fcer1ahu/hu mice, and single cell suspensions were stained with antibodies for the basophil marker CD49b and either mouse (top plots) or human (bottom plots) FcεR1α. (C) PCA response of WT or Fcer1ahu/hu mice sensitized with an intradermal injection with a cocktail of two allergen-specific human IgE antibodies or an irrelevant IgG antibody (negative control). (D to G) Ears of FcεRIahu/hu were sensitized by intradermal injection of sera from nonallergic or cat-allergic donors (D), BM supernatant from nonallergic or cat-allergic donors (E), sera from nonallergic or olive-allergic donors [(F), right graph], or BM supernatant from nonallergic or olive-allergic donors [(G), right graph]. Plots show Evans blue dye extravasation as nanograms of Evans blue per milligram of tissue. Left graphs on (F) and (G) show IgE levels in serum and BM supernatant in olive-allergic and nonallergic donors, respectively. *P ≤ 0.05, **P ≤ 0.01, ****P ≤ 0.0001.

Supplementary Materials

  • immunology.sciencemag.org/cgi/content/full/5/43/eaav8402/DC1

    Fig. S1. Chronic HDM exposure induces PC accumulation in the BM.

    Fig. S2. Generation and characterization of IgEVenus/Blimp-1mCherry reporter mice.

    Fig. S3. IgE persists in HDM-exposed mice during rest.

    Fig. S4. IgE+ PCs are detected in the lung but not the spleen of mice exposed to HDM for 15 weeks and rested for 8 months.

    Fig. S5. IgE generated after chronic HDM exposure can drive systemic anaphylaxis.

    Fig. S6. Characterization of allergic and nonallergic donors.

    Fig. S7. Validation of FcεRIα humanized mice.

    Fig. S8. Summary model.

    Table S1. Sγ1 switch region sequence alignment of IgE+ clones from spleen of 4-week HDM-exposed mice and BM of 15-week HDM-exposed mice.

    Table S2. Mouse antibodies used in the study.

    Table S3. Human antibodies used in the study.

    Data file S1. Raw data in Excel spreadsheet.

  • Supplementary Materials

    The PDF file includes:

    • Fig. S1. Chronic HDM exposure induces PC accumulation in the BM.
    • Fig. S2. Generation and characterization of IgEVenus/Blimp-1mCherry reporter mice.
    • Fig. S3. IgE persists in HDM-exposed mice during rest.
    • Fig. S4. IgE+ PCs are detected in the lung but not the spleen of mice exposed to HDM for 15 weeks and rested for 8 months.
    • Fig. S5. IgE generated after chronic HDM exposure can drive systemic anaphylaxis.
    • Fig. S6. Characterization of allergic and nonallergic donors.
    • Fig. S7. Validation of FcεRIα humanized mice.
    • Fig. S8. Summary model.
    • Table S1. Sγ1 switch region sequence alignment of IgE+ clones from spleen of 4-week HDM-exposed mice and BM of 15-week HDM-exposed mice.
    • Table S2. Mouse antibodies used in the study.
    • Table S3. Human antibodies used in the study.

    Download PDF

    Other Supplementary Material for this manuscript includes the following:

    • Data file S1 (Microsoft Excel format). Raw data in Excel spreadsheet.

    Files in this Data Supplement:

Stay Connected to Science Immunology

Navigate This Article