1. Rapid polyclonal desensitization with antibodies to IgE and FcεRIα
Marat V. Khodoun, PhD, Zeynep Yesim Kucuk, MD, Richard T. Strait, Durga Krishnamurthy, MS, Kevin Janek, BS, Ian Lewkowich, PhD, Suzanne C. Morris, PhD, Fred D. Finkelman, MD 
Journal of Allergy and Clinical Immunology 
Volume 131, Issue 6, Pages e7 (June 2013)
DOI: /j.jaci
Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

2. Fig 1
Rapid desensitization with an activating anti-IgE mAb. A, In 3 separate experiments, BALB/c mice (4 per group) were injected intravenously every 90 minutes with doubling or tripling doses of anti-IgE mAb (EM-95) or an isotype-matched control mAb for a total of 8 doses, starting with 0.05 μg. Mice were then challenged the next day intravenously with 36 μg of anti-IgE mAb. Rectal temperatures were determined during the 90 minutes after challenge. Means ± SEs of maximum decreases in temperature are shown. B, The lowest temperature after each intraperitoneal dose of anti-IgE mAb is shown for each of the 3 experiments. Additional experiments that varied dose number and dose increment did not reliably avoid temperature drops >0.5°C. *P < .05 compared with mice desensitized with control mAb. †P < .05 compared with temperature of unchallenged mice. Expt, Experiment.
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3. Fig 2
Anti-FcεRIα mAb induces anaphylaxis that can be blocked by rapid desensitization or antihistamine. A, Mice were serially injected intraperitoneally every 60 to 90 minutes with the doses of MAR-1 or control mAb shown, starting with 0.05 μg. The mean ± SE maximum decrease in temperature during the 60 minutes after each injection is shown. Results are representative of 2 experiments, 4 mice per group. B, Mice were mock-desensitized with control mAb or desensitized with MAR-1 as in A, then challenged intravenously with 40 μg of MAR-1. Rectal temperatures were followed for the next 60 minutes. Results are representative of 2 experiments, 4 mice per group. C, Mice were injected intraperitoneally with IL-4C that contained 1 μg of IL-4, then the next day serially injected intraperitoneally every 60 to 90 minutes with the doses of MAR-1 or control mAb shown, starting with 0.05 μg. The mean ± SE maximum decrease in temperature during the 60 minutes after each injection is shown. Results are pooled from 2 experiments, total of 8 mice per group for MAR-1–treated mice and 6 mice per group for control mAb-treated mice. D, In a separate experiment, mice that had been treated with IL-4C and desensitized with MAR-1 or mock-desensitized with control mAb, as in Fig 4C, were challenged intravenously with 40 μg of MAR-1. E, Mice were injected intravenously with a single 40-μg dose of MAR-1 45 minutes after pretreatment with saline, antihistamine (triprolidine), dexamethasone, or antihistamine plus dexamethasone. Rectal temperatures were determined during the subsequent 60 minutes. Results were pooled from 2 experiments, total of 8 mice per group.
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4. Fig 3
Mechanism of MAR-1 rapid desensitization. A, Peritoneal mast cells and blood basophils from IgE-deficient mice were incubated on ice with 20 μg of IgE anti-TNP mAb, MAR-1, or control mAb, then washed, and incubated with 1 of the 3 same mAbs. Cells were washed again and stained for FcεRIα (left panels), IgE (middle panels), or hamster IgG (right panels) and analyzed for surface fluorescence after gating on basophil or mast cell markers. Results are representative from 1 of 2 experiments, 4 mice per group. B, WT and FcεRIα− mice were injected intravenously once or twice with saline or 200 μg of mouse IgE at 2-hour intervals, then challenged intravenously 1 hour later with 40 μg of MAR-1. Rectal temperatures were determined. Results are representative from 1 of 2 experiments, 4 mice per group. C, IL-4 secretion was determined by IVCCA for 4 hours after challenge with MAR-1 in the same experiment shown in B. Results are representative from 1 of 2 experiments. D, Peritoneal mast cells from wild-type BALB/c mice were cultured for 1 hour at 4°C or 37°C with 20 μg/mL MAR-1 or control mAb, then stained for IgE and analyzed by flow cytometry. Results are representative from 1 of 2 experiments, 4 mice per group. E, BALB/c mice were injected intravenously with 10 μg of IgE anti-TNP mAb, then rapidly desensitized with MAR-1 or mock-desensitized with control mAb. Next, mice were injected intravenously with 50 μg of TNP-OVA or 100 μg of EM-95 2 hours after the last dose of MAR-1 or control mAb. Rectal temperatures were determined. Results are representative from 1 of 2 experiments, 4 mice per group. F, BALB/c mice were left untreated or were rapidly desensitized with MAR-1. Some mice were injected intravenously 2 hours after the last MAR-1 dose with 100 μg of anti-IgE mAb. Serum MMCP1 and IL-4 production were evaluated. Results are representative from 1 of 2 experiments, 4 mice per group. G, BALB/c mice were rapidly desensitized with MAR-1 or mock-desensitized with control mAb, then injected intravenously 2 days later with 100 μg of anti-IgE mAb. Rectal temperatures were determined. Results are representative from 1 of 2 experiments, 4 mice per group. H and I, BALB/c mice were injected intraperitoneally with 10 μg of IgE anti-TNP mAb. Mice were rapidly desensitized 24 hours later with MAR-1 or treated with equal doses of normal hamster IgG. Mice were re-injected with 10 μg of IgE anti-TNP mAb 2, 5, and 11 days after the initial IgE anti-TNP mAb treatment and with 40 μg of MAR-1 or control mAb 2, 5, 8, and 11 days after the initial MAR-1 treatment. Mice were challenged intravenously with 50 μg of TNP-OVA on the days shown. The mean maximum decrease in rectal temperature (H) and the mean serum MMCP1 level (I) 4 hours after challenge are shown for mice challenged 2 hours or 2, 4, 8, or 14 days after desensitization. Similar results were obtained in a second experiment in which MAR-1 or control mAb was injected without rapid desensitization, 4 mice per group. J, Peritoneal lavage cells from mice rapidly desensitized with MAR-1 or control mAb 2 hours or 2 days before cell collection were loaded with Fluo-4. Relative levels of intracellular Ca+2 ([Ca+2]i) were determined at baseline and immediately after in vitro challenge with anti-IgE mAb. Results are pooled from 2 experiments, total of 8 mice per group. †P < .05 for decrease compared with control. K, BALB/c mice were left untreated or rapidly desensitized with MAR-1, then injected intravenously with 3.5 mg of histamine or 500 ng of PAF. Rectal temperatures were determined. Results are pooled from 2 experiments, total of 8 mice per group. IVCCA, In vivo cytokine capture assay; MFI, mean fluorescent intensity; MMCP1, mouse mast cell protease 1; PAF, platelet-activating factor; TNP, trinitrophenyl; WT, wild-type.
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5. Fig 4
Suppression of FcεRI expression by anti-FcεRIα mAb. A, WT BALB/c mice were rapidly desensitized with MAR-1 or mock-desensitized with control mAb (maximum dose 40 μg), then treated every 2 days for 8 days with 40 μg of MAR-1 or control mAb. Peritoneal nucleated cells were then incubated for 30 minutes on ice with no mAb, 12.5 μg/mL MAR-1, or 12.5 μg/mL IgE anti-TNP mAb, then stained for IgE or hamster IgG. Average mean fluorescence is shown. Results are from 1 experiment, 4 mice per group. B, WT BALB/c mice were treated with IgE anti-TNP mAb (10 μg on days 0, 4, 6, and 9) and rapidly desensitized with MAR-1 or isotype control mAb on day 1. Mice were re-injected with 40 μg of MAR-1 or isotype control mAb on days 5, 7, and 10. Peritoneal mast cells obtained on the days shown were stained for FcεRIa, IgE, or hamster IgG. In addition, peritoneal mast cells obtained on days 0 and 16 from control mAb-treated mice were incubated with MAR-1 at 4°C, then stained for hamster IgG (right panel). Average mean fluorescence is shown. Results are representative of 2 experiments, 4 mice per group. C, Naive WT, naive IL-4 transgenic (TG.UG), and OVA-immune WT mice were left untreated or rapidly desensitized with MAR-1 or control mAb. Peritoneal mast cells obtained before or on 1 or 4 days after MAR-1 injection were stained for IgE and analyzed by flow cytometry. Peripheral blood was analyzed for percentage of basophils, 4 mice per group. Similar results were obtained in a second experiment in which mice were injected intraperitoneally with a single 40-μg dose of MAR-1 or control mAb. D, Serum IgE levels were determined by ELISA for naive WT and TG.UG C57BL/6 mice (left) and for naive and OVA-immune BALB/c mice (right). Results are from 1 experiment, 4 mice per group. E, BALB/c peritoneal mast cells were incubated on ice with pHRhodo-labeled MAR-1 or control mAb. BALB/c mice were rapidly desensitized with the same mAbs, and peritoneal mast cells were obtained 3 hours later. Cells were analyzed for pHRodo fluorescence by flow cytometry. Results are representative of 2 experiments, 4 mice per group. F, IgE-deficient mice were injected intravenously with 500 μg of anti-CD23 mAb to block FcεRII, then with 10 μg of IgE anti-TNP mAb. The next day, mice were injected with 50 μg of MAR-1 or control mAb. Peritoneal mast cells obtained before or 1 or 4 days after MAR-1 or control mAb injection were analyzed for membrane IgE. Results are representative of 2 experiments, 4 mice per group. G, WT BALB/c mice were rapidly desensitized with MAR-1 or control mAb, then injected intraperitoneally with 50 μg of the same mAb on days 4, 8, and 12. Rectal temperatures were obtained 1 hour after each repeated mAb dose. Means ± SEs of temperatures after each dose are shown. Data were pooled from 2 experiments, total 7 to 8 mice per group. *P < .05 for increase compared with control. †P < .05 for decrease compared with control. MFI, Mean fluorescent intensity; TNP, trinitrophenyl; WT, wild-type.
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6. Fig 5
Suppression of IgE-mediated anaphylaxis with MAR-1. A, BALB/c mice were rapidly desensitized with MAR-1 or control mAb, then treated with 40 μg of MAR-1 or control mAb on days 5, 12, and 18 and injected on day 22 with antihistamine or saline and challenged with anti-IgE mAb. Rectal temperature was determined. Sera obtained 5 minutes and 4 hours later were assayed for histamine and MMCP1, respectively. Data were pooled from 2 experiments, total of 8 mice per group. B, BALB/c mice were initially injected intravenously with 10 μg of IgE anti-TNP mAb, followed by MAR-1 or control mAb, given intraperitoneally initially as a single 40-μg injection or by rapid desensitization. All mice then were injected intraperitoneally every 7 days for 21 days with 40-μg doses of MAR-1 or control mAb. All mice received a second 10-μg dose of IgE anti-TNP mAb on day 23 and were challenged on day 24 with TNP-OVA. Rectal temperatures were determined. Results are pooled from 2 experiments, 8 mice per group; similar results were obtained when mice were initially injected once or rapidly desensitized with MAR-1. C, In the same experiments shown in B, IL-4 and IL-13 production were determined by IVCCA for the 4 hours after TNP-OVA challenge. Results are pooled from 2 experiments, total 8 mice per group. D, Mice immunized intraperitoneally with goat anti-mouse IgD antiserum on day 0 were rapidly desensitized with MAR-1 or control mAb intraperitoneally on day 8 and re-injected with the same mAb on day 12. All mice were injected intraperitoneally on day 14 with 500 μg of anti-FcγRIIb/RIII mAb to block IgG-mediated anaphylaxis (2.4G2 has no effect on IgE-mediated histamine responses; data not shown). Mice were challenged intravenously with saline or with 5 mg of goat IgG on day 15. Rectal temperatures and IL-4, IL-13, and MMCP1 secretion were determined. Data were pooled from 2 experiments, total of 8 mice per group. E, BALB/c mice were immunized intraperitoneally with OVA/alum on days 0 and 12, then rapidly desensitized intraperitoneally with MAR-1 on day 14 and injected intraperitoneally on day 16 and 18 with 40 μg of MAR-1 or control mAb. Mice were injected intraperitoneally with 500 μg of 2.4G2 on day 19 to block IgG-mediated anaphylaxis and challenged intravenously with 200 μg of OVA the next day. Changes in rectal temperature and IL-4, IL-13, and MMCP1 secretion were determined. Data were pooled from 2 experiments, total of 8 mice per group. †P < .05 for decrease compared with control. **P < .05 for decrease compared with control mAb-treated mice. Desens, Desensitization; GaMD, goat anti-mouse IgD antibody; GIgG, goat IgG; IVCCA, in vivo cytokine capture assay; MMCP1, mouse mast cell protease 1; TNP, trinitrophenyl.
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7. Fig 6
Suppression of active anaphylaxis with anti-FcεRIα mAb. A, BALB/c mice were immunized twice intraperitoneally with EW/alum, then treated intraperitoneally with IL-4C that contained 1 μg of IL-4. The next day, mice were rapidly desensitized with EW (6 doubling doses, starting at 6 μg) or MAR-1 (8 doubling doses, starting at 0.4 μg), or mock-desensitized with BSA (negative control, 6 doubling doses, starting at 6 μg). The minimum rectal temperature for each mouse during rapid desensitization is shown, along with the mean ± SEM of minimum rectal temperature for the group. Data were pooled from 2 experiments except 1 experiment for the BSA (negative control) group, 4 mice per group; total of 8 mice per group, except 4 mice for the BSA group. *Significant lowering in rectal temperature compared with BSA group. B, BALB/c mice were immunized with EW as in Fig 6, A, then injected intraperitoneally 17 days after the initial immunization with 500 μg of 2.4G2 to block IgG-mediated anaphylaxis. Mice were then rapidly desensitized with EW or MAR-1 or mock-rapidly desensitized and challenged intravenously with 200 μg of EW 2, 24, or 48 hours after rapid desensitization. All mice were challenged with EW 1 to 2 days after 2.4G2 injection. Results are from 2 experiments, total of 8 mice per group. Mean maximum temperature drop and percentage of survival for >2 hours is shown for each group. †Significantly lower decrease in temperature drop and/or mortality for a group above the box compared with a group to the right of the box. *Significantly greater decrease in temperature drop and/or mortality for a group above the box compared with a group to the right of the box. Colors code for comparisons for groups challenged 2 hours (black), 24 hours (red), or 48 hours (green) after rapid desensitization. BSA, Bovine serum albumin.
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8. Fig E1
FcεRIα expression. Fluorescence histograms of the following cell types stained with MAR-1 anti-FcεRIα mAb (open red histograms) or with a hamster IgG control mAb (filled purple histograms) are shown: Peripheral blood basophils (IL-3R+CD49b+B220−FSCloSSClo), peritoneal mast cells (c-kit+IL-3R+FSChiSSChi), peripheral blood eosinophils (CCR3hiFSCintSSChi), splenic CD8+ T cells (CD3+CD4−CD8+FSCloSSClo), splenic CD4+ T cells (CD3+CD4+CD8−FSCloSSClo), splenic B cells (B220+CD19+CD3−FSCloSSClo), peripheral blood monocytes (CD11b+Ly6G−F4/80loFSCloSSClo), peripheral blood neutrophils (CD11b+Ly6GhiFSCintSSCint), and a subset of splenic dendritic cells that includes an FcεRI+ population (FSC and SSC low, CD11cint, CD11bint, CD317−, CD8α−, Gr1−). Numbers above each set of histograms indicate the mean percentage of cells positive for FcεRIα ± SEM (blue) and the mean fluorescence intensity ± SEM of FcεRIα+ cells (green). Dendritic cells were analyzed on a different flow cytometer than the other cells; hence, mean fluorescence intensities of positive cells cannot be compared. Results are pooled from 2 experiments, total 8 mice per group.
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9. Fig E2
Anti-FcεRIα mAb induces anaphylaxis. A, BALB/c mice were injected intravenously with 50 μg of hamster anti-mouse FcεRIα mAb (MAR-1) or a hamster IgG control mAb. Rectal temperature was followed for 45 minutes. Results are representative of 2 experiments, 4 mice per group. B, Serum MMCP1 4 hours after mAb injection was determined by ELISA. Results are representative of 2 experiments, 4 mice per group. C, Serum histamine 5 minutes after mAb injection was determined by ELISA. Results are representative of 2 experiments, 4 mice per group. D, IL-4 secretion during the 4 hours after injection of the indicated MAR-1 dose was determined by IVCCA. Results are pooled from 2 experiments, total 8 mice per group. E, Rectal temperature was followed for 1 hour after a single intravenous injection of the doses of MAR-1 shown; the average maximum temperature decrease for the mice in each group is indicated. Results are pooled from 2 experiments, total 8 mice per group. IVCCA, In vivo cytokine capture assay; MMCP1, mouse mast cell protease 1. ∗P < .05 for increase compared to control mAb or saline group. †P < .05 for increase compared to Anti-FcεRIα, 10 μg group.
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10. Fig E3
IgE blocks MAR-1 binding to mast cells. BALB/c peritoneal wash cells were treated with 6 μg/mL of an IgE mAb (IGEL2a) or an IgG2b mAb (negative control), washed, stained with 6 μg/mL MAR-1 or normal hamster IgG, washed again, and stained for hamster IgG, c-kit, and IL-3R. Cells were gated on high forward and side scatter, c-kit, and IL-3R and were analyzed for hamster IgG staining (ie, MAR-1 binding) on the gated cells. Results are representative of 3 mice per group.
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11. Fig E4
Effects of anti-IgE, anti-FcεRIα mAb on cell numbers. BALB/c mice were rapidly desensitized with MAR-1 or isotype control mAb on day 0. mAbs (40 μg) were also injected intraperitoneally on days 4, 8, and 12. The percentages or numbers of the cell populations shown in peritoneal lavage, spleen, and peripheral blood were determined 3 days (A) or 14 days (B) after rapid desensitization. Cell types were identified and enumerated by flow cytometry, using the strategies shown in Fig E1. In addition, tongue mast cell numbers per 10 high-powered fields were determined. In separate experiments (C), peripheral blood basophil and peritoneal lavage mast cells numbers were determined 3 days after rapid desensitization with EM-95 anti-IgE mAb. Results were pooled from 2 experiments, total 8 mice per group. †P < .05 for decrease compared with control. DC, Dendritic cell; HPF, high-powered field; NK, natural killer; PBNC, peripheral blood nucleated cell.
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12. Fig E5
MAR-1 eliminates FcεRI+ DCs. Upper panels, Gating strategy to identify DC subpopulation that contains FcεRIα+ DCs. Lower panels, Treatment with MAR-1 for 3 days caused loss of staining for both FcεRI and IgE. Results are representative of 2 experiments, 4 mice per group per experiment. DC, Dendritic cell; FSC, forward scatter.
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13. Fig E6
MAR-1 progressively decreases mast cell staining for IgE and depletes basophils. Treatment with MAR-1 for 1 or 4 days causes progressive loss of IgE staining on peritoneal mast cells and disappearance of blood basophils. Mast cells gated on high forward and side scatter, IL-3R+kit+ cells; basophils gated on low-intermediate forward and side scatter, IL-3R+CD49b+ cells. Results are representative of 2 experiments, 4 mice per group per experiment.
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