Changes in antigen-specific T-cell number and function during oral desensitization in cow's milk allergy enabled with omalizumab

Abstract

Food allergy is a major public health problem, for which there is no effective treatment. We examined the immunological changes that occurred in a group of children with significant cow's milk allergy undergoing a novel and rapid high-dose oral desensitization protocol enabled by treatment with omalizumab (anti-immunoglobulin (Ig)E monoclonal antibodies). Within a week of treatment, the CD4(+) T-cell response to milk was nearly eliminated, suggesting anergy in, or deletion of, milk-specific CD4(+) T cells. Over the following 3 months while the subjects remained on high doses of daily oral milk, the CD4(+) T-cell response returned, characterized by a shift from interleukin-4 to interferon-γ production. Desensitization was also associated with reduction in milk-specific IgE and a 15-fold increase in milk-specific IgG4. These studies suggest that high-dose oral allergen desensitization may be associated with deletion of allergen-specific T cells, without the apparent development of allergen-specific Foxp3(+) regulatory T cells.

Trial registration: ClinicalTrials.gov NCT00968110.

Figure 1. Milk-induced CD4⁺ T cell proliferation is greatly reduced during the rush desensitization phase

(a) Frozen PBMC, isolated from 5 milk allergic patients undergoing rush milk desensitization, were thawed and labeled with CFSE, and cultured in the presence of tetanus toxoid (TT) or milk proteins (MP) for 7 days. Cells were then collected, stained with anti-CD4 mAb, and analyzed by flow cytometry. The number in each panel represents the fraction of total CD4⁺ cells that are within the CFSE^low CD4⁺ gate (antigen-specific proliferating cells) on day 0. Mean CD4⁺ milk-specific proliferating cells=7.75%; mean CD4⁺ tetanus specific cells=24.6%. Mean CD4⁺ proliferating cells without antigen=0.62%. (b) Each line represents data for all 5 patients combined (solid line, milk protein (MP); dashed line, tetanus toxoid (TT)). Data represent mean % antigen-specific cells (CFSE^low CD4⁺ cells) of total CD4⁺ cells, normalized to day 0 for each patient ± SEM, over the course of the study. ** p < 0.01, *** p < 0.001 versus baseline and week 8, determined using paired t-test. ° p < 0.05 versus TT response, determined using two-way ANOVA for matched values with Bonferroni’s post hoc test. (c) Each line represents data for an individual patient, of % milk-specific CFSE^low CD4⁺ cells of total CD4⁺ cells, normalized to day 0 for each patient, over the course of the study.

Figure 2. Milk specific Foxp3⁺ T cells are not detected during the milk desensitization

(a–c) CFSE-labeled PBMC isolated from 5 milk allergic patients undergoing milk desensitization were cultured in the presence of milk proteins for 7 days. Cells were then collected, fixed and permeabilized, stained with anti-CD4 and anti–Foxp3, and analyzed by flow cytometry. (a–b) Figures represent combined percentages of CD4⁺ Foxp3⁺ cells of total cells normalized to day 0. Data represent mean ± SEM. (a) Normalized CD4⁺ CFSE^low cells (proliferating cells, representing milk-specific T_Reg cells). (b) Normalized CD4⁺ CFSE^high cells (nonproliferating cells, representing natural T_Reg cells). (c) Individual dot plots of flow cytometry analysis are shown. CD4⁺CFSE^low T cells (left panels) represent milk-specific T_Reg cells, which did not increase over time. CD4⁺CFSE^high T cells (right panels) represent natural T_Reg cells, which did not change over time.

Figure 3. Functional regulatory T cells are not detected during the milk desensitization

(a–b) CFSE-labeled PBMC isolated from 5 patients undergoing milk desensitization were cultured in the presence of milk (MP) for 7 days. Cells were then collected, stained with anti-CD4 mAb, and analyzed by flow cytometry. (a) Frozen samples from multiple time points were thawed and cultured with or without anti-IL-10 mAb and milk proteins. Percentages of the milk-specific cell populations (CFSE^low) within the CD4⁺ gate are shown. Results with and without anti-IL-10 mAb overlap, indicating an absence of IL-10 mediated suppression. (b) PBMC from week 12 were cultured with or without anti-IL-10, anti-TGF-β mAb or both and compared to baseline PBMC. Figures represent percentages of the milk-specific cell populations (CFSE^low) within the CD4⁺ gate. No increase in proliferation was observed with the mAbs, indicating an absence of IL-10 or TGF-β mediated suppression. (c) CD4⁺ T cells and regulatory T cells were purified from week 12 and week 14 PBMC, respectively. 1× 10⁴ (+) or 5 × 10⁴ (++) CD4⁺ CD25⁺ cells, or 1.5 × 10⁴ (+) or 7.5 × 10⁴ (++) CD4⁺ cells were then cocultured with 2 × 10⁵ baseline PBMC in the presence of milk proteins. The proliferation was measured as [³H]thymidine incorporation during the last 16 hours of a 6-day culture.

Figure 4. Evidence of milk-specific CD4⁺ T cell anergy

(a). PBMC were isolated from week 12 samples of 5 milk allergic patients undergoing milk desensitization protocol, labeled with CFSE, cultured in the presence of milk proteins (MP) for 7 days and treated with or without IL-2. Cells were then collected and stained with anti-CD4 mAb. Proliferation was analyzed by flow cytometry and compared to PBMC proliferation from baseline samples. Figure shows percentages of the milk-specific cells (CFSE^low) within the CD4⁺ gate as mean ± SEM for 5 patients. ** p < 0.01, *** p < 0.001. Significance was determined using paired t-test. (b–d). Phenotype of the milk specific CD4⁺ T cells during desensitization. CFSE-labeled PBMC isolated from all 5 of the milk allergic patients undergoing milk desensitization were cultured in the presence of milk proteins for 7 days, then analyzed by flow cytometry. (b) Data represent CD25 expression (mean CD25 MFI (mean fluorescence intensity)) by milk specific (CD4⁺ CFSE^low) cells ± SEM for the 5 patients evaluated, normalized to results on day 0. (c) Solid line represents CD25 expression (mean MFI) for milk specific cells from the 4 patients who were desensitized and who passed the DBPCFC, normalized to results on day 0. Dashed line represents CD25 expression (MFI) by milk specific cells from patient #3, who was only partially desensitized, normalized to results on day 0. * p < 0.05 versus baseline and week 8, using paired t-test. (d) Each line represents CD25 expression (MFI) on milk specific cells from each patient normalized to results on day 0. Patient #3 was only partially desensitized.

Figure 5. Evidence for immune deviation during milk desensitization

(a–c) PBMC isolated from 5 milk allergic patients undergoing milk desensitization, labeled with CFSE, and cultured in the presence of antigens for 7 days, were restimulated with PMA and ionomycin in the presence of monensin for 5 h. Cytokine production was then assessed using intracellular IL-4, IFN-γ and IL-13 staining. (a) Data for all 5 patients. IFN-γ/IL-4 production ratio for the antigen-specific (CFSE^low) CD4⁺ T cells, normalized to day 0 is shown (mean ± SEM). (b) Solid line represents data from (A), for desensitized patients (patients #1, 2, 4 and 5), and dotted line represents data from (A), for the partially desensitized patient (patient #3). * p < 0.05 versus baseline and week 8, using paired t-test. (c) Each line represents data for an individual patient, representing IFN-γ/IL-4 production ratio by the antigen-specific CD4⁺ T cells, normalized to day 0. (d) IL-13 production was also assessed in CD4⁺ T cells from patient #5. Data represents IFN-γ/IL-13 production by milk-specific CD4⁺ T cells. (e–f) Serum milk-specific IgE (e) and IgG4 (f) levels were determined for the 10 patients who completed the study. Points represent data from different individuals and bars show mean values ± SEM. * p < 0.05, ** p < 0.01 versus baseline, determined using nonparametric Wilcoxon matched-pairs test.

Figure 6. Milk skin test reactivity and in vitro basophil activation are reduced by oral milk desensitization

(a) Skin prick test wheal with cow’s milk allergen extract (n=10) was reduced at week 52 compared to week 0. ** P < 0.01 determined using paired t-test. ° patient #3, who was slow to become desensitized. (b) Skin prick test wheal with cashew or ovalbumin did not change, comparing week 0 and week 52 time points in 4 patients known to have those allergies. (c) Basophils in whole blood, taken on day 0, but not subsequently, when stimulated ex vivo for 20 minutes with cow’s milk (CM), were activated, as assessed by expression of CD203c, CD63 and histamine release (n=5). Treatment with vehicle/glycerine showed no shift in CD203c or CD63 expression (data not shown). MFI = median fluorescence intensity. (d) Basophil activation with cashew or ovalbumin was blocked by in vivo treatment with omalizumab, as assessed on weeks 8, 9, 10 and 16. Prior to and after omalizumab treatment, basophil activation with cashew or ovalbumin was robust, as assessed by expression of CD203c, CD63 and histamine release.