Synchronous immune alterations mirror clinical response during allergen immunotherapy

Abstract

Background: Three years of treatment with either sublingual or subcutaneous allergen immunotherapy has been shown to be effective and to induce long-term tolerance. The Gauging Response in Allergic Rhinitis to Sublingual and Subcutaneous Immunotherapy (GRASS) trial demonstrated that 2 years of treatment through either route was effective in suppressing the response to nasal allergen challenge, although it was insufficient for inhibition 1 year after discontinuation.

Objective: We sought to examine in the GRASS trial the time course of immunologic changes during 2 years of sublingual and subcutaneous immunotherapy and for 1 year after treatment discontinuation.

Methods: We performed multimodal immunomonitoring to assess allergen-specific CD4 T-cell properties in parallel with analysis of local mucosal cytokine responses induced by nasal allergen exposure and humoral immune responses that included IgE-dependent basophil activation and measurement of serum inhibitory activity for allergen-IgE binding to B cells (IgE-facilitated allergen binding).

Results: All 3 of these distinct arms of the immune response displayed significant and coordinate alterations during 2 years of allergen desensitization, followed by reversal at 3 years, reflecting a lack of a durable immunologic effect. Although frequencies of antigen-specific T_H2 cells in peripheral blood determined by using HLA class II tetramer analysis most closely paralleled clinical outcomes, IgE antibody-dependent functional assays remained inhibited in part 1 year after discontinuation.

Conclusion: Two years of allergen immunotherapy were effective but insufficient for long-term tolerance. Allergen-specific T_H2 cells most closely paralleled the transient clinical outcome, and it is likely that recurrence of the T-cell drivers of allergic immunity abrogated the potential for durable tolerance. On the other hand, the persistence of IgE blocking antibody 1 year after discontinuation might be an early indicator of a protolerogenic mechanism.

Keywords: Allergy; T(H)2 cells; allergen desensitization; immune tolerance; immunotherapy.

Fig. 1

Analysis of allergen-specific CD4 T cells and relationship to the clinical parameters measured during the GRASS trial. (A) Representative flow cytometry analysis of tetramer bound allergen-specific T cells from a study subject receiving subcutaneous allergen immunotherapy. Lymphocytes that bind the pooled tetramers are displayed on the y axis and expression of phenotypic cell surface markers CD4, CRTH2, CD27, CD161 and CCR4 are displayed on the x axis at baseline, at 2 years after continuous SCIT therapy and at 3 years after one year off therapy. (B) Frequencies of allergen-specific CD4 T cells were determined for 53 HLA-DR4 subjects in the GRASS trial, by tetramer binding (left panel). Frequencies of Th2 cells are identified by CD161+CRTH2+CD27−CCR4+T4+/CD45RA−T4+ phenotypic marker expression (right panel). (C) The total nasal symptom score (TNSS) average AUC (left panel) and the peak nasal inspiratory flow (PNIF) average AUC (right panel) for 0–10 hours following allergen challenge were measured at baseline and years 1–3 for all participants treated with sublingual immunotherapy (green), subcutaneous immunotherapy (red), and placebo (blue). Significant differences are indicated by * (p<.05), ** (p<.01). Data are shown as means with 95% confidence intervals, for the 84 per-protocol subjects enrolled in the GRASS trial.

Fig. 2

Cytokine levels of nasal fluids following nasal allergen challenge. The levels of cytokines IL-4, IL-5 and IL-13 in nasal fluids for 10 hours following nasal allergen challenge are displayed from study subjects at baseline, after 2 years of desensitization and at 3 years, one year after discontinuation of desensitization therapy. Participants treated with sublingual immunotherapy are displayed in green, subcutaneous immunotherapy in red and placebo in blue. Data are shown as means with 95% confidence intervals.

Fig. 3

Allergen-specific IgE-dependent functional assays. (A) The impact of the IgG4 increase on the ability of allergen-IgE complexes to bind B cells was measured by IgE-FAB assay. The serum from patients was evaluated for ability to inhibit allergen-IgE complex binding at baseline and years 1–3. (B) Grass pollen-specific IgG4 and IgE were monitored and the IgG4/IgE ratio is displayed for subjects at baseline, after years 1 and 2 during desensitization therapy and at 3 years after discontinuation of therapy. (C) Basophil surface activation markers from whole blood of participants treated with sublingual immunotherapy (green), subcutaneous immunotherapy (red) and placebo (blue) at baseline and years 1–3 after incubation with grass pollen allergen. Significant differences are indicated by * (p<.05), ** (p<.01). Data are shown as means with 95% confidence intervals.

Fig. 4

Relationship between nasal cytokine measurements and lower peripheral blood antigen-specific T cells (memory tetramer + cells) and serum antigen-specific IgE-FAB for each individual studied. Data displayed are expressed as fold changes from the baseline at year 2 (on treatment) for each immunological parameter measured for participants treated with Sublingual immunotherapy (green diamonds), Subcutaneous immunotherapy (red triangles), and placebo (blue circles), so values <1 on each axis represent reduction (improvement) in the parameters shown. Nasal cytokine measurements are the summation of area under curve (AUC) from 2 to 10 hours post-challenge for Th2 cytokines (IL-4, IL-5 and IL-13). Lower peripheral blood antigen-specific T cells are measured as the frequency of memory tetramer + cells per million CD4+ cells. Serum antigen-specific IgE-FAB is measured as the percentage of allergen-IgE binding to B cells. Cluster distributions were compared using a Hotelling T-square test, as follows: Placebo vs. SCIT: p<0.001; Placebo vs. SLIT: p<0.001; SLIT vs. SCIT: p=0.31. An online interactive version of this Figure is available at https://www.itntrialshare.org/GRASSmech_fig4.url.