METHOD FOR CLASSIFYING AN ALLERGIC PATIENT AS ELIGIBLE TO ALLERGEN IMMUNOTHERAPY

Abstract

Disclosed is a method of improving global response to allergen desensitization, by allergen immunotherapy, which includes classifying allergic patients as eligible to allergen immunotherapy based on the patients' allergen-specific T cell reactivity against the allergen.

Claims

1. A method for classifying an allergic patient as eligible to allergen immunotherapy, said method comprising screening the T cell reactivity of said patient to an allergen, said screening comprising the following steps: a) contacting with an allergen an isolated biological sample of the patient containing T cells, b) measuring T cell reactivity to said allergen, c) calculating a stimulation index as the ratio of the T cell reactivity measured in step b) on T cell reactivity of the isolated biological sample of the patient containing T cells unstimulated by the allergen, and d) classifying said patient as being eligible to treatment with a composition comprising said allergen if the stimulation index is equal or above a threshold value.

2. The method of claim 1, wherein the threshold value is at least 10.

3. The method of claim 1, wherein measuring T cell reactivity comprises measuring the proliferative response of T cells of the patient specific for the allergen.

4. The method of claim 1, wherein measuring T cell reactivity comprises measuring the number of T cells secreting one or more cytokine(s) selected from the group consisting of IL-4, IL-5, IL-9, IL-13, and IL-17E/IL-25.

5. The method of claim 4, wherein measuring T cell reactivity comprises measuring the number of T cells secreting IL-4 and/or IL-13.

6. The method of claim 1, wherein step a) comprises culturing peripheral blood mononuclear cells (PBMCs) of the patient with the allergen.

7. The method of claim 1, wherein, in step c), T cell reactivity of the isolated biological sample of the patient containing T cells unstimulated by the allergen is measured after culturing the isolated biological sample of the patient containing T cells in a culture medium in absence of the allergen.

8. The method according to claim 1, wherein the allergen comprises or consist of an allergen extract or a mixture of allergenic peptides.

9. The method according to claim 1, wherein the allergen is from a source of allergens to which the patient is allergic.

10. The method according to claim 1, wherein the allergen is an allergen from pollen (from tree, herb, weed or grass), from food, from a house dust mite, or from an animal or insect.

11. The method according to claim 1, which further comprises administering the patient with a composition comprising the allergen if the patient is classified as being eligible to treatment with a composition comprising said allergen.

12. The method according to claim 1, which further comprises, if the patient is not classified as eligible to treatment with a composition comprising said allergen: e) Determining the patient's sensitivity to individual allergenic proteins of the allergen; f) Spiking the allergen with the allergenic protein(s) to which the patient is sensitized; and g) Administering the patient with the spiked allergen.

13. The method of claim 2, wherein measuring T cell reactivity comprises measuring the proliferative response of T cells of the patient specific for the allergen.

14. The method of claim 2, wherein measuring T cell reactivity comprises measuring the number of T cells secreting one or more cytokine(s) selected from the group consisting of IL-4, IL-5, IL-9, IL-13, and IL-17E/IL-25.

15. The method of claim 3, wherein measuring T cell reactivity comprises measuring the number of T cells secreting one or more cytokine(s) selected from the group consisting of IL-4, IL-5, IL-9, IL-13, and IL-17E/IL-25.

16. The method of claim 2, wherein step a) comprises culturing peripheral blood mononuclear cells (PBMCs) of the patient with the allergen.

17. The method of claim 3, wherein step a) comprises culturing peripheral blood mononuclear cells (PBMCs) of the patient with the allergen.

18. The method of claim 4, wherein step a) comprises culturing peripheral blood mononuclear cells (PBMCs) of the patient with the allergen.

19. The method of claim 5, wherein step a) comprises culturing peripheral blood mononuclear cells (PBMCs) of the patient with the allergen.

20. The method of claim 2, wherein, in step c), T cell reactivity of the isolated biological sample of the patient containing T cells unstimulated by the allergen is measured after culturing the isolated biological sample of the patient containing T cells in a culture medium in absence of the allergen.

Description

BRIEF DESCRIPTION OF THE SOLE DRAWING

[0091] FIG. 1 is an illustration of the rationale of the method of classifying patients according to the invention.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

EXAMPLE 1

T Cell Proliferation Assay

[0092] T cell proliferation assays is performed as described in Tscheppe et al. J Allergy Clin Immunol 2020;145:229-38.

[0093] PBMCs are isolated from the blood of allergic patients and cultivated with 1 to 10 μg/mL of the allergen. Proliferation is measured through incorporation of tritiated thymidine within 16 hours. Results are expressed as stimulation indices (Sls), which is calculated as the ratio of the amounts of radioactivity in allergen-stimulated PBMCs and unstimulated cells.

[0094] For confirming allergen-specific proliferation of T CD4.sup.+ cells, carboxyfluorescein N-succinimidyl ester (CFSE) staining is performed, and expression of cell-surface markers is measured by using flow cytometry. In details, freshly isolated PBMCs of patient are cultivated in the presence of CFSE with 6 μg/mL of the allergen. As positive and negative controls 2U of IL-2 and medium are used. On day eight, PBMCs are restimulated with phorbol 12-myristate 13-acetate and ionomycin. Following 2 hours of stimulation, PBMCs are treated with brefeldin A for 5 hours and subsequently stained with Viability Dye 780. After fixing, cells are stained with anti-human CD8 antibody and anti-human CD3 antibody. Viable lymphocytes are gated for CD3.sup.+, CD8.sup.− and CFSE.sup.low. The percentage of proliferating (carboxyfluorescein N-succinimidyl ester-low) cells among CD4.sup.+ T cells (CD3.sup.+CD8.sup.− cells) is thereby evaluated.

EXAMPLE 2

ELISPOT for Assaying IL-4 and/or IL-13 Secretion

[0095] The production of IL-4 and/or IL-13 by PBMCs post-stimulation with the allergen in analyzed in dual ELISPOT assays. Flat-bottom 96-well nitrocellulose plates are coated with either 10 μg/ml of both anti-human IL-4 and anti-human IL-13. PBMC are then incubated at a density of 1×10.sup.5/well either with 1 to 10 μg/ml of the allergen (either allergenic peptide pools or allergen extract), or control medium. After 24 h, cells are removed, and plates are incubated with a cocktail containing biotinylated anti-human IL-4 and horseradish peroxidase (HRP)-conjugated anti-human IL-13 at 37° C. After 2 h, spots corresponding to the biotinylated IL-4 Ab are developed by incubation with Alkaline-phosphatase-Complex (Vector Laboratories, Burlingame, Calif.) and then visualized by applying the Vector Blue Alkaline Phosphatase Substrate Kit III (Vector Laboratories, Burlingame, CA) according to the manufacturer's instructions. Spots corresponding to the HRP-conjugated anti-human IL-13 Ab are visualized by incubation with 3-amino-9-ethylcarvazole solution (Sigma-Aldrich, St. Louis, Mo.). Spots are counted by computer-assisted image analysis (KS-ELISPOT reader, Zeiss, Munich). Each assay is performed in triplicate. The level of statistical significance is determined with a Student t test using the mean of triplicate values of the response against the allergen versus the response against the control. Criteria for response positivity were 20 spot-forming cells (SFCs)/10.sup.6 PBMC, p <0.05, and a stimulation index (SI)•2. SFCs are measured per 10.sup.5 PBMC, subsequently readings are background subtracted and multiplied by 10 to be expressed as SFC per million PBMC.