Oral myeloid cells uptake allergoids coupled to mannan driving Th1/Treg responses upon sublingual delivery in mice

I Soria, J López-Relaño, M Viñuela, J-I Tudela, A Angelina, C Benito-Villalvilla, C M Díez-Rivero, B Cases, A I Manzano, E Fernández-Caldas, M Casanovas, O Palomares, J L Subiza, I Soria, J López-Relaño, M Viñuela, J-I Tudela, A Angelina, C Benito-Villalvilla, C M Díez-Rivero, B Cases, A I Manzano, E Fernández-Caldas, M Casanovas, O Palomares, J L Subiza

Abstract

Background: Polymerized allergoids coupled to nonoxidized mannan (PM-allergoids) may represent novel vaccines targeting dendritic cells (DCs). PM-allergoids are better captured by DCs than native allergens and favor Th1/Treg cell responses upon subcutaneous injection. Herein we have studied in mice the in vivo immunogenicity of PM-allergoids administered sublingually in comparison with native allergens.

Methods: Three immunization protocols (4-8 weeks long) were used in Balb/c mice. Serum antibody levels were tested by ELISA. Cell responses (proliferation, cytokines, and Tregs) were assayed by flow cytometry in spleen and lymph nodes (LNs). Allergen uptake was measured by flow cytometry in myeloid sublingual cells.

Results: A quick antibody response and higher IgG2a/IgE ratio were observed with PM-allergoids. Moreover, stronger specific proliferative responses were seen in both submandibular LNs and spleen cells assayed in vitro. This was accompanied by a higher IFNγ/IL-4 ratio with a quick IL-10 production by submandibular LN cells. An increase in CD4+ CD25high FOXP3+ Treg cells was detected in LNs and spleen of mice treated with PM-allergoids. These allergoids were better captured than native allergens by antigen-presenting (CD45+ MHC-II+ ) cells obtained from the sublingual mucosa, including DCs (CD11b+ ) and macrophages (CD64+ ). Importantly, all the differential effects induced by PM-allergoids were abolished when using oxidized instead of nonoxidized PM-allergoids.

Conclusion: Our results demonstrate for the first time that PM-allergoids administered through the sublingual route promote the generation of Th1 and FOXP3+ Treg cells in a greater extent than native allergens by mechanisms that might well involve their better uptake by oral antigen-presenting cells.

Keywords: allergoid; glycoconjugate; immunotherapy; mannan; sublingual.

© 2018 The Authors. Allergy Published by John Wiley & Sons Ltd.

Figures

Figure 1
Figure 1
Sublingual immunizations with Phleum pratense pollen allergens and assayed immunological parameters. (A) Schematic diagrams of the three different protocols used. The immunogens were as follows: N (native allergen); PM (PM‐allergoids); PM‐OX (PM‐allergoids further oxidized); PBS (phosphate‐buffered saline as a negative control). (B) After the last immunization, all animals were killed. Blood was collected for the measurement of specific antibodies in serum. Spleen and submandibular LN cells were isolated and cultured in vitro for assessing cytokines production and cell proliferation. In some experiments, FOXP3+ cells were determined in freshly isolated spleen and submandibular LNs
Figure 2
Figure 2
Serum antibody response in mice after sublingual immunization with Phleum pratense pollen allergens. The immunogens were as follows: N (native allergen); PM (PM‐allergoids); PM‐OX (PM‐allergoids further oxidized); PBS (phosphate‐buffered saline as a negative control). (A) Serum levels of IgG1, IgG2a, and IgE were measured by ELISA against native allergens (Phleum pratense) in mice immunized with different protocols. (B) IgG2a/IgE ratios of antibody levels from each individual mouse immunized with different protocols. Results are the mean ± SEM of 6 mice. Statistical differences were analyzed with Student's t test. *P < .05, **P < .01, ***P < .001
Figure 3
Figure 3
Proliferative T‐cell response in submandibular LNs and spleen from mice after sublingual immunization with Phleum pratense pollen allergens. The immunogens were as follows: N (native allergen); PM (PM‐allergoids); PM‐OX (PM‐allergoids further oxidized); PBS (phosphate‐buffered saline as a negative control). Proliferation was measured by flow cytometry (CFSE assay) in response to the native allergens (Phleum pratense) after 5‐day culture. The results obtained with submandibular LN (A) or spleen (B) cells, from mice immunized with different protocols, are the mean ± SEM of 6 mice per group. Statistical differences were analyzed with Student's t test. *P < .05, **P < .01, ***P < .001
Figure 4
Figure 4
Cytokine production in submandibular LNs and spleen from mice after sublingual immunization with Phleum pratense pollen allergens. The immunogens were as follows: N (native allergen); PM (PM‐allergoids); PM‐OX (PM‐allergoids further oxidized); PBS (phosphate‐buffered saline as a negative control). Cytokines were measured in the culture supernatant by flow cytometry (CBA) in response to the native allergens (Phleum pratense) after 48‐h culture. The results obtained with submandibular LN (A) or spleen (B) cells, from mice immunized with different protocols, are the mean ± SEM of 6 mice per group. N.d. (nondetermined). Statistical differences were analyzed with Student's t test. *P < .05, **P < .01, ***P < .001
Figure 5
Figure 5
FOXP3+ Treg cell induction in submandibular LNs and spleen from mice after sublingual immunization with Phleum pratense pollen allergens. The immunogens were as follows: N (native allergen); PM (PM‐allergoids); PM‐OX (PM‐allergoids further oxidized). Percentage of CD4+ CD25high FOXP3+ Treg cells measured by flow cytometry in submandibular LNs (A) or spleen (B) from mice immunized with different protocols. A representative flow cytometry dot plot is also displayed for each assayed condition. Results are the mean ± SEM of 6 mice. Statistical differences were analyzed with Student's t test. *P < .05
Figure 6
Figure 6
Allergen uptake by antigen‐presenting (CD45+ MHC‐II +) cells isolated from the mouse sublingual mucosa. (A) Phenotypic characterization of the different cell subsets by flow cytometry: CD45+ MHC‐II + cells were gated and analyzed for CD64, CD11b, and CD207 cell surface markers. (B) Uptake of fluorescence‐labeled Phleum pratense allergen preparations after 30 min of incubation with CD45+/MHC‐II + cells. N (native allergen); PM (PM‐allergoids); PM‐OX (PM‐allergoids further oxidized). Representative histograms for each assayed condition are also displayed. (C) Percentage of CD64+ and CD11b+ cells within the CD45+ MHC‐II + cells fraction that uptake PM‐allergoids or N allergens. A representative example out of two independent experiments is shown. Data are the mean ± SEM of 4 independent experiments in A and B. Statistical differences were analyzed with Student's t test. **P < .01, ***P < .001

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