Inflammatory and tolerogenic myeloid cells determine outcome following human allergen challenge

Innate mononuclear phagocytic system (MPS) cells preserve mucosal immune homeostasis. We investigated their role at nasal mucosa following allergen challenge with house dust mite. We combined single-cell proteome and transcriptome profiling on nasal immune cells from nasal biopsies cells from 30 allergic rhinitis and 27 non-allergic subjects before and after repeated nasal allergen challenge. Biopsies of patients showed infiltrating inflammatory HLA-DRhi/CD14+ and CD16+ monocytes and proallergic transcriptional changes in resident CD1C+/CD1A+ conventional dendritic cells (cDC)2 following challenge. In contrast, non-allergic individuals displayed distinct innate MPS responses to allergen challenge: predominant infiltration of myeloid-derived suppressor cells (MDSC: HLA-DRlow/CD14+ monocytes) and cDC2 expressing inhibitory/tolerogenic transcripts. These divergent patterns were confirmed in ex vivo stimulated MPS nasal biopsy cells. Thus, we identified not only MPS cell clusters involved in airway allergic inflammation but also highlight novel roles for non-inflammatory innate MPS responses by MDSC to allergens in non-allergic individuals. Future therapies should address MDSC activity as treatment for inflammatory airway diseases.</p

Gene expression of MHC Class II components and costimulatory molecules.

<p>RT-PCR of RNA from freshly isolated basophils (1), MHC Class II negative (2) and MHC Class II positive basophils (3) after 72 hours of culture with IL-3, IFN-γ, GM-CSF and HDM extract, compared with CD123⁺ IgE^low pDC (4), CD19⁺ B cells from freshly isolated PBMC (5) and CD19⁺ B cells from CpG stimulated PBMC (6) as positive controls, and a RT negative control (7). Data for a non-atopic donor are shown, representative of 5 separate experiments.</p

Costimulatory molecule expression on stimulated basophils.

<p>CD40 (A), CD80 (B), CD86 (C) and MHC Class II (D) expression on IL-3, IFN-γ and GM-CSF stimulated basophils of 2 atopic HDM-allergic (i,ii) and 1 non-atopic (iii) donor. CpG-stimulated B cells served as a positive control for costimulatory molecule detection (iv). Gates were determined using corresponding isotype controls.</p

MHC Class II expression on stimulated basophils.

<p>Representative dot plots showing MHC Class II expression on basophils freshly isolated (A), and after 72 hours culture with medium alone (B) or IL-3 (10 ng/ml), IFN-γ and GM-CSF (100 ng/ml) (C). Corresponding isotype control (D). Percentage of MHC Class II positive basophils for individual atopic HDM-allergic (open shapes) and non-atopic (closed shapes) donors after 72 hours culture with various stimuli (E). Differences between groups were calculated with One-way ANOVA and Dunett's Multiple comparison post-test with IL-3 as the control group for complete data sets of the 6 donors, * p&lt;0.05, ** p&lt;0.01.</p

Isolated basophil purity and viability.

<p>Representative dot plots showing proportion of basophils in PBMC and after isolation assessed by antibodies specific for (A) IgE and CD123, (B) FcεRI and CD203c, (C) FcεRI and Lineage-1. Example of basophil viability after culture for 72 hours with (D) IL-3 (10 ng/ml) or (E) medium alone. (F) Percentage of viable basophils (7AAD⁻, Annexin V⁻) after 72 hours of culture with a panel of cytokines and TLR ligands. Representative of 2 separate experiments.</p

T cell proliferation assay.

<p>(i) Percentage of MHC Class II positive basophils after 72 hours culture with IL-3, IFN-γ and GM-CSF prior to co-culture with T cells. HDM extract (10 µg/ml) was also added to the basophil stimulation culture for A. (ii) Percentage of CD25⁺ CFSE^low proliferating CD3⁺CD4⁺ T cells after culture of T cells alone or with autologous basophils or monocytes in the presence of 10 µg/ml HDM (A, non-atopic donor; similar results for 2 HDM-allergic donors) for 72 hours or 10 µg/ml peptide (B) for 120 hours. T cell: APC ratio indicated below. (C) Representative dot plot showing gating strategy for identification of proliferating CD3⁺CD4⁺ T cells.</p

Primer sequences used for RT-PCR.

<p>Primer sequences used for RT-PCR.</p

Engineered systems

The antigen-presenting abilities of basophils and their role in initiating a Th2 phenotype is a topic of current controversy. We aimed to determine whether human basophils can be induced to express MHC Class II and act as antigen presenting cells for T cell stimulation. Isolated human basophils were exposed to a panel of cytokines and TLR-ligands and assessed for MHC Class II expression. MHC Class II was expressed in up to 17% of isolated basophils following incubation with a combination of IL-3, IFN-γ and GM-CSF for 72 hours. Costimulatory molecules (CD80 and CD86) were expressed at very low levels after stimulation. Gene expression analysis of MHC Class II-positive basophils confirmed up-regulation of HLA-DR, HLA-DM, CD74 and Cathepsin S. However, MHC Class II expressing basophils were incapable of inducing antigen-specific T cell activation or proliferation. This is the first report of significant cytokine-induced MHC Class II up-regulation, at both RNA and protein level, in isolated human basophils. By testing stimulation with relevant T cell epitope peptide as well as whole antigen, the failure of MHC Class II expressing basophils to induce T cell response was shown not to be solely due to inefficient antigen uptake and/or processing