Profound capacity of Bet-specific iTreg to suppress allergen-specific and cross-reactive T cell responses in vitro
In order to analyze the function of iTreg with regard to their suppressive capacity on allergen-specific and cross-reactive responses, we investigated the suppressive capacity of iTregBet and iTreg0 on Bet- or Cor-stimulated responder T cells (Tresp) obtained from birch-pollen allergic patients with associated hazelnut allergy in a flow cytometry-based suppressor assay.35 (see Methods and Supplementary Methods; gating strategy in Supplementary Figure 2).
Coculture with iTregBet resulted in a significantly reduced proliferation of allergen (Bet) -specific, and cross-reactive, Cor- specific Tresp, respectively, indicating the induction of allergen-specific (birch) and cross-reactive (hazelnut) tolerance (Figure 3A). Further analysis revealed that higher numbers of iTreg (ratio 1:2) led to a significantly more impaired Tresp proliferation regardless of the allergen-specificity, demonstrating a dose-dependency of the iTreg suppressive capacity (Figure 3A). We also listed the results from each individual allergic donor (Supplementary Figure 3A).
We further compared the suppressive activity of iTregBetwith non-specific iTreg0 on antigen-specific and cross-reactive T cell immune responses (Figure 3B). The experiments revealed a moderately impaired Tresp proliferation after coculture with iTreg0. However, iTregBet exhibit a significantly higher suppressive potential for both Bet- and Cor-specific T cell responses, indicating the strong potential of antigen-specific stimulated iTregBet as inducers of allergen-specific and cross-reactive tolerance in birch pollen- and hazelnut-allergic patients. Further evidence is depicted in Supplementary Figure 3B, which presents the corresponding data of each individual donor. In 8 out of 10 (80%) experiments with Bet-stimulation, and 6 out of 8 (75%) with Cor-stimulation, iTregBet showed a stronger capacity to reduce Tresp proliferation compared to iTreg0, suggesting their specific and cross-reactive suppressive capacity as superior to non-specific iTreg. Thus, the data demonstrated the high, specific and dose-dependent suppressive capacity of iTregBet to suppress allergen-specific (birch) as well as cross-reactive (hazelnut) T cell responses, highlighting the importance of allergen-specific iTreg induction.
To support our findings, we harvested the supernatants of suppressor assay samples after Bet- or Cor-specific mDC-stimulation and analyzed the T cell cytokine profile (Figure 3C,D). The presence of iTregBet – but not non-specific iTreg0- resulted in significantly reduced levels of the TH2 cytokine IL-13 (Figure 3C) compared to pronounced cytokine levels produced by control allergen-specific (both, Bet or Cor-stimulated) responder T cells. These results strongly confirmed our T cell proliferation data (Figure 3A,B) and indicated an allergen-specific and cross\sout-reactive downregulation of TH2 immunity mediated by Bet-specific iTreg (iTregBet).
We also found a profound and significant upregulation of the immunosuppressive cytokine IL-10 in suppressor assays with cocultured Bet-specific, but not with non-specific iTreg, when compared to control (Figure 3D). Production of additional TH2 (IL-5, IL-9) and TH1 (IFN-y, TNF-α) cytokines were mostly unchanged (see Supplementary Figure 4).
In addition to T cell proliferation and cytokine production, we analyzed the phenotype of Tresp after presence or absence of iTregBet in suppressor assays (Supplementary Figure 5A). Coculture of Bet-stimulated Tresp with iTregBet in suppressor assays resulted in a significantly impaired activation (reduction of CD25, HLA-DR) and differentiation (reduced CD45RO / increased CD45RA expression) of Bet-stimulated Tresp. Similar results were observed for Cor-stimulated Tresp (Supplementary Figure 5B), confirming the data of the allergen-specific as well as cross-reactive suppressive capacity of iTregBet on Tresp proliferation and IL-13 production.