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.