We have identified a novel immune pathway that amplifies Th2 responses in humans. Specifically, we have shown that targeting cat allergen to FcγRI upregulates expression of TSLPr in atopic moDCs. Upregulation of TSLPr was most pronounced in moDCs from atopic patients and Th2 responses triggered by H22-Fel d 1 were markedly amplified in the presence of TSLP in these individuals. These findings indicated that receptor-targeted allergen potentiates the Th2-promoting effect of TSLP through TSLPr upregulation. In support of this theory, we further showed that TSLPr upregulation was regulated by FcR signaling components, and inhibition of TSLPr upregulation abolished the Th2-promoting effect of TSLP elicited by H22-Fel d 1. This study is the first report of an immune pathway linking Fc receptor-mediated events to TSLPr expression and consequent TSLP-driven Th2 responses.
While TSLP was previously shown to be sufficient to induce asthma-like disease in mouse models and to drive Th2 responses in human-based in vitro
systems, the role of allergen in those studies was largely ignored 1–6
. In contrast to previous reports, TSLP alone was actually a weak inducer of Th2 responses in our system, irrespective of allergic status. By contrast, receptor-targeted allergen and TSLP acted coordinately to exert a potent Th2-promoting effect. This phenomenon was restricted to atopic individuals and was dependent upon the capacity to upregulate TSLPr. These observations are in line with a recent report that both antigen and
TSLP are required for the development of full airway inflammation in a mouse asthma model 23
The Th2-promoting effect of TSLP that was triggered by H22-Fel d 1 was most pronounced in cat-allergic patients with AD as judged by a synergistic induction of IL-4+ T cells. Notably, H22-Fel d 1-primed moDCs induced only a weak T cell response in these patients (see ). Since T cell anergy can be a feature of AD, we postulate that co-priming moDCs with H22-Fel d 1 and TSLP surpasses a stimulation threshold required for optimal T-cell activation in these patients.
H22-Fel d 1 has the capacity to rapidly upregulate TSLPr expression in both blood DCs and moDCs and expression of TSLPr appears to be modulated in a similar manner in both cell types. Upregulation of TSLPr in atopic moDCs induced by H22-Fel d 1 was positively regulated by PI3-kinase, which acts downstream of Syk in FcγRI signaling 24
. Thus, we expected that inhibition of Syk and its upstream activating components (SRTKs) would yield similar results to those for PI3-kinase. However, inhibition of SRTKs actually enhanced expression of TSLPr in atopic cells, suggesting that ITAM-associated molecules provide a “braking mechanism” for TSLPr upregulation. Since PI3-kinase is shared among different signaling pathways, the effects of LY294002 could reflect engagement of this molecule in pathways not related to FcγRI. Arguing against this, LY294002 did not alter TSLPr expression in non-primed moDCs suggesting that its effects were specifically linked to FcγRI-triggered events (data not shown). Another important consideration is that inhibitors may act on other molecules through non-kinase “off-target” effects. Such off-target effects may occur in the concentration range for LY294002 commonly used to inhibit PI3-kinase25
. In our study, LY294002 was used at 25μM; however, inhibition of H22-Fel d 1-induced TSLPr expression was evident at concentrations of LY294002 as low as 0.25μM with marked inhibition at 2.5μM suggesting that the effects of LY294002 were specific (data not shown). Moreover, the SRTK inhibitor, PP2, had no effect on constitutive levels of TSLPr on moDCs, providing further evidence that inhibitory molecules acted on the FcγRI pathway in H22-Fel d 1-primed moDCs (data not shown).
Interestingly, TSLPr expression was also upregulated by non-receptor-targeted allergen; however, this process was highly variable, less efficient than for H22-Fel d 1, and restricted to atopic cells. This effect is likely mediated through ligation of Fc receptors expressed at high density on atopic moDCs, by IgE- or IgG-bound allergen. It was previously reported that moDCs from atopic patients express increased levels of FcεRI, and this is driven by enhanced expression of the γ chains of the receptor 26
. Notably, these γ chains can be shared among FcεRI and FcγRI. Moreover, high expression of the high affinity IgE receptor, FcεRI, on DCs from atopic subjects is pivotal to allergen uptake and T cell activation 27
. Thus, DCs from atopic individuals possess the molecular machinery necessary to upregulate TSLPr through Fc receptor ligation upon allergen encounter. With this in mind, the present study provides insight into how allergens may induce potent Th2 responses in humans, despite their relatively weak Th2-inducing properties ex vivo
. In line with this view, we have recently confirmed that moDCs primed with dust mite allergen (Der p 1) in the presence of TSLP, can drive a robust Th2 response in a subset of highly atopic subjects (Hulse and Woodfolk, unpublished data).
A surprising feature of our study was the relatively weak Th2-promoting capacity of TSLP. In addition, the TSLP-mediated amplification of Th2 responses triggered through FcγRI did not appear to involve OX40L or increased production of the Th2-attracting chemokine, TARC/CCL17. This is in contrast to previous reports on the effects of TSLP mediated through CD11c+
blood DCs 1, 2
. One possible explanation is that TSLP-primed moDCs, unlike TSLP-primed blood DCs, secrete IL-12 which may counter-regulate the Th2-promoting effects of TSLP 1
; however, no IL-12 was detected in our system. It should be pointed out that while no increase in TARC was noted for primed versus non-primed moDCs in our system, background levels were high. This could reflect exhaustion of TARC/CCL17 production during generation of moDCs in vitro
. Nevertheless, our findings are in line with recent findings in a TSLP-driven mouse model of AD showing that Th2 polarization mediated by Langerhans cells did not involve secretion of TARC/CCL17 by these cells and was OX40L-independent. 28
. Consistent with activation of an alternate TSLP-mediated pathway triggered through FcγRI, cells expressing IFN-γ constituted up to 37% of IL-4+
T cells induced by co-primed atopic moDCs.
From a clinical standpoint, our findings question whether DC-targeted allergens will be clinically efficacious in atopic individuals. The route of vaccine administration will no doubt be a key factor to consider if TSLP-mediated effects are to be avoided. On the other hand, our results suggest that targeting Fc receptor signaling components may provide a useful strategy for modulating Th2 responses.