By showing the presence and antigen uptake of pDCs in the lung, by demonstrating direct priming to antigen in the absence of pDCs, and by demonstrating transfer of the tolerizing effect through injection of pDCs, we believe that pDCs have an essential immunoregulatory role in the lung that protects against development of airway inflammation to harmless antigens.
To our knowledge, this is the first description of pDCs in the lung of mice. These cells demonstrate intermediate expression of CD11c, express Gr-1, B220, and CD45RB, and they produce large amounts of IFN-α after stimulation with CpG motifs. These cells take up inhaled FITC-OVA protein and migrate to the MLNs draining the lung carrying their fluorescent cargo inside vacuoles. We believe that these cells are most likely related to the pDCs also described in the draining LNs and spleen of mice (35
), and are distinct from the CD45RB+
DCs recently described by Wakkach et al. (39
) by their expression of Gr-1. We are currently studying if these CD45RB+
DCs are also present in the lung.
In the absence of pDCs during primary OVA exposure to the lung, sensitization was induced, which led to features of Th2 cell–associated asthma after inhaling harmless antigen. This is a remarkable observation, as primary exposure to inhaled OVA in our system usually leads to inhalational tolerance, even when mice are immunized with OVA in alum (Fig. S2; references 18
). For our studies in which we depleted pDCs, we have used Gr-1 Abs that also deplete neutrophils (24
). We do not believe that absence of neutrophils had a predominant effect on T cell priming, as Gr-1+
neutrophils did not carry Ag to the node. However, to more directly prove a role for pDCs as tolerogenic cells, we used another, more selective pDC-depleting Ab, 120G8, and found identical results, arguing against a predominant role for the neutrophil (25
). Moreover, we performed adoptive transfer experiments before a fully immunogenic asthma protocol. Adoptive transfer of OVA-pulsed BM-derived pDCs (BM-pDCs) before induction of asthma completely inhibited airway inflammation. This response required the presence of OVA, as unpulsed pDCs did not suppress airway inflammation. Remarkably, under the same conditions, BM-mDCs did not exert this effect and even strongly enhanced the cardinal features of asthma (unpublished data and 13).
Next, we performed experiments to elucidate the mechanism by which absence of pDCs might lead to sensitization. It is known that inhalational tolerance to harmless antigen is accompanied by a significant amount of primary T cell activation in the draining nodes, and therefore it was suggested that depletion of pDCs might influence T cell division. To our surprise, however, we noticed no difference in the primary division of naive T cells when the outcome was either immunity (i.e., in the case of depletion of pDCs) or tolerance (treatment with control Abs), illustrating that the strength of primary T cell division does not predict the functional outcome of a T cell response. Moreover, removing pDCs during OVA inhalation resulted in higher levels of effector Th2 (IL-5, IL-10, IL-13) and Th1 cell cytokines (IFN-γ) being produced by T cells in the draining MLNs. Indirectly, these data show that the mDC subset is the predominant APC responsible for inducing naive T cell proliferation in the draining nodes. The uncoupling of T cell proliferation from generation of effector potential and from differentiation into memory cells is emerging as a new concept in T cell biology (4
). Lanzavecchia and Sallusto (41
) recently proposed that T cell stimulation of insufficient strength or duration leads to proliferation of T cells that are “unfit” to respond to T cell survival signals and do not produce effector cytokines. The fact that in our experiments T cell proliferation was accompanied by proinflammatory effector T cell generation only when pDCs were depleted, suggests that pDCs are inefficient APCs that compete with immunogenic mDCs for antigen presentation, thus effectively reducing the overall strength or duration of T cell stimulation. From our and previous studies there is evidence that pDCs are weak APCs. Only when pDCs were pulsed with various doses of OVA peptide in vitro, could either Th1 or Th2 cell responses be induced (23
). The fact that lung mDCs and pDCs take up fluorescent OVA does not imply that both cells provide an identical TCR ligand density, as there might be significant differences in antigen processing and presentation efficiency (32
). Alternatively, pDCs in the lung and MLNs are immature, expressing low levels of MHC II and costimulatory molecules, yet high levels of the inhibitory B7 family member PD-L1 molecule, known to suppress T cell activation through interaction with PD-1 (42
). This profile of high inhibitory and low costimulatory molecule expression together with the poor T cell stimulatory capacity ex vivo, point out to the “tolerogenic” potential of pDCs in the lung. It remains to be demonstrated whether the depletion of pDCs during priming would also lead to longer survival of responding T cells as would be predicted from the “T cell fitness” model (40
). Alternative explanations could be that immature lung pDCs are important for generating T reg cells after exposure of harmless antigen, as proposed in vitro by others (27
) and also shown indirectly ex vivo here ( C). In this scenario, in the absence of pDCs, T reg cells would no longer be induced from naive T cells or expanded from a pool of naturally occurring OVA-responsive CD4+
regulatory cells (44
). The exact phenotype and function of these T reg cells induced by lung pDCs remains to be determined.
Concomitant exposure of harmless antigen with signals that activate innate immune responses such as viral infection, bacterial LPS, or GM-CSF, lead to a break in inhalational tolerance presumably through activation of mDCs (4
). Supporting this idea, injection of mature mDCs but not pDCs indeed leads to Th2 cell sensitization in the lung (unpublished data and 13). We observed induction of Th2 cell sensitization in response to LPS-free OVA inhalation, which was shown by us (Fig. S2) and by others (47
) to lead to tolerance and did not induce any form of mDC activation (unpublished data). Therefore, we believe that modulation of the intrinsic tolerogenic function of pDCs represents a second mechanism by which “adjuvant factors” lead to Th2 cell sensitization to harmless antigens. In this context, there is a known association between respiratory viral infections such as respiratory syncytial virus at a young age and the development of asthma (48
). As viruses strongly interact with pDCs, we propose that inhibition of the normal tolerogenic activities of lung pDCs by viral infection could be the mechanism behind this association. Viral infections induce maturation of pDCs in vivo and this might explain the breaking of inhalational tolerance (37
). Some “adjuvants” might also alter the ratio between immunogenic mDCs and pDCs in the lung and thus influence the decision between immunity and tolerance. This was nicely shown in the case of adenoviral transfection of the lung with GM-CSF, a predominant growth factor for mDCs but not pDCs, which led to sensitization (46
). It will be interesting to study if increasing pDCs would have the opposite effect. It is striking that administration of CpG motifs to the lung leads to strong accumulation of lung pDCs (unpublished data) and to protection from asthma through unclear mechanisms not mediated by Th1 cell cytokines (49
The discovery that pDCs influence the generation of T cell effector responses has extensive implications for our understanding of immunoregulation in a wide range of T cell–mediated diseases. Inhibition of the tolerogenic capacity of pDCs could thus offer an explanation why some viral infections lead to a break in peripheral self-tolerance in a number of autoimmune diseases such as diabetes (50
). In light of our findings, it will be very interesting to see whether the function of lung pDCs in humans with allergy or asthma would also be reduced, thus favoring Th2 cell sensitization to inhaled antigen (52
). One recent report showed that the number of circulating pDCs in children with atopic asthma was reduced compared with healthy controls (53
In conclusion, we have identified a role for endogenous pDCs in the protection against asthmatic reactions to harmless antigens by demonstrating that asthma develops in the absence of pDCs. Therefore, aberrant function of pDCs might contribute to allergic sensitization. Therapies targeted at amplifying the intrinsic tolerogenic capacity of lung pDCs may limit the development of asthma.