We initiated this study with the objective of evaluating the Ag-presenting capacity of pDC. The extraordinary ability of myDC to present and cross-present Ags is well established (14
). However, evidence for exogenous Ag presentation by pDC has been limited.
Our data show that pDC, like myDC (16
), are capable of acquiring opsonized Ags through their IgG1-binding FcR, leading to Ag presentation and cross-presentation in vivo. Recent reports describe a new type of lectin-binding receptor, Siglec-H, which is specifically expressed on pDC and can function in Ag uptake, leading to Ag presentation and cross-presentation (17
). It seems likely that Siglec-H and FcγR mediate the uptake of different types of Ags, thus providing pDC with multiple mechanisms for acquiring Ag and modulating the immune response.
Expression of FcγR was higher on pDC derived from cultured bone marrow than on freshly prepared spleen or lymph node cells, and this may partly explain why FcγR on murine pDC has not been described previously. In addition, we deliberately avoided the use of FcR-blocking Ab (clone 2.4G2) in our FACS-staining protocols, which would have prevented detection of FcR. Moreover, we focused our study on stimulating FcγRIII/FcγRII by using an Ab, 2.4G2, that recognizes an epitope shared by CD16 and CD32. This receptor mainly binds Abs of the IgG1, IgG2a, and IgG2b subclasses contained in IC (19
). We used an IgG1 anti-OVA Ab to prepare OVA plus anti-OVA-IC, in part, because the IgG1 Abs consistently bind and stimulate other cells in vivo (20
). Although both pDC and mDC express CD64 (FcγRI) at low levels, this receptor does not bind IgG1 Abs well in the mouse, in contrast to human CD64, which binds IgG1 with high affinity (19
). Exposure of pDC to IC not only resulted in Ag uptake, but also cellular activation, as indicated by up-regulation of MHC class II and accessory molecules. Such activated pDC-IC, in turn, induced the growth and activation of Ag-specific CD4+
T cells. myDC, like pDC, acquired opsonized Ag, became activated, and stimulated Ag-specific T cells, confirming a previous report (16
). MyDC also acquired and processed free protein relatively efficiently. By contrast, pDC that had been pulsed with free protein failed to stimulate T cell responses. This is probably explained both by the reduced ability of pDC to take up free Ag as opposed to opsonized Ag, and by their reduced cellular activation relative to myDC-IC. The mechanism responsible for IC-induced activation of pDC remains to be explored.
Surprisingly, the cytokine profile of pDC-IC-activated CD4+
T cells varied, depending on whether the cells were activated in vitro or in vivo. In vitro, pDC-IC induced mainly IFN-γ-producing Th1 cells, whereas in vivo pDC-IC induced mainly IL-4-producing Th2 cells. By contrast, myDC-IC promoted mainly Th1 cells in vitro and in vivo. The reason for this discrepancy is not clear from our data, but it is apparent that cells and soluble factors in addition to those present in our in vitro culture system can modulate the effects of pDC-IC in vivo. The absence of Th1-biasing factors could also favor the generation of a Th2 response by pDC-IC. In this regard, we were not able to detect IFN-α in supernatants from pDC cultured in the presence of IC (), and pDC are more likely to polarize T cells toward a Th2 profile in the absence of this cytokine (21
). Interactions between OX40L on pDC and OX40 on T cells may also contribute to a Th2 bias, as suggested by in vitro studies of human pDC (22
pDC-IC activated Ag-specific CD8+
T cells as well as Ag-specific CD4+
T cells. Although a relatively high proportion of CD8+
T cells activated in vitro by pDC-IC produced IFN-γ, they did not differentiate into CTL effectors unless LPS, a TLR4 agonist, was added to the cultures. Of note, mouse pDC express almost all TLRs (23
), whereas human pDC have a more restricted TLR repertoire, expressing mainly TLR 1, 2, 7, and 9 (1
). One possible explanation for the failure of CTL development in our pDC cultures is that in the absence of TLR stimulation, IL-10-secreting CD8+
T cells, which were also induced in abundance by pDC-IC, blocked the development of CTL. IL-10 is known to inhibit CD8+
T cell activation and CTL formation (24
). Moreover, human pDC cross-linked with CD40L have been shown to induce IL-10-secreting CD8+
T cells in vitro, which inhibit the activation of naive CD8+
T cells (26
). Peptide-specific IL-10-secreting CD8+
regulatory T cells have been described in humans following immunization with peptide-pulsed immature myDC (28
), and in patients with advanced HIV disease (29
). It remains to be determined whether the IL-10-secreting CD8+
T cells generated in our pDC-IC cultures were responsible for preventing development of CTL and, if so, whether these cells have features in common with previously described CD8+
What are the implications of these findings? Although pDC respond rapidly to invading pathogens by secreting IFN-α, and hence are a key component of the innate immune system, our data suggest that pDC can play an important role in adaptive immunity as well. The results show clearly that pDC have the capacity to function as true Ag-processing and -presenting cells, inducing effector as well as memory T cells. On the one hand, by promoting immune effector activity, pDC-IC may play an important role in the clearance of pathogens or tumors. In addition, through their induction of Th2 and regulatory T cells, pDC-IC can modulate the immune response, potentially averting excessive cellular immunity and related tissue damage. In contrast, inopportune inhibition of the immune system by pDC-IC-activated regulatory cells could prevent clearance of pathogens or tumors, whereas induction of immunity to allergens or autoantigens might contribute to allergic or autoimmune disorders. For example, patients with systemic lupus erythematosus have circulating IC containing small nuclear RNA and anti-small nuclear RNA autoantibody; recent studies have shown that pDC can acquire such IC via their FcγR, resulting in stimulation of TLR7 and 8 and production of IFN-α (30
). Because IFN-α is thought to play a fundamental role in the pathogenesis of systemic lupus erythematosus, IC-mediated activation of pDC may be a key initiation factor in this disease. Thus, the consequences of IC uptake by pDC appear to depend on the nature of the Ag as well as the presence or absence of other factors (e.g., TLR ligands) in the environment that help determine whether these cells induce a proinflammatory or anti-inflammatory/tolerogenic response.