Previous studies have demonstrated the utility of using flt3 ligand and described its function for enhancing the innate immune responses through the expansion of DC subsets 
. In vitro
it is known that flt3 ligand can induce the maturation of cDC, but unlike GM-CSF also induces the differentiation of pDCs. The present studies verify these observations by demonstrating expansion of cDC and pDC in the lungs of naive animals treated with flt3 ligand. The objectives of these studies were to not only expand the DC populations for enhanced anti-viral responses, but also to examine whether the altered immune response was dependent upon the pDC subset. Although the 120G8 did not completely remove all pDCs, the treatment significantly reduced pDC numbers while allowing the increased numbers of cDC to be maintained and respond to the virus infection. These data point out several important aspects. Firstly, the results verify the importance of pDC during viral infections for promoting the appropriate and non-pathogenic responses. Secondly, it indicates that providing more cDCs within the lung is not by itself sufficient for enhancing the anti-viral responses. Thirdly, it suggests that pDC have a role in enhancing CD8 T cell maturation and/or differentiation during the viral responses. This latter issue may be both direct and indirect through APC function and type I IFN production. While these studies allow a better understanding of the relative role of the pDC subset functions during RSV infection, they do not fully define how the individual subsets contribute. In fact, numerous studies have offered data suggesting that one important role of pDCs during anti-viral or anti-tumor immunity may be for shaping the utility of the cDC for proper APC function 
. By examining the cytokine profiles of the two DC subsets in response to RSV a picture might be drawn on how these two subsets differentially contribute to the immune phenotype. While the cDC are capable of producing significant levels of IL-12 that would enhance IFNγ production from T and NK cells, the pDC preferentially produces type I IFN that may augment NK cell and CD8 T cell differentiation into a cytotoxic phenotype 
. Interestingly, our previous studies demonstrated that depletion of pDC in normal mice led to an increase in IFNγ production, whereas in this study with Flt3L treatment the reduction in pDC led to a decrease in IFNγ. While we have not explored this further, one explanation may be the relationship to the fluctuation in the CD8 T cell population displayed in related to the Flt3L treatment. Related to this latter issue, the observation that the RSV infection significantly upregulated CD80 and CD86 expression on the pDC may also contribute to this observation. A recent study demonstrated using animals devoid of cDC subsets that pDC could participate in APC function in lymph nodes for CD4 T cell activation 
, a notion suggested by other previous studies in vitro 
. Together, this may suggest a direct or complimentary role for pDC, along with the cDC, to function as APCs locally and/or in the lymph node during RSV infection and enhance the anti-viral responses, including IFNγ production.
Classically, pDC are considered the primary source of IFN-α. Although type I IFN can impact numerous cells involved in the immune response 
, our previous reconstitution studies with type I IFN after pDC depletion did not demonstrate an effect on the nature of the immune response 
. Other recent studies have also indicated that cellular contact was necessary for pDC to influence cDC ability to alter T cell responsiveness and soluble factors were not sufficient 
. Thus, we are presently considering a reasonable model that includes both cDC and pDC interaction with T cells in order to initiate the proper immune response. It is unclear from our present data whether the altered CD8 T cell responses are a direct result of reduced pDC participation or from the altered CD4 T cell cytokine response that would normally influence the differentiation of the cytotoxic T cells. Although it is unclear what the necessary profile of molecules from pDC signal cDC to instruct anti-viral T cell responses, they may include direct interaction with the T cells via CD80 and CD86 upregulated on pDC after RSV infection. Altogether, these data clearly demonstrate the importance of pDC for directing the type of response to RSV. Expansion of both pDC and cDC resulted in a decrease in type 2, but an increase in type 1 T cell response to RSV and to a general lower immunopathology after RSV infection. In contrast, removal of pDC and expansion of only cDC significantly enhanced Th2 type responses to RSV and led to more inflammation and a higher airway response to RSV. As suggested also by others 
, this demonstrates the highly immunogenic role of cDC and the more regulatory role of pDC in the lung.
The coordination of the pulmonary immune response to a viral infection appears to be primarily controlled in part by the presence of the proper APC subsets. Previous studies have defined that specific DC subsets can skew the immune response. Using defined antigen models, early studies defined the nature of the local cDC subset for preferential skewing toward Th2 responses 
. However, these latter studies were performed in the absence of pDC populations that were subsequently shown to regulate the development of Th2 responses in lung 
. The regulation and coordination of the response by these two subsets may indeed depend upon how they recognize a pathogen or antigen. The cDC subset preferentially expresses a specific profile of TLRs that are relevant to RSV infection, including TLR3 (dsRNA) and TLR4 (F protein of RSV) 
. In contrast, pDC preferentially expresses TLR7 (ssRNA) that would promote type I IFN production 
. In our own studies with RSV we have found that in the absence of TLR3 there is a skewed response toward increased IL-13 and mucus overproduction, while deletion of MyD88 led to a fully Th2 skewed system that appeared to depend upon IL-12 and other Th1-mediated responses associated with cDC populations 
. Thus, multiple pathways and cell types are necessary for the most appropriate non-pathogenic response in the lung. Altogether, these studies represent additional evidence that the pDC population can provide additional protection from adverse pathologic outcomes and further support the use of Flt3 ligand as an effective mediator to alter the pulmonary immune response.
Future vaccine strategies for RSV should focus on the role of pDC during the immune response as well as a balance between cDC and pDC in the lung and draining lymph nodes that seems to be crucial for the outcome of a pulmonary infection. One limitation of using Flt3L for expansion of pDC subsets during specific diseases will be when and in what situations will it be most relevant to use such a treatment modality. Finally, these studies represent additional evidence that the pDC subset can initiate an environment that is protected from adverse pathologic outcomes during a pulmonary immune response.