We first sought to analyze the antiviral IFN-α response of cord blood cells to TLR7 dependent IAV 
and HIV 
as well as in response to TLR9 dependent HSV 
and CpGA activation (). High levels of IFN-α secretion were observed in all conditions for neonatal whole blood and CBMC. No statistical differences were observed between neonatal and adult samples for all virus stimulation, but CpG/TLR9 displayed discrepancies. Neonatal pDCs were shown to be defective for IFN-α response to CpG/TLR9 activation, but these early studies were performed with pDCs of 50% purity 
. pDCs can be clearly identified by the expression of BDCA4 and BDCA2, as well as the high expression of CD45RA and CD123. Therefore we further investigated functions of cord blood pDCs to >99% purity by magnetic bead enrichement of BDCA4 cells followed by FACS sorting on CD123 and CD45RA expression (Fig. S1
) and then exposed these cells to viruses. pDCs purified from adult and neonatal blood responded similarly (). These IFN-α responses along with the production of TNF-α and chemokines were as efficient as those obtained with adult samples, except for CpGA/TLR9 activation (). We observed similar IFN-α responses to heat inactivated IAV in adult and neonatal samples tested as WB, MNC and purified pDCs (). For neonatal whole blood exposed to live IAV, pDCs were mainly responsible for IFN-α secretion, whereas monocyte only poorly contributed to this response (). Similarly, following depletion of pDCs, but not ofmonocytes, neonatal MNC were strongly impaired or decreased in their capacity to produce IFN-α in response to CpGA, live and HI- IAV (). Interestingly, all cord blood samples responded to IAV or HIV, whereas several samples were unresponsive to CpGA as observed with both WB and isolated pDC stimulation. Indeed, we observed a lack of cell survival in several neonatal pDCs samples treated with CpGA that was not observed following viral stimulation (). Non responders and low responders to CpGA for IFN-α (<100 pg/ml) was associated with poor cell survival (). This phenomenon may come from the defective signaling observed by others 
. In line with this, cord blood pDC may require stronger signaling to survive which CpGA stimulation cannot fully provide, although this was only observed in about 15% of samples. Any correlation could be made with the mode of birth delivery (natural delivery vs. caesarian), the gestational age at birth, or the sex of the neonate (Fig. S2
). Alternatively, some unknown factor(s) related to development may render neonatal pDCs slightly more vulnerable to apoptosis. The underlying mechanism awaits further investigation. Cell survival as well as IFN-α and TNF-α secretion () were restored by adding IL-3 
or GM-CSF 
as survival factors. In contrast to previous reports 
, we failed to detect unresponsive pDCs during HSV/TLR9 stimulation, as both neonatal MNC and purified pDCs produced IFN-α to HSV-1 stimulation () indicating that the unresponsive phenotype is not associated with a defective TLR9 pathway in cord pDCs. Our results are supported by an earlier report in which both cord blood and fetal liver pDCs responded normally to HSV stimulation by producing high amount of IFN-α 
. In addition, this report also provided strong evidence that at earlier developmental stage pDCs expressing CD34 already have the capacity to produce IFN-α in response to HSV 
Innate responses of cord blood pDCs to viruses.
Cord blood pDCs are the main contributor for IFN-α response.
Survival fate of neonatal pDC following CpG activation.
Mouse pDCs are clearly described for the production of both IFN-α and IL-12 following TLR7/9 activation by viruses and synthetic agonists. Human pDCs seem to be much more restricted in their antiviral responses by producing IFN-α but not IL-12 
. Nevertheless, a small fraction of mature pDC can produce IL12 but not IFN-α 
. Recently, CD2 was highlighted as a potential marker for human pDCs subsets 
, and CD2+pDCs were shown to produce IL-12p40 in addition to other properties including IFN-α production. Importantly, the frequency of CD2+ and CD2- pDCs differed markedly between neonates and adult (), therefore we assessed the capacity of both subsets to respond to CpGA or IAV and HIV viruses (). Both neonatal and adult pDCs subsets similarly produced IFN-α. Non responders to CpGA was not associated with a particular subset and could respond to HIV and IAV (). We further investigated IL-12p40 production by CD2+ vs. CD2- pDCs, however we identified among CD2+pDCs that only a minor population expressing CD5 did produce IL-12p40 in response to CpGA and to HI-IAV (). Steady state CD5+CD2+ pDC produced much less IFN-α following TLR activation as compared to other pDC subsets (). These results are in agreement with the dichotomy between IFN-α and IL-12 producing cells 
. Interestingly, CD5+CD2+ pDC at steady state have a mature phenotype with increased CD86 and HLADR expression as compared to other pDCs () and thus probably more prone to play antigen-presenting cell functions. Again no difference was observed between neonatal and adult pDCs. IL-12p40 was not induced in myeloid DC and monocytes following CpGA activation () in accordance with the lack of TLR 9 expression in these cells 
. IL-12p70 and IL-23 were not detected in agreement with previous studies 
. IL-12p40 has been suggested to be a chemoattractant for macrophages in the context of bacterial infection, whether it may play a similar function in viral infection or in antigen-presenting cell related functions of pDCs remains to be clarified 
. Altogether, these results show that neonatal pDCs potently produce IFN-α in response to live and inactivated viruses. Recent publications highlighted the age dependent maturation of TLR responses 
. We provide evidence that this is not the case at the level of antiviral responses of isolated pDC and for bulk mononuclear cells for a variety of viruses. This points out the fact that age-dependent maturation may not occur at the level of innate cells, but may rather be controlled at the level of their environment to express optimal functions. Therefore, age-dependent regulatory phenomenon rather than innate deficiencies should be considered for limited inflammatory responses and susceptibility to infections seen in earlylife. We demonstrated that neonatal pDCs can potently participate in innate immunity in the context of viral infection, indicating that pDCs do not contribute to early life susceptibility to infections.
IL-12p40 producing pDCs represent a small subset of CD2+pDC.