Compared to HPIV3 or IAV, stimulation of human MDDC with HRSV or HMPV in vitro resulted in inefficient maturational changes in chemokine receptor usage – namely down-regulation of CCR1, CCR2, and CCR5 and up-regulation of CCR7 – that are necessary for DC migration in vivo following antigen uptake. MDDC stimulated with HRSV or HMPV did not migrate efficiently towards a CCL19 gradient in an in vitro assay, compared to HPIV3 or IAV, confirming that the poor surface expression of CCR7 had functional consequences. The weak chemokine receptor modulation and migration by MDDC exposed to HMPV and HRSV, viruses that are thought to induce incomplete immunity, was particularly evident compared to MDDC exposed to IAV, a virus that induces effective immunity.
, maturing, antigen-bearing DC migrate from peripheral tissue to secondary lymphatic tissue and localize in defined lymphoid compartments, where they present antigens to CD4+ and CD8+ T lymphocytes, initiating and polarizing the T cell response 
. DC migration to and positioning within lymphatic tissue are critical towards mounting an effective adaptive immune response 
. While there are multiple chemokine receptors that direct immature DCs towards peripheral sites, CCR7 is the only receptor that mediates migration toward and positioning within lymphatic compartments for interaction with T lymphocytes 
. Thus, differential effects of pathogens on CCR7 expression in particular could be functionally relevant for differences in the immune response to these pathogens. Accordingly, the reduced migration observed in our in vitro
assay for HMPV- and HRSV-treated MDDC following stimulation with HRSV and HMPV suggests that, during an HMPV or HRSV infection in vivo
, maturing DC migrate with reduced efficiency from the infected mucosa towards secondary lymphatic tissues. This might lead to reduced adaptive immune responses that could explain the greater ability of HMPV and HRSV to reinfect humans throughout life without need for significant antigenic change.
The present study was done with primary human cells from multiple donors. While the use of cells from an outbred population provides data with substantial individualistic differences and reduced statistical significance compared to convenient, uniform hosts like inbred mice, it is important to note that the natural host of the viruses in the present study is the human and not the mouse. Direct in vivo
studies of virus-specific effects on DC migration during respiratory infections of humans are difficult, especially in children. Gill et al 
noted that DC persisted in the lungs of children hospitalized for HRSV infection for as long as 8 weeks following the resolution of infection 
. Resorting to data from mice, sustained increases in pulmonary DC have also been observed following HRSV infection 
. Lucken et al 
tracked the migration of mouse DC following HRSV infection and showed that the increase in DC numbers in the mouse mediastinal lymph node was slower compared to IAV or Sendai virus infection 
. These observations would be consistent with inefficient migration from the lung to lymphoid tissue. Our in vitro
studies now provide a mechanism for these previous in vivo
observations. In addition, we provided data that MDDC maturation also was reduced with HMPV compared to HPIV3 and IAV.
We previously provided data indicating that the level of MDDC maturation in response to exposure to HMPV and HRSV is lower compared to HPIV3 
and IAV (not shown). In vivo
, the combination of these two factors, namely reduced overall maturation and inefficient CCR7-CCL19 driven migration, might result in additive net effects that could affect both the magnitude and the quality of the adaptive immune response. Compared to infection with IAV, HRSV and HMPV infections may yield lower overall numbers of virus-stimulated mature DC in the afferent lymphatics. Reduced expression of co-stimulatory surface molecules and reduced cytokine expression could affect the quality of the response as well as its magnitude. In addition, the inefficient migration of maturing DCs may also play a role in viral pathogenesis: specifically, the sustained presence of mature DC in the mouse lung has been suggested to contribute to airway inflammation 
Another paramyxovirus, measles virus (MeV), was recently shown to inhibit CCR7-driven DC migration. Interference with DC maturation and function is considered to be central to MeV-induced immunosuppression. Compared to LPS, MeV infection failed to promote the switch from CCR5 to CCR7 expression, and MeV-matured DC exhibited chemotactic responses to CCL3 rather than to CCL19 
. Inhibition of CCR7-driven migration was also described for vaccinia virus and for herpes simplex virus type 1 
. However, the effects of reduced DC maturation and migration on long-term protection might be particularly significant for respiratory viruses such as HMPV and HRSV. Both of these viruses are restricted in tropism to the superficial cell layer of the respiratory tract, and protection against re-infection has reduced effectiveness (compared to viremic viruses, for example) due to the short-lived nature of local IgA antibodies, the inefficiency with which serum antibodies access the respiratory lumen, and the down-regulation of virus-specific CD8+ T cell functionality in the respiratory tract 
. Thus, even modest decreases in the magnitude of the adaptive response could result in decreases in viral clearance and protection against re-infection.
We used recombinant GFP-expressing viruses to distinguish between effects in robustly infected (GFP-positive) and uninfected/abortively-infected (GFP-negative cells) MDDC. This revealed additional differences between the viruses. For MDDC infected with HMPV or HPIV3, the GFP-positive population expressed significantly more surface CCR7 than the GFP-negative population. In contrast, for MDDC infected with HRSV, the GFP-positive subpopulation resembled the GFP-negative population in having very low CCR7 surface expression. Thus, whereas robust infection with HMPV and HPIV3 stimulated expression of CCR7, robust infection with HRSV did not. Furthermore, GFP-positive cells infected with HRSV showed no down-regulation of CCR1, 2, and 5 surface expression. Thus, compared to HMPV or HPIV3, even the subpopulation of DC that is robustly infected with HRSV and contains abundant intracellular antigen would not be mobilized for migration. This would impede the delivery of HRSV antigen from the periphery to lymphoid tissue. Furthermore, DC that are robustly infected with a virus can readily process newly synthesized viral antigens for display on MHC class I molecules and presentation to CD8+ T cells. Reduced migration of DC that are robustly infected with HRSV to lymphoid tissue would reduce this activity. This would make activation of CD8+ T cells more dependent on cross-presentation by non-infected DC, and could reduce the efficiency of CD8+ T cell activation during HRSV infection, reducing viral clearance and the disease-sparing regulatory effects of HRSV-specific CD8+ T cells 
Secondary stimulation of HRSV- or HMPV-stimulated MDDC with LPS, a strong DC activator, resulted in up-regulation of CCR7 expression on both GFP-negative and GFP-positive cells and increased in vitro
migration. In contrast, with vaccinia virus or human cytomegalovirus, a secondary stimulation of the infected DC with LPS failed to up-regulate the CCR7 chemokine receptor 
. LPS is a strong NFκ-B and AP-1 dependent DC activator 
. Secondary stimulation of HRSV- and HMPV-infected MDDC with the NFκ-B/AP-1-dependent pro-inflammatory cytokines TNF-α, IL-1α and IL-6, at concentrations comparable to those induced by LPS treatment, up-regulated CCR7 expression and was pro-migratory. This suggests that, in contrast to MeV, vaccinia virus, or herpes simplex virus, suboptimal stimulation, rather than inhibition, is responsible for the poor-migration phenotype of pneumovirus-exposed MDDC.
In summary, compared to HPIV3 and, in particular, IAV, the pneumoviruses HMPV and HRSV were inefficient in inducing the maturation-related changes in cell surface chemokine receptor expression in MDDC that are necessary in vivo to re-direct DC from the periphery to lymphoid tissue. Consistent with this, both HRSV and HMPV were poor inducers of MDDC maturation and migration in vitro. These effects could be contributing factors in the incomplete nature of protection induced by HRSV infection in humans.