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Immune responses at mucosal sites are thought to be initiated in the draining lymph nodes, where dendritic cells present viral antigens and induce naive T cells to proliferate and to become effectors. Formal proof that antigen-presenting cells (APC) do indeed localize to the regional lymph nodes has been lacking for viral infections of the respiratory tract. Influenza virus was detected in the draining mediastinal lymph nodes (MLN) early after intranasal inoculation, with peak virus titers in this tissue measured at 2 days postinfection. Virus-specific cytotoxic T-lymphocyte responses were first detected in the MLN 1 day later. Macrophages, dendritic cells, and B lymphocytes were isolated from influenza virus-infected mice and assayed for the capacity to stimulate a major histocompatibility complex class I-restricted virus-specific T-cell hybridoma. All APC populations from lungs and MLN contained virus and thus had the potential to present antigen to CD8+ T cells. The APC recovered from the lungs of influenza virus-infected mice and dendritic cells from the MLN were able to stimulate virus-specific responses. The lack of a virus-specific T-cell response to B cells corresponds to the small number of virus-positive B lymphocytes in the MLN. These results indicate that dendritic cells and macrophages are antigen positive in mice acutely infected with an influenza A virus and that dendritic cells are probably responsible for initiating the cytotoxic T-lymphocyte response to influenza virus in the draining lymph nodes.

To determine site and mechanism of suppression, regulatory T cell (Treg) migration and function were investigated in an islet allograft model. Treg first migrated from blood to the inflammed allografts, this depended on CCR2, CCR4, CCR5, and P- and E-selectin ligands, and was essential for suppression of alloimmunity. In the allograft, Treg were activated, upregulated effector molecules, migrated to the draining lymph nodes (dLN) in a CCR2, CCR5, and CCR7 fashion, and this movement was essential for optimal suppression. Treg inhibited dendritic cell migration in a TGFβ and IL-10 dependent fashion; and suppressed antigen specific T effector cell migration, accumulation, and proliferation in dLNs and allografts. These results showed that sequential migration from blood to the target tissue and then to dLNs were required for nTreg to differentiate and execute fully their suppressive function, by inhibiting DC in the peripheral tissue, and T effector cell responses in dLN and allografts.

Antiinfluenza type 2 (T2) immunity contributes to both immunopathology and immunoprotection, yet the underlying mechanisms modulating T2 immunity remain ill defined. We describe a novel mouse antigen (Ag)-presenting cell (APC), designated late-activator APC (LAPC). After pulmonary influenza A (H1N1) virus infection, LAPCs enter the lungs, capture viral Ag, and subsequently migrate to the draining lymph node (DLN) and spleen, with delayed kinetics relative to dendritic cells (DCs). In the DLN, influenza virus–activated LAPCs present Ag and selectively induce T helper type 2 (Th2) effector cell polarization by cell–cell contact–mediated modulation of GATA-3 expression. In adoptive transfer experiments, influenza virus–activated LAPCs augmented Th2 effector T cell responses in the DLN, increased production of circulating antiinfluenza immunoglobulin, and increased levels of T2 cytokines in bronchoalveolar lavage fluid in recipient influenza virus–infected mice. LAPC-recipient mice exhibited exacerbated pulmonary pathology, with delayed viral clearance and enhanced pulmonary eosinophilia. Collectively, our results identify and highlight the importance of LAPCs as immunomodulators of T2 immunity during influenza A virus infection.

Severe Acute Respiratory Syndrome caused substantial morbidity and mortality during the 2002–2003 epidemic. Many of the features of the human disease are duplicated in BALB/c mice infected with a mouse-adapted version of the virus (MA15), which develop respiratory disease with high morbidity and mortality. Here, we show that severe disease is correlated with slow kinetics of virus clearance and delayed activation and transit of respiratory dendritic cells (rDC) to the draining lymph nodes (DLN) with a consequent deficient virus-specific T cell response. All of these defects are corrected when mice are treated with liposomes containing clodronate, which deplete alveolar macrophages (AM). Inhibitory AMs are believed to prevent the development of immune responses to environmental antigens and allergic responses by interacting with lung dendritic cells and T cells. The inhibitory effects of AM can also be nullified if mice or AMs are pretreated with poly I:C, which directly activate AMs and rDCs through toll-like receptors 3 (TLR3). Further, adoptive transfer of activated but not resting bone marrow–derived dendritic cells (BMDC) protect mice from lethal MA15 infection. These results may be relevant for SARS in humans, which is also characterized by prolonged virus persistence and delayed development of a SARS-CoV-specific immune response in individuals with severe disease.

Author Summary

Severe Acute Respiratory Syndrome (SARS) occurred in human populations in 2002–2003 and was caused by a novel coronavirus (CoV). Human SARS was characterized by prolonged virus excretion, lymphopenia and delayed adaptive immune responses in patients with severe disease. Recently, small animal models have been developed that mimic some of the features of the human disease. Specifically, BALB/c mice infected with mouse-adapted SARS-CoV develop severe respiratory disease. Here, we show that the T cell response is defective in these mice and that this results from inefficient activation of the initial immune response to the virus. This defect can be corrected by several treatments, including depletion of inhibitory macrophages from the lungs and direct activation of respiratory dendritic cells, important in initiating the immune response or transfer of activated dendritic cells prior to infection. All of these modalities result in improved initiation of the immune response and an enhanced anti-virus T cell response. Inefficient activation of the immune response may play a role in human SARS, and our results suggest possible strategies that might be used to develop novel anti-viral therapies.

Migration of dendritic cells (DCs) to the draining lymph node (DLN) is required for the activation of naive T cells. We show here that migration of DCs from the lung to the DLN after Mycobacterium tuberculosis (Mtb) exposure is defective in mice lacking interleukin (IL)-12p40. This defect compromises the ability of IL-12p40–deficient DCs to activate naive T cells in vivo; however, DCs that express IL-12p40 alone can activate naive T cells. Treatment of IL-12p40–deficient DCs with IL-12p40 homodimer (IL-12(p40)2) restores Mtb-induced DC migration and the ability of IL-12p40–deficient DCs to activate naive T cells. These data define a novel and fundamental role for IL-12p40 in the pathogen-induced activation of pulmonary DCs.

During pulmonary mycobacterial infection, there is increased trafficking of dendritic cells from the lungs to the draining lymph nodes. We hypothesized that ongoing mycobacterial infection would modulate recruitment and activation of antigen-specific naive CD4+ T cells after airway antigen challenge. BALB/c mice were infected by aerosol with Mycobacterium bovis BCG. At peak bacterial burden in the lungs (4 to 6 weeks postinfection), carboxy-fluorescein diacetate succinimidyl ester-labeled naive ovalbumin-specific DO11.10 T cells were adoptively transferred into infected and uninfected mice. Recipient mice were challenged intranasally with soluble ovalbumin (OVA), and OVA-specific T-cell responses were measured in the lungs, draining mediastinal lymph nodes (MLN), and spleens. OVA challenge resulted in increased activation and proliferation of OVA-specific T cells in the draining MLN of both infected and uninfected mice. However, only BCG-infected mice had prominent OVA-specific T-cell activation, proliferation, and Th1 differentiation in the lungs. BCG infection caused greater distribution of airway OVA to pulmonary dendritic cells and enhanced presentation of OVA peptide by lung CD11c+ cells. Together, these data suggest that an existing pulmonary mycobacterial infection alters the phenotype of lung dendritic cells so that they can activate antigen-specific naive CD4+ T cells in the lungs in response to airway antigen challenge.

Trafficking of lung dendritic cells (DCs) to the draining lymph node (dLN) is a crucial step for the initiation of T cell responses upon pathogen challenge. However, little is known about the factors that regulate lung DC migration to the dLN. In this study, using a model of influenza infection, we demonstrate that complement component C3 is critically required for efficient emigration of DCs from the lung to the dLN. C3 deficiency affect lung DC-mediated viral antigen transport to the dLN, resulting in severely compromised priming of virus-specific T cell responses. Consequently, C3-deficient mice lack effector T cell response in the lungs that affected viral clearance and survival. We further show that direct signaling by C3a and C5a through C3aR and C5aR respectively expressed on lung DCs is required for their efficient trafficking. However, among lung DCs, only CD103+ DCs make a significant contribution to lung C5a levels and exclusively produce high levels of C3 and C5 during influenza infection. Collectively, our findings show that complement has a profound impact on immune regulation by controlling tissue DC trafficking and highlights a potential utility for complement as an adjuvant in novel vaccine strategies.

Author Summary

Influenza is a global health problem frequented by epidemics and pandemics. Current vaccines against influenza offer limited protection hence the need for reformulation and repeated vaccination. There is a pressing need to develop newer vaccines that are able to generate T cell response. In order to develop such vaccines, there is a need to understand how T cell responses are generated during influenza infection. Influenza specific T cell responses are generated by the dendritic cells (DCs) in the lung. Upon influenza infection, DCs in the lung carry viral peptides to the draining lymph node (dLN) to initiate an immune response. Thus, migration of DCs from the lung to the dLN is an important step in the initiation of influenza specific T cell response. We now show that activation products of the complement system interact with their receptors on the DCs, which signals for the DCs to migrate from the lung to the dLN. Thus, our results reveal a previously unknown function for complement in mediating lung DC migration during influenza infection and highlight its potential as an adjuvant in novel vaccine strategies.

Suppression of immune responses by regulatory T cells (Tregs) is thought to limit late stages of pathogen-specific immunity as a means of minimizing associated tissue damage. We examined a role for Tregs during mucosal herpes simplex virus infection in mice, and observed an accelerated fatal infection with increased viral loads in the mucosa and central nervous system after ablation of Tregs. Although augmented interferon production was detected in the draining lymph nodes (dLNs) in Treg-deprived mice, it was profoundly reduced at the infection site. This was associated with a delay in the arrival of natural killer cells, dendritic cells, and T cells to the site of infection and a sharp increase in proinflammatory chemokine levels in the dLNs. Our results suggest that Tregs facilitate early protective responses to local viral infection by allowing a timely entry of immune cells into infected tissue.

Initiation of an adaptive cellular immune response depends on intimate interactions with antigen-presenting cells and naive T lymphocytes. We have previously reported that activation of naive Mycobacterium tuberculosis-specific CD4+ T cells depends on dendritic cell (DC) transport of live bacteria from the lungs to the mediastinal lymph node (MDLN). Since the migratory paths of DCs are largely governed by the chemokine receptor CCR7, which is expressed on DCs upon maturation by proinflammatory stimuli, we examined the quantitative contribution of CCR7-dependent DC migration in the context of tuberculosis, and found that early trafficking of DCs from the lungs to the MDLN depended on CCR7-mediated signaling, but alternative mechanism(s) are employed later in infection. Impaired migration of DCs in CCR7−/− mice resulted in delayed dissemination of bacteria to MDLN and spleen, and in delayed kinetics of activation of adoptively-transferred Ag85B-specific CD4+ T cells. Furthermore, in contrast to control mice, we found that naive Ag85B-specific CD4+ T cells are activated to proliferate in the lungs of CCR7−/− mice and, when infected with higher doses of bacteria, resistance to M. tuberculosis infection in CCR7−/− mice is compromised compared to wild type mice.

Acute viral infections induce robust adaptive immune responses resulting in virus clearance. Recent evidence suggests that there may be depots of viral antigen that persist in draining lymph nodes (DLNs) after virus clearance and could, therefore, affect the adaptive immune response and memory T cell formation. The nature of these residual antigen depots, the mechanism of antigen persistence, and the impact of the persistent antigen on memory T cells remain ill defined. Using a mouse model of influenza virus infection of the respiratory tract, we identified respiratory dendritic cells (RDCs) as essential for both sampling and presenting residual viral antigen. RDCs in the previously infected lung capture residual viral antigen deposited in an irradiation-resistant cell type. RDCs then transport the viral antigen to the LNs draining the site of infection, where they present the antigen to T cells. Lastly, we document preferential localization of memory T cells to the DLNs after virus clearance as a consequence of presentation of residual viral antigen by the migrant RDC.

Mature dendritic cells (DCs) are powerful antigen presenting cells that have the unique capacity to migrate to the T cell zone of draining lymph nodes after subcutaneous injection. Here we report that treatment of antigen-pulsed mature DCs with tumor necrosis factor (TNF)-related activation-induced cytokine (TRANCE), a TNF family member, before immunization enhances their adjuvant capacity and elicits improved T cell priming in vivo, such that both primary and memory T cell immune responses are enhanced. By enumerating migratory DCs in the draining lymph nodes and by studying their function in stimulating naive T cells, we show that one of the underlying mechanisms for enhanced T cell responses is an increase in the number of ex vivo antigen-pulsed DCs that are found in the T cell areas of lymph nodes. These results suggest that the longevity and abundance of mature DCs at the site of T cell priming influence the strength of the DC-initiated T cell immunity in situ. Our findings have the potential to improve DC-based immunotherapy; i.e., the active immunization of humans with autologous DCs that have been pulsed with clinically significant antigens ex vivo.

There is growing evidence that chemokines and their receptors regulate the movement and interaction of antigen-presenting cells such as dendritic cells (DCs) and T cells. We tested the hypothesis that the CC chemokine receptor (CCR)2 and CCR5 and the chemokine macrophage inflammatory protein (MIP)-1α, a ligand for CCR5, influence DC migration and localization. We found that deficiency of CCR2 but not CCR5 or MIP-1α led to distinct defects in DC biology. Langerhans cell (skin DC) density in CCR2-null mice was normal, and their ability to migrate into the dermis was intact; however, their migration to the draining lymph nodes was markedly impaired. CCR2-null mice had lower numbers of DCs in the spleen, and this was primarily due to a reduction in the CD8α1 T helper cell type 1 (Th1)-inducing subset of DCs. Additionally, there was a block in the Leishmania major infection–induced relocalization of splenic DCs from the marginal zone to the T cell areas. We propose that these DC defects, in conjunction with increased expression of B lymphocyte chemoattractant, a B cell–specific chemokine, may collectively contribute to the striking B cell outgrowth and Th2 cytokine–biased nonhealing phenotype that we observed in CCR2-deficient mice infected with L. major. This disease phenotype in mice with an L. major–resistant genetic background but lacking CCR2 is strikingly reminiscent of that observed typically in mice with an L. major–susceptible genetic background. Thus, CCR2 is an important determinant of not only DC migration and localization but also the development of protective cell-mediated immune responses to L. major.

Summary

Activated virus-specific CD8 T cells remain in the lung airways for several months after influenza virus infection. We show that maintenance of this cell population is dependent upon the route of infection and prolonged presentation of viral antigen in the draining lymph nodes (DLN) of the respiratory tract. The local effects on T cell migration have been examined. We show retention of virus-specific CD8 T cells in the mediastinal lymph node (MLN) and continuing recruitment of blood-borne migrants into the lung airways during antigen presentation. These data show that antigen that is retained after pulmonary influenza virus infection controls the migratory pattern and activation state of virus-specific CD8 T cells near the site of virus amplification.

Prior ultraviolet (UV) irradiation of the site of application of hapten on murine skin reduces contact sensitization, impairs the ability of dendritic cells in the draining lymph nodes (DLN) to present antigen, and leads to development of hapten-specific suppressor T lymphocytes. We tested the hypothesis that UV-induced DNA damage plays a role in the impaired antigen-presenting activity of DLN cells. First, we assessed the location and persistence of cells containing DNA damage. A monoclonal antibody specific for cyclobutyl pyrimidine dimers (CPD) was used to identify UV-damaged cells in the skin and DLN of C3H mice exposed to UV radiation. Cells containing CPD were present in the epidermis, dermis, and DLN and persisted, particularly in the dermis, for at least 4 d after UV irradiation. When fluorescein isothiocyanate (FITC) was applied to UV-exposed skin, the DLN contained cells that were Ia+, FITC+, and CPD+; such cells from mice sensitized 3 d after UV irradiation exhibited reduced antigen-presenting function in vivo. We then assessed the role of DNA damage in UV-induced modulation of antigen-presenting cell (APC) function by using a novel method of increasing DNA repair in mouse skin in vivo. Liposomes containing T4 endonuclease V (T4N5) were applied to the site of UV exposure immediately after irradiation. This treatment prevented the impairment in APC function and reduced the number of CPD+ cells in the DLN of UV- irradiated mice. Treatment of unirradiated skin with T4N5 in liposomes or treatment of UV-irradiated skin with liposomes containing heat- inactivated T4N5 did not restore immune function. These studies demonstrate that cutaneous immune cells sustain DNA damage in vivo that persists for several days, and that FITC sensitization causes the migration of these to the DLN, which exhibits impaired APC function. Further, they support the hypothesis that DNA damage is an essential initiator of one or more of the steps involved in impaired APC function after UV irradiation.

While prions probably interact with the innate immune system immediately following infection, little is known about this initial confrontation. Here we investigated incunabular events in lymphotropic and intranodal prion trafficking by following highly enriched, fluorescent prions from infection sites to draining lymph nodes. We detected biphasic lymphotropic transport of prions from the initial entry site upon peripheral prion inoculation. Prions arrived in draining lymph nodes cell autonomously within two hours of intraperitoneal administration. Monocytes and dendritic cells (DCs) required Complement for optimal prion delivery to lymph nodes hours later in a second wave of prion trafficking. B cells constituted the majority of prion-bearing cells in the mediastinal lymph node by six hours, indicating intranodal prion reception from resident DCs or subcapsulary sinus macrophages or directly from follicular conduits. These data reveal novel, cell autonomous prion lymphotropism, and a prominent role for B cells in intranodal prion movement.

SUMMARY

Aspergillus fumigatus, a ubiquitous fungus, causes invasive disease in immunocompromised humans. Although monocytes and antigen-specific CD4 T cells contribute to defense against inhaled fungal spores, how these cells interact during infection remains undefined. Investigating the role of inflammatory monocytes and monocyte-derived dendritic cells during fungal infection, we find that A. fumigatus infection induces an influx of chemokine receptor CCR2- and Ly6C-expressing inflammatory monocytes into lungs and draining lymph nodes. Depletion of CCR2+ cells reduced A. fumigatus conidial transport from lungs to draining lymph nodes, abolished CD4 T cell priming following respiratory challenge, and impaired pulmonary fungal clearance. In contrast, depletion of CCR2+Ly6Chi monocytes during systemic fungal infection did not prevent CD4 T cell priming in the spleen. Our findings demonstrate that pulmonary CD4 T cell responses to inhaled spores require CCR2+Ly6Chi monocytes and their derivatives, revealing a compartmentally restricted function for these cells in adaptive respiratory immune responses.

Secondary lymphoid organ chemokine (SLC) is expressed in high endothelial venules and in T cell zones of spleen and lymph nodes (LNs) and strongly attracts naive T cells. In mice homozygous for the paucity of lymph node T cell (plt) mutation, naive T cells fail to home to LNs or the lymphoid regions of spleen. Here we demonstrate that expression of SLC is undetectable in plt mice. In addition to the defect in T cell homing, we demonstrate that dendritic cells (DCs) fail to accumulate in spleen and LN T cell zones of plt mice. DC migration to LNs after contact sensitization is also substantially reduced. The physiologic significance of these abnormalities in plt mice is indicated by a markedly increased sensitivity to infection with murine hepatitis virus. The plt mutation maps to the SLC locus; however, the sequence of SLC introns and exons in plt mice is normal. These findings suggest that the abnormalities in plt mice are due to a genetic defect in the expression of SLC and that SLC mediates the entry of naive T cells and antigen-stimulated DCs into the T cell zones of secondary lymphoid organs.

Naive T cells recirculate mainly within the secondary lymphoid compartment, but once activated they can enter peripheral tissues and perform effector functions. To activate naive T cells, foreign antigens must traffic from the site of infection to the draining lymph nodes, where they can be presented by professional antigen presenting cells. For major histocompatibility complex class I–restricted presentation to CD8+ T cells, this can occur via the cross-presentation pathway. Here, we investigated the conditions allowing antigen access to this pathway. We show that the level of antigen expressed by peripheral tissues must be relatively high to facilitate cross-presentation to naive CD8+ T cells. Below this level, peripheral antigens did not stimulate by cross-presentation and were ignored by naive CD8+ T cells, although they could sensitize tissue cells for destruction by activated cytotoxic T lymphocytes (CTLs). Interestingly, CTL-mediated tissue destruction facilitated cross-presentation of low dose antigens for activation of naive CD8+ T cells. This represents the first in vivo evidence that cellular destruction can enhance access of exogenous antigens to the cross-presentation pathway. These data indicate that the cross-presentation pathway focuses on high dose antigens and those released during tissue destruction.

Mouse mammary tumor virus (MMTV[SW]) encodes a superantigen expressed by infected B cells. It evokes an antibody response specific for viral envelope protein, indicating selective activation of antigen-specific B cells. The response to MMTV(SW) in draining lymph nodes was compared with the response to haptenated chicken gamma globulin (NP-CGG) using flow cytometry and immunohistology. T cell priming occurs in both responses, with T cells proliferating in association with interdigitating dendritic cells in the T zone. T cell proliferation continues in the presence of B cells in the outer T zone, and B blasts then undergo exponential growth and differentiation into plasma cells in the medullary cords. Germinal centers develop in both responses, but those induced by MMTV(SW) appear later and are smaller. Most T cells activated in the T zone and germinal centers in the MMTV(SW) response are superantigen specific and these persist for weeks in lymph nodes draining the site MMTV(SW) injection; this contrasts with the selective loss of superantigen-specific T cells from other secondary lymphoid tissues. The results indicate that this viral superantigen, when expressed by professional antigen-presenting cells, drives extrafollicular and follicular B cell differentiation leading to virus-specific antibody production.

The immune response of naive CD4 T cells to influenza virus is initiated in the draining lymph nodes and spleen, and only after effectors are generated do antigen-specific cells migrate to the lung which is the site of infection. The effector cells generated in secondary organs appear as multiple subsets which are a heterogeneous continuum of cells in terms of number of cell divisions, phenotype and function. The effector cells that migrate to the lung constitute the more differentiated of the total responding population, characterized by many cell divisions, loss of CD62L, down-regulation of CCR7, stable expression of CD44 and CD49d, and transient expression of CCR5 and CD25. These cells also secrete high levels of interferon γ and reduced levels of interleukin 2 relative to those in the secondary lymphoid organs. The response declines rapidly in parallel with viral clearance, but a spectrum of resting cell subsets reflecting the pattern at the peak of response is retained, suggesting that heterogeneous effector populations may give rise to corresponding memory populations. These results reveal a complex response, not an all-or-none one, which results in multiple effector phenotypes and implies that effector cells and the memory cells derived from them can display a broad spectrum of functional potentials.

The initial steps of Venezuelan equine encephalitis virus (VEE) spread from inoculation in the skin to the draining lymph node have been characterized. By using green fluorescent protein and immunocytochemistry, dendritic cells in the draining lymph node were determined to be the primary target of VEE infection in the first 48 h following inoculation. VEE viral replicon particles, which can undergo only one round of infection, identified Langerhans cells to be the initial set of cells infected by VEE directly following inoculation. These cells are resident dendritic cells in the skin, which migrate to the draining lymph node following activation. A point mutation in the E2 glycoprotein gene of VEE that renders the virus avirulent and compromises its ability to spread beyond the draining lymph blocked the appearance of virally infected dendritic cells in the lymph node in vivo. A second-site suppressor mutation that restores viral spread to lymphoid tissues and partially restore virulence likewise restored the ability of VEE to infect dendritic cells in vivo.

The paucity of lymph node T cells (plt) mutation leads to a loss of CCL21 and CCL19 expression in secondary lymphoid organs. plt mice have defects in the migration of naive T cells and activated dendritic cells into the T cell zones of lymphoid organs, suggesting that they would have defects in T cell immune responses. We now demonstrate T cell responses in plt mice are delayed but ultimately enhanced. Responses to contact sensitization are decreased at day 2 after priming but increased at day 6. After subcutaneous immunization, antigen-specific T cell proliferation and cytokine production in plt mice are increased and remain markedly elevated for at least 8 wk. Compared with wild-type mice, a proportion of T cell response in plt mice are shifted to the spleen, and prior splenectomy reduces the T cell response in draining lymph nodes. After immunization of plt mice, T cells and dendritic cells colocalize in the superficial cortex of lymph nodes and in splenic bridging channels, but not in T cell zones. These results demonstrate that plt mice mount robust T cell responses despite the failure of naive T cells and activated dendritic cells to enter the thymus dependent areas of secondary lymphoid organs.

West Nile virus (WNV) is transmitted during mosquito bloodfeeding. Consequently, the first vertebrate cells to contact WNV are cells in the skin, followed by those in the draining lymph node. Macrophages and dendritic cells are critical early responders in host defense against WNV infection, not just because of their role in orchestrating the immune response, but also because of their importance as sites of early peripheral viral replication. Antigen-presenting cell (APC) signals have a profound effect on host antiviral responses and disease severity. During transmission, WNV is intimately associated with mosquito saliva. Due to the ability of mosquito saliva to affect inflammation and immune responses, and the importance of understanding early events in WNV infection, we investigated whether mosquito saliva alters APC signaling during arbovirus infection, and if alterations in cell recruitment occur when WNV infection is initiated with mosquito saliva. Accordingly, experiments were performed with cultured dendritic cells and macrophages, flow cytometry was used to characterize infiltrating cell types in the skin and lymph nodes during early infection, and real-time RT-PCR was employed to evaluate virus and cytokine levels. Our in vitro results suggest that mosquito saliva significantly decreases the expression of interferon-β and inducible nitric oxide synthase in macrophages (by as much as 50 and 70%, respectively), whilst transiently enhancing interleukin-10 (IL-10) expression. In vivo results indicate that the predominate effect of mosquito feeding is to significantly reduce the recruitment of T cells, leading the inoculation site of mice exposed to WNV alone to have up to 2.8 fold more t cells as mice infected in the presence of mosquito saliva. These shifts in cell population are associated with significantly elevated IL-10 and WNV (up to 4.0 and 10 fold, respectively) in the skin and draining lymph nodes. These results suggest that mosquito saliva dysregulates APC antiviral signaling, and reveal a possible mechanism for the observed enhancement of WNV disease mediated by mosquito saliva via a reduction of T lymphocyte and antiviral activity at the inoculation site, an elevated abundance of susceptible cell types, and a concomitant increase in immunoregulatory activity of IL-10.

The recruitment of antigen-specific T lymphocytes to the intestinal mucosa is central to the development of an effective mucosal immune response, yet the mechanism by which this process occurs remains to be fully defined. Here we show that the CC chemokine receptor 9 (CCR9) is selectively and functionally expressed on murine αEβ7+ naive CD8αβ+ lymphocytes and a subset of recently activated CD69+ CD8αβ+ lymphocytes. Using a T cell receptor transgenic transfer model, we demonstrate that CCR9 expression is functionally maintained on CD8αβ+ lymphocytes following activation in mesenteric lymph nodes but rapidly downregulated on CD8αβ+ lymphocytes activated in peripheral lymph nodes. These recently activated CCR9+ CD8αβ+ lymphocytes selectively localized to the small-intestinal mucosa, and in vivo neutralization of the CCR9 ligand, CCL25, reduced the ability of these cells to populate the small-intestinal epithelium. Together these results demonstrate an important role for chemokines in the localization of T lymphocytes to the small-intestinal mucosa and suggest that targeting CCL25 and/or CCR9 may provide a means to selectively modulate small-intestinal immune responses.

Mycobacterium tuberculosis infection results in the generation of protective cellular immunity and formation of granulomatous structures in the lung. CXC chemokine ligand (CXCL)-13, CC chemokine ligand (CCL)-21 and CCL19 are constitutively expressed in the secondary lymphoid organs and play a dominant role in the homing of lymphocytes and dendritic cells. Although it is known that dendritic cell transport of M. tuberculosis from the lung to the draining lymph node is dependent on CCL19/CCL21, we show here that CCL19/CCL21 is also important for the accumulation of antigen-specific IFNγ-producing T cells in the lung, development of the granuloma, and control of mycobacteria. Importantly, we also show that CXCL13 is not required for generation of IFNγ responses, but is essential for the spatial arrangement of lymphocytes within granulomas, optimal activation of phagocytes and subsequent control of mycobacterial growth. Further, we show that these chemokines are also induced in the lung during the early immune responses following pulmonary M. tuberculosis infection. These results demonstrate that homeostatic chemokines perform distinct functions that cooperate to mediate effective expression of immunity against M. tuberculosis infection.