Novel approach to visualize the inter-dependencies between maternal sensitization, breast milk immune components and human milk oligosaccharides in the LIFE Child cohort.

BACKGROUND: Numerous studies have shown that specific components of breast milk, considered separately, are associated with disease status in the mother or the child using univariate analyses. However, very few studies have considered multivariate analysis approaches to evaluate the relationship between multiple breast milk components simultaneously. AIM: Here we aimed at visualizing breast milk component complex interactions in the context of the allergy status of the mother or the child. METHODS: Milk samples were collected from lactating mothers participating in the Leipziger Forschungszentrum für Zivilisationskrankheiten (LIFE) Child cohort in Leipzig, Germany. A total of 156 breast milk samples, collected at 3 months after birth from mother/infant pairs, were analyzed for 51 breast milk components. Correlation, principal component analysis (PCA) and graphical discovery analysis were used. RESULT: Correlations ranging from 0.40 to 0.96 were observed between breast milk fatty acid and breast milk phospholipids levels and correlations ranging from 0 to 0.76 between specific human milk oligosaccharides (HMO) were observed. No separation of the data based on the risk of allergy in the infants was identified using PCA. When graphical discovery analysis was used, dependencies between maternal plasma immunoglobulin E (IgE) level and the breast milk immune marker transforming growth factor-beta 2 (TGF-ß2), between TGF-ß2, breast milk immunoglobulin A (IgA) and TGF-ß1 as well as between breast milk total protein and birth weight were observed. Graphical discovery analysis also exemplifies a possible competition for the fucosyl group between 2'FL, LNFP-I and 3'FL in the HMO group. Additionally, dependencies between immune component IgA and specific HMO (6'SL and blood group A antigen tetraose type 5 or PI-HMO) were identified. CONCLUSION: Graphical discovery analysis applied to complex matrices such as breast milk composition can aid in understanding the complexity of interactions between breast milk components and possible relations to health parameters in the mother or the infant. This approach can lead to novel discoveries in the context of health and diseases such as allergy. Our study thus represents the first attempt to visualize the complexity and the inter-dependency of breast milk components.

A Rapid Chemiluminescence Assay for Measurement of Folate in Small Volumes of Breast Milk.

Early life exposure to folate has long lasting effects on development and health. Newborns obtain part of their folate from maternal milk. Studies on health effects of milk folate require rapid, affordable and reliable measurements in large numbers of samples from cohort studies. Recently, a competitive chemiluminescence assay for quantification of folate has become available for automated diagnostic measurement of folate in human serum or plasma. We tested if this method ("FOLA" from Siemens Healthcare) could also be used for human milk. To minimize interference and matrix effects, samples had to be skimmed, diluted seven times with demineralized water, and heated for 5 min at 90 °C. Folate could thus be measured in a linear range between 8.4 and 111.7 nM, with recoveries for the most relevant form, 5-methyltetrahydrofolate (5-MeTHF), of 96%-107%. Results were comparable to those with a recently validated Liquid Chromatography/Mass Spectrometry method (Y = 0.998X - 0.2; R2 = 0.807). The FOLA method was subsequently used for samples from the LIFE Child cohort in Germany, providing first data of breast milk folate in this country (range: 6.2-100.7 nM). This technique could indeed prove useful for large cohorts with multiple samplings.

Oral administration of Lactobacillus paracasei NCC 2461 for the modulation of grass pollen allergic rhinitis: a randomized, placebo-controlled study during the pollen season.

BACKGROUND: The efficacy of Lactobacillus paracasei NCC 2461 in modulating allergic rhinitis was previously evaluated in two exploratory clinical studies. Oral administration with NCC 2461 reduced specific subjective symptoms following nasal provocation tests with controlled grass pollen allergen concentrations. Our aim was to confirm the anti-allergic effect of NCC 2461 in grass pollen allergic subjects exposed to natural doses of allergens during the pollen season. FINDINGS: A double-blind, randomized, placebo-controlled, parallel study was conducted with 131 grass pollen allergic subjects from May to July 2012 in concomitance with the pollen season in Berlin. NCC 2461 or placebo was administered daily for an 8-week period to adult subjects with clinical history of allergic rhinitis to grass pollen, positive skin prick test and IgE to grass pollen. During the 8 weeks, symptoms and quality of life questionnaires were filled out, and plasma was collected for IgE analysis at screening and at the end of the intervention. All subjects were included within a 5-day interval, ensuring exposure to similar air pollen counts for each individual during the trial period. The results obtained show that symptoms increased with pollen loads, confirming a natural exposure to the allergen and presence of pollen-induced allergic rhinitis in the subjects. However, no significant differences were observed in allergic rhinitis symptoms scores, quality of life, or specific IgE levels between subjects receiving NCC 2461 as compared to placebo administration. CONCLUSION: In contrast to previous findings, oral administration of NCC 2461 did not show a beneficial effect on allergic rhinitis in a field trial. The influence of study design, allergen exposure and intervention window on the efficacy of NCC 2461 in modulating respiratory allergy should be further evaluated.

Nanoparticle conjugation enhances the immunomodulatory effects of intranasally delivered CpG in house dust mite-allergic mice.

An emerging strategy in preventing and treating airway allergy consists of modulating the immune response induced against allergens in the lungs. CpG oligodeoxynucleotides have been investigated in airway allergy studies, but even if promising, efficacy requires further substantiation. We investigated the effect of pulmonary delivery of nanoparticle (NP)-conjugated CpG on lung immunity and found that NP-CpG led to enhanced recruitment of activated dendritic cells and to Th1 immunity compared to free CpG. We then evaluated if pulmonary delivery of NP-CpG could prevent and treat house dust mite-induced allergy by modulating immunity directly in lungs. When CpG was administered as immunomodulatory therapy prior to allergen sensitization, we found that NP-CpG significantly reduced eosinophilia, IgE levels, mucus production and Th2 cytokines, while free CpG had only a moderate effect on these parameters. In a therapeutic setting where CpG was administered after allergen sensitization, we found that although both free CpG and NP-CpG reduced eosinophilia and IgE levels to the same extent, NP conjugation of CpG significantly enhanced reduction of Th2 cytokines in lungs of allergic mice. Taken together, these data highlight benefits of NP conjugation and the relevance of NP-CpG as allergen-free therapy to modulate lung immunity and treat airway allergy.

Nanoparticle conjugation of CpG enhances adjuvancy for cellular immunity and memory recall at low dose.

In subunit vaccines, strong CD8(+) T-cell responses are desired, yet they are elusive at reasonable adjuvant doses. We show that targeting adjuvant to the lymph node (LN) via ultrasmall polymeric nanoparticles (NPs), which rapidly drain to the LN after intradermal injection, greatly enhances adjuvant efficacy at low doses. Coupling CpG-B or CpG-C oligonucleotides to NPs led to better dual-targeting of adjuvant and antigen (codelivered on separate NPs) in cross-presenting dendritic cells compared with free adjuvant. This led to enhanced dendritic cell maturation and T helper 1 (Th1)-cytokine secretion, in turn driving stronger effector CD8(+) T-cell activation with enhanced cytolytic profiles and, importantly, more powerful memory recall. With only 4 µg CpG, NP-CpG-B could substantially protect mice from syngeneic tumor challenge, even after 4 mo of vaccination, compared with free CpG-B. Together, these results show that nanocarriers can enhance vaccine efficacy at a low adjuvant dose for inducing potent and long-lived cellular immunity.

A high-throughput nanoimmunoassay chip applied to large-scale vaccine adjuvant screening.

Large-scale experimentation is becoming instrumental in enabling new discoveries in systems biology and personalized medicine. We developed a multiplexed high-throughput nanoimmunoassay chip capable of quantifying four biomarkers in 384 5 nL samples, for a total of 1536 assays. Our platform, compared to conventional methods, reduces volume and reagent cost by ~1000-fold. We applied our platform in the context of systems vaccinology, to assess the synergistic production of inflammatory cytokines from dendritic cells (DCs) stimulated with 10 different adjuvants that target members of the Toll-like receptor (TLR) family. We quantified these adjuvants both alone and in all pairwise combinations, for a total of 435 conditions, revealing numerous synergistic pairs. We evaluated two synergistic interactions, MPLA + Gardiquimod and MPLA + CpG-B, in a mouse model, where we measured the same inflammatory cytokines in bronchoalveolar lavage and in blood serum at 4 different time points using our chip, and observed similar synergistic effects in vivo, demonstrating the potential of our microfluidic platform to predict agonistic immunogenicity. More generally, a high-throughput, matrix-insensitive, low sample volume technology can play an important role in the discovery of novel therapeutics and research areas requiring large-scale biomarker quantitation.

Nanoparticle size influences the magnitude and quality of mucosal immune responses after intranasal immunization.

BACKGROUND: The development of nanoparticulate antigen-delivery systems is an important emerging area of vaccinology, being sought to amplify immune responses to recombinant antigens that are poorly immunogenic. Nanoparticle size may play an important role in influencing the activity of such particulate-based adjuvants. METHODS: To explore how the size of nanoparticles that are in the range of many common viruses can modulate the magnitude and quality of mucosal immune responses, the model antigen ovalbumin (OVA) was conjugated to 30 nm or 200 nm polypropylene sulfide nanoparticles (NPs) and administered intranasally to C57BL/6 mice. RESULTS: We show that by increasing the size of the NPs from 30 to 200 nm, OVA was more effectively delivered into both MHC class I and MHC class II-presentation pathways. Intranasal immunization with the 200 nm NPs increased the magnitude of CD4(+) T cell responses in the lungs, as well as systemic and mucosal humoral responses. Most importantly, 200 nm NPs increased the proportion of antigen-specific polyfunctional CD4(+) T cells as compared to 30 nm NPs. CONCLUSIONS: The 200 nm NPs are a very interesting antigen nanocarrier for prophylactic vaccines against mucosal pathogens that require multifunctional CD4(+) T cells for protection. These results contribute to our understanding of how the size of an antigen-conjugated nanoparticle modulates mucosal immune responses to a protein antigen and may be useful to engineer subunit vaccines able to elicit appropriate mucosal immune responses that correlate with protection.

VEGF-C promotes immune tolerance in B16 melanomas and cross-presentation of tumor antigen by lymph node lymphatics.

Tumor expression of the lymphangiogenic factor VEGF-C is correlated with metastasis and poor prognosis, and although VEGF-C enhances transport to the draining lymph node (dLN) and antigen exposure to the adaptive immune system, its role in tumor immunity remains unexplored. Here, we demonstrate that VEGF-C promotes immune tolerance in murine melanoma. In B16 F10 melanomas expressing a foreign antigen (OVA), VEGF-C protected tumors against preexisting antitumor immunity and promoted local deletion of OVA-specific CD8(+) T cells. Naive OVA-specific CD8(+) T cells, transferred into tumor-bearing mice, were dysfunctionally activated and apoptotic. Lymphatic endothelial cells (LECs) in dLNs cross-presented OVA, and naive LECs scavenge and cross-present OVA in vitro. Cross-presenting LECs drove the proliferation and apoptosis of OVA-specific CD8(+) T cells ex vivo. Our findings introduce a tumor-promoting role for lymphatics in the tumor and dLN and suggest that lymphatic endothelium in the local microenvironment may be a target for immunomodulation.

Nanoparticle conjugation of antigen enhances cytotoxic T-cell responses in pulmonary vaccination.

The ability of vaccines to induce memory cytotoxic T-cell responses in the lung is crucial in stemming and treating pulmonary diseases caused by viruses and bacteria. However, most approaches to subunit vaccines produce primarily humoral and only to a lesser extent cellular immune responses. We developed a nanoparticle (NP)-based carrier that, upon delivery to the lung, specifically targets pulmonary dendritic cells, thus enhancing antigen uptake and transport to the draining lymph node; antigen coupling via a disulfide link promotes highly efficient cross-presentation after uptake, inducing potent protective mucosal and systemic CD8(+) T-cell immunity. Pulmonary immunization with NP-conjugated ovalbumin (NP-ova) with CpG induced a threefold enhancement of splenic antigen-specific CD8(+) T cells displaying increased CD107a expression and IFN-γ production compared with immunization with soluble (i.e., unconjugated) ova with CpG. This enhanced response was accompanied by a potent Th17 cytokine profile in CD4(+) T cells. After 50 d, NP-ova and CpG also led to substantial enhancements in memory CD8(+) T-cell effector functions. Importantly, pulmonary vaccination with NP-ova and CpG induced as much as 10-fold increased frequencies of antigen-specific effector CD8(+) T cells to the lung and completely protected mice from morbidity following influenza-ova infection. Here, we highlight recruitment to the lung of a long-lasting pool of protective effector memory cytotoxic T-cells by our disulfide-linked antigen-conjugated NP formulation. These results suggest the reduction-reversible NP system is a highly promising platform for vaccines specifically targeting intracellular pathogens infecting the lung.

Nano-sized drug-loaded micelles deliver payload to lymph node immune cells and prolong allograft survival.

By delivering immunomodulatory drugs in vivo directly to lymph nodes draining an injection site, an opportunity exists to increase drug bioavailability to local immune cells. Importantly, particles smaller than 100 nm are efficiently transported through lymphatic vessels to draining lymph nodes. To investigate whether this approach could be used for local delivery of immunomodulatory drugs, amphiphilic poly(ethylene glycol)-bl-poly(propylene sulfide) (PEG-bl-PPS) block copolymers forming 50 nm micelles were used to encapsulate hydrophobic drugs. Micelle drainage was determined using fluorescent micelles and showed effective targeting of multiple immune cell subsets in lymph nodes. For functional studies of our formulations, two approaches were considered. To evaluate the efficacy of anti-inflammatory drug delivery, dendritic cell activation was shown to be prevented when mice were pretreated with micelles loaded with the glucocorticoid mometasone and then challenged with the TLR9 ligand, CpG. To evaluate whether immunosuppressive drug-loaded micelles were effective in prolonging MHC-mismatched allograft survival, BALB/c mice were treated for 14 consecutive days with drug-loaded micelles following transplantation of allogenic C57BL/6 tail skin. Micelles loaded with a mixture of rapamycin and tacrolimus prolonged allograft survival by 2-fold. Our results indicate that the drug-loaded micelle approach effectively targets the draining lymph nodes and exhibits proper immune regulation.

Nanoparticle conjugation and pulmonary delivery enhance the protective efficacy of Ag85B and CpG against tuberculosis.

Vaccines that drive robust T-cell immunity against Mycobacterium tuberculosis (Mtb) are needed both for prophylactic and therapeutic purposes. We have recently developed a synthetic vaccine delivery platform with Pluronic-stabilized polypropylene sulfide nanoparticles (NPs), which target lymphoid tissues by their small size (∼ 30 nm) and which activate the complement cascade by their surface chemistry. Here we conjugated the tuberculosis antigen Ag85B to the NPs (NP-Ag85B) and compared their efficacy in eliciting relevant immune responses in mice after intradermal or pulmonary administration. Pulmonary administration of NP-Ag85B with the adjuvant CpG led to enhanced induction of antigen-specific polyfunctional Th1 responses in the spleen, the lung and lung-draining lymph nodes as compared to soluble Ag85B with CpG and to the intradermally-delivered formulations. Mucosal and systemic Th17 responses were also observed with this adjuvanted NP formulation and vaccination route, especially in the lung. We then evaluated protection induced by the adjuvanted NP formulation following a Mtb aerosol challenge and found that vaccination with NP-Ag85B and CpG via the pulmonary route displayed a substantial reduction of the lung bacterial burden, both compared to soluble Ag85B with CpG and to the corresponding intradermally delivered formulations. These findings highlight the potential of administrating NP-based formulations by the pulmonary route for TB vaccination.

Bacterial-induced protection against allergic inflammation through a multicomponent immunoregulatory mechanism.

BACKGROUND: Airborne microbial products have been reported to promote immune responses that suppress asthma, yet how these beneficial effects take place remains controversial and poorly understood. METHODS: We exposed mice to the bacterium Escherichia coli and subsequently induced allergic airway inflammation through sensitization and intranasal challenge with ovalbumin. RESULTS: Pulmonary exposure to the bacterium Escherichia coli leads to a suppression of allergic airway inflammation. This immune modulation was neither mediated by the induction of a T helper 1 (Th1) response nor regulatory T cells; however, it was dependent on Toll-like receptor 4 (TLR4) but did not involve TLR desensitisation. Dendritic cell migration to the draining lymph nodes and activation of T cells was unaffected by prior exposure to E. coli, while dendritic cells in the lung displayed a less activated phenotype and had impaired antigen presentation capacity. Consequently, in situ Th2 cytokine production was abrogated. The suppression of airway hyper-responsiveness was mediated through the recruitment of gd T cells; however, the suppression of dendritic cells and T cells was mediated through a distinct mechanism that could not be overcome by the local administration of activated dendritic cells, or by the in vivo administration of tumour necrosis factor a. CONCLUSION: Our data reveal a localized immunoregulatory pathway that acts to protect the airways from allergic inflammation.

The kinase activity of Rip2 determines its stability and consequently Nod1- and Nod2-mediated immune responses.

Rip2 (RICK, CARD3) has been identified as a key effector molecule downstream of the pattern recognition receptors, Nod1 and Nod2; however, its mechanism of action remains to be elucidated. In particular, it is unclear whether its kinase activity is required for signaling or for maintaining protein stability. We have investigated the expression level of different retrovirally expressed kinase-dead Rip2 mutants and the role of Rip2 kinase activity in the signaling events that follow Nod1 and Nod2 stimulation. We show that in primary cells expressing kinase-inactive Rip2, protein levels were severely compromised, and stability could not be reconstituted by the addition of a phospho-mimetic mutation in its autophosphorylation site. Consequently, inflammatory cytokine production in response to Nod1 and Nod2 ligands was abrogated both in vitro and in vivo in the absence of Rip2 kinase activity. Our results highlight the central role that Rip2 kinase activity plays in conferring stability to the protein and thus in the preservation of Nod1- and Nod2-mediated innate immune responses.

IL-17-producing T cells in lung immunity and inflammation.

T(H)17 cells are a recently described effector CD4 T-cell subset characterized by the production of IL-17A, IL-17F, and IL-22, which have been implicated in the pathogenesis of several autoimmune diseases. T(H)17 and other IL-17A-producing T cells, including a population of gammadelta T cells and natural killer T cells, have also been associated with the development of skin, intestinal, and lung inflammatory diseases, such as asthma, granulomatous disease, chronic obstructive pulmonary disease, and cystic fibrosis. On the other hand, IL-17-producing T cells play important roles in protective immunity against some bacterial infections, mainly through the recruitment and activation of neutrophils. Thus, their regulation appears to be critical, and excess or deficient IL-17 elaboration leads either to deficient responses or disease. This review will summarize T(H)17 cell differentiation and discuss the host beneficial and detrimental function of IL-17A and related cytokines produced by different subpopulations of T cells.

Effective T-cell immune responses in the absence of the serine/threonine kinase RIP2.

The serine/threonine kinase RIP2 has been reported to be essential for Nod1 and Nod2 mediated cell activation, and has been suggested to play a role in the signaling cascade downstream of the T-cell receptor. We sought to ascertain the exact role of RIP2 in T-helper cell differentiation and CD8+ T-cell effector function in vivo and in vitro. In contrast to previous reports, we found that RIP2-deficient T cells did not exhibit impaired proliferation upon TCR engagement in vitro, and differentiation to cytokine producing Th1 or Th2 cells was normal in the absence of RIP2. These results were confirmed in vivo, as wild-type and RIP2-deficient virus-specific CD8+ T cells expanded comparably in mice after LCMV infection. Wild-type and RIP2-deficient CD4+ and CD8+ T cells from infected mice also showed similar proliferation and cytokine production when restimulated with full or partial agonist peptides ex vivo. Furthermore, no significant difference in adaptive T-cell responses could be observed between wild-type and RIP2-deficient mice after Listeria monocytogenes infection. Thus contrary to early reports, our data show that RIP2 is not an essential component of the TCR signaling machinery.

TLR ligands act directly upon T cells to restore proliferation in the absence of protein kinase C-theta signaling and promote autoimmune myocarditis.

The serine/threonine kinase, protein kinase C-theta (PKC-theta), plays a central role in the activation and differentiation of Th2 cells while being redundant in CD4+ and CD8+ antiviral responses. Recent evidence indicates that PKC-theta may however be required for some T cell-driven autoimmune responses. We have investigated the role of PKC-theta in the induction of autoimmune myocarditis induced by either Coxsackie B3 virus infection or immunization with alpha-myosin/CFA (experimental autoimmune myocarditis (EAM)). PKC-theta-deficient mice did not develop EAM as shown by impaired inflammatory cell infiltration into the heart, reduced CD4+ T cell IL-17 production, and the absence of a myosin-specific Ab response. Comparatively, PKC-theta was not essential for both early and late-phase Coxsackie virus-induced myocarditis. We sought to find alternate pathways of immune stimulation that might reconcile the differential requirements for PKC-theta in these two disease models. We found systemic administration of the TLR ligand CpG restored EAM in PKC-theta-deficient mice. CpG could act directly upon TLR9-expressing T cells to restore proliferation and up-regulation of Bcl-x(L), but exogenous IL-6 and TGF-beta was required for Th17 cell differentiation. Taken together, these results indicate that TLR-mediated activation of T cells can directly overcome the requirement for PKC-theta signaling and, combined with the dendritic cell-derived cytokine milieu, can promote the development of autoimmunity.

Mechanisms of neonatal mucosal antibody protection.

Following an abrupt transition at birth from the sterile uterus to an environment with abundant commensal and pathogenic microbes, neonatal mammals are protected by maternal Abs at mucosal surfaces. We show in mice that different Ab isotypes work in distinct ways to protect the neonatal mucosal surface. Secretory IgA acts to limit penetration of commensal intestinal bacteria through the neonatal intestinal epithelium: an apparently primitive process that does not require diversification of the primary natural Ab repertoire. In contrast, neonatal protection against the exclusively luminal parasite Heligmosomoides polygyrus required IgG from primed females. This immune IgG could either be delivered directly in milk or retrotransported via neonatal Fc receptor from the neonatal serum into the intestinal lumen to exert its protective effect.

Strong TCR signaling, TLR ligands, and cytokine redundancies ensure robust development of type 1 effector T cells.

T cell effector function is a central mechanism of adaptive immunity, and accordingly, protection of the host against pathogens. One of the primary effector molecules produced by T cells in response to such pathogens is the cytokine, IFN-gamma. Although the signaling pathways associated with the production of IFN-gamma are well established, disparate in vivo and in vitro results indicate that distinct pathways may become more prominent dependent upon the nature of the infection, inflammatory milieu and tissue localization. We have examined the roles and requirements of the major IFN-gamma-inducing pathways in vivo and in vitro, specifically: strength of TCR signal; paracrine release of IL-12, IL-23, and IL-18; and autocrine production of IFN-gamma. Our data show a dynamic interaction between these activation pathways, which allows the host a degree of flexibility and redundancy in the induction of IFN-gamma. Upon strong signaling through the TCR, IL-12, IL-18, and IL-23 play negligible roles in the induction of IFN-gamma, whereas autocrine IFN-gamma is an important component in sustaining its own secretion. However, the absence of any one of these factors during a weaker TCR signal, results in strikingly impaired T cell IFN-gamma production. Of note, TLR-activated dendritic cells (DCs) were capable of overcoming the absence of a strong TCR signal, IL-12, IL-23, or IL-18 revealing an important additional mechanism for ensuring a robust IFN-gamma response. Our findings clarify the hierarchical requirements of the major IFN-gamma inducing pathways and highlight the important role TLR ligand-activated DCs have to preserve them.

Innate signals compensate for the absence of PKC-{theta} during in vivo CD8(+) T cell effector and memory responses.

PKC- is central to T-helper (Th) 2 cell differentiation and effector function; however, its importance for antiviral effector, and in particular memory CD8(+) T cell responses, remains unclear. We have investigated the role of PKC- during in vivo and in vitro responses against influenza virus, lymphocytic choriomeningitis virus, vaccinia virus, and replication-deficient virus-like particles. In the absence of PKC-, antiviral CD8(+) T cells presented an unresponsive phenotype in vitro, which could be restored with exogenous IL-2 or by Toll-like receptor ligand-activated dendritic cells. In striking contrast, PKC- appeared to be superfluous for in vivo antiviral responses irrespective of whether the virus infected systemically, was localized to the lung, or did not replicate. In addition, CD8(+) CCR7-effector memory responses were normal in PKC--deficient mice, both in lymphoid and peripheral tissues. Our data show that increased activation signals delivered in vivo by highly activated dendritic cells, as present during viral infections, overcome the requirement for PKC- during CD8(+) T cell antiviral responses.

CCL19 and CCL21 induce a potent proinflammatory differentiation program in licensed dendritic cells.

Dendritic cells (DCs) are key instigators of adaptive immune responses. Using an alphaviral expression cloning technology, we have identified the chemokine CCL19 as a potent inducer of T cell proliferation in a DC-T cell coculture system. Subsequent studies showed that CCL19 enhanced T cell proliferation by inducing maturation of DCs, resulting in upregulation of costimulatory molecules and the production of proinflammatory cytokines. Moreover, CCL19 programmed DCs for the induction of T helper type (Th) 1 rather than Th2 responses. Importantly, only activated DCs that migrated from the periphery to draining lymph nodes, but not resting steady-state DCs residing within lymph nodes, expressed high levels of CCR7 in vivo and responded to CCL19 with the production of proinflammatory cytokines. Migrating DCs isolated from mice genetically deficient in CCL19 and CCL21 (plt/plt) presented an only partially mature phenotype, highlighting the importance of these chemokines for full DC maturation in vivo. Our findings indicate that CCL19 and CCL21 are potent natural adjuvants for terminal activation of DCs and suggest that chemokines not only orchestrate DC migration but also regulate their immunogenic potential for the induction of T cell responses.