Search

Murine T cell subsets differ in their expression level of P2X7. Depending on several parameters like extracellular NAD+ , P2X7 can be ADP-ribosylated rapidly by adjacent ARTC2.2 resulting in susceptibilities to apoptosis to a varying extent. This detrimental effect can be prevented when drugs like KN-62 are present during cell preparations.

Background To successfully limit pathogen dissemination, an immunological link between the entry tissue of the pathogen and the underlying secondary lymphoid organs (SLOs) needs to be established to prime adaptive immune responses. Here, the prerequisite of CCR7 to mount host immune responses within SLOs during gastrointestinal Yersinia pseudotuberculosis infection to limit pathogen spread was investigated. Methods Survival, bacterial dissemination, and intestinal and systemic pathology of wild-type and CCR7-/- mice were assessed and correlated to the presence of immune cell subsets and cytokine responses throughout the course of infection. Results The CCR7-/- mice show a significantly higher morbidity and are more prone to pathogen dissemination and intestinal and systemic inflammation during the oral route of infection. Significant impact of CCR7 deficiency over the course of infection on several immunological parameters were observed (ie, elevated neutrophil-dominated innate immune response in Peyer's patches, limited dendritic cell migration to mesenteric lymph nodes [mLNs] causing reduced T cell-mediated adaptive immune responses (in particular Th17-like responses) in mLNs). Conclusions Our work indicates that CCR7 is required to mount a robust immune response against enteropathogenic Y. pseudotuberculosis by promoting Th17-like responses in mLNs.

The chemokine receptor CXCR5 is primarily expressed on B cells and Tfh cells and facilitates their migration towards B cell follicles. In the present study we investigated the role of the CXCL13/CXCR5 axis in the pathogenesis of rheumatoid arthritis (RA) and specifically addressed the impact of CXCR5-mediated T and B cell migration in this disease. Employing collagen-induced arthritis (CIA) we identify CXCR5 as an absolutely essential factor for the induction of inflammatory autoimmune arthritis. Cxcr5-deficient mice and mice selectively lacking Cxcr5 on T cells were completely resistant to CIA, showed impaired germinal center responses and failed to mount an IgG1 antibody response to collagen II. Selective ablation of CXCR5 expression in B cells also led to suppression of CIA owing to diminished GC responses in secondary lymphoid organs (SLO) and impaired anti-collagen II antibody production. Chimeric mice harboring Cxcr5-proficient and Cxcr5-deficient immune cells revealed SLO and not the synovial tissue as the compartment where CXCR5-mediated cell migration induces autoimmune inflammation in arthritis. Thus our data demonstrate that CXCR5-mediated co-localization of Tfh cells and B cells in SLOs is absolutely essential for the induction of RA and identify CXCR5 and Tfh cells as promising therapeutic targets for the treatment of RA.

Bronchus-associated lymphoid tissue (BALT) forms spontaneously in the lung after pulmonary infection and has been identified as a highly organized lymphoid structure supporting the efficient priming of T cells in the lung. To explore the mechanisms and instructive signals controlling BALT neogenesis we used both, a single dose of vaccinia virus MVA and repeated inhalations of heat-inactivated Pseudomonas aeruginosa (P. aeruginosa). Intranasal administration of both pathogens induces highly organized BALT but distinct pathways and molecules are used to promote the development of BALT. Here, we describe the induction and phenotype of the distinct types of BALT as well as the immunofluorescence microscopy-based analysis of the induced lymphoid tissue in the lung.

Bronchus-associated lymphoid tissue (BALT) forms spontaneously in the lung after pulmonary infection and has been identified as a highly organized lymphoid structure supporting the efficient priming of T cells in the lung. To explore the mechanisms and instructive signals controlling BALT neogenesis we used both, a single dose of vaccinia virus MVA and repeated inhalations of heat-inactivated Pseudomonas aeruginosa (P. aeruginosa). Intranasal administration of both pathogens induces highly organized BALT but distinct pathways and molecules are used to promote the development of BALT. Here, we describe the induction and phenotype of the distinct types of BALT as well as the immunofluorescence microscopy-based analysis of the induced lymphoid tissue in the lung.

Ectopic lymphoid tissue, such as bronchus-associated lymphoid tissue (BALT) in the lung, develops spontaneously at sites of chronic inflammation or during infection. The molecular mechanisms underlying the neogenesis of such tertiary lymphoid tissue are still poorly understood. We show that the type of inflammation-inducing pathogen determines which key factors are required for the formation and maturation of BALT. Thus, a single intranasal administration of the poxvirus modified vaccinia virus Ankara (MVA) is sufficient to induce highly organized BALT with densely packed B cell follicles containing a network of CXCL13-expressing follicular DCs (FDCs), as well as CXCL12-producing follicular stromal cells. In contrast, mice treated with P. aeruginosa (P.a.) develop BALT but B cell follicles lack FDCs while still harboring CXCL12-positive follicular stromal cells. Furthermore, in IL-17–deficient mice, P.a.-induced BALT largely lacks B cells as well as CXCL12-expressing stromal cells, and only loose infiltrates of T cells are present. We show that Toll-like receptor pathways are required for BALT induction by P.a., but not MVA, and provide evidence that IL-17 drives the differentiation of lung stroma toward podoplanin-positive CXCL12-expressing cells that allow follicle formation even in the absence of FDCs. Taken together, our results identify distinct pathogen-dependent induction and maturation pathways for BALT formation.

αβ T-cell development and selection proceed while thymocytes successively migrate through distinct regions of the thymus. For γδ T cells, the interplay of intrathymic migration and cell differentiation is less well understood. Here, we crossed C-C chemokine receptor (CCR)7-deficient (Ccr7(-/-) ) and CCR9-deficient mice (Ccr9(-/-) ) to mice with a TcrdH2BeGFP reporter background to investigate the impact of thymic localization on γδ T-cell development. γδ T-cell frequencies and numbers were decreased in CCR7-deficient and increased in CCR9-deficient mice. Transfer of CCR7- or CCR9-deficient BM into irradiated C57BL/6 WT recipients reproduced these phenotypes, pointing toward cell-intrinsic migration defects. Monitoring recent thymic emigrants by intrathymic labeling allowed us to identify decreased thymic γδ T-cell output in CCR7-deficient mice. In vitro, CCR7-deficient precursors showed normal γδ T-cell development. Immunohistology revealed that CCR7 and CCR9 expression was important for γδ T-cell localization within thymic medulla or cortex, respectively. However, γδ T-cell motility was unaltered in CCR7- or CCR9-deficient thymi. Together, our results suggest that proper intrathymic localization is important for normal γδ T-cell development.

Invariant natural killer T (iNKT) cells comprise a subpopulation of innate lymphocytes developing in thymus. A new model proposes subdividing murine iNKT cells into iNKT1, 2 and 17 cells. Here, we use transcriptome analyses of iNKT1, 2 and 17 subsets isolated from BALB/c and C57BL/6 thymi to identify candidate genes that may affect iNKT cell development, migration or function. We show that Fcɛr1γ is involved in generation of iNKT1 cells and that SerpinB1 modulates frequency of iNKT17 cells. Moreover, a considerable proportion of iNKT17 cells express IL-4 and IL-17 simultaneously. The results presented not only validate the usefulness of the iNKT1/2/17-concept but also provide new insights into iNKT cell biology., A recent advance in invariant natural killer T cell (iNKT) cell biology is their classification into iNKT1, iNKT2 and iNKT17 subsets. Here the authors provide a transcriptomic analysis of these thymic subsets from Balb/c and C57Bl/6 mice that supports and extends the categorization.

International audience; Lymphatic collecting vessels direct lymph into and from lymph nodes (LNs) and can become hyperpermeable as the result of a previous infection. Enhanced permeability has been implicated in compromised immunity due to reduced flow of lymph and immune cells to LNs, which are the primary site of antigen presentation to T cells. Presently, very little is known about the molecular signals that affect lymphatic collecting vessel permeability. Here, we have shown that lymphatic collecting vessel permeability is controlled by CCR7 and that the chronic hyperpermeability of collecting vessels observed in Ccr7–/– mice is followed by vessel fibrosis. Reexpression of CCR7 in DCs, however, was sufficient to reverse the development of such fibrosis. IFN regulatory factor 4–positive (IRF4+) DCs constitutively interacted with collecting lymphatics, and selective ablation of this DC subset in Cd11c-Cre Irf4fl/fl mice also rendered lymphatic collecting vessels hyperpermeable and fibrotic. Together, our data reveal that CCR7 plays multifaceted roles in regulating collecting vessel permeability and fibrosis, with one of the key players being IRF4-dependent DCs.

Diffusion of chemokines away from their site of production results in the passive formation of chemokine gradients. We have recently shown that chemokine gradients can also be formed in an active manner, namely by atypical chemokine receptors (ACKRs) that scavenge chemokines locally. Here, we describe an advanced method that allows the visualization of leukocyte migration in a three-dimensional environment along a chemokine gradient that is actively established by cells expressing an ACKR. Initially developed to visualize the migration of dendritic cells along gradients of CCL19 or CCL21 that were actively shaped by an ACKR4-expressing cell line, we expect that this chamber system can be exploited to study many other combinations of atypical and conventional chemokine receptor-expressing cells.

The continuous migration of immune cells between lymphoid and nonlymphoid organs is a key feature of the immune system, facilitating the distribution of effector cells within nearly all compartments of the body. Furthermore, reaching their correct position within primary, secondary, or tertiary lymphoid organs is a prerequisite to ensure immune cells’ unimpaired differentiation, maturation, and selection, as well as their activation or functional silencing. The superfamilies of chemokines and chemokine receptors are of major importance in guiding immune cells to and within lymphoid and nonlymphoid tissues. In this review we focus on the role of the chemokine system in the migration dynamics of immune cells within lymphoid organs at the steady state and on how these dynamics are affected by infectious and inflammatory processes.

Summary According to in vitro assays, T cells are thought to kill rapidly and efficiently, but the efficacy and dynamics of cytotoxic T lymphocyte (CTL)-mediated killing of virus-infected cells in vivo remains elusive. We used two-photon microscopy to quantify CTL-mediated killing in mice infected with herpesviruses or poxviruses. On average, one CTL killed 2–16 virus-infected cells per day as determined by real-time imaging and by mathematical modeling. In contrast, upon virus-induced MHC class I downmodulation, CTLs failed to destroy their targets. During killing, CTLs remained migratory and formed motile kinapses rather than static synapses with targets. Viruses encoding the calcium sensor GCaMP6s revealed strong heterogeneity in individual CTL functional capacity. Furthermore, the probability of death of infected cells increased for those contacted by more than two CTLs, indicative of CTL cooperation. Thus, direct visualization of CTLs during killing of virus-infected cells reveals crucial parameters of CD8+ T cell immunity., Graphical Abstract, Highlights • Two-photon imaging indicates that CTLs kill 2–16 virus-infected cells per day • CTLs form kinapses rather than stable synapses when killing virus-infected cells • Some CTL contacts trigger long-lasting calcium fluxes in virus-infected cells • CTLs can cooperate during killing of virus-infected cells, According to in vitro assays, T cells are thought to kill rapidly and efficiently. Using two-photon microscopy, Forster and colleagues have found that killing capacities of single cytotoxic T lymphocytes (CTLs) in vivo are heterogeneous and limited. Quantification of target-cell-death probabilities identified efficient cooperative killing when multiple CTLs attacked a virus-infected cell.

Diffusion of chemokines away from their site of production results in the passive formation of chemokine gradients. We have recently shown that chemokine gradients can also be formed in an active manner, namely by atypical chemokine receptors (ACKRs) that scavenge chemokines locally. Here, we describe an advanced method that allows the visualization of leukocyte migration in a three-dimensional environment along a chemokine gradient that is actively established by cells expressing an ACKR. Initially developed to visualize the migration of dendritic cells along gradients of CCL19 or CCL21 that were actively shaped by an ACKR4-expressing cell line, we expect that this chamber system can be exploited to study many other combinations of atypical and conventional chemokine receptor-expressing cells.

In search of foreign antigens, lymphocytes recirculate from the blood, through lymph nodes, into lymphatics and back to the blood. Dendritic cells also migrate to lymph nodes for optimal interaction with lymphocytes. This continuous trafficking of immune cells into and out of lymph nodes is essential for immune surveillance of foreign invaders. In this article, we review our current understanding of the functions of high endothelial venules (HEVs), stroma and lymphatics in the entry, positioning and exit of immune cells in lymph nodes during homeostasis, and we highlight the unexpected role of dendritic cells in the control of lymphocyte homing through HEVs.

It is emerging that CD4+Foxp3+ regulatory T (Treg) cells can produce the proinflammatory cytokine IFN-γ when stimulated in a Th1 cytokine environment. In this study, we report that Foxp3+ Treg cells readily produced IFN-γ in vivo in a highly inflammatory model of graft-versus-host disease (GVHD) and during a Th1-dominated immune response to intracellular bacteria. Moreover, stimulation in vitro via TCR in the presence of IL-12 alone was sufficient to induce IFN-γ production by Treg cells in a dose-dependent manner. Transfer of donor Treg cells can prevent lethal GVHD; therefore, we used this model as a robust readout for in vivo Treg function. Interestingly, >50% of allogeneic donor, but not residual recipient Foxp3+ Treg cells produced IFN-γ after transplantation, suggesting that this cytokine production was alloantigen specific. These IFN-γ producers were stable Foxp3+ Treg cells because methylation analysis of the Foxp3 gene locus of transferred and reisolated Treg cells during GVHD showed a fully demethylated Treg-specific–demethylated region. Next, we addressed whether IFN-γ production was supporting or rather impairing the immunosuppressive function of Treg cells during GVHD. Blocking of IFN-γ with specific mAb completely abolished the beneficial effect of donor Treg cells. We could further show that only wild-type Treg cells, but not Treg cells from IFN-γ–deficient donor mice, prevented GVHD. This indicated that Treg cell-intrinsic IFN-γ production was required for their protective function. In conclusion, our data show that IFN-γ produced by Foxp3+ Treg cells has essential immune-regulatory functions that are required for prevention of experimental GVHD.

CCR7 is a homeostatically expressed chemokine receptor that is known to regulate the homing of various types of immune cells to primary, secondary, and tertiary lymphoid organs. Recent evidence suggests that, in addition to controlling cell migration, CCR7-mediated signals affect T-cell homeostasis in lymph nodes at various levels and also influence T-cell activation and polarization. In this review, we highlight these findings and discuss recently proposed functions of the CCR7 pathway in the induction and maintenance of chronic inflammation.

Beyond providing a scaffold for immune cells, recent studies indicate that lymph node stromal cells provide potent regulatory capacities that affect the quality of adaptive immune responses. In this study, we provide evidence that neonatal lymph node stromal cells (nnLNSCs) consistently promote the differentiation of macrophage dendritic cell progenitors as well as mature and immature dendritic cells into a distinct population of CX3CR1+ CD11b+F4/80+ regulatory macrophages (regMΦ). These cells possess remarkably low levels of T cell costimulatory molecules as well as MHC class II molecules. regMΦ do not interfere with early T-cell activation but, via nitric oxide secretion, efficiently suppress T-cell proliferation. Furthermore, CD4+ T cells proliferating in the presence of regMΦ gain immunosuppressive capacity and MΦ isolated from day 3 nnLNs are T-cell immunosuppressive. Adoptive transfer of antigen-loaded regMΦ induce a profound antigen-specific immune suppression in vivo. Together our data show that nnLNSCs skew the differentiation of dendritic cells and their progenitors toward regMΦ, thus revealing a novel mechanism for local immune regulation.

The β2-integrin lymphocyte function-associated antigen-1 (LFA-1) plays a crucial role within the immune system. It regulates the interaction between T cells and antigen-presenting cells and facilitates T-cell adhesion to the endothelium, a process that is important for lymphocyte extravasation and homing. Signals mediated via the T-cell receptor and the chemokine receptor CCR7 activate LFA-1 through processes known as inside-out signaling. The molecular mechanisms underlying inside-out signaling are not completely understood. Here, we have assessed the role of the ADAP/SKAP55 module for CCR7-mediated signaling. We show that loss of the module delays homing and reduces intranodal T-cell motility in vivo. This is probably because of a defect in CCR7-mediated adhesion that affects both affinity and avidity regulation of LFA-1. Further analysis of how the ADAP/SKAP55 module regulates CCR7-induced integrin activation revealed that 2 independent pools of the module are expressed in T cells. One pool interacts with a RAPL/Mst1 complex, whereas the other pool is linked to a RIAM/Mst1/Kindlin-3 complex. Importantly, both the RAPL/Mst1 and the RIAM/Mst1/Kindlin-3 complexes require ADAP/SKAP55 for binding to LFA-1 upon CCR7 stimulation. Hence, 2 independent ADAP/SKAP55 modules are essential components of the signaling machinery that regulates affinity and avidity of LFA-1 in response to CCR7.

The spondyloarthritides (SpA) are a group of rheumatic diseases characterized by ossification and inflammation of entheseal tissue, the region where tendon attaches to bone. Interleukin-23 (IL-23) is involved in the pathogenesis of SpA by acting on IL-23 receptor (IL-23R) expressed on enthesis-resident lymphocytes. Upon IL-23 binding, CD3+CD4-CD8- tissue-resident lymphocytes secrete IL-17A and IL-22, leading to inflammation, bone loss, and ossification. Knowledge about enthesis-resident lymphocytes remains fragmentary, and the contribution of entheseal γ/δ T cells in particular is not clear. This study was undertaken to investigate the presence of γ/δ T cells in the enthesis.We used 2-photon microscopy and flow cytometry to analyze entheseal lymphocytes from C57BL/6, Tcrd-H2BeGFP, Rorc-GFP, and IL-23R-eGFP mice. To analyze entheseal γ/δ T cells in IL-23-induced inflammation, Tcrd-H2BeGFP mice were crossed with mice of the susceptible B10.RIII background. Hydrodynamic injection of IL-23 minicircle DNA was performed for overexpression of IL-23 and induction of inflammation. Light-sheet fluorescence microscopy was used to visualize arthritic inflammation.Activated Vγ6+CD27- γ/δ T cells were abundant in uninflamed entheseal tissue and constituted the large majority of retinoic acid receptor-related orphan nuclear receptor γt (RORγt)+IL-23R+ enthesis-resident lymphocytes. Fetal thymus-dependent γ/δ T cells were the main source of IL-17A at the enthesis. Under inflammatory conditions, γ/δ T cells increased in number at the Achilles tendon enthesis, aortic root, and adjacent to the ciliary body.Entheseal γ/δ T cells are derived from fetal thymus and are maintained as self-renewing tissue-resident cells. As main IL-17A producers within tissues exposed to mechanical stress including enthesis, γ/δ T cells are key players in the pathogenesis of IL-23-induced local inflammation.

Protective, high-affinity antibodies are a crucial component of immune defense against pathogens. They are produced by plasma cells that originate from antigen-specific B cells, which get activated and selected in the germinal centers (GCs) of lymph nodes. Despite advances in our understanding of GC biology in recent years—facilitated in particular by the use of two-photon microscopy—several aspects of the spatiotemporal dynamics of cellular interaction in the GC are still unclear. On page 716 of this issue, Lu et al. ( 1 ) recognize a role for the cell surface protein ephrin B1 in regulating the longevity of interactions between B cells and follicular T helper (TFH) cells in GCs. This may control T cell homeostasis in GCs as well as support the selection of high-affinity antibody-producing plasma cells.

SummaryLittle is known about mechanisms determining the homeostasis of lymphocytes within lymphoid organs. Applying different mouse models, including conditionally proficient Ccr7 gene-targeted mice, we now show that semimature steady state dendritic cells (sDCs) constitutively trafficking into lymph nodes (LNs) were essential contributors to T cell homeostasis in these organs. sDCs provided vascular endothelial growth factor known to support high endothelial venule formation, thus facilitating enhanced homing of T cells to LNs. The presence of sDCs led to increased CCL21 production in T-zone fibroblastic reticular cells. CCL21 is a ligand for CCR7 known to regulate homing as well as retention of T cells in LNs. In addition, we provide evidence that CCL21 binds to the surface of DCs via its heparin-binding domain, further explaining why T cells leave LNs more rapidly in the absence of sDCs. Together, these data reveal multiple roles for sDCs in regulating T cell homeostasis in LNs.

The chemokine receptor CCR7 has a central role in regulating homing and positioning of T cells and DCs to lymph nodes (LNs) and participates in T-cell development and activation. In this study, we addressed the role of CCR7 signaling in TH2 polarization and B-cell activation. We provide evidence that the lack of CCR7 drives the capacity of naive CD4+ T cells to polarize toward TH2 cells. This propensity contributes to a lymph node environment in CCR7-deficent mice characterized by increased expression of IL-4 and increased frequency of TH2 cells. We show that elevated IL-4 levels lead to B-cell activation characterized by up-regulated expression of MHC class II, CD23 and CD86. Activated B cells are in turn highly efficient in presenting antigen to CD4+ T cells and thus potentially contribute to the TH2 microenvironment. Taken together, our results support the idea of a CCR7-dependent patterning of TH2 responses, with absent CCR7 signaling favoring TH2 polarization, dislocation of T helper cells into the B-cell follicles and, as a consequence, B-cell activation.

Dendritic cells (DCs) are the most potent antigen-presenting cells of the immune system. Depending on their maturation status, they prime T cells to induce adaptive immunity or tolerance. DCs express CD155, an immunoglobulin-like receptor binding CD226 present on T and natural killer (NK) cells. CD226 represents an important co-stimulator during T cell priming but also serves as an activating receptor on cytotoxic T and NK cells. Here, we report that cells of the T and NK cell lineage of CD155(-/-) mice express markedly elevated protein levels of CD226 compared with wild type (WT). On heterozygous CD155(+/-) T cells, CD226 up-regulation is half-maximal, implying an inverse gene-dosis effect. Moreover, CD226 up-regulation is independent of antigen-driven activation because it occurs already in thymocytes and naïve peripheral T cells. In vivo, neutralizing anti-CD155 antibody elicits up-regulation of CD226 on T cells demonstrating, that the observed modulation can be triggered by interrupting CD155-CD226 contacts. Adoptive transfers of WT or CD155(-/-) T cells into CD155(-/-) or WT recipients, respectively, revealed that CD226 modulation is accomplished in trans. Analysis of bone marrow chimeras showed that regulators in trans are of hematopoietic origin. We demonstrate that DCs are capable of manipulating CD226 levels on T cells in vivo but not in vitro, suggesting that the process of T cells actively scanning antigen-presenting DCs inside secondary lymphoid organs is required for CD226 modulation. Hence, a CD226 level divergent from WT may be exploited as a sensor to detect abnormal DC/T-cell cross-talk as illustrated for T cells in mice lacking CCR7.

Little is known about the molecular mechanisms that determine the entry into the lymph node and intranodal positioning of lymph-derived cells. By injecting cells directly into afferent lymph vessels of popliteal lymph nodes, we demonstrate that lymph-derived T cells entered lymph-node parenchyma mainly from peripheral medullary sinuses, whereas dendritic cells (DCs) transmigrated through the floor of the subcapsular sinus on the afferent side. Transmigrating DCs induced local changes that allowed the concomitant entry of T cells at these sites. Signals mediated by the chemokine receptor CCR7 were absolutely required for the directional migration of both DCs and T cells into the T cell zone but were dispensable for the parenchymal entry of lymph-derived T cells and dendrite probing of DCs. Our findings provide insight into the molecular and structural requirements for the entry into lymph nodes and intranodal migration of lymph-derived cells of the immune system.

The molecular mechanisms leading to reactivation of latent cytomegalovirus are not well understood. To study reactivation, the few cells in an organ tissue that give rise to reactivated virus need to be identified, ideally at the earliest possible time point in the process. To this end, mouse cytomegalovirus (MCMV) reporter mutants were designed to simultaneously express the red fluorescent protein mCherry and the secreted Gaussia luciferase (Gluc). Whereas Gluc can serve to assess infection at the level of individual mice by measuring luminescence in blood samples or by in vivo imaging, mCherry fluorescence offers the advatage of detection of infection at the single cell level. To visualize cells in which MCMV was being reactivated, precision-cut lung slices (PCLS) that preserve tissue microanatomy were prepared from the lungs of latently infected mice. By day 3 of cultivation of the PCLS, reactivation was revealed by Gluc expression, preceding the detection of infectious virus by approximately 4 days. Reactivation events in PCLS could be identified when they were still confined to single cells. Notably, using fractalkine receptor–GFP reporter mice, we never observed reactivation originating from CX3CR1+ monocytes or pulmonary dendritic cells derived therefrom. Furthermore, latent viral genome in the lungs was not enriched in sorted bone-marrow-derived cells expressing CD11b. Taken together, these complementary approaches suggest that CD11b+ and CX3CR1+ subsets of the myeloid differentiation lineage are not the main reservoirs and cellular sites of MCMV latency and reactivation in the lungs.

The chemokine receptor CCR7 represents an important determinant for circulating lymphocytes to enter lymph nodes (LN) via high endothelial venules. High endothelial venules also represent the major site of entry for plasmacytoid dendritic cells (pDC). In the steady-state, murine pDC have been suggested to home to LN engaging the chemokine receptors CXCR3, CXCR4, and CCR5, whereas responsiveness to CCR7 ligands is thought to be acquired only upon activation. In this study, we show that already resting pDC express minute amounts of CCR7 that suffice to trigger migration to CCL19/CCL21 in vitro. Upon activation with TLR ligands, CCR7 levels on pDC are strongly increased. Notably, CCR7-deficient mice display substantially reduced pDC counts in LN but not in bone marrow and spleen. Adoptive cell transfer experiments revealed that under both steady-state as well as inflammatory conditions, the homing of CCR7-deficient pDC is severely impaired, indicating that the reduced cell counts of naive pDC observed in CCR7−/− mice reflect an intrinsic homing defect of pDC. Together, these observations provide strong evidence that similar to naive lymphocytes, nonstimulated pDC exploit CCR7 to gain entry into LN. This adds to the repertoire of chemokine receptors permitting them to enter diverse tissues.

Mouse CMV (MCMV) infection rapidly induces the proliferation of NK cells, which correlates with immunological protection. Whether NK cells primed during acute response against MCMV are maintained for the long term is not known. In this study, we used TcrdH2BeGFP mice in which maturing NK cells are genetically labeled with a pulse of very stable histone-2B–eGFP. In this system, we found that the reporter protein was diluted out upon NK cell division during acute MCMV infection. At the same time, mature NK cells in uninfected mice showed only very limited turnover in vivo. Three months after primary infection when MCMV latency was established, the majority of peripheral NK cells still displayed a higher record of proliferation than NK cells in mock-infected controls. This observation included both Ly49H+ and Ly49H– NK cells. Conversely, naive NK cells did not show more proliferation after transfer into latently MCMV-infected mice than that after transfer into mock-infected control mice. This indicated that the observed alterations of the NK cell compartment in MCMV latency were “legacy” (i.e., resulting from prior events during the initial immune response). Together, these results suggest that antiviral immune responses induce sustained alterations of innate lymphocyte populations that extend far beyond the first days of acute infection.

SummaryChemokines orchestrate immune cell trafficking by eliciting either directed or random migration and by activating integrins in order to induce cell adhesion. Analyzing dendritic cell (DC) migration, we showed that these distinct cellular responses depended on the mode of chemokine presentation within tissues. The surface-immobilized form of the chemokine CCL21, the heparan sulfate-anchoring ligand of the CC-chemokine receptor 7 (CCR7), caused random movement of DCs that was confined to the chemokine-presenting surface because it triggered integrin-mediated adhesion. Upon direct contact with CCL21, DCs truncated the anchoring residues of CCL21, thereby releasing it from the solid phase. Soluble CCL21 functionally resembles the second CCR7 ligand, CCL19, which lacks anchoring residues and forms soluble gradients. Both soluble CCR7 ligands triggered chemotactic movement, but not surface adhesion. Adhesive random migration and directional steering cooperate to produce dynamic but spatially restricted locomotion patterns closely resembling the cellular dynamics observed in secondary lymphoid organs.PaperClip

T-cell development depends on recruitment of bone marrow–derived precursor cells to the thymus via a multistep adhesion cascade involving the chemokine receptor CCR9. However, CCR9 deficiency does not result in complete abrogation of progenitor entry into the adult thymus. Therefore, we tested the hypothesis that additional chemokine/chemokine receptor systems might play a role in this process. To this end, we generated mice deficient in both CCR9 and CCR7. Deficiency in both chemokine receptors resulted in severely reduced numbers of early T-cell progenitors and in near-complete abrogation of thymus reconstitution. Progenitors in bone marrow and peripheral blood remained largely unaffected in CCR7−/−CCR9−/− mice, and direct intrathymic transfer of precursors from CCR7−/−CCR9−/− mice as well as single-mutant mice showed that intrathymic differentiation of these precursors remained functional. Thus, our data reveal a previously unrecognized role of CCR7 in progenitor seeding of the adult thymus, which is largely masked by compensatory effects of CCR9 signals. In turn, CCR7 signals can partially compensate for CCR9 signals, thus explaining the rather mild phenotype of CCR9−/− mice with respect to progenitor seeding.

In the version of this article initially published, a source of funding (Deutsche Jose Carreras Leukamie-Stiftung e.V. (DJCLS R12/29 to C.K. and I.P.)) was not included in the Acknowledgments section. The correct statement is as follows: “Supported by Deutsche Forschungsgemeinschaft, (SFB900/B8 to C.K. and I.P.; and PR727/4-1 to I.P.), Deutsche Jose Carreras Leukamie-Stiftung e.V. (DJCLS R12/29 to C.K. and I.P.) and the German Federal Ministry of Education and Research (01EO1302 to C.S.-F., C.K. and I.P.).” The error has been corrected in the HTML and PDF versions of the article.

Eosinophils are potent effector cells that are recruited to sites of inflammation. However, in some tissues, in particular in the gastrointestinal tract, eosinophils constitute an abundant leukocyte population also under homeostatic conditions. The lack of suitable isolation protocols restricted the analysis of these cells to histological assessment of cell numbers while important aspects of their phenotype, turnover, and functions remain unresolved. In this study, we report a protocol that allows the quantitative isolation of intestinal eosinophils. We characterized small intestinal eosinophils by flow cytometry as SSChighCD11b+CD11c+CCR3+Siglec-F+ cells. Intestinal eosinophils resembled eosinophils isolated from thymus and uterus but differed from eosinophils isolated from lung or blood. Eosinophils in intestine, thymus, and uterus showed in vivo a markedly higher life time compared with eosinophils present in lung and blood measured by incorporation of BrdU. This indicates that under steady-state conditions homeostasis of eosinophils is controlled by regulation of cell survival. Intestinal eosinophils are severely reduced in the intestines of Rag-2/common γ-chain double-deficient mice but not Rag-2-deficient mice, correlating with differential expression of GM-CSF and CCL11 in both mouse strains. Moreover, under steady-state conditions, intestinal eosinophils constitutively express high levels of the common γ-chain transcripts compared with lung eosinophils as well as eosinophils present under inflammatory conditions. These observations reveal a hitherto unrecognized diversity in phenotypic and functional properties of eosinophils and suggest that tissue-specific common γ-chain-dependent signals might profoundly affect eosinophil function and homeostasis.

The secondary humoral immune response is characterized by plasma B cells secreting isotype-switched and affinity-matured antibodies. The efficient generation of plasma B cells in the GC depends on the presence of follicular helper T (T(FH)) cells, a cell type thought to arise from naive CD4-positive T cells by a hitherto unresolved differentiation pathway. Mice deficient for CD155, an adhesion receptor of the immunoglobulin superfamily, are impaired to mount a secondary humoral immune response upon oral administration of antigen, while the primary IgM response is unaffected. Here, we show that mice lacking CD155 harbor significantly reduced numbers of T(FH) cells in their Peyer's patches. This was paralleled by a decreased frequency of T(FH) cells in the GC. Moreover, the CD155 ligand CD226, which is involved in T-cell activation, is down-regulated during T(FH) cell differentiation, resulting in a complete absence of CD226 on those T(FH) cells residing in the GC. Concurrently, the expression of TIGIT/WUCAM, a newly discovered CD155 ligand, is induced in T(FH) cells. Thus, these cells replace an activating by a putative inhibitory CD155-binding partner during their differentiation.

Gammadelta T cells are a potent source of innate IL-17A and IFN-gamma, and they acquire the capacity to produce these cytokines within the thymus. However, the precise stages and required signals that guide this differentiation are unclear. Here we show that the CD24(low) CD44(high) effector gammadelta T cells of the adult thymus are segregated into two lineages by the mutually exclusive expression of CCR6 and NK1.1. Only CCR6+ gammadelta T cells produced IL-17A, while NK1.1+ gammadelta T cells were efficient producers of IFN-gamma but not of IL-17A. Their effector phenotype correlated with loss of CCR9 expression, particularly among the NK1.1+ gammadelta T cells. Accordingly, both gammadelta T-cell subsets were rare in gut-associated lymphoid tissues, but abundant in peripheral lymphoid tissues. There, they provided IL-17A and IFN-gamma in response to TCR-specific and TCR-independent stimuli. IL-12 and IL-18 induced IFN-gamma and IL-23 induced IL-17A production by NK1.1+ or CCR6+ gammadelta T cells, respectively. Importantly, we show that CCR6+ gammadelta T cells are more responsive to TCR stimulation than their NK1.1+ counterparts. In conclusion, our findings support the hypothesis that CCR6+ IL-17A-producing gammadelta T cells derive from less TCR-dependent selection events than IFN-gamma-producing NK1.1+ gammadelta T cells.

Nose-associated lymphoid tissue (NALT) in the rodent upper respiratory tract develops postnatally and is considered to be independent of several factors known to be involved in the organogenesis of LN and Peyer's patches (PP). In this study we demonstrate that at least two different pathways result in NALT development. Following NALT anlage formation the intrinsic pathway relies on a signaling cascade including those mediated through the chemokine receptor CXCR5 and the lymphotoxin beta receptor (LTbetaR). This allows for the formation of high endothelial venules and thereby the recruitment of lymphocytes into NALT. Alternatively, high endothelial venule formation and lymphocyte recruitment can be induced in the NALT anlage by environmental signals, which are independent of LT-betaR and chemokine receptor CXCR5 signaling but in part rely on CD40 ligand. Thus, our study identifies a novel mechanism that facilitates the rescue of NALT development at late stages in adult life independent of the canonical LTbetaR-CXCR5 signaling axis.

In humans with typhoid fever or in mouse strains susceptible toSalmonella entericaserovar Typhimurium (S. Typhimurium) infection, bacteria gain access to extraintestinal tissues, causing severe systemic disease. Here we show that in the gut-draining mesenteric lymph nodes (MLN), the majority ofS.Typhimurium-carrying cells show dendritic-cell (DC) morphology and express the DC marker CD11c, indicating thatS. Typhimurium bacteria are transported to the MLN by migratory DCs. In vivo FLT-3L-induced expansion of DCs, as well as stimulation of DC migration by Toll-like receptor agonists, results in increased numbers ofS. Typhimurium bacteria reaching the MLN. Conversely, genetically impaired DC migration in chemokine receptor CCR7-deficient mice reduces the number ofS. Typhimurium bacteria reaching the MLN. This indicates that transport ofS. Typhimurium from the intestine into the MLN is limited by the number of migratory DCs carryingS. Typhimurium bacteria. In contrast, modulation of DC migration does not affect the number ofS. Typhimurium bacteria reaching systemic tissues, indicating that DC-bound transport ofS. Typhimurium does not substantially contribute to systemicS. Typhimurium infection. Surgical removal of the MLN results in increased numbers ofS. Typhimurium bacteria reaching systemic sites early after infection, thereby rendering otherwise resistant mice susceptible to fatal systemic disease development. This suggests that the MLN provide a vital barrier shielding systemic compartments from DC-mediated dissemination ofS. Typhimurium. Thus, confinement ofS. Typhimurium in gut-associated lymphoid tissue and MLN delays massive extraintestinal dissemination and at the same time allows for the establishment of protective adaptive immune responses.

Adhesion and motility of mammalian leukocytes are essential requirements for innate and adaptive immune defense mechanisms. We show here that the guanine nucleotide exchange factor cytohesin-1, which had previously been demonstrated to be an important component of beta-2 integrin activation in lymphocytes, regulates the activation of the small GTPase RhoA in primary dendritic cells (DCs). Cytohesin-1 and RhoA are both required for the induction of chemokine-dependent conformational changes of the integrin beta-2 subunit of DCs during adhesion under physiological flow conditions. Furthermore, use of RNAi in murine bone marrow DCs (BM-DCs) revealed that interference with cytohesin-1 signaling impairs migration of wild-type dendritic cells in complex 3D environments and in vivo. This phenotype was not observed in the complete absence of integrins. We thus demonstrate an essential role of cytohesin-1/RhoA during ameboid migration in the presence of integrins and further suggest that DCs without integrins switch to a different migration mode.

Murine γ-herpes virus 68 is a natural rodent pathogen closely related to the human γ-herpes viruses Kaposi’s sarcoma-associated herpes virus and EBV. By intranasally infecting wild-type and CCR7-deficient mice, we investigated whether CCR7 is necessary for viral clearance from the lung and the establishment of latency. We found during the lytic phase of infection that inflammation in lungs of CCR7−/− mice was more severe and viral load significantly higher compared with wild-type littermates. In addition, activation of T cells was delayed and clearance of the inflammation was retarded in mutant lungs, demonstrating that CCR7 is necessary for a rapid and efficient immune response. However, for the establishment of splenomegaly and latency, the presence of CCR7 was dispensable. Finally, by microdissecting BALT, we could demonstrate that these ectopic lymphoid structures are a place in the lung where virus resides during latency.

Background: The pathological features of inflammatory bowel disease (IBD) are associated with leukocyte cell infiltrates, which contribute to disease progression and persistence by production of proinflammatory mediators. Recruiting leukocytes to the gut involves local expression of chemokines that interact with receptors on the leukocytes' surface. Specific antagonists may interfere with leukocyte recruitment to the intestine. The chemokine receptor CCR9 is one of the key molecules in leukocyte homing to gut mucosa. CCR9 antagonists have been shown to retard progression in patients with IBD. Objective: To discuss CCR9 as a potential target for the treatment of IBD. Methods: A literature review. Results/conclusions: The therapeutic effects of CCR9 antagonists, in combination with established therapies, should be evaluated in an attempt to slow down leukocyte recruitment at early stages of IBD.

mAb targeting the gammadelta TCR have been used for gammadelta T-cell depletion with varying success. Although the depletion-capacity of the anti-gammadelta TCR mAb clone GL3 has been disputed repeatedly, many groups continue to use gammadelta T-cell depletion protocols involving the mAb clone UC7-13D5 and find significant biological effects. We show here that treatment with both GL3 and UC7-13D5 antibodies does not deplete gammadelta T cells in vivo, but rather leads to TCR internalization and thereby generates "invisible" gammadelta T cells. We addressed this issue using anti-gammadelta TCR mAb injections into WT mice as well as into reporter TCR delta locus-histone 2B enhanced GFP knock-in mice, in which gammadelta T cells can be detected based on an intrinsic green fluorescence. Importantly, the use of TCR delta locus-histone 2B enhanced GFP mice provided here for the first time direct evidence that the "depleted" gammadelta T cells were actually still present. Our results show further that GL3 and UC7-13D5 mAb are in part cross-competing for the same epitope. Assessed by activation markers, we observed in vitro and in vivo activation of gammadelta T cells through mAb. We conclude that gammadelta T-cell depletion experiments must be evaluated with caution and discuss the implications for future studies on the physiological functions of gammadelta T cells.

Mucosal vaccination via the respiratory tract can elicit protective immunity in animal infection models, but the underlying mechanisms are still poorly understood. We show that a single intranasal application of the replication-deficient modified vaccinia virus Ankara, which is widely used as a recombinant vaccination vector, results in prominent induction of bronchus-associated lymphoid tissue (BALT). Although initial peribronchiolar infiltrations, characterized by the presence of dendritic cells (DCs) and few lymphocytes, can be found 4 d after virus application, organized lymphoid structures with segregated B and T cell zones are first observed at day 8. After intratracheal application, in vitro–differentiated, antigen-loaded DCs rapidly migrate into preformed BALT and efficiently activate antigen-specific T cells, as revealed by two-photon microscopy. Furthermore, the lung-specific depletion of DCs in mice that express the diphtheria toxin receptor under the control of the CD11c promoter interferes with BALT maintenance. Collectively, these data identify BALT as tertiary lymphoid structures supporting the efficient priming of T cell responses directed against unrelated airborne antigens while crucially requiring DCs for its sustained presence.

Inactivation of members of the nuclear factor-kappaB (NF-kappaB) family results in the decrease or defect of marginal zone B (MZB) cells. It is not known which inhibitors of the NF-kappaB family (IkappaB) are required for MZB cell development. Here, we show that mice with B cell-specific inactivation of the main NF-kappaB inhibitor IkappaBalpha have a marked decrease of MZB cells and their presumed precursors. They exhibited increased mortality rates after blood-borne bacterial infection, indicating the importance of MZB cells for bacterial clearance. In contrast, response to T cell-dependent and -independent antigens resulted only in minor changes in immunoglobulin production. Our data demonstrate the importance of the intact NF-kappaB/IkappaBalpha pathway for proper MZB cell development.

In the past decade accumulating evidence supported the view that the immune system should be regarded as trust consisting of several branches. In this review, we will first introduce the architectural features comprising the intestinal immune system emphasising its plasticity and subsequently discuss the concepts describing its development. We then focus on the chemokine/receptor system as a key integrator managing coordinated migration of and communication among the cells mediating intestinal immunity. Thus, chemokines control development and maintain functionality of the intestinal immune system that is required to perform the unique balancing act between tolerating food, curtailing commensals activities and eliminating pathogenic infections.

A key feature of the immune system is its ability to induce protective immunity against pathogens while maintaining tolerance towards self and innocuous environmental antigens. Recent evidence suggests that by guiding cells to and within lymphoid organs, CC-chemokine receptor 7 (CCR7) essentially contributes to both immunity and tolerance. This receptor is involved in organizing thymic architecture and function, lymph-node homing of naive and regulatory T cells via high endothelial venules, as well as steady state and inflammation-induced lymph-node-bound migration of dendritic cells via afferent lymphatics. Here, we focus on the cellular and molecular mechanisms that enable CCR7 and its two ligands, CCL19 and CCL21, to balance immunity and tolerance.

The constitutive migration of B cells from the circulation into the peritoneal cavity and back is essential for peritoneal B cell homeostasis and function. However, the molecular machinery and the anatomical basis for these migratory processes have hardly been investigated. In this study, we analyze the role of integrins as well as the role of the omentum for B2 cell migration into and out of the peritoneal cavity of mice. We demonstrate that α4β7 integrin-mucosal addressin cell adhesion molecule 1 interaction enables B2 cell migration from the circulation into omental milky spots but not into the peritoneum. In contrast, α4β1 integrin mediates direct entry of B2 cells into the peritoneal cavity as well as their retention at that site, limiting B2 cell egress via the draining parathymic lymph nodes. Surgical removal of the omentum results in a 40% reduced immigration of B2 cells from the circulation into the peritoneum but does not impair B cell exit from this compartment. In conclusion, these data reveal the existence of alternative routes for B2 cell entry into the peritoneal cavity and identify integrins as key factors for peritoneal B2 cell homeostasis, mediating B2 cell migration into and out of the peritoneal cavity as well as their retention at this site.

CCR7 and its ligands, CCL19 and CCL21, are responsible for directing the migration of T cells and dendritic cells into lymph nodes, where these cells play an important role in the initiation of the immune response. Recently, we have shown that systemic application of CCL19-IgG is able to inhibit the colocalization of T cells and dendritic cells within secondary lymphoid organs, resulting in pronounced immunosuppression with reduced allograft rejection after organ transplantation. In this study, we demonstrate that the application of sustained high concentrations of either soluble or immobilized CCL19 and CCL21 elicits an inhibitory program in T cells. We show that these ligands specifically interfere with cell proliferation and IL-2 secretion of CCR7+ cells. This could be demonstrated for human and murine T cells and was valid for both CD4+ and CD8+ T cells. In contrast, CCL19 had no inhibitory effect on T cells from CCR7 knockout mice, but CCR7−/− T cells showed a proliferative response upon TCR-stimulation similar to that of CCL19-treated wild-type cells. Furthermore, the inhibition of proliferation is associated with delayed degradation of the cyclin-dependent kinase (CDK) inhibitor p27Kip1 and the down-regulation of CDK1. This shows that CCR7 signaling is linked to cell cycle control and that sustained engagement of CCR7, either by high concentrations of soluble ligands or by high density of immobilized ligands, is capable of inducing cell cycle arrest in TCR-stimulated cells. Thus, CCR7, a chemokine receptor that has been demonstrated to play an essential role during activation of the immune response, is also competent to directly inhibit T cell proliferation.

Dendritic cells (DC) are able to capture, process, and present exogenous Ag to CD8+ T lymphocytes through MHC class I, a process referred to as cross-presentation. In this study, we demonstrate that CD103+ (CD11chighCD11blow) and CD103− (CD11cintCD11bhigh) DC residing in the lung-draining bronchial lymph node (brLN) have evolved to acquire opposing functions in presenting innocuous inhaled Ag. Thus, under tolerogenic conditions, CD103− DC are specialized in presenting innocuous Ag to CD4+ T cells, whereas CD103+ DC, which do not express CD8α, are specialized in presenting Ag exclusively to CD8+ T cells. In CCR7-deficient but not in plt/plt mice, Ag-carrying CD103+ DC are largely absent in the brLN, although CD103+ DC are present in the lung of CCR7-deficient mice. As a consequence, adoptively transferred CD8+ T cells can be activated under tolerizing conditions in plt/plt but not in CCR7-deficient mice. These data reveal that CD103+ brLN DC are specialized in cross-presenting innocuous inhaled Ag in vivo. Because these cells are largely absent in CCR7−/− mice, our findings strongly suggest that brLN CD103+ DC are lung-derived and that expression of CCR7 is required for their migration from the lung into its draining lymph node.

Oral infection of mice with Salmonella enterica serovar Typhimurium results in the colonization of Peyer's patches, triggering a vigorous inflammatory response and immunopathology at these sites. Here we demonstrate that in parallel to Peyer's patches a strong inflammatory response occurs in the intestine, resulting in the appearance of numerous inflammatory foci in the intestinal mucosa. These foci surround small lymphoid cell clusters termed solitary intestinal lymphoid tissue (SILT). Salmonella can be observed inside SILT at early stages of infection, and the number of infected structures matches the number of inflammatory foci arising at later time points. Infection leads to enlargement and morphological destruction of SILT but does not trigger de novo formation of lymphoid tissue. In conclusion, SILT, a lymphoid compartment mostly neglected in earlier studies, represents a major site for Salmonella invasion and ensuing mucosal pathology.

Presence and extent of bronchus-associated lymphoid tissue (BALT) is subject to considerable variations between species and is only occasionally observed in lungs of mice. Here we demonstrate that mice deficient for the chemokine receptor CCR7 regularly develop highly organized BALT. These structures were not present at birth but were detectable from day 5 onwards. Analyzing CCR7−/−/wild-type bone marrow chimeras, we demonstrate that the development of BALT is caused by alterations of the hematopoietic system in CCR7-deficient mice. These observations together with the finding that CCR7-deficient mice posses dramatically reduced numbers of regulatory T cells (T reg cells) in the lung-draining bronchial lymph node suggest that BALT formation might be caused by disabled in situ function of T reg cells. Indeed, although adoptive transfer of wild-type T reg cells to CCR7-deficient recipients resulted in a profound reduction of BALT formation, neither naive wild-type T cells nor T reg cells from CCR7−/− donors impair BALT generation. Furthermore, we provide evidence that CCR7-deficient T reg cells, although strongly impaired in homing to peripheral lymph nodes, are fully effective in vitro. Thus our data reveal a CCR7-dependent homing of T reg cells to peripheral lymph nodes in conjunction with a role for these cells in controlling BALT formation.