Dynamics of immune cell recruitment during West Nile encephalitis and identification of a new CD19+B220-BST-2+ leukocyte population

West Nile virus (WNV) is an emerging neurotropic flavivirus. We investigated the dynamics of immune cell recruitment in peripheral tissues and in the CNS during WNV encephalitis in an immunocompetent mouse model. In the periphery, immune cell expansion can successfully limit viremia and lymphoid tissue infection. However, viral clearance in the periphery is too late to prevent viral invasion of the CNS. In the CNS, innate immune cells, including microglia/macrophages, NK cells, and plasmacytoid dendritic cells, greatly expand as the virus invades the brain, whereas B and T cells are recruited after viral invasion, and fail to control the spread of the virus. Thus, the onset of WNV encephalitis was correlated both with CNS viral infection and with a large local increase of innate immune cells. Interestingly, we identify a new immune cell type: CD19(+)B220(-) BST-2(+), which we name G8-ICs. These cells appear during peripheral infection and enter the CNS. G8-ICs express high levels of MHC class II, stain for viral Ag, and are localized in the paracortical zone of lymph nodes, strongly suggesting they are previously unidentified APCs that appear in response to viral infection.     

Activation, differentiation, and migration of naive virus-specific CD8+ T cells during pulmonary influenza virus infection

The low precursor frequency of individual virus-specific CD8(+) T cells in a naive host makes the early events of CD8(+) T cell activation, proliferation, and differentiation in response to viral infection a challenge to identify. We have therefore examined the response of naive CD8(+) T cells to pulmonary influenza virus infection with a murine adoptive transfer model using hemagglutinin-specific TCR transgenic CD8(+) T cells. Initial activation of CD8(+) T cells occurs during the first 3 days postinfection exclusively within the draining lymph nodes. Acquisition of CTL effector functions, including effector cytokine and granule-associated protease expression, occurs in the draining lymph nodes and differentially correlates with cell division. Division of activated CD8(+) T cells within the draining lymph nodes occurs in an asynchronous manner between days 3 and 4 postinfection. Despite the presence of Ag for several days within the draining lymph nodes, dividing T cells do not appear to maintain contact with residual Ag. After multiple cell divisions, CD8(+) T cells exit the draining lymph nodes and migrate to the infected lung. Activated CD8(+) T cells also disseminate throughout lymphoid tissue including the spleen and distal lymph nodes following their emigration from draining lymph nodes. These results demonstrate an important role for draining lymph nodes in orchestrating T cell responses during a local infection of a discrete organ to generate effector CD8(+) T cells capable of responding to infection and seeding peripheral lymphoid tissues.     

Disruption of innate-mediated proinflammatory cytokine and reactive oxygen species third signal leads to antigen-specific hyporesponsiveness

Successful Ag activation of naive T helper cells requires at least two signals consisting of TCR and CD28 on the T cell interacting with MHC II and CD80/CD86, respectively, on APCs. Recent evidence demonstrates that a third signal consisting of proinflammatory cytokines and reactive oxygen species (ROS) produced by the innate immune response is important in arming the adaptive immune response. In an effort to curtail the generation of an Ag-specific T cell response, we targeted the synthesis of innate immune response signals to generate Ag-specific hyporesponsiveness. We have reported that modulation of redox balance with a catalytic antioxidant effectively inhibited the generation of third signal components from the innate immune response (TNF-alpha, IL-1beta, ROS). In this study, we demonstrate that innate immune-derived signals are necessary for adaptive immune effector function and disruption of these signals with in vivo CA treatment conferred Ag-specific hyporesponsiveness in BALB/c, NOD, DO11.10, and BDC-2.5 mice after immunization. Modulating redox balance led to decreased Ag-specific T cell proliferation and IFN-gamma synthesis by diminishing ROS production in the APC, which affected TNF-alpha levels produced by CD4(+) T cells and impairing effector function. These results demonstrate that altering redox status can be effective in T cell-mediated diseases such as autoimmune diabetes to generate Ag-specific immunosuppression because it inhibits the third signal necessary for CD4(+) T cells to transition from expansion to effector function.     

Variable requirement of dendritic cells for recruitment of NK and T cells to different TLR agonists

TLRs initiate the host immune response to microbial pathogens by activating cells of the innate immune system. Dendritic cells (DCs) can be categorized into two major groups, conventional DCs (including CD8(+) and CD8(-) DCs) and plasmacytoid DCs. In mice, these subsets of DCs express a variety of TLRs, with conventional DCs responding in vitro to predominantly TLR3, TLR4, TLR5, and TLR9 ligands, and plasmacytoid DCs responding mainly to TLR7 and TLR9 ligands. However, the in vivo requirement of DCs to initiate immune responses to specific TLR agonists is not fully known. Using mice depleted of >90% of CD11c(+) MHC class II(+) DCs, we demonstrate that cellular recruitment, including CD4(+) T cell and CX5(+)DX5(+) NK cell recruitment to draining lymph nodes following the footpad administration of TLR4 and TLR5 agonists, is dramatically decreased upon reduction of DC numbers, but type I IFN production can partially substitute for DCs in response to TLR3 and TLR7 agonists. Interestingly, TLR ligands can activate T cells and NK cells in the draining lymph nodes, even with reduced DC numbers. The findings reveal considerable plasticity in the response to TLR agonists, with TLR4 and TLR5 agonists sharing the requirement of DCs for subsequent lymph node recruitment of NK and T cells.     

Innate immune activation of CD4 T cells in salmonella-infected mice is dependent on IL-18

Production of IFN-gamma by CD4 T cells is generally thought to be mediated by TCR triggering, however, Ag-nonspecific activation of effector CD8 T cells has been reported in infection models. In this study, we demonstrate that Ag-experienced CD4 T cells in the spleen of Salmonella-infected mice acquire the capacity to rapidly secrete IFN-gamma in response to stimulation with bacterial lysate or LPS. This innate responsiveness of T cells was transient and most apparent during, and immediately following, active Salmonella infection. Furthermore, innate T cell production of IFN-gamma in response to bacterial lysate or LPS was Ag independent and could be induced in Listeria-infected mice and in the absence of MHC class II expression. IL-18 was required for maximal innate responsiveness of CD4 T cells in Salmonella-infected mice and for optimal bacterial clearance in vivo. These data demonstrate that CD4 T cells acquire the capacity to respond to innate stimuli during active bacterial infection, a process that may contribute significantly to amplifying effector responses in vivo.     

CD4+ T cells recognizing a single self-peptide expressed by APCs induce spontaneous autoimmune arthritis

We have examined processes leading to the spontaneous development of autoimmune inflammatory arthritis in transgenic mice containing CD4+ T cells targeted to a nominal Ag (hemagglutinin (HA)) and coexpressing HA driven by a MHC class II promoter. Despite being subjected to multiple tolerance mechanisms, autoreactive CD4+ T cells accumulate in the periphery of these mice and promote systemic proinflammatory cytokine production. The majority of mice spontaneously develop inflammatory arthritis, which is accompanied by an enhanced regional immune response in lymph nodes draining major joints. Arthritis development is accompanied by systemic B cell activation; however, neither B cells nor Ab is required for arthritis development, since disease develops in a B cell-deficient background. Moreover, arthritis also develops in a recombinase activating gene-deficient background, indicating that the disease process is driven by CD4+ T cells recognizing the neo-self HA Ag. These findings show that autoreactive CD4+ T cells recognizing a single self-Ag, expressed by systemically distributed APCs, can induce arthritis via a mechanism that is independent of their ability to provide help for autoantibody production.     

Microbial and T cell-derived stimuli regulate antigen presentation by dendritic cells in vivo

B cells and dendritic cells (DC) internalize and degrade exogenous Ags and present them as peptides bound to MHC class II molecules for scrutiny by CD4(+) T cells. Here we use an Ab specific for a processed form of the model Ag, hen egg lysozyme (HEL), to demonstrate that this protein is not efficiently presented by lymph node DC following s.c. immunization. HEL presentation by the DC can be dramatically enhanced upon coinjection of a microbial adjuvant, which appears to act by enhancing peptide loading onto MHC class II. CD40 cross-linking or the presence of a high frequency of T cells specific for HEL can similarly improve presentation by DC in vivo. For any of these activating stimuli, CD8alpha(+) DC consistently display the highest proportion of HEL-loaded MHC class II molecules. These data indicate that exogenous Ags can be displayed to T cells in lymphoid tissues by a large cohort of resident DC whose presentation is regulated by innate and adaptive stimuli. Our data further reveal the existence of a feedback mechanism that augments Ag presentation during cognate APC-T cell interactions.     

Visualization of antigen presentation by actin-mediated targeting of glycolipid-enriched membrane domains to the immune synapse of B cell APCs

Glycolipid-enriched membrane (GEM) domains, or lipid rafts, function in signaling in immune cells, but their properties during Ag presentation are less clear. To address this question, GEM domains were studied using fluorescence cell imaging of mouse CH27 B cells presenting Ag to D10 T cells. Our experiments showed that APCs were enriched with GEM domains in the immune synapse, and this occurred in an actin-dependent manner. This enrichment was specific to GEM domains, because a marker for non-GEM regions of the membrane was excluded from the immune synapse. Furthermore, fluorescence photobleaching experiments showed that protein in the immune synapse was dynamic and rapidly exchanged with that in other compartments of CH27 cells. To identify the signals for targeting GEM domains to the immune synapse in APCs, capping of the domains was measured in cells after cross-linking surface molecules. This showed that co-cross-linking CD48 with MHC class II was required for efficient capping and intracellular signaling. Capping of GEM domains by co-cross-linking CD48 and MHC class II occurred with co-capping of filamentous actin, and both domain capping and T cell-CH27 cell conjugation were inhibited by pretreating CH27 cells with latrunculin B. Furthermore, disruption of the actin cytoskeleton of the CH27 cells also inhibited formation of a mature immune synapse in those T cells that did conjugate to APCs. Thus, Ag presentation and efficient T cell stimulation occur by an actin-dependent targeting of GEM domains in the APC to the site of T cell engagement.     

Type I IFN receptor signals directly stimulate local B cells early following influenza virus infection

Rapidly developing Ab responses to influenza virus provide immune protection even during a primary infection. How these early B cell responses are regulated is incompletely understood. In this study, we show that the first direct stimulatory signal for local respiratory tract B cells during influenza virus infection is provided through the type I IFNR. IFNR-mediated signals were responsible for the influenza infection-induced local but not systemic up-regulation of CD69 and CD86 on virtually all lymph node B cells and for induction of a family of IFN-regulated genes within 48 h of infection. These direct IFNR-mediated signals were shown to affect both the magnitude and quality of the local virus-specific Ab response. Thus, ligand(s) of the type I IFNR are direct nonredundant early innate signals that regulate local antiviral B cell responses.     

TLR9 is required for the gut-associated lymphoid tissue response following oral infection of Toxoplasma gondii

TLRs expressed by a variety of cells, including epithelial cells, B cells, and dendritic cells, are important initiators of the immune response following stimulation with various microbial products. Several of the TLRs require the adaptor protein, MyD88, which is an important mediator for the immune response following Toxoplasma gondii infection. Previously, TLR9-mediated innate immune responses were predominantly associated with ligation of unmethylated bacterial CpG DNA. In this study, we show that TLR9 is required for the Th1-type inflammatory response that ensues following oral infection with T. gondii. After oral infection with T. gondii, susceptible wild-type (WT; C57BL/6) but not TLR9(-/-) (B6 background) mice develop a Th1-dependent acute lethal ileitis; TLR9(-/-) mice have higher parasite burdens than control WT mice, consistent with depressed IFN-gamma-dependent parasite killing. A reduction in the total T cell and IFN-gamma-producing T cell frequencies was observed in the lamina propria of the TLR9(-/-) parasite-infected mice. TLR9 and type I IFN production was observed by cells from infected intestines in WT mice. TLR9 expression by dendritic cell populations is essential for their expansion in the mesenteric lymph nodes of infected mice. Infection of chimeric mice deleted of TLR9 in either the hemopoietic or nonhemopoietic compartments demonstrated that TLR9 expression by cells from both compartments is important for efficient T cell responses to oral infection. These observations demonstrate that TLR9 mediates the innate response to oral parasite infection and is involved in the development of an effective Th1-type immune response.     

The pathway of antigen uptake and processing dictates MHC class II-mediated B cell survival and activation

The influence of the pathway of Ag uptake and processing on MHC class II (CII)-mediated B cell function is unknown. In this study, we investigate in resting and activated (via the BCR or CD40) B cells the biological properties of CII-peptide complexes (CII-peptide) generated by either the BCR-mediated Ag processing (type I complex) or fluid phase Ag processing (type II complex). Compared with type I complex, ligation of type II complex by either specific Ab or the TCR in Ag-presenting assay results in significant decreases in B cell survival rate (50-100%) and expression levels of CII, CD86, and CD54. Loss of B cells following ligation of type II complex occurs in the presence of a comparatively good level of specific CD4(+) T cell division, indicating that B cell loss is a late event following T cell stimulation. Comparative analysis of T and B cell conjugates after Ab ligation of type I or II complex reveals decreased efficiency of the latter in forming conjugates. Neither initial differential levels of CII and other studied surface markers, B cell type inherent differences, BCR signaling, T cell proliferation, nor initial density of CII-peptide complexes could explain the T cell-induced B cell loss. We propose that the context in which CII-peptide complexes are present in the membrane following BCR uptake and processing leads to B cell survival. Thus, appropriate targeting of Ag ensures generation of relevant immune responses.     

Bovine plasmacytoid dendritic cells are the major source of type I interferon in response to foot-and-mouth disease virus in vitro and in vivo

Type I interferons (alpha/beta interferons [IFN-α/β]) are the main innate cytokines that are able to induce a cellular antiviral state, thereby limiting viral replication and disease pathology. Plasmacytoid dendritic cells (pDCs) play a crucial role in the control of viral infections, especially in response to viruses that have evolved mechanisms to block the type I IFN signal transduction pathway. Using density gradient separation and cell sorting, we have highly enriched a population of bovine cells capable of producing high levels of biologically active type I IFN. These cells represented less than 0.1% of the total lymphocyte population in blood, pseudoafferent lymph, and lymph nodes. Phenotypic analysis identified these cells as bovine pDCs (CD3(-) CD14(-) CD21(-) CD11c(-) NK(-) TCRδ(-) CD4(+) MHC II(+) CD45RB(+) CD172a(+) CD32(+)). High levels of type I IFN were generated by these cells in vitro in response to Toll-like receptor 9 (TLR-9) agonist CpG and foot-and-mouth disease virus (FMDV) immune complexes. In contrast, immune complexes formed with UV-inactivated FMDV or FMDV empty capsids failed to elicit a type I IFN response. Depletion of CD4 cells in vivo resulted in levels of type I IFN in serum early during FMDV infection that were significantly lower than those for control animals. In conclusion, pDCs interacting with immune-complexed virus are the major source of type I interferon production during acute FMDV infection in cattle.     

Incomplete activation of CD4 T cells by antigen-presenting transitional immature B cells: implications for peripheral B and T cell responsiveness

B cells leave the bone marrow as transitional B cells. Transitional B cells represent a target of negative selection and peripheral tolerance, both of which are abrogated in vitro by mediators of T cell help. In vitro, transitional and mature B cells differ in their responses to B cell receptor ligation. Whereas mature B cells up-regulate the T cell costimulatory molecule CD86 (B7.2) and are activated, transitional B cells do not and undergo apoptosis. The ability of transitional B cells to process and present Ag to CD4 T cells and to elicit protective signals in the absence of CD86 up-regulation was investigated. We report that transitional B cells can process and present Ag as peptide:MHC class II complexes. However, their ability to activate T cells and elicit help signals from CD4-expressing Th cells was compromised compared with mature B cells, unless exogenous T cell costimulation was provided. A stringent requirement for CD28 costimulation was not evident in interactions between transitional B cells and preactivated CD4-expressing T cells, indicating that T cells involved in vivo in an ongoing immune response might rescue Ag-specific transitional B cells from negative selection. These data suggest that during an immune response, immature B cells may be able to sustain the responses of preactivated CD4(+) T cells, while being unable to initiate activation of naive T cells. Furthermore, the ability of preactivated, but not naive T cells to provide survival signals to B cell receptor-engaged transitional immature B cells argues that these B cells may be directed toward activation rather than negative selection when encountering Ag in the context of a pre-existing immune response.     

Cross presentation of antigen on MHC class II via the draining lymph node after corneal transplantation in mice

We investigated Ag trafficking from the cornea and T effector cell activation in secondary lymphoid tissue after corneal transplantation. In preliminary experiments, the central cornea was shown to contain a population of CD45(+), CD11b(+), CD11c- cells, with a few MHC class II(+) cells, and F4/80(+) cells. However, MHC class II(+) passenger leukocytes in donor cornea after allografting did not traffic to the draining lymph node. Instead, Ag (plasmid) delivered to the eye via the donor cornea during allograft was detected in host CD11c(+) and F4/80(+) APC in the draining lymph nodes and spleen. The earliest detection of APC-associated Ag was at 6 h in the draining lymph node and 24 h in the spleen. After 48 h Ag was not detected in the draining lymph node but was still present in the spleen. Ag applied to the donor corneal epithelium before allografting induced Ag-specific T cell activation and expansion in the draining lymph node with a peak response at 4-6 days, indicating that cross-presentation of Ag had occurred. We conclude therefore, that Ag is transported from the donor cornea within host APC and that this event occurs within hours after grafting. Ag is cross-presented to host CD4(+) T cells on MHC class II and leads to the activation of Ag-specific effector T cells and clonal expansion in the draining lymph node.     

Blockade of secondary lymphoid tissue chemokine exacerbates Propionibacterium acnes-induced acute lung inflammation

Chemokine-chemokine receptor interaction plays an essential role in leukocyte/dendritic cell (DC) trafficking in inflammation and immune responses. We investigated the pathophysiological roles of secondary lymphoid tissue chemokine (SLC; CCL21) and macrophage inflammatory protein-2 (MIP-2) in the development of acute pulmonary inflammation induced by an intratracheal injection of Propionibacterium acnes in mice. Immunohistochemical studies revealed that SLC was constitutively expressed in the peribronchial areas and perivascular lymphatics in normal mice. MIP-2-positive cells were observed in alveolar spaces in mice challenged with P. acnes. Both neutralization Abs against MIP-2 and CXC chemokine receptor 2 alleviated the P. acnes-induced pulmonary inflammation when injected before P. acnes Ag challenge. On the other hand, polyclonal anti-SLC Abs (pAbs) exacerbated the pulmonary inflammation. The numbers of mature DCs (MHC class II +, CD11c+, and CD86+) as well as macrophages and neutrophils in the P. acnes Ag-challenged lungs were increased, whereas the number of CD4+ T cells, including memory T cells, was decreased. The numbers of mature and proliferating CD4+ T cells (bromodeoxyuridine(+)CD4+) in regional lymph nodes were decreased in mice injected with anti-SLC pAbs compared with those in mice treated with control Abs. An in vitro proliferation assay confirmed the impairment of the Ag-specific T cell response in regional lymph nodes of mice treated with anti-SLC pAbs. These results indicate for the first time a regulatory role for SLC-recruited mature DCs in bridging an acute inflammatory response (innate immunity) and acquired immunity in the lung.     

Intestinal epithelial antigen induces mucosal CD8 T cell tolerance, activation, and inflammatory response

Intestinal autoimmune diseases are thought to be associated with a breakdown in tolerance, leading to mucosal lymphocyte activation perhaps as a result of encounter with bacterium-derived Ag. To study mucosal CD8(+) T cell activation, tolerance, and polarization of autoimmune reactivity to self-Ag, we developed a novel (Fabpl(4x at -132)-OVA) transgenic mouse model expressing a truncated form of OVA in intestinal epithelia of the terminal ileum and colon. We found that OVA-specific CD8(+) T cells were partially tolerant to intestinal epithelium-derived OVA, because oral infection with Listeria monocytogenes-encoding OVA did not elicit an endogenous OVA-specific MHC class I tetramer(+)CD8(+) T cell response and IFN-gamma-, IL-4-, and IL-5-secreting T cells were decreased in the Peyer's patches, mesenteric lymph nodes, and intestinal mucosa of transgenic mice. Adoptive transfer of OVA-specific CD8(+) (OT-I) T cells resulted in their preferential expansion in the Peyer's patches and mesenteric lymph nodes and subsequently in the epithelia and lamina propria but failed to cause mucosal inflammation. Thus, CFSE-labeled OT-I cells greatly proliferated in these tissues by 5 days posttransfer. Strikingly, OT-I cell-transferred Fabpl(4x at -132)-OVA transgenic mice underwent a transient weight loss and developed a CD8(+) T cell-mediated acute enterocolitis 5 days after oral L. monocytogenes-encoding OVA infection. These findings indicate that intestinal epithelium-derived "self-Ag" gains access to the mucosal immune system, leading to Ag-specific T cell activation and clonal deletion. However, when Ag is presented in the context of bacterial infection, the associated inflammatory signals drive Ag-specific CD8(+) T cells to mediate intestinal immunopathology.     

Absence of cross-presenting cells in the salivary gland and viral immune evasion confine cytomegalovirus immune control to effector CD4 T cells

Horizontal transmission of cytomegaloviruses (CMV) occurs via prolonged excretion from mucosal surfaces. We used murine CMV (MCMV) infection to investigate the mechanisms of immune control in secretory organs. CD4 T cells were crucial to cease MCMV replication in the salivary gland (SG) via direct secretion of IFNγ that initiated antiviral signaling on non-hematopoietic cells. In contrast, CD4 T cell helper functions for CD8 T cells or B cells were dispensable. Despite SG-resident MCMV-specific CD8 T cells being able to produce IFNγ, the absence of MHC class I molecules on infected acinar glandular epithelial cells due to viral immune evasion, and the paucity of cross-presenting antigen presenting cells (APCs) prevented their local activation. Thus, local activation of MCMV-specific T cells is confined to the CD4 subset due to exclusive presentation of MCMV-derived antigens by MHC class II molecules on bystander APCs, resulting in IFNγ secretion interfering with viral replication in cells of non-hematopoietic origin.     

Mycobacterium tuberculosis infects dendritic cells with high frequency and impairs their function in vivo

Mycobacterium tuberculosis (Mtb) is thought to reside in macrophages, although infected dendritic cells (DCs) have been observed. Thus, although cellular associations have been made, global characterization of the cells harboring Mtb is lacking. We have performed temporal and quantitative characterization of the cells harboring Mtb following aerosol infection of mice by using GFP-expressing bacteria and flow cytometry. We discovered that Mtb infects phagocytic cells of diverse phenotypes, that the predominant infected cell populations change with time, and that myeloid DCs are the major cell population infected with Mtb in the lungs and lymph nodes. We also found that the bacteria in the lung-draining lymph node are transported there from the lungs by a CCL19/21-dependent mechanism and that the transport of bacteria to the lymph node is a transient phenomenon despite chronic infection. In addition, we found that the lymph node cell subsets that are most efficacious in stimulating Mtb-specific, TCR-transgenic CD4(+) T lymphocytes are not infected with the bacteria and are scarce or absent from the lungs of infected mice. Finally, we found that the lung cell populations that are infected with Mtb at high frequency are relatively ineffective at stimulating Ag-specific CD4(+) T lymphocytes, and we have obtained evidence that live Mtb can inhibit MHC class II Ag presentation without a decrease in the surface expression of MHC class II. These results indicate that Mtb targets DC migration and Ag presentation in vivo to promote persistent infection.     

Protection against influenza A virus by memory CD8 T cells requires reactivation by bone marrow-derived dendritic cells

Influenza A virus is the causative agent of an acute inflammatory disease of the airway. Although Abs can prevent infection, disease and death can be prevented by T cell-mediated immunity. Recently, we showed that protection against lethal influenza A (PR8/34) virus infection is mediated by central memory CD8 T cells (T(CM)). In this study, using relB(-/-) mice we began to investigate the role of bone marrow (BM)-derived dendritic cells (DCs) in the mechanism of protection. We found that in the absence of functional DCs, memory CD8 T cells specific for the nucleoprotein epitope (NP(366-374)) fail to protect even after adoptive transfer into naive recipients. Through an analysis of Ag uptake, activation of memory CD8 T cells, and display of peptide/MHC complex by DCs in draining LNs and spleen early after virus infection, we established that lack of protection is associated with defective Ag presentation by BM-derived DCs and defective homing of memory T cells in the lymph nodes draining the airway tract. Collectively, the data suggest that protection against the influenza A virus requires that memory CD8 T cells be reactivated by Ag presented by BM-derived DCs in the lymph nodes draining the site of infection. They also imply that protection depends both on the characteristics of systemic adaptive immunity and on the coordinated interplay between systemic and local immunity.