In Situ Microscopy Analysis Reveals Local Innate Immune Response Developed around Brucella Infected Cells in Resistant and Susceptible Mice

Brucella are facultative intracellular bacteria that chronically infect humans and animals causing brucellosis. Brucella are able to invade and replicate in a broad range of cell lines in vitro, however the cells supporting bacterial growth in vivo are largely unknown. In order to identify these, we used a Brucella melitensis strain stably expressing mCherry fluorescent protein to determine the phenotype of infected cells in spleen and liver, two major sites of B. melitensis growth in mice. In both tissues, the majority of primary infected cells expressed the F4/80 myeloid marker. The peak of infection correlated with granuloma development. These structures were mainly composed of CD11b⁺ F4/80⁺ MHC-II⁺ cells expressing iNOS/NOS2 enzyme. A fraction of these cells also expressed CD11c marker and appeared similar to inflammatory dendritic cells (DCs). Analysis of genetically deficient mice revealed that differentiation of iNOS⁺ inflammatory DC, granuloma formation and control of bacterial growth were deeply affected by the absence of MyD88, IL-12p35 and IFN-γ molecules. During chronic phase of infection in susceptible mice, we identified a particular subset of DC expressing both CD11c and CD205, serving as a reservoir for the bacteria. Taken together, our results describe the cellular nature of immune effectors involved during Brucella infection and reveal a previously unappreciated role for DC subsets, both as effectors and reservoir cells, in the pathogenesis of brucellosis.     

Preferential infection of dendritic cells during human immunodeficiency virus type 1 infection of blood leukocytes

Human immunodeficiency virus type 1 (HIV-1) transmission by the parenteral route is similar to mucosal transmission in the predominance of virus using the CCR5 coreceptor (R5 virus), but it is unclear whether blood dendritic cells (DCs), monocytes, or T cells are the cells initially infected. We used ex vivo HIV-1 infection of sorted blood mononuclear cells to model the in vivo infection of blood leukocytes. Using quantitative real-time PCR to detect full-length HIV-1 DNA, both sorted CD11c⁺ myeloid and CD11c⁻ plasmacytoid DCs were more frequently infected than other blood mononuclear cells, including CD16⁺ or CD14⁺ monocytes or resting CD4⁺ T cells. There was a strong correlation between CCR5 coreceptor use and preferential DC infection across a range of HIV-1 isolates. After infection of unsorted blood mononuclear cells, HIV-1 was initially detected in the CD11c⁺ DCs and later in other leukocytes, including clustering DCs and activated T cells. DC infection with R5 virus was productive, as shown by efficient transmission to CD4⁺ T cells in coculture. Blood DCs infected with HIV-1 in vitro and cultured alone expressed only low levels of multiply spliced HIV-1 RNA unless cocultured with CD4⁺ T cells. Early selective infection of immature blood DCs by R5 virus and upregulation of viral expression during DC-T-cell interaction and transmission provide a potential pathway for R5 selection following parenteral transmission.     

Addition of deoxynucleosides enhances human immunodeficiency virus type 1 integration and 2LTR formation in resting CD4+ T cells

Resting CD4⁺ T cells restrict human immunodeficiency virus (HIV) infection at a step prior to integration. Despite this restriction, we showed previously that HIV integration occurs in resting CD4⁺ T cells in vitro, albeit at lower levels than in activated CD4⁺ T cells. Here we show that addition of deoxynucleosides enhanced integration and 2LTR formation in resting CD4⁺ T cells but that the kinetics were still significantly delayed compared to those of activated T cells. Thus, deoxynucleoside addition partially overcomes the restriction to HIV infection in resting CD4⁺ T cells.     

Functional Divergence among CD103+ Dendritic Cell Subpopulations following Pulmonary Poxvirus Infection

A large number of dendritic cell (DC) subsets have now been identified based on the expression of a distinct array of surface markers as well as differences in functional capabilities. More recently, the concept of unique subsets has been extended to the lung, although the functional capabilities of these subsets are only beginning to be explored. Of particular interest are respiratory DCs that express CD103. These cells line the airway and act as sentinels for pathogens that enter the lung, migrating to the draining lymph node, where they add to the already complex array of DC subsets present at this site. Here we assessed the contributions of these individual populations to the generation of a CD8⁺ T-cell response following respiratory infection with poxvirus. We found that CD103⁺ DCs were the most effective antigen-presenting cells (APC) for naive CD8⁺ T-cell activation. Surprisingly, we found no evidence that lymph node-resident or parenchymal DCs could prime virus-specific cells. The increased efficacy of CD103⁺ DCs was associated with the increased presence of viral antigen as well as high levels of maturation markers. Within the CD103⁺ DCs, we observed a population that expressed CD8α. Interestingly, cells bearing CD8α were less competent for T-cell activation than their CD8α⁻ counterparts. These data show that lung-migrating CD103⁺ DCs are the major contributors to CD8⁺ T-cell activation following poxvirus infection. However, the functional capabilities of cells within this population differ with the expression of CD8, suggesting that CD103⁺ cells may be divided further into distinct subsets.In order for the body to mount an adaptive immune response to a pathogen, T cells circulating through lymph nodes (LN) must be alerted to the presence of infection in the periphery. This occurs as a result of presentation of pathogen-derived epitopes on professional antigen-presenting cells (APC), primarily dendritic cells (DC). The DC that reside in the tissue continually sample the local environment for the presence of foreign/pathogenic antigens. In a noninfected tissue, DC exist in an immature state, i.e., they are highly phagocytic and have low levels of expression of costimulatory molecules (3). Following an encounter with infection-associated signals, e.g., pathogen-associated molecular patterns (PAMPs) and/or inflammatory cytokines, DC undergo maturation (3). This process results in upregulation of chemokine receptors, which promotes trafficking to the lymph node, as well as increased expression of costimulatory molecules and cytokines, which are necessary accessory signals for the activation of naive T cells (2, 3).Unlike many other tissues, the lung is constantly assaulted with foreign antigens, both environmental and infectious. This includes a large number of viruses which spread via aerosolized droplets. As such, it is critical to understand how the immune system detects these infections and subsequently elicits an efficacious adaptive CD8⁺ T-cell response. While an important role for DC as the activators of naive T cells is clear, the contribution of distinct DC subsets in this process is less understood. Multiple DC subsets are present within the lung draining lymph nodes, and as such, all are potential regulators of T-cell activation (for a review, see references 14 and 32). These subsets are either resident in the lymph node or present at this site as a result of migration from the periphery, in this case, the lung. These DC subsets are defined by the array of molecules expressed at their surface. Among the subsets resident within the lymph nodes are those which express CD8α or CD4 or are double negative (express neither CD4 nor CD8α) (32). These subsets appear to be segregated in their capabilities to elicit T-cell responses. For example, previous studies have suggested that CD8α⁺ DC are the predominant DC subset involved in priming CD8⁺ T cells (4), while CD8α ⁻ CD4⁺ DC are more important in the regulation of CD4⁺ T cells (31). Further, CD8α⁺ DC are efficient at cross-presentation, a property shown to be critical in the generation of CD8⁺ T-cell responses in a number of infectious models (24, 33).In addition to LN-resident populations, lung-resident DC that have migrated to the lymph node following infection make up a significant portion of LN DC. CD103 (an α^E integrin)-expressing DC reside at the airway mucosa and surrounding pulmonary vessels (35). In contrast, CD103⁻ CD11b⁺ DC are restricted to the lung parenchyma. Given the relatively recent identification of these distinct lung-resident DC populations, there is limited information available regarding their role in T-cell activation following infection. However, they have been assessed in several models, including influenza virus, respiratory syncytial virus (RSV), and Bordetella pertussis (1, 5, 15, 19, 23, 26, 37). At present, the relative contributions of migrating versus resident DC populations remain controversial. Earlier studies reported a role for LN-resident CD8α⁺ DC in priming naive CD8⁺ T cells in addition to lung-migrating DC (5). More recently, however, studies have suggested that activating potential is restricted primarily to lung-migrating DC (1, 23). The underlying cause of these discrepancies is currently unknown but may reflect differences in the markers used to identify the DC subsets or in the individual infection models. Regardless, our understanding of the role of these subsets remains incomplete.We have analyzed the migration and maturation of DC following respiratory infection with the orthopoxvirus vaccinia virus (VV). These studies revealed that airway-resident CD103⁺ DC were the most efficient activators of virus-specific CD8⁺ T cells. Further studies determined that this was the result of both increased access to viral antigen and increased maturation within this subset. In our analyses, we found no evidence to support a role for LN-resident CD8α⁺ DC or lung-migrating CD11b⁺ DC in T-cell activation. Further, we found that CD103⁺ DC were heterogeneous with regard to their functional capabilities. Interestingly, this correlated with the expression of CD8α. While more-recent studies have found CD8α expression on CD103⁺ DC (30), none have looked at the functional capabilities of these cells separately from those of CD8α⁻ CD103⁺ DC. Our findings are the first to suggest that CD8α expression within the CD103⁺ population may identify a distinct subset that differs in its functional capabilities.     

Yersinia enterocolitica Targets Cells of the Innate and Adaptive Immune System by Injection of Yops in a Mouse Infection Model

Yersinia enterocolitica (Ye) evades the immune system of the host by injection of Yersinia outer proteins (Yops) via a type three secretion system into host cells. In this study, a reporter system comprising a YopE-β-lactamase hybrid protein and a fluorescent staining sensitive to β-lactamase cleavage was used to track Yop injection in cell culture and in an experimental Ye mouse infection model. Experiments with GD25, GD25-β1A, and HeLa cells demonstrated that β1-integrins and RhoGTPases play a role for Yop injection. As demonstrated by infection of splenocyte suspensions in vitro, injection of Yops appears to occur randomly into all types of leukocytes. In contrast, upon infection of mice, Yop injection was detected in 13% of F4/80⁺, 11% of CD11c⁺, 7% of CD49b⁺, 5% of Gr1⁺ cells, 2.3% of CD19⁺, and 2.6% of CD3⁺ cells. Taking the different abundance of these cell types in the spleen into account, the highest total number of Yop-injected cells represents B cells, particularly CD19⁺CD21⁺CD23⁺ follicular B cells, followed by neutrophils, dendritic cells, and macrophages, suggesting a distinct cellular tropism of Ye. Yop-injected B cells displayed a significantly increased expression of CD69 compared to non-Yop-injected B cells, indicating activation of these cells by Ye. Infection of IFN-γR (receptor)- and TNFRp55-deficient mice resulted in increased numbers of Yop-injected spleen cells for yet unknown reasons. The YopE-β-lactamase hybrid protein reporter system provides new insights into the modulation of host cell and immune responses by Ye Yops.     

Dendritic Cell Migration Limits the Duration of CD8+ T-Cell Priming to Peripheral Viral Antigen

CD8⁺ T cells (T_CD8⁺) play a crucial role in immunity to viruses. Antiviral T_CD8⁺ are initially activated by recognition of major histocompatibility complex (MHC) class I-peptide complexes on the surface of professional antigen-presenting cells (pAPC). Migration of pAPC from the site of infection to secondary lymphoid organs is likely required during a natural infection. Migrating pAPC can be directly infected with virus or may internalize antigen derived from virus-infected cells. The use of experimental virus infections to assess the requirement for pAPC migration in initiation of T_CD8⁺ responses has proven difficult to interpret because injected virus can readily drain to secondary lymphoid organs without the need for cell-mediated transport. To overcome this ambiguity, we examined the generation of antigen-specific T_CD8⁺ after immunization with recombinant adenoviruses that express antigen driven by skin-specific or ubiquitous promoters. We show that the induction of T_CD8⁺ in response to tissue-targeted antigen is less efficient than the response to ubiquitously expressed antigen and that the resulting T_CD8⁺ fail to clear all target cells pulsed with the antigenic peptide. This failure to prime a fully functional T_CD8⁺ response results from a reduced period of priming to peripherally expressed antigen versus ubiquitously expressed antigen and correlated with a brief burst of pAPC migration from the skin, a requirement for induction of the response to peripheral antigen. These results indicate that a reduced duration of pAPC migration after virus infection likely reduces the amplitude of the T_CD8⁺ response, allowing persistence of the peripheral virus.The induction of effector CD8⁺ T cells (T_CD8⁺) is a vital step in the eradication or control of many viral infections. The induction of antiviral T_CD8⁺ requires the presentation of virally derived peptides in complex with major histocompatibility complex (MHC) class I on the surface of specialized professional antigen-presenting cells (pAPC), most commonly a subset of dendritic cells (DC) that bear the CD8α chain (1, 29). The CD8α⁺ DC reside only in secondary lymphoid organs and not in the tissues, implying that cell-mediated transport or drainage of virus particles to a lymph node is required for initiation of a T_CD8⁺ response. Partial inhibition of DC migration from the skin can impair the initiation of a T_CD8⁺ response (2). After influenza infection in the lungs, there is a burst of DC migration, followed by a refractory period in which no DC migration occurs (19). The functional consequences of this refractory period of DC migration have not been explored.A number of viruses, particularly human papillomaviruses, infect the skin and are ignored by the immune response for extended periods of time (31). We sought to explore the possibility that, after a low-level peripheral virus infection of the skin, changes in DC migration may limit the availability of antigen in the draining lymph node and thus the induction of a T_CD8⁺ response. There are a number of confounding factors that make the study of DC migration in the initiation of an antiviral T_CD8⁺ response difficult. Virus particles may directly drain to the lymph node within seconds (11, 13, 25). In addition, many viruses will alter DC functions, including migration, after infection of the DC itself. This may occur via specific viral modulation of DC function (16) or via nonspecific shut down of host protein synthesis (26), both of which will affect migration. Thus, it is often not possible to distinguish between the effects of virus infection upon DC migration, drainage of virus directly to the lymph node, and the natural response that follows migration of DC responding to a peripheral virus infection.There is currently no mouse model of a peripheral virus infection that is confined to the skin, as no natural mouse papillomavirus has ever been isolated. Therefore, to address these issues, we have made use of another small DNA virus, namely, an adenovirus vector that is replication deficient (rAd). These vectors express influenza virus nucleoprotein (NP) under the control of a ubiquitous (cytomegalovirus [CMV] immediate-early) or tissue-targeted promoter (K14, targeted to keratinocytes, the site of papillomavirus replication). Antigen driven by the K14 promoter is expressed only in skin cells, so only uninfected DC can present antigen in this system, removing the need to account for modulation of the function of virus-infected DC.We demonstrate that when antigen is expressed in only keratinocytes in the skin, the efficiency of T_CD8⁺ induction is reduced and the time period for which antigen is available to prime effector cells is reduced dramatically. DC-mediated transport is required for antigen to reach the lymph node where a T_CD8⁺ response is initiated. The reduced time period of antigen presentation is the result of a transient blockade in DC migration from the site of infection. The blockade in DC migration reduced the delivery of viral antigen to the lymph node needed to induce a T_CD8⁺ response. The resulting T_CD8⁺ response to peripheral viral antigen is not capable of clearing all target cells presenting a viral peptide, thus allowing the persistence of peripheral virus-infected cells. These results provide a potential mechanism for the long-term evasion of the immune response by papillomaviruses following natural infection and also have important implications for tissue targeted gene therapy vectors.     

Dendritic Cells Transmit Human Immunodeficiency Virus Type 1 to Monocytes and Monocyte-Derived Macrophages

Previous studies have shown that human immunodeficiency virus type 1 (HIV-1) exploits dendritic cells (DC) to replicate and spread among CD4⁺ T cells. To explain the predominance of non-syncytium-inducing (NSI) over syncytium-inducing (SI) strains during the initial viremia of HIV, we investigated the ability of blood monocyte (Mo)-derived DC to transmit HIV-1 to CD4⁺ cells of the monocytoid lineage. First, we demonstrate that in our system, DC are able to transmit NSI strains, but not SI strains, of HIV-1 to fresh blood Mo and to Mo-derived macrophages (MDM). To establish a productive infection, a 10-fold-lower amount of virus was necessary for DC-mediated transmission of HIV-1 to Mo than in case of cell-free infection. Second, immature CD83⁻ DC (imDC) transmit virus to Mo and MDM with higher efficacy compared to mature CD83⁺ DC (maDC); this finding is in contrast to data previously obtained with CD4⁺ T cells. Third, maturation from imDC to maDC efficiently silenced expression of β₂-integrins CD11b, CD11c, and CD18 by maDC. Moreover, monoclonal antibody against CD18 inhibited transmission of HIV-1 from imDC to Mo. We propose that the adhesion molecules of the CD11/CD18 family, involved in cell-cell interactions of DC with the microenvironment, may play a major role in imDC-mediated HIV-1 infection of Mo and MDM.     

Both Conventional and Interferon Killer Dendritic Cells Have Antigen-Presenting Capacity during Influenza Virus Infection

Natural killer cells are innate effector cells known for their potential to produce interferon-γ and kill tumour and virus-infected cells. Recently, B220⁺CD11c^intNK1.1⁺ NK cells were found to also have antigen-presenting capacity like dendritic cells (DC), hence their name interferon-producing killer DC (IKDC). Shortly after discovery, it has already been questioned if IKDC really represent a separate subset of NK cells or merely represent a state of activation. Despite similarities with DCs, in vivo evidence that they behave as bona fide APCs is lacking. Here, using a model of influenza infection, we found recruitment of both conventional B220⁻ NK cells and IKDCs to the lung. To study antigen-presenting capacity of NK cell subsets and compare it to cDCs, all cell subsets were sorted from lungs of infected mice and co-cultured ex vivo with antigen specific T cells. Both IKDCs and conventional NK cells as well as cDCs presented virus-encoded antigen to CD8 T cells, whereas only cDCs presented to CD4 T cells. The absence of CD4 responses was predominantly due to a deficiency in MHCII processing, as preprocessed peptide antigen was presented equally well by cDCs and IKDCs. In vivo, the depletion of NK1.1-positive NK cells and IKDCs reduced the expansion of viral nucleoprotein-specific CD8 T cells in the lung and spleen, but did finally not affect viral clearance from the lung. In conclusion, we found evidence for APC function of lung NK cells during influenza infection, but this is a feature not exclusive to the IKDC subset.     

FoxP3+ CD25+ CD8+ T-cell induction during primary simian immunodeficiency virus infection in cynomolgus macaques correlates with low CD4+ T-cell activation and high viral load

The early immune response fails to prevent the establishment of chronic human immunodeficiency virus (HIV) infection but may influence viremia during primary infection, thereby possibly affecting long-term disease progression. CD25⁺ FoxP3⁺ regulatory T cells may contribute to HIV/simian immunodeficiency virus (SIV) pathogenesis by suppressing efficient antiviral responses during primary infection, favoring high levels of viral replication and the establishment of chronic infection. In contrast, they may decrease immune activation during chronic infection. CD4⁺ regulatory T cells have been studied in the most detail, but CD8⁺ CD25⁺ FoxP3⁺ T cells also have regulatory properties. We monitored the dynamics of CD25⁺ FoxP3⁺ T cells during primary and chronic SIVmac251 infection in cynomolgus macaques. The number of peripheral CD4⁺ CD25⁺ FoxP3⁺ T cells paralleled that of memory CD4⁺ T cells, with a rapid decline during primary infection followed by a rebound to levels just below baseline and gradual depletion during the course of infection. No change in the proportion of CD25⁺ FoxP3⁺ T cells was observed in peripheral lymph nodes. A small number of CD4⁺ CD25⁺ FoxP3⁺ T cells at set point was associated with a high plasma viral load. In contrast, peripheral CD8⁺ CD25⁺ FoxP3⁺ T cells were induced a few days after peak plasma viral load during primary infection. The number of these cells was positively correlated with viral load and negatively correlated with CD4⁺ T-cell activation, SIV antigen-specific proliferative responses during primary infection, and plasma viral load at set point, with large numbers of CD8⁺ CD25⁺ FoxP3⁺ T cells being indicative of a poor prognosis.     

Neutralizing Monoclonal Antibodies Block Human Immunodeficiency Virus Type 1 Infection of Dendritic Cells and Transmission to T Cells

Prevention of the initial infection of mucosal dendritic cells (DC) and interruption of the subsequent transmission of HIV-1 from DC to T cells are likely to be important attributes of an effective human immunodeficiency virus type 1 (HIV-1) vaccine. While anti-HIV-1 neutralizing antibodies have been difficult to elicit by immunization, there are several human monoclonal antibodies (MAbs) that effectively neutralize virus infection of activated T cells. We investigated the ability of three well-characterized neutralizing MAbs (IgG1b12, 2F5, and 2G12) to block HIV-1 infection of human DC. DC were generated from CD14⁺ blood cells or obtained from cadaveric human skin. The MAbs prevented viral entry into purified DC and the ensuing productive infection in DC/T-cell cultures. When DC were first pulsed with HIV-1, MAbs blocked the subsequent transmission to unstimulated CD3⁺ T cells. Thus, neutralizing antibodies can block HIV-1 infection of DC and the cell-to-cell transmission of virus from infected DC to T cells. These data suggest that neutralizing antibodies could interrupt the initial events associated with mucosal transmission and regional spread of HIV-1.     

Respiratory Syncytial Virus-Induced Activation and Migration of Respiratory Dendritic Cells and Subsequent Antigen Presentation in the Lung-Draining Lymph Node

In the respiratory tract, different dendritic cell (DC) populations guard a tight balance between tolerance and immunity to infectious or harmless materials to which the airways are continuously exposed. For infectious and noninfectious antigens administered via different routes, different subsets of DC might contribute during the induction of T-cell tolerance and immunity. We studied the impact of primary respiratory syncytial virus (RSV) infection on respiratory DC composition in C57BL/6 mice. We also tracked the migration of respiratory DC to the lymph nodes and studied antigen presentation by lung-derived and lymph node-resident DC to CD4⁺ and CD8⁺ T cells. We observed a massive influx of mainly CD103⁻ CD11b^high CD11c⁺ conventional DC (cDC) and plasmacytoid DC during the first 7 days of RSV infection, while CD103⁺ CD11b^low CD11c⁺ cDC disappeared from the lung. The two major subsets of lung tissue DC, CD103⁺ CD11b^low CD11c⁺ and CD103⁻ CD11b^high CD11c⁺ cDC, both transported RSV RNA to the lung-draining lymph node. Furthermore, these lung-derived cDC subsets as well as resident LN DC, which did not contain viral RNA, displayed viral antigen by major histocompatibility complex class I and class II to CD8⁺ and CD4⁺ T cells. Taken together, our data indicate that during RSV infections, at least three DC subsets might be involved during the activation of lymph node-homing naïve and memory CD4⁺ and CD8⁺ T cells.Respiratory syncytial virus (RSV) constitutes a major health burden for infants, elderly people, and immunocompromised individuals (16, 19). The virus infects most children in their first year of life and is the main cause of severe lower respiratory tract infections in infants (19). Despite many decades of research, the immune response to RSV is still not completely understood. Infection with RSV leads to poor development of immunity, and recurrent infections are common (23). In mice, it was found that RSV induces virus-specific CD8⁺ T-cell responses in the lung that are functionally impaired (10). It has been suggested that a functional inactivation of CD8⁺ T cells by RSV could be a reason for the short-lived immune response. Furthermore, we and others have previously shown that human monocyte-derived dendritic cells (DC) can be infected with RSV, which results in a strong inhibition of their ability to support proliferative responses and induction of effector function in naïve T cells (11, 12). An early vaccine trial with formalin-inactivated RSV in alum administered intramuscularly elicited a memory immune response that caused a strong aberrant secondary immune response in vaccinees upon natural exposure with live virus. This resulted in a high rate of morbidity in the vaccinated children (31). These observations underscore the necessity to understand the components of the immune response that are protective during RSV infections and the need to understand the mechanism by which protective immunity can be elicited for the development of an effective and safe vaccine.DC play an important role in the initiation of both the innate and adaptive immune responses to pathogens including RSV (3). They are a heterogeneous population of cells represented by two main subsets, the myeloid or “conventional” CD11c⁺ DC (cDC) and the CD11c^low/mPDCA-1⁺ plasmacytoid DC (pDC) (47, 52). cDC can be further divided based on the expression of surface markers and anatomic location. cDC in the tissue and cDC in lymph nodes (LN) appear to be different subsets arising from different pools of progenitor cells and with specialized functions (13, 17, 30, 33, 46). In the mouse lung, two major cDC populations are derived from blood monocytes. CD11c⁺ major histocompatibility complex class II (MHC-II)-positive (MHC-II⁺) CD103⁻ CD11b^high cDC (CD11b^hi cDC) are localized in the parenchyma. These cells are the main producers of chemokines and are important for the recruitment of leukocytes (4). A second cDC population, CD11c⁺ MHC-II⁺ CD103⁺ CD11b^low cDC (CD103⁺ cDC), is located directly underneath the airway epithelium. These CD103⁺ cDC express the integrin α_Eβ₇; therefore, they are found mainly at the basal lamina of the bronchial epithelia and arterioles, which express E-cadherin, the ligand for α_Eβ₇. Furthermore, CD103⁺ cDC express the tight-junction proteins ZO-2 and claudin-7, which enables them to sample the airways with their extensions (45). In the lung-draining LN, in addition to pDC, at least two steady-state populations of cDC are present, which are characterized by the expression or absence of CD8α. In contrast to the lung tissue DC, these cells enter the LN from the blood, and they are directly derived from a bone marrow precursor (38, 39, 41). In addition, minor fractions of tissue-derived cDC also access draining LN in the steady state (28). Several studies have addressed the roles of different DC subsets that are present in the tissue and LN draining the infection site. In spleen and skin-draining LN, the role of CD8α⁺ cDC seems to be important for the initiation of anti-ovalbumin and antiviral CD8⁺ T-cell responses (6, 26, 35). In mice exposed to innocuous (ovalbumin) or infectious (influenza virus) antigen, functional specialization was described for CD103⁺ and CD11b^hi lung cDC subsets. CD11b^hi cDC presented intranasally administered ovalbumin or influenza virus antigen mainly to naïve CD4⁺ T cells, while CD103⁺ cDC were important for the induction of CD8⁺ T-cell responses (14, 32).The ability of DC to present or cross-present antigens depends on the type of antigenic materials and the uptake mechanism used by antigen-presenting cells. Hence, different pathogens and innocuous antigens might be differently presented by different DC subsets. We studied the kinetics of lung DC migration and repopulation during primary RSV infection in C57BL/6 mice. We found that upon RSV infection, CD103⁺ cDC disappeared from the lung, while there was a net increase in numbers of CD11b^hi cDC, pDC, and macrophages. Within the first 48 h after virus exposure, both CD103⁺ and CD11b^hi cDC rapidly migrated to the lung-draining mediastinal LN (MLN), while this accumulation was absent in the non-lung-draining axillary LN. The migrating cDC showed the highest level of expression of the costimulatory molecules CD40, CD80, and CD86, which are necessary for T-cell stimulation, compared to the MLN-resident cDC. Furthermore, the migrating cDC transported viral RNA to the MLN and were capable of stimulating RSV-specific CD4⁺ and CD8⁺ T-cell responses. Resident cDC in the LN were uniformly negative for viral RNA. However, resident cDC in the LN did present viral antigen to CD8⁺ and CD4⁺ T cells via MHC-I and MHC-II, respectively.     

Visualizing Early Splenic Memory CD8+ T Cells Reactivation against Intracellular Bacteria in the Mouse

Memory CD8⁺ T cells represent an important effector arm of the immune response in maintaining long-lived protective immunity against viruses and some intracellular bacteria such as Listeria monocytogenes (L.m). Memory CD8⁺ T cells are endowed with enhanced antimicrobial effector functions that perfectly tail them to rapidly eradicate invading pathogens. It is largely accepted that these functions are sufficient to explain how memory CD8⁺ T cells can mediate rapid protection. However, it is important to point out that such improved functional features would be useless if memory cells were unable to rapidly find the pathogen loaded/infected cells within the infected organ. Growing evidences suggest that the anatomy of secondary lymphoid organs (SLOs) fosters the cellular interactions required to initiate naive adaptive immune responses. However, very little is known on how the SLOs structures regulate memory immune responses. Using Listeria monocytogenes (L.m) as a murine infection model and imaging techniques, we have investigated if and how the architecture of the spleen plays a role in the reactivation of memory CD8⁺ T cells and the subsequent control of L.m growth. We observed that in the mouse, memory CD8⁺ T cells start to control L.m burden 6 hours after the challenge infection. At this very early time point, L.m-specific and non-specific memory CD8⁺ T cells localize in the splenic red pulp and form clusters around L.m infected cells while naïve CD8⁺ T cells remain in the white pulp. Within these clusters that only last few hours, memory CD8⁺ T produce inflammatory cytokines such as IFN-γ and CCL3 nearby infected myeloid cells known to be crucial for L.m killing. Altogether, we describe how memory CD8⁺ T cells trafficking properties and the splenic micro-anatomy conjugate to create a spatio-temporal window during which memory CD8⁺ T cells provide a local response by secreting effector molecules around infected cells.     

Respiratory Dendritic Cell Subsets Differ in Their Capacity to Support the Induction of Virus-Specific Cytotoxic CD8+ T Cell Responses

Dendritic cells located at the body surfaces, e.g. skin, respiratory and gastrointestinal tract, play an essential role in the induction of adaptive immune responses to pathogens and inert antigens present at these surfaces. In the respiratory tract, multiple subsets of dendritic cells (RDC) have been identified in both the normal and inflamed lungs. While the importance of RDC in antigen transport from the inflamed or infected respiratory tract to the lymph nodes draining this site is well recognized, the contribution of individual RDC subsets to this process and the precise role of migrant RDC within the lymph nodes in antigen presentation to T cells is not clear. In this report, we demonstrate that two distinct subsets of migrant RDC - exhibiting the CD103⁺ and CD11b^hi phenotype, respectively - are the primary DC presenting antigen to naïve CD4⁺ and CD8⁺ T lymphocytes in the draining nodes in response to respiratory influenza virus infection. Furthermore, the migrant CD103⁺ RDC subset preferentially drives efficient proliferation and differentiation of naive CD8⁺ T cells responding to infection into effector cells, and only the CD103⁺ RDC subset can present to naïve CD8⁺ T cells non-infectious viral vaccine introduced into the respiratory tract. These results identify CD103⁺ and CD11b^hi RDC as critical regulators of the adaptive immune response to respiratory tract infection and potential targets in the design of mucosal vaccines.     

CD8+ DC,but Not CD8?DC,Isolated from BCG-Infected Mice Reduces Pathological Reactions Induced by Mycobacterial Challenge Infection

Background

Tuberculosis is a mycobacterial infection causing worldwide public health problems but the available vaccine is far from ideal. Type-1 T cell immunity has been shown to be critical for host defence against tuberculosis infection, but the role of dendritic cell (DC) subsets in pathogenesis of mycobacterial infection remains unclear.

Methodology/Principal Findings

We examined the effectiveness of dendritic cell (DC) subsets in BCG-infected mice in generating immune responses beneficial for pathogen clearance and reduction of pathological reactions in the tissues following challenge infection. Our data showed that only the adoptive transfer of the subset of CD8α⁺ DC isolated from infected mice (iCD8⁺ DC) generated significant protection, demonstrated by less mycobacterial growth and pathological changes in the lung and liver tissues in iCD8⁺ DC recipients than sham-treated control mice. The adoptive transfer of the CD8α⁻DC from the infected mice (iCD8⁻ DC) not only failed to reduce bacterial growth, but enhanced inflammation characterized by diffuse heavy cellular infiltration. Notably, iCD8⁻ DC produced significantly higher levels of IL-10 than iCD8⁺ DC and promoted more Th2 cytokine responses in in vitro DC-T cell co-culture and in vivo adoptive transfer experiments.

Conclusions/Significance

The data indicate that in vivo BCG-primed CD8⁺ DC is the dominant DC subset in inducing protective immunity especially for reducing pathological reactions in infected tissues. The finding has implications for the rational improvement of the prophylactic and therapeutic approaches for controlling tuberculosis infection and related diseases.     

Peripheral NKT Cells in Simian Immunodeficiency Virus-Infected Macaques

NKT cells are a specialized population of T lymphocytes that have an increasingly recognized role in immunoregulation, including controlling the response to viral infections. The characteristics of NKT cells in the peripheral blood of macaques during simian immunodeficiency virus (SIV) or chimeric simian/human immunodeficiency virus (HIV) (SHIV) infection were assessed. NKT cells comprised a mean of 0.19% of peripheral blood lymphocytes across the 64 uninfected macaques studied. Although the range in the percentages of NKT cells was large (0 to 2.2%), levels were stable over time within individual macaques without SIV/SHIV infection. The majority of NKT cells in macaques were CD4⁺ (on average 67%) with smaller populations being CD8⁺ (21%) and CD4/CD8 double positive (13%). A precipitous decline in CD4⁺ NKT cells occurred in all six macaques infected with CXCR4-tropic SHIV_mn229 early after infection, with a concomitant rise in CD8⁺ NKT cells in some animals. The depletion of CD4⁺ NKT cells was tightly correlated with the depletion of total CD4⁺ T cells. R5-tropic SIV_mac251 infection of macaques resulted in a slower and more variable decline in CD4⁺ NKT cells, with animals that were able to control SIV virus levels maintaining higher levels of CD4⁺ NKT cells. An inverse correlation between the depletion of total and CD4⁺ NKT cells and SIV viral load during chronic infection was observed. Our results demonstrate the infection-driven depletion of peripheral CD4⁺ NKT cells during both SHIV and SIV infection of macaques. Further studies of the implications of the loss of NKT cell subsets in the pathogenesis of HIV disease are needed.     

Suppression of acute anti-friend virus CD8+ T-cell responses by coinfection with lactate dehydrogenase-elevating virus

Friend virus (FV) and lactate dehydrogenase-elevating virus (LDV) are endemic mouse viruses that can cause long-term chronic infections in mice. We found that numerous mouse-passaged FV isolates also contained LDV and that coinfection with LDV delayed FV-specific CD8⁺ T-cell responses during acute infection. While LDV did not alter the type of acute pathology induced by FV, which was severe splenomegaly caused by erythroproliferation, the immunosuppression mediated by LDV increased both the severity and the duration of FV infection. Compared to mice infected with FV alone, those coinfected with both FV and LDV had delayed CD8⁺ T-cell responses, as measured by FV-specific tetramers. This delayed response accounted for the prolonged and exacerbated acute phase of FV infection. Suppression of FV-specific CD8⁺ T-cell responses occurred not only in mice infected concomitantly with LDV but also in mice chronically infected with LDV 8 weeks prior to infection with FV. The LDV-induced suppression was not mediated by T regulatory cells, and no inhibition of the CD4⁺ T-cell or antibody responses was observed. Considering that most human adults are carriers of chronically infectious viruses at the time of new virus insults and that coinfections with viruses such as human immunodeficiency virus and hepatitis C virus are currently epidemic, it is of great interest to determine how infection with one virus may impact host responses to a second infection. Coinfection of mice with LDV and FV provides a well-defined, natural host model for such studies.     

Dendritic Cells in Chronic Mycobacterial Granulomas Restrict Local Anti-Bacterial T Cell Response in a Murine Model

Background

Mycobacterium-induced granulomas are the interface between bacteria and host immune response. During acute infection dendritic cells (DCs) are critical for mycobacterial dissemination and activation of protective T cells. However, their role during chronic infection in the granuloma is poorly understood.

Methodology/Principal Findings

We report that an inflammatory subset of murine DCs are present in granulomas induced by Mycobacteria bovis strain Bacillus Calmette-guerin (BCG), and both their location in granulomas and costimulatory molecule expression changes throughout infection. By flow cytometric analysis, we found that CD11c⁺ cells in chronic granulomas had lower expression of MHCII and co-stimulatory molecules CD40, CD80 and CD86, and higher expression of inhibitory molecules PD-L1 and PD-L2 compared to CD11c⁺ cells from acute granulomas. As a consequence of their phenotype, CD11c⁺ cells from chronic lesions were unable to support the reactivation of newly-recruited, antigen 85B-specific CD4⁺IFNγ⁺ T cells or induce an IFNγ response from naïve T cells in vivo and ex vivo. The mechanism of this inhibition involves the PD-1:PD-L signaling pathway, as ex vivo blockade of PD-L1 and PD-L2 restored the ability of isolated CD11c⁺ cells from chronic lesions to stimulate a protective IFNγ T cell response.

Conclusions/Significance

Our data suggest that DCs in chronic lesions may facilitate latent infection by down-regulating protective T cell responses, ultimately acting as a shield that promotes mycobacterium survival. This DC shield may explain why mycobacteria are adapted for long-term survival in granulomatous lesions.     

Expression of nonstructural rotavirus protein NSP4 mimics Ca2+ homeostasis changes induced by rotavirus infection in cultured cells

Rotavirus infection modifies Ca²⁺ homeostasis, provoking an increase in Ca²⁺ permeation, the cytoplasmic Ca²⁺ concentration ([Ca²⁺]_cyto), and total Ca²⁺ pools and a decrease in Ca²⁺ response to agonists. A glycosylated viral protein(s), NSP4 and/or VP7, may be responsible for these effects. HT29 or Cos-7 cells were infected by the SA11 clone 28 strain, in which VP7 is not glycosylated, or transiently transfected with plasmids coding for NSP4-enhanced green fluorescent protein (EGFP) or NSP4. The permeability of the plasma membrane to Ca²⁺ and the amount of Ca²⁺ sequestered in the endoplasmic reticulum released by carbachol or ATP were measured in fura-2-loaded cells at the single-cell level under a fluorescence microscope or in cell suspensions in a fluorimeter. Total cell Ca²⁺ pools were evaluated as ⁴⁵Ca²⁺ uptake. Infection with SA11 clone 28 induced an increase in Ca²⁺ permeability and ⁴⁵Ca²⁺ uptake similar to that found with the normally glycosylated SA11 strain. These effects were inhibited by tunicamycin, indicating that inhibition of glycosylation of a viral protein other than VP7 affects the changes of Ca²⁺ homeostasis induced by infection. Expression of NSP4-EGFP or NSP4 in transfected cells induced the same changes observed with rotavirus infection, whereas the expression of EGFP or EGFP-VP4 showed the behavior of uninfected and untransfected cells. Increased ⁴⁵Ca²⁺ uptake was also observed in cells expressing NSP4-EGFP or NSP4, as evidenced in rotavirus infection. These results indicate that glycosylated NSP4 is primarily responsible for altering the Ca²⁺ homeostasis of infected cells through an initial increase of cell membrane permeability to Ca²⁺.     

CD4+ CCR5+ T-cell dynamics during simian immunodeficiency virus infection of Chinese rhesus macaques

Simian immunodeficiency virus (SIV) infection of rhesus macaques (RMs) provides a reliable model to study the relationship between lentivirus replication, cellular immune responses, and CD4⁺ T-cell dynamics. Here we investigated, using SIVmac251-infected RMs of a Chinese genetic background (which experience a slower disease progression than Indian RMs), the dynamics of CD4⁺ CCR5⁺ T cells, as this subset of memory/activated CD4⁺ T cells is both a preferential target of virus replication and a marker of immune activation. As expected, we observed that the number of circulating CD4⁺ CCR5⁺ T cells decreases transiently at the time of peak viremia. However, at 60 days postinfection, i.e., when set-point viremia is established, the level of CD4⁺ CCR5⁺ T cells was increased compared to the baseline level. Interestingly, this increase correlated with faster disease progression, higher plasma viremia, and early loss of CD4⁺ T-cell function, as measured by CD4⁺ T-cell count, the fraction of memory CD4⁺ T cells, and the recall response to purified protein derivative. Taken together, these data show a key difference between the dynamics of the CD4⁺ CCR5⁺ T-cell pool (and its relationship with disease progression) in Chinese RMs and those described in previous reports for Indian SIVmac251-infected RMs. As the SIV-associated changes in the CD4⁺ CCR5⁺ T-cell pool reflect the opposing forces of SIV replication (which reduces this cellular pool) and immune activation (which increases it), our data suggest that in SIV-infected Chinese RMs the impact of immune activation is more prominent than that of virus replication in determining the size of the pool of CD4⁺ CCR5⁺ T cells in the periphery. As progression of HIV infection in humans also is associated with a relative expansion of the level of CD4⁺ CCR5⁺ T cells, we propose that SIV infection of Chinese RMs is a very valuable and important animal model for understanding the pathogenesis of human immunodeficiency virus infection.     

Neonatal neural progenitor cells and their neuronal and glial cell derivatives are fully permissive for human cytomegalovirus infection

Congenital human cytomegalovirus (HCMV) infection causes central nervous system structural abnormalities and functional disorders, affecting both astroglia and neurons with a pathogenesis that is only marginally understood. To better understand HCMV's interactions with such clinically important cell types, we utilized neural progenitor cells (NPCs) derived from neonatal autopsy tissue, which can be differentiated down either glial or neuronal pathways. Studies were performed using two viral isolates, Towne (laboratory adapted) and TR (a clinical strain), at a multiplicity of infection of 3. NPCs were fully permissive for both strains, expressing the full range of viral antigens (Ags) and producing relatively large numbers of infectious virions. NPCs infected with TR showed delayed development of cytopathic effects (CPE) and replication centers and shed less virus. This pattern of delay for TR infections held true for all cell types tested. Differentiation of NPCs was carried out for 21 days to obtain either astroglia (>95% GFAP⁺) or a 1:5 mixed neuron/astroglia population (β-tubulin III⁺/GFAP⁺). We found that both of these differentiated populations were fully permissive for HCMV infection and produced substantial numbers of infectious virions. Utilizing a difference in plating efficiencies, we were able to enrich the neuron population to ~80% β-tubulin III⁺ cells. These β-tubulin III⁺-enriched populations remained fully permissive for infection but were very slow to develop CPE. These infected enriched neurons survived longer than either NPCs or astroglia, and a small proportion were alive until at least 14 days postinfection. These surviving cells were all β-tubulin III⁺ and showed viral Ag expression. Surprisingly, some cells still exhibited extended processes, similar to mock-infected neurons. Our findings strongly suggest neurons as reservoirs for HCMV within the developing brain.