CD1d-independent regulation of NKT cell migration and cytokine production upon Listeria monocytogenes infection

Natural killer T (NKT) cells are a unique T-cell population that is positively selected by CD1d-expressing cells. In this study, we examined the kinetics of conventional CD4+TCRbeta+ and CD4-TCRbeta+ cells along with various NKT cell populations from WT and CD1d KO mice after oral Listeria monocytogenes (Lm) infection at different time points in tissue compartments. We found that CD4+TCRbeta+ cells expressing NK1.1+ (NKT) were constitutively expressed in the lung of both strains of mice, but disappeared after infection. In contrast, CD4-TCRbeta+ NK1.1+ cells migrated to the spleen. Here, we demonstrated that endogenous IL-12 was predominantly expressed in the spleen of CD1d KO mice 2 days after infection, whereas IL-4 was predominantly expressed in the liver of WT mice. Higher levels of IFN-gamma were expressed in MLN of CD1d KO but not in WT mice on day 5. Thus, tissue-specific ligands orchestrate the localization and activation of NKT cells to control immune response to Listeria, which may explain the difference in disease susceptibility.     

The role of CD1d in the immune response against Listeria infection

To address the role of CD1d in mucosal immune regulation in bacterial infection, we infected CD1d KO mice with Listeria monocytogenes (Lm). A higher systemic bacterial burden associated with inflammatory lymphocytic infiltrations within the intestine was found in CD1d KO compared with wild type (WT) mice. Lm induced strong IFN-gamma mRNA expression in the liver of WT and the intestine of CD1d KO mice, thus demonstrating the dual, opposing immune activities of IFN-gamma in Lm infection that is dependent on CD1d and/or NKT cells. Analysis of hepatic T cell population demonstrated a reduction of NK1.1(+)TCRbeta+ cells in both mice, followed by recovery only in WT mice. Last, the proportion of alpha4beta1 integrin on lung lymphocytes from CD1d KO was dramatically increased compared with WT mice. Thus, the absence of CD1d resulted in increased susceptibility towards Listeria infection, induced changes in NKT cells, and increased trafficking of alpha4beta1 molecule to inflamed lung.     

NKT cells inhibit antigen-specific effector CD8 T cell induction to skin viral proteins

We recently demonstrated that CD1d-restricted NKT cells resident in skin can inhibit CD8 T cell-mediated graft rejection of human papillomavirus E7-expressing skin through an IFN-γ-dependent mechanism. In this study, we examined the role of systemically derived NKT cells in regulating the rejection of skin grafts expressing viral proteins. In lymph nodes draining transplanted skin, Ag-specific CD8 T cell proliferation, cytokine production, and cytotoxic activity were impaired by NKT cells. NKT cell suppression was mediated via CD11c(+) dendritic cells. Inhibition of CD8 T cell function did not require Foxp3(+) regulatory T cells or NKT cell-secreted IFN-γ, IL-10, or IL-17. Thus, following skin grafting or immunization with human papillomavirus-E7 oncoprotein, NKT cells reduce the capacity of draining lymph node-resident APCs to cross-present Ag to CD8 T cell precursors, as evidenced by impaired expansion and differentiation to Ag-specific CD8 T effector cells. Therefore, in the context of viral Ag challenge in the skin, systemic NKT cells limit the capacity for effective priming of adaptive immunity.     

Persistent antigen presentation after acute vesicular stomatitis virus infection

Long-term antigen expression is believed to play an important role in modulation of T-cell responses to chronic virus infections. However, recent studies suggest that immune responses may occur late after apparently acute infections. We have now analyzed the CD8 T-cell response to vesicular stomatitis virus (VSV), which is thought to cause to an infection characterized by rapid virus clearance by innate and adaptive immune system components. Unexpectedly, virus-encoded antigen was detectable more than 6 weeks after intranasal VSV infection in both draining and nondraining lymph nodes by adoptively transferred CD8 T cells. Infection with Listeria monocytogenes expressing the same antigen did not result in prolonged antigen presentation. Weeks after VSV infection, discrete T-cell clustering with dendritic cells within the lymph node was observed after transfer of antigen-specific CD8 T cells. Moreover, memory CD8 T cells as defined by phenotype and function were generated from na?ve CD8 T cells entering the response late after infection. These findings suggested that protracted antigen presentation after an apparently acute virus infection may contribute to an ongoing antiviral immune response.     

PDL-1 blockade impedes T cell expansion and protective immunity primed by attenuated Listeria monocytogenes

Infection with attenuated Listeria monocytogenes (Lm) is a robust in vivo model for examining how Ag-specific T cells are primed, and subsequent challenge with virulent Lm allows for the protective effects of T cell priming to be quantified. Herein, we investigated the role of programmed death ligand 1 (PDL-1) in T cell priming and immunity conferred after primary infection with Lm DeltaactA followed by virulent Lm challenge. In striking contrast to the inhibitory role of PDL-1 on T cell immunity in other infection models, marked reductions in the magnitude of T cell expansion and the kinetics of T cell proliferation were observed with PDL-1 blockade after primary Lm DeltaactA infection. More importantly, PDL-1 blockade beginning before primary infection and maintained throughout the experiment resulted in delayed bacterial clearance and T cell expansion after secondary challenge with virulent Lm. These results indicate that for immunity to intracellular bacterial infection, PDL-1 plays an important stimulatory role for priming and expansion of protective T cells.     

Chronic alcohol consumption decreases the percentage and number of NK cells in the peripheral lymph nodes and exacerbates B16BL6 melanoma metastasis into the draining lymph nodes

NK cells in the lymph nodes play important roles in inhibiting tumor metastasis into draining lymph nodes. Previously, we reported that chronic alcohol consumption interferes with NK cell trafficking from the bone marrow to the spleen. Herein, we found that alcohol consumption decreases the numbers of NK cells in lymph nodes. Adoptive transfer experiments indicated that continued exposure of donor splenocytes to alcohol inhibits NK but not T cell trafficking to lymph nodes. Alcohol did not negatively affect CCR7⁺ and CXCR3⁺ NK cells, but decreased the percentage and number of CD62L⁺ NK cells in the spleen, which are an important source of NK cell trafficking into the lymph nodes. These data suggest that modulation of the microenvironment associated with alcohol consumption impairs the trafficking of NK cells to lymph nodes. The decreased number of NK cells in the lymph nodes was associated with increased melanoma metastasis into the draining lymph nodes.     

CD4(+) type II NKT cells mediate ICOS and programmed death-1-dependent regulation of type 1 diabetes

Type 1 diabetes (T1D) is a chronic autoimmune disease that results from T cell-mediated destruction of pancreatic β cells. CD1d-restricted NKT lymphocytes have the ability to regulate immunity, including autoimmunity. We previously demonstrated that CD1d-restricted type II NKT cells, which carry diverse TCRs, prevented T1D in the NOD mouse model for the human disease. In this study, we show that CD4(+) 24αβ type II NKT cells, but not CD4/CD8 double-negative NKT cells, were sufficient to downregulate diabetogenic CD4(+) BDC2.5 NOD T cells in adoptive transfer experiments. CD4(+) 24αβ NKT cells exhibited a memory phenotype including high ICOS expression, increased cytokine production, and limited display of NK cell markers, compared with double-negative 24αβ NKT cells. Blocking of ICOS or the programmed death-1/programmed death ligand 1 pathway was shown to abolish the regulation that occurred in the pancreas draining lymph nodes. To our knowledge, these results provide for the first time cellular and molecular information on how type II CD1d-restricted NKT cells regulate T1D.     

Dynamic imaging of the effector immune response to listeria infection in vivo

Host defense against the intracellular pathogen Listeria monocytogenes (Lm) requires innate and adaptive immunity. Here, we directly imaged immune cell dynamics at Lm foci established by dendritic cells in the subcapsular red pulp (scDC) using intravital microscopy. Blood borne Lm rapidly associated with scDC. Myelomonocytic cells (MMC) swarmed around non-motile scDC forming foci from which blood flow was excluded. The depletion of scDC after foci were established resulted in a 10-fold reduction in viable Lm, while graded depletion of MMC resulted in 30-1000 fold increase in viable Lm in foci with enhanced blood flow. Effector CD8+ T cells at sites of infection displayed a two-tiered reduction in motility with antigen independent and antigen dependent components, including stable interactions with infected and non-infected scDC. Thus, swarming MMC contribute to control of Lm prior to development of T cell immunity by direct killing and sequestration from blood flow, while scDC appear to promote Lm survival while preferentially interacting with CD8+ T cells in effector sites.     

Oral immunization with recombinant listeria monocytogenes controls virus load after vaginal challenge with feline immunodeficiency virus

Recombinant Listeria monocytogenes has many attractive characteristics as a vaccine vector against human immunodeficiency virus (HIV). Wild-type and attenuated Listeria strains expressing HIV Gag have been shown to induce long-lived mucosal and systemic T-cell responses in mice. Using the feline immunodeficiency virus (FIV) model of HIV we evaluated recombinant L. monocytogenes in a challenge system. Five cats were immunized with recombinant L. monocytogenes that expresses the FIV Gag and delivers an FIV Env-expressing DNA vaccine (LMgag/pND14-Lc-env). Control cats were either sham immunized or immunized with wild-type L. monocytogenes (LM-wt). At 1 year after vaginal challenge, provirus could not be detected in any of the nine tissues evaluated from cats immunized with the recombinant bacteria but was detected in at least one tissue in 8 of 10 control animals. Virus was isolated from bone marrow of four of five LMgag/pND14-Lc-env-immunized cats by use of a stringent coculture system but required CD8(+) T-cell depletion, indicating CD8(+) T-cell suppression of virus replication. Control animals had an inverted CD4:CD8 ratio in mesenteric lymph node and were depleted of both CD4(+) and CD8(+) intestinal epithelial T cells, while LMgag/pND14-Lc-env-immunized animals showed no such abnormalities. Vaginal FIV-specific immunoglobulin A was present at high titer in three LMgag/pND14-Lc-env-immunized cats before challenge and in all five at 1 year postchallenge. This study demonstrates that recombinant L. monocytogenes conferred some control of viral load after vaginal challenge with FIV.     

Differential effect of Listeria monocytogenes infection on cytokine production and cytotoxicity of CD8 T cells

Bacterial infection induces a shift to type 1 CD4 T cell subset in an infected host and this shift is important for protection of the host from disease development. Many researchers think that the shift is antigen-dependent, but we previously demonstrated an initial induction step for CD4 T cell subsets during Listeria monocytogenes (Lm) infection is antigen-independent. Although Listeria is a TLR2 ligand, the immune system of the Lm-infected host responded to the pathogen to induce expression of CD69 but not CD25 on CD4 T cells, CD8 T cells and B cells even in the absence of TLR2 or MyD88. The antigen-independent activation of type 1 CD4 T cells accelerate the clearance of pathogens by activating innate immune cells with type 1 cytokines. Type 1 CD4 T cells and CD8 T cells also collaborate to protect the host from intracellular Lm infection. Since CD8 T cells function mainly as cytotoxic T cells and CD69-positive CD8 T cells increase during Lm-infection, cytotoxic activity of CD8 T cells was evaluated during Lm-infection. Although CD8 T cells were activated to produce IFN-gamma, the cytotoxic function of CD8 T cells in Lymphocytic choriomeningitis virus (LCMV) p14 TCR-transgenic mouse was not augmented by Lm-infection. Therefore, Lm-infection differentially influences on cytokine production and cytotoxicity of CD8 T cells.     

Dendritic cell editing by activated natural killer cells results in a more protective cancer-specific immune response

Over the last decade, several studies have extensively reported that activated natural killer (NK) cells can kill autologous immature dendritic cells (DCs) in vitro, whereas they spare fully activated DCs. This led to the proposal that activated NK cells might select a more immunogenic subset of DCs during a protective immune response. However, there is no demonstration that autologous DC killing by NK cells is an event occurring in vivo and, consequently, the functional relevance of this killing remains elusive. Here we report that a significant decrease of CD11c(+) DCs was observed in draining lymph nodes of mice inoculated with MHC-devoid cells as NK cell targets able to induce NK cell activation. This in vivo DC editing by NK cells was perforin-dependent and it was functionally relevant, since residual lymph node DCs displayed an improved capability to induce T cell proliferation. In addition, in a model of anti-cancer vaccination, the administration of MHC-devoid cells together with tumor cells increased the number of tumor-specific CTLs and resulted in a significant increase in survival of mice upon challenge with a lethal dose of tumor cells. Depletion of NK cells or the use of perforin knockout mice strongly decreased the tumor-specific CTL expansion and its protective role against tumor cell challenge. As a whole, our data support the hypothesis that NK cell-mediated DC killing takes place in vivo and is able to promote expansion of cancer-specific CTLs. Our results also indicate that cancer vaccines could be improved by strategies aimed at activating NK cells.     

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.     

IFN-gamma-dependent recruitment of mature CD27(high) NK cells to lymph nodes primed by dendritic cells

NK cells have been proposed to be an initial source of IFN-gamma that supports either Th1 or CTL priming. Although NK cells reside in naive lymph nodes (LN) at a very low frequency, they can be recruited into LN draining sites of infection, inflammation, or immunization where they potentially influence adaptive immunity. In this study, we report that mature CD27(high) NK cells are predominantly recruited into the draining LN following dendritic cell (DC) challenge. Importantly, the recruitment of the CD27(high) NK cell subset in the draining LN was dependent on host IFN-gamma and the activation status of NK cells. Endogenous epidermal DC migration induced by hapten challenge also triggers NK cell recruitment to the draining LN in an IFN-gamma-dependent mechanism. Thus, our results identify that CD27(high) NK cells are the dominant population recruited to the draining LN and NK cell recruitment requires endogenous IFN-gamma in coordinating with DC migration.     

Distinct regulation of H2-M3-restricted memory T cell responses in lymph node and spleen

CD8 T cell populations restricted by H2-M3 MHC class Ib molecules expand rapidly during primary Listeria monocytogenes infection but only minimally upon reinfection. In contrast, CD8 T cells restricted by MHC class Ia molecules undergo more delayed expansion during primary infection but rapid and robust expansion following reinfection. In this study we demonstrate that primary H2-M3-restricted CD8 T cell responses are unaffected by the frequency of naive MHC class Ia-restricted T cells during L. monocytogenes infection. The magnitude of H2-M3-restricted memory responses, in contrast, is down-modulated by increasing frequencies of MHC class Ia-restricted effector T cells following secondary systemic infection. Suppression by MHC class Ia-restricted T cells, however, is not a universal feature of MHC class Ib-restricted memory responses. Primary systemic L. monocytogenes infection followed by secondary tissue infection, for example, results in robust expansion of H2-M3-restricted memory T cells in draining lymph nodes, despite the activation of MHC class Ia-restricted memory T cell responses. Thus, whereas MHC class Ia-restricted memory T cell populations predominate in spleens following systemic reinfection, H2-M3-restricted memory T cell responses remain prominent in lymph nodes draining localized infections. Our studies demonstrate that interactions between CD8 T cell populations can differ, depending on the status of the responding T cells (naive vs memory) and the route of reinfection. These results may have important implications for prime-boost vaccination strategies.     

Infiltration of a mesothelioma by IFN-gamma-producing cells and tumor rejection after depletion of regulatory T cells

Depletion of CD4+CD25+Foxp3+ regulatory T cells (CD25+ T(reg)) with an anti-CD25 Ab results in immune-mediated rejection of tolerogenic solid tumors. In this study, we have examined the immune response to a mesothelioma tumor in mice after depletion of CD25+ cells to elucidate the cellular mechanisms of CD25+ T(reg), a subject over which there is currently much conjecture. Tumor rejection was found to be primarily due to the action of CD8+ T cells, although CD4+ cells appeared to play some role. Depletion of CD25+ cells resulted in an accumulation in tumor tissue of CD4+ and CD8+ T cells and NK cells that were producing the potent antitumor cytokine IFN-gamma. Invasion of tumors by CD8+ T cells was partially dependent on the presence of CD4+ T cells. Although a significant increase in the proliferation and number of tumor-specific CD8+ T cells was observed in lymph nodes draining the tumor of anti-CD25-treated mice, this effect was relatively modest compared with the large increase in IFN-gamma-producing T cells found in tumor tissue, which suggests that the migration of T cells into tumor tissue may also have been altered. Depletion of CD25+ cells did not appear to modulate antitumor CTL activity on a per cell basis. Our data suggests that CD25+ T(reg) limit the accumulation of activated T cells producing IFN-gamma in the tumor tissue and, to a lesser extent, activation and/or rate of mitosis of tumor-specific T cells in lymph nodes.     

Stimulation of enhanced CD8 T cell responses following immunization with a hyper-antigen secreting intracytosolic bacterial pathogen

The intracytosolic niche for replication of Listeria monocytogenes (Lm) facilitates delivery of bacteria-derived Ags into the MHC class I pathway for subsequent stimulation of CD8 effector T cells. Using Lm strains that are equivalent for in vivo virulence yet express marked differences in the level of secretion of a protective target Ag, we have evaluated how these specific differences in secretion levels influences the magnitude and effector function of Ag-specific CD8 T cell responses following Lm injection. Immunization with low doses of a hyperantigen-secreting Lm strain stimulated enhanced target-Ag specific CD8 T cell responses compared with the magnitude stimulated following immunization with the same dose of wild-type Lm. The enhanced determinant-specific response was also evident by in vivo CTL activity, increased numbers of memory cells 4 wk following immunization, and enhanced antilisterial protection following a challenge infection. Initiation of antibiotic treatment 24 h following infection with wild-type Lm markedly reduced the magnitude of the effector CD8 T cell response. In contrast, antibiotic treatment initiated 24 h following immunization with the hyperantigen secreting strain of Lm did not impact the frequency of the target-Ag specific CD8 T cells. Thus, immunization with a low dose of a hyperantigen secreting Lm strain, followed by antibiotic treatment to limit the extent of the infection, may represent a safe strategy for the stimulation of enhanced effector CD8 T cell responses to specific Ag by a rLm vaccine.     

The Initial Draining Lymph Node Primes the Bulk of the CD8 T Cell Response and Influences Memory T Cell Trafficking after a Systemic Viral Infection

Lymphocytic choriomeningitis virus (LCMV) causes a systemic infection in mice with virus replication occurring in both peripheral tissues and secondary lymphoid organs. Because of the rapid systemic dissemination of the virus, the secondary lymphoid organs responsible for the induction of the LCMV-specific CD8 T cell response are poorly defined. We show that the mediastinal lymph node (MedLN) serves as the primary draining lymph node following LCMV infection. In addition, we demonstrate that the MedLN is responsible for priming the majority of the virus-specific CD8 T cell response. Following resolution of the acute infection, the draining MedLN exhibits characteristics of a reactive lymph node including an increased presence of germinal center B cells and increased cellularity for up to 60 days post-infection. Furthermore, the reactive MedLN harbors an increased frequency of CD62L⁻ effector memory CD8 T cells as compared to the non-draining lymph nodes. The accumulation of LCMV-specific CD62L⁻ memory CD8 T cells in the MedLN is independent of residual antigen and is not a unique feature of the MedLN as footpad infection with LCMV leads to a similar increase of virus-specific CD62L⁻ effector memory CD8 T cells in the draining popliteal lymph node. Our results indicate that CD62L⁻ effector memory CD8 T cells are granted preferential access into the draining lymph nodes for an extended time following resolution of an infection.     

NK cells are effectors for resolvin E1 in the timely resolution of allergic airway inflammation

Immune responses are pathologically sustained in several common diseases, including asthma. To determine endogenous proresolving mechanisms for adaptive immune responses, we used a murine model of self-limited allergic airway inflammation. After cessation of allergen exposure, eosinophils and T cells were cleared concomitant with the appearance of increased numbers of NK cells in the lung and mediastinal lymph nodes. The mediastinal lymph node NK cells were activated, expressing CD27, CD11b, CD69, CD107a, and IFN-γ. NK cell depletion disrupted the endogenous resolution program, leading to delayed clearance of airway eosinophils and Ag-specific CD4(+) T cells. NK cell trafficking to inflamed tissues for resolution was dependent upon CXCR3 and CD62L. During resolution, eosinophils and Ag-specific CD4(+) T cells expressed NKG2D ligands, and a blocking Ab for the NKG2D receptor delayed clearance of these leukocytes. Of interest, NK cells expressed CMKLR1, a receptor for the proresolving mediator resolvin E1, and depletion of NK cells decreased resolvin E1-mediated resolution of allergic inflammation. Resolvin E1 regulated NK cell migration in vivo and NK cell cytotoxicity in vitro. Together, these findings indicate new functions in catabasis for NK cells that can also serve as targets for proresolving mediators in the resolution of adaptive immunity.     

Loss of IL-4 secretion from human type 1a diabetic pancreatic draining lymph node NKT cells

Altered frequency and function of peripheral invariant NKT (iNKT) cells have been implicated in the regulation of murine and human type 1a diabetes. To examine regulatory cells from the site of drainage of autoinflammatory tissue and autoantigenic T cell priming in diabetes, we directly cloned iNKT cells from human pancreatic draining lymph nodes (PLN). From 451 T cell clones from control and diabetic PLN, we derived 55 iNKT cells by two methods and analyzed function by cytokine secretion. iNKT cell clones isolated from control PLN secreted IL-4 and IFN-gamma upon TCR stimulation. For type 1a diabetic subjects, PLN iNKT cell clones from three samples secreted IFN-gamma and no IL-4. In a rare recent onset diabetic sample with islet-infiltrating CD4+ T cells, the phenotype of PLN iNKT cell clones was mixed. From normal and diabetic PLN, one-third of CD1d tetramer+-sorted T cell clones were reactive with CD1d transfectants or proliferated/secreted cytokine in response to alpha-galactosylceramide-pulsed PBMCs; tetramer-staining T cell clones from diabetic PLN did not secrete IL-4. This is the first report directly examining iNKT cells from lymph nodes draining the site of autoimmunological attack in humans; iNKT cells were altered in cytokine secretion as previously reported for circulating iNKT cells in human type 1a diabetes.