Competitive but Not Allosteric mTOR Kinase Inhibition Enhances Tumor Cell Radiosensitivity

The mechanistic target of rapamycin (mTOR) is a critical kinase in the regulation of gene translation and has been suggested as a potential target for radiosensitization. The goal of this study was to compare the radiosensitizing activities of the allosteric mTOR inhibitor rapamycin with that of the competitive mTOR inhibitor PP242. On the basis of immunoblot analyses, whereas rapamycin only partially inhibited mTOR complex 1 (mTORC1) activity and had no effect on mTOR complex 2 (mTORC2), PP242 inhibited the activity of both mTOR-containing complexes. Irradiation alone had no effect on mTORC1 or mTORC2 activity. Clonogenic survival was used to define the effects of the mTOR inhibitors on in vitro radiosensitivity. In the two tumor cell lines evaluated, PP242 treatment 1 hour before irradiation increased radiosensitivity, whereas rapamycin had no effect. Addition of PP242 after irradiation also enhanced the radiosensitivity of both tumor lines. To investigate the mechanism of radiosensitization, the induction and repair of DNA double-strand breaks were evaluated according yH2AX foci. PP242 exposure did not influence the initial level of yH2AX foci after irradiation but did significantly delay the dispersal of radiationinduced yH2AX foci. In contrast to the tumor cell lines, the radiosensitivity of a normal human fibroblast cell line was not influenced by PP242. Finally, PP242 administration to mice bearing U251 xenografts enhanced radiationinduced tumor growth delay. These results indicate that in a preclinical tumor model PP242 enhances tumor cell radiosensitivity both in vitro and in vivo and suggest that this effect involves an inhibition of DNA repair.     

Antiviral T Cell Response Triggers Cytomegalovirus Hepatitis in Mice

One common sign of human cytomegalovirus infection is altered liver function. Murine cytomegalovirus strain v70 induces a rapid and severe hepatitis in immunocompetent mice that requires the presence of T cells in order to develop. v70 exhibits approximately 10-fold-greater virulence than the commonly used strain K181, resulting in a more severe, sustained, and lethal hepatitis but not dramatically higher viral replication levels. Hepatitis and death are markedly delayed in immunodeficient SCID compared to immunocompetent BALB/c mice. Transfer of BALB/c splenocytes to SCID mice conferred rapid disease following infection, and depletion of either CD4 or CD8 T cells in BALB/c mice reduced virus-induced hepatitis. The frequency of CD8 T cells producing gamma interferon and tumor necrosis factor in response to viral antigen was higher in settings where more severe disease occurred. Thus, virus-specific effector CD8 T cells appear to contribute to lethal virus-induced hepatitis, contrasting their protective role during sublethal infection. This study reveals how protection and disease during cytomegalovirus infection depend on viral strain and dose, as well as the quality of the T cell response.     

Latent Adeno-Associated Virus Infection Elicits Humoral but Not Cell-Mediated Immune Responses in a Nonhuman Primate Model

Latent infection with wild-type (wt) adeno-associated virus (AAV) was studied in rhesus macaques, a species that is a natural host for AAV and that has some homology to humans with respect to the preferred locus for wt AAV integration. Each of eight animals was infected with an inoculum of 10(10) IU of wt AAV, administered by either the intranasal, intramuscular, or intravenous route. Two additional animals were infected intranasally with wt AAV and a helper adenovirus (Ad), while one additional animal was inoculated with saline intranasally as a control. There were no detectable clinical or histopathologic responses to wt AAV administration. Molecular analyses, including Southern blot, PCR, and fluorescence in situ hybridization, were performed 21 days after infection. These studies indicated that AAV DNA sequences persisted at the sites of administration, albeit at low copy number, and in peripheral blood mononuclear cells. Site-specific integration into the AAVS1-like locus was observed in a subset of animals. All animals, except those infected by the intranasal route with wt AAV alone, developed a humoral immune response to wt AAV capsid proteins, as evidenced by a >/=fourfold rise in anti-AAV neutralizing titers. However, only animals infected with both wt AAV and Ad developed cell-mediated immune responses to AAV capsid proteins. These findings provide some insights into the nature of anti-AAV immune responses that may be useful in interpreting results of future AAV-based gene transfer studies.     

Antiapoptotic Role for Lifeguard in T Cell Mediated Immune Response

Anti-apoptotic protein Lifeguard (LFG) is upregulated on T cells upon in vitro activation. To investigate its role in T cell immunity we infected wild type and LFG knockout bone marrow chimaeras mice with LCMV. We observed a decreased number of LFG KO activated CD8 and CD4 T cells throughout the infection and a marked decrease in LFG KO LCMV specific memory T cells. WT and KO T cells proliferated at the same rate, however, LFG KO CD44^hi T cells showed increased cell death during the initial phase of the immune response. LFG KO and WT T cells were equally sensitive to the FAS antibody Jo-2 in ex vivo cultures, and blocking extrinsic pathways of cell death in vivo with Fas L or caspase 8 inhibitors did not rescue the increased apoptosis in LFG KO T cells. Our data suggest that LFG plays a role in T cell survival during the initial phase of anti-viral immune response by protecting pre-existing memory T cells and possibly newly activated T cells resulting in a diminished immune response and a decreased number of LCMV specific memory T cells.     

Environmental Enrichment Alters Splenic Immune Cell Composition and Enhances Secondary Influenza Vaccine Responses in Mice

Chronic stress has deleterious effects on immune function, which can lead to adverse health outcomes. However, studies investigating the impact of stress reduction interventions on immunity in clinical research have yielded divergent results, potentially stemming from differences in study design and genetic heterogeneity, among other clinical research challenges. To test the hypothesis that reducing glucocorticoid levels enhances certain immune functions, we administered influenza vaccine once (prime) or twice (boost) to mice housed in either standard control caging or environmental enrichment (EE) caging. We have shown that this approach reduces mouse corticosterone production. Compared with controls, EE mice had significantly lower levels of fecal corticosterone metabolites (FCMs) and increased splenic B and T lymphocyte numbers. Corticosterone levels were negatively associated with the numbers of CD19⁺ (r² = 0.43, p = 0.0017), CD4⁺ (r² = 0.28, p = 0.0154) and CD8⁺ cells (r² = 0.20, p = 0.0503). Vaccinated mice showed nonsignificant differences in immunoglobulin G (IgG) titer between caging groups, although EE mice tended to exhibit larger increases in titer from prime to boost than controls; the interaction between the caging group (control versus EE) and vaccine group (prime versus boost) showed a strong statistical trend (cage-group*vaccine-group, F = 4.27, p = 0.0555), suggesting that there may be distinct effects of EE caging on primary versus secondary IgG vaccine responses. Vaccine-stimulated splenocytes from boosted EE mice had a significantly greater frequency of interleukin 5 (IL-5)-secreting cells than boosted controls (mean difference 7.7, IL-5 spot-forming units/10⁶ splenocytes, 95% confidence interval 0.24–135.1, p = 0.0493) and showed a greater increase in the frequency of IL-5–secreting cells from prime to boost. Our results suggest that corticosterone reduction via EE caging was associated with enhanced secondary vaccine responses, but had little effect on primary responses in mice. These findings help identify differences in primary and secondary vaccine responses in relationship to stress mediators that may be relevant in clinical studies.     

Evasion by Stealth: Inefficient Immune Activation Underlies Poor T Cell Response and Severe Disease in SARS-CoV-Infected Mice

Severe Acute Respiratory Syndrome caused substantial morbidity and mortality during the 2002–2003 epidemic. Many of the features of the human disease are duplicated in BALB/c mice infected with a mouse-adapted version of the virus (MA15), which develop respiratory disease with high morbidity and mortality. Here, we show that severe disease is correlated with slow kinetics of virus clearance and delayed activation and transit of respiratory dendritic cells (rDC) to the draining lymph nodes (DLN) with a consequent deficient virus-specific T cell response. All of these defects are corrected when mice are treated with liposomes containing clodronate, which deplete alveolar macrophages (AM). Inhibitory AMs are believed to prevent the development of immune responses to environmental antigens and allergic responses by interacting with lung dendritic cells and T cells. The inhibitory effects of AM can also be nullified if mice or AMs are pretreated with poly I:C, which directly activate AMs and rDCs through toll-like receptors 3 (TLR3). Further, adoptive transfer of activated but not resting bone marrow–derived dendritic cells (BMDC) protect mice from lethal MA15 infection. These results may be relevant for SARS in humans, which is also characterized by prolonged virus persistence and delayed development of a SARS-CoV-specific immune response in individuals with severe disease.     

Dietary Supplementation with Lactobacilli Improves Emergency Granulopoiesis in Protein-Malnourished Mice and Enhances Respiratory Innate Immune Response

This work studied the effect of protein malnutrition on the hemato-immune response to the respiratory challenge with Streptococcus pneumoniae and evaluated whether the dietary recovery with a probiotic strain has a beneficial effect in that response. Three important conclusions can be inferred from the results presented in this work: a) protein-malnutrition significantly impairs the emergency myelopoiesis induced by the generation of the innate immune response against pneumococcal infection; b) repletion of malnourished mice with treatments including nasally or orally administered Lactobacillus rhamnosus CRL1505 are able to significantly accelerate the recovery of granulopoiesis and improve innate immunity and; c) the immunological mechanisms involved in the protective effect of immunobiotics vary according to the route of administration. The study demonstrated that dietary recovery of malnourished mice with oral or nasal administration of L. rhamnosus CRL1505 improves emergency granulopoiesis and that CXCR4/CXCR12 signaling would be involved in this effect. Then, the results summarized here are a starting point for future research and open up broad prospects for future applications of probiotics in the recovery of immunocompromised malnourished hosts.     

大剂量HBsAg对HBV转基因小鼠T细胞免疫调节的研究

目的：研究大剂量HBsAg对HBV转基因小鼠其T细胞免疫效果的影响。方法：用大剂量血源性HBsAg免疫HBV转基因小鼠,采用ELISA方法观察转基因小鼠所诱生的HBsAg特异性Th1类细胞因子的水平,ELISPOT方法检测不同免疫方案对小鼠HBsAg特异性分泌IFN-γT细胞数量的影响,同时检测对小鼠淋巴细胞增殖的影响。结果：HBsAg组免疫后脾细胞产生的Th1类细胞因子（IFN-γ、IL-2）、HBsAg特异性分泌IFN-γT细胞及T细胞增殖水平较对照组显著增加（P〈0.05）。结论：大剂量的HBs-Ag可以诱导乙肝转基因小鼠产生高水平Th1类细胞因子并打破免疫耐受。     

Critical Role of SAP in Progression and Reactivation but Not Maintenance of T Cell-Dependent Humoral Immunity

Signaling lymphocytic activation molecule (SLAM)-associated protein (SAP) is a small adaptor molecule mutated in X-linked lymphoproliferative disease, a human immunodeficiency. SAP plays a critical role in the initiation of T cell-dependent B cell responses leading to germinal center reaction, the production of high-affinity antibodies, and B cell memory. However, whether SAP has a role in these responses beyond their initiation is not known. It is important to address this matter not only for mechanistic reasons but also because blockade of the SAP pathway is being contemplated as a means to treat autoimmune diseases in humans. Using an inducibly SAP deficient mouse, we found that SAP was required not only for the initiation but also for the progression of primary T cell-driven B cell responses to haptens. It was also necessary for the reactivation of T cell-dependent B cell immunity during secondary immune responses. These activities consistently correlated with the requirement of SAP for full expression of the lineage commitment factor Bcl-6 in follicular T helper (T_FH) cells. However, once memory B cells and long-lived antibody-secreting cells were established, SAP became dispensable for maintaining T cell-dependent B cell responses. Thus, SAP is pivotal for nearly all phases, but not for maintenance, of T cell-driven B cell humoral immunity. These findings may have implications for the treatment of immune disorders by targeting the SAP pathway.     

Interleukin-7, but Not Thymic Stromal Lymphopoietin,Plays a Key Role in the T Cell Response to Influenza A Virus

The immune response to viral infection is ideally rapid and specific, resulting in viral clearance and establishment of immune memory. Some viruses such as HIV can evade such responses leading to chronic infection, while others like Influenza A can elicit a severe inflammatory response with immune-related complications including death. Cytokines play a major role in shaping the appropriate outcomes to infection. While Interleukin-7 (IL-7) has a critical role in T and B cell development, treatment with IL-7 has recently been shown to aid the adaptive T cell response in clearance of chronic viral infection. In contrast, the IL-7-related cytokine thymic stromal lymphopoietin (TSLP) has a limited role in lymphocyte development but is important in the immune response to parasitic worms and allergens. The role for these cytokines in the immune response to an acute viral infection is unclear. IL-7 and TSLP share IL-7Rα as part of their heterodimeric receptors with the gamma common chain (γc) and TSLPR, respectively. We investigated the role of IL-7 and TSLP in the primary immune response to influenza A infection using hypomorphic IL-7Rα (IL-7Rα^449F) and TSLPR^−/− mice. We found that IL-7, but not TSLP, plays an important role in control of influenza A virus. We also showed that IL-7 signaling was necessary for the generation of a robust influenza A-specific CD4 and CD8 T cell response and that this requirement is intrinsic to CD8 T cells. These findings demonstrate a significant role for IL-7 during acute viral infection.     

Original Encounter with Antigen Determines Antigen-Presenting Cell Imprinting of the Quality of the Immune Response in Mice

Background

Obtaining a certain multi-functionality of cellular immunity for the control of infectious diseases is a burning question in immunology and in vaccine design. Early events, including antigen shuttling to secondary lymphoid organs and recruitment of innate immune cells for adaptive immune response, determine host responsiveness to antigens. However, the sequence of these events and their impact on the quality of the immune response remain to be elucidated. Here, we chose to study Modified Vaccinia virus Ankara (MVA) which is now replacing live Smallpox vaccines and is proposed as an attenuated vector for vaccination strategies against infectious diseases.

Methodology/Principal findings

We analyzed in vivo mechanisms triggered following intradermal (i.d.) and intramuscular (i.m.) Modified Vaccinia virus Ankara (MVA) administration. We demonstrated significant differences in the antigen shuttling to lymphoid organs by macrophages (MΦs), myeloid dendritic cells (DCs), and neutrophils (PMNs). MVA i.d. administration resulted in better antigen distribution and more sustained antigen-presenting cells (APCs) recruitment into draining lymph nodes than with i.m. administration. These APCs, which comprise both DCs and MΦs, were differentially involved in T cell priming and shaped remarkably the quality of cytokine-producing virus-specific T cells according to the entry route of MVA.

Conclusions/Significance

This study improves our understanding of the mechanisms of antigen delivery and their consequences on the quality of immune responses and provides new insights for vaccine development.     

Agonistic Anti-CD40 Enhances the CD8+ T Cell Response during Vesicular Stomatitis Virus Infection

Intracellular pathogens are capable of inducing vigorous CD8⁺ T cell responses. However, we do not entirely understand the factors driving the generation of large pools of highly protective memory CD8⁺ T cells. Here, we studied the generation of endogenous ovalbumin-specific memory CD8⁺ T cells following infection with recombinant vesicular stomatitis virus (VSV) and Listeria monocytogenes (LM). VSV infection resulted in the generation of a large ovalbumin-specific memory CD8⁺ T cell population, which provided minimal protective immunity that waned with time. In contrast, the CD8⁺ T cell population of LM-ova provided protective immunity and remained stable with time. Agonistic CD40 stimulation during CD8⁺ T cell priming in response to VSV infection enabled the resultant memory CD8⁺ T cell population to provide strong protective immunity against secondary infection. Enhanced protective immunity by agonistic anti-CD40 was dependent on CD70. Agonistic anti-CD40 not only enhanced the size of the resultant memory CD8⁺ T cell population, but enhanced their polyfunctionality and sensitivity to antigen. Our data suggest that immunomodulation of CD40 signaling may be a key adjuvant to enhance CD8⁺ T cell response during development of VSV vaccine strategies.     

Sm29, but Not Sm22.6 Retains its Ability to Induce a Protective Immune Response in Mice Previously Exposed to a Schistosoma mansoni Infection

Background

A vaccine against schistosomiasis would have a great impact in disease elimination. Sm29 and Sm22.6 are two parasite tegument proteins which represent promising antigens to compose a vaccine. These antigens have been associated with resistance to infection and reinfection in individuals living in endemic area for the disease and induced partial protection when evaluated in immunization trials using naïve mice.

Methodology/principals findings

In this study we evaluated rSm29 and rSm22.6 ability to induce protection in Balb/c mice that had been previously infected with S. mansoni and further treated with Praziquantel. Our results demonstrate that three doses of the vaccine containing rSm29 were necessary to elicit significant protection (26%–48%). Immunization of mice with rSm29 induced a significant production of IL-2, IFN-γ, IL-17, IL-4; significant production of specific antibodies; increased percentage of CD4+ central memory cells in comparison with infected and treated saline group and increased percentage of CD4+ effector memory cells in comparison with naïve Balb/c mice immunized with rSm29. On the other hand, although immunization with Sm22.6 induced a robust immune response, it failed to induce protection.

Conclusion/significance

Our results demonstrate that rSm29 retains its ability to induce protection in previously infected animals, reinforcing its potential as a vaccine candidate.     

4-1BBL Enhances CD8+ T Cell Responses Induced by Vectored Vaccines in Mice but Fails to Improve Immunogenicity in Rhesus Macaques

T cells play a central role in the immune response to many of the world’s major infectious diseases. In this study we investigated the tumour necrosis factor receptor superfamily costimulatory molecule, 4-1BBL (CD137L, TNFSF9), for its ability to increase T cell immunogenicity induced by a variety of recombinant vectored vaccines. To efficiently test this hypothesis, we assessed a number of promoters and developed a stable bi-cistronic vector expressing both the antigen and adjuvant. Co-expression of 4-1BBL, together with our model antigen TIP, was shown to increase the frequency of murine antigen-specific IFN-γ secreting CD8⁺ T cells in three vector platforms examined. Enhancement of the response was not limited by co-expression with the antigen, as an increase in CD8⁺ immunogenicity was also observed by co-administration of two vectors each expressing only the antigen or adjuvant. However, when this regimen was tested in non-human primates using a clinical malaria vaccine candidate, no adjuvant effect of 4-1BBL was observed limiting its potential use as a single adjuvant for translation into a clinical vaccine.     

Quantifying the Early Immune Response and Adaptive Immune Response Kinetics in Mice Infected with Influenza A Virus

Seasonal and pandemic influenza A virus (IAV) continues to be a public health threat. However, we lack a detailed and quantitative understanding of the immune response kinetics to IAV infection and which biological parameters most strongly influence infection outcomes. To address these issues, we use modeling approaches combined with experimental data to quantitatively investigate the innate and adaptive immune responses to primary IAV infection. Mathematical models were developed to describe the dynamic interactions between target (epithelial) cells, influenza virus, cytotoxic T lymphocytes (CTLs), and virus-specific IgG and IgM. IAV and immune kinetic parameters were estimated by fitting models to a large data set obtained from primary H3N2 IAV infection of 340 mice. Prior to a detectable virus-specific immune response (before day 5), the estimated half-life of infected epithelial cells is ∼1.2 days, and the half-life of free infectious IAV is ∼4 h. During the adaptive immune response (after day 5), the average half-life of infected epithelial cells is ∼0.5 days, and the average half-life of free infectious virus is ∼1.8 min. During the adaptive phase, model fitting confirms that CD8⁺ CTLs are crucial for limiting infected cells, while virus-specific IgM regulates free IAV levels. This may imply that CD4 T cells and class-switched IgG antibodies are more relevant for generating IAV-specific memory and preventing future infection via a more rapid secondary immune response. Also, simulation studies were performed to understand the relative contributions of biological parameters to IAV clearance. This study provides a basis to better understand and predict influenza virus immunity.Current strategies for preventing or decreasing the severity of influenza infection focus on increasing virus-neutralizing antibody titers through vaccination, as experience indicates that this is the best way to prevent morbidity and mortality. Influenza A virus (IAV) undergoes mutations of the genes encoding the hemagglutinin (HA) and neuraminidase (NA) proteins that the neutralizing antibodies are directed against. When the variation is low (antigenic drift), prior vaccination often confers substantial heterologous immunity against a new seasonal IAV strain. In contrast, major genetic changes (antigenic shift) can result in pandemic IAV strains, since for novel strains, the humoral immune response is a primary response, and heterologous immunity is lacking. The emergence of such pandemic strains and the fact that young children are more vulnerable to influenza diseases highlight the need to better understand which viral and immune parameters determine the outcome of infection with viruses novel to the individual.Conventional experimental methods to measure influenza virus immunity have been limited to animal models and studies of adult human peripheral blood leukocytes. The advantages of using animal models include the ability to intensively sample multiple tissues and to utilize genetic and other interventions, such as blocking or depleting antibodies, to dissect the contribution of individual arms of the immune system. However, it is easy to question the relevance of these experiments to humans because of the many important biological differences between human and murine immune systems (29). In both the animal and human systems, we are limited to measuring those parameters and variables for which assays are available, most of them being ex vivo. Parameters such as cell-to-cell spread of the virus in vivo, trafficking of immune cells to the lung, and the in vivo interactions in an intact immune system are much more difficult or impossible to measure with contemporary techniques, particularly in humans. Computational approaches have the potential to offset some of these limitations and provide additional insight into the kinetics of the IAV infection and the associated immune response.Animal models of influenza virus infection in which different arms of the immune system have been suppressed suggest that some components of the adaptive immune system are required for complete viral clearance, often termed a sterilizing immune response. For example, abrogation of the CD4 T-cell response by cytotoxic antibody therapy or through knockout of major histocompatibility complex (MHC) class II slightly delays viral clearance but has little overall effect on the ability to control the infection (21, 54, 55). Elimination of the CD8 T-cell response typically results in delayed viral clearance (12, 20, 47), although animals with intact CD4 T-cell and B-cell compartments are able to control the infection in the absence of CD8 T cells. Presumably, this occurs through antibody-mediated mechanisms (54). Most animals depleted of both CD8 T cells and B cells are not able to clear the virus, which results in death (14, 32, 53). CD4⁺ T cells certainly contribute to the control of IAV infection, although cytotoxic CD4 T cells are not frequently observed unless cultured in vitro (8, 22, 45). Thus, it is generally accepted that CD8 T cells and/or antibodies are sufficient for timely and complete IAV clearance. Studies that strictly separate the relative roles of CD8 T cells and virus-specific antibodies are less satisfying. Animals depleted of both CD4 and CD8 T cells generally do not control the infection, despite substantial production of anti-IAV IgM antibodies (4, 23, 33, 34). However, adoptive transfer of IAV-specific IgM or IgG antibodies is protective (40, 51), suggesting that the timing and magnitude of the antibody response, i.e., the affinity, avidity, and antibody isotype, are important protective factors.While murine gene knockout or lymphocyte depletion studies are highly informative, they also have a number of limitations. Most importantly, the near-complete ablation of one component of the adaptive immune system often causes profound and unpredictable effects on many other immune components. Although the reported experimental measurements are highly quantitative, they often focus only on a limited number of time points or measurements and do not capture the complexity of the altered, or intact, immune response. In contrast, high-frequency experimental sampling, coupled with mathematical modeling techniques and new statistical approaches, can give insights into the complex biology of IAV infection and test the assumptions inherent in the model. We have learned from other systems, particularly HIV (19, 35, 37, 38, 56), that quantitative analysis of the biology can reveal important factors that are not intuitively obvious. For example, our current estimates for the rates of HIV production and the life span of productively infected cells in vivo were obtained via mathematical modeling (35).Mathematical models have long been used to investigate viral dynamics and immune responses to viral infections, including influenza A virus (3, 5, 7, 15, 16, 31, 36, 48). We recently described a complex differential equation model to simulate and predict the adaptive immune response to IAV infection (24). This model involves 15 equations and 48 parameters, and because of its complexity, many of the parameter values that could not be directly measured were unidentifiable. Thus, it is difficult to estimate all model parameters by fitting experimental data directly to this complex model, although the model can be used to perform simulation predictions (25). This issue can, however, be addressed by reducing the model into smaller submodels with smaller but identifiable sets of parameters, which can be estimated from experimental data. In this paper, we describe such an approach which focuses on IAV infection and the immune response solely within the lung.In the present report, we have fitted a model of primary murine influenza virus infection to data. In naïve subjects, our data suggested that there is no adaptive immune response (e.g., IAV-specific CD8⁺ T cells or antibodies) detectable in the spleen, lymph nodes, or lung until approximately 5 days after infection; therefore, we have divided the analysis into the following two phases: the initial preadaptive (innate) phase and the later adaptive phase. We use direct experimental data from infection of mice with the H3N2 influenza virus A/X31 strain (2, 24) to obtain key kinetic parameters. The model fitting has revealed that the duration of the infection depends on a small set of immune components, and even large fluctuations in other arms of the immune system or IAV behavior have surprisingly little impact on the outcome of the infection.     

Vaccination Method Affects Immune Response and Bacterial Growth but Not Protection in the Salmonella Typhimurium Animal Model of Typhoid

Understanding immune responses elicited by vaccines, together with immune responses required for protection, is fundamental to designing effective vaccines and immunisation programs. This study examines the effects of the route of administration of a live attenuated vaccine on its interactions with, and stimulation of, the murine immune system as well as its ability to increase survival and provide protection from colonisation by a virulent challenge strain. We assess the effect of administration method using the murine model for typhoid, where animals are infected with S. Typhimurium. Mice were vaccinated either intravenously or orally with the same live attenuated S. Typhimurium strain and data were collected on vaccine strain growth, shedding and stimulation of antibodies and cytokines. Following vaccination, mice were challenged with a virulent strain of S. Typhimurium and the protection conferred by the different vaccination routes was measured in terms of challenge suppression and animal survival. The main difference in immune stimulation found in this study was the development of a secretory IgA response in orally-vaccinated mice, which was absent in IV vaccinated mice. While both strains showed similar protection in terms of challenge suppression in systemic organs (spleen and liver) as well as survival, they differed in terms of challenge suppression of virulent pathogens in gut-associated organs. This difference in gut colonisation presents important questions around the ability of vaccines to prevent shedding and transmission. These findings demonstrate that while protection conferred by two vaccines can appear to be the same, the mechanisms controlling the protection can differ and have important implications for infection dynamics within a population.     

Substance P Enhances Desensitization of the Nicotinic Response in Bovine Chromaffin Cells but Enhances Secretion upon Removal

A fundamental process in neurosecretion is desensitization, or a declining response to a stimulus. The response of chromaffin cells to continuous nicotinic stimulation, secretion of catecholamines, desensitizes within a few minutes. The neuropeptide substance P (SP) has been reported to prevent desensitization in culture dish experiments and to enhance desensitization in patch clamp studies. In the present study, these contradictory responses have been demonstrated and the apparent contradictions resolved. We have measured catecholamine secretion by on-line electrochemical detection in a constant-pressure flow system. Isolated chromaffin cells cultured on quartz plates were stimulated with the nicotinic agonist 1,1-dimethyl-4-phenylpiperazinium (DMPP) in the presence and absence of SP. SP inhibited secretion and increase the rate of desensitization compared with stimulation by DMPP alone. However, when the cells were stimulated a second time with DMPP alone immediately after 5-min stimulation with SP + DMPP, the rate of desensitization was markedly lower than the control. Removal of SP after a desensitizing stimulation with SP + DMPP caused a slow secondary release of catecholamine in response to the continued stimulation with DMPP. The kinetic analysis of the secretory response shows that the primary response to SP is enhanced desensitization, but that upon removal of SP the response to DMPP desensitizes less rapidly. We suggest that SP protects some receptors from nicotinic desensitization while holding them in an inactive state, and that upon removal of SP these receptors can slowly respond to DMPP.     

Commitment to the Regulatory T Cell Lineage Requires CARMA1 in the Thymus but Not in the Periphery

Regulatory T (T_reg) cells expressing forkhead box P3 (Foxp3) arise during thymic selection among thymocytes with modestly self-reactive T cell receptors. In vitro studies suggest Foxp3 can also be induced among peripheral CD4⁺ T cells in a cytokine dependent manner. T_reg cells of thymic or peripheral origin may serve different functions in vivo, but both populations are phenotypically indistinguishable in wild-type mice. Here we show that mice with a Carma1 point mutation lack thymic CD4⁺Foxp3⁺ T_reg cells and demonstrate a cell-intrinsic requirement for CARMA1 in thymic Foxp3 induction. However, peripheral Carma1-deficient T_reg cells could be generated and expanded in vitro in response to the cytokines transforming growth factor beta (TGFβ) and interleukin-2 (IL-2). In vivo, a small peripheral T_reg pool existed that was enriched at mucosal sites and could expand systemically after infection with mouse cytomegalovirus (MCMV). Our data provide genetic evidence for two distinct mechanisms controlling regulatory T cell lineage commitment. Furthermore, we show that peripheral T_reg cells are a dynamic population that may expand to limit immunopathology or promote chronic infection.