The effects of vaccination and immunity on bacterial infection dynamics in vivo.

Salmonella enterica infections are a significant global health issue, and development of vaccines against these bacteria requires an improved understanding of how vaccination affects the growth and spread of the bacteria within the host. We have combined in vivo tracking of molecularly tagged bacterial subpopulations with mathematical modelling to gain a novel insight into how different classes of vaccines and branches of the immune response protect against secondary Salmonella enterica infections of the mouse. We have found that a live Salmonella vaccine significantly reduced bacteraemia during a secondary challenge and restrained inter-organ spread of the bacteria in the systemic organs. Further, fitting mechanistic models to the data indicated that live vaccine immunisation enhanced both the bacterial killing in the very early stages of the infection and bacteriostatic control over the first day post-challenge. T-cell immunity induced by this vaccine is not necessary for the enhanced bacteriostasis but is required for subsequent bactericidal clearance of Salmonella in the blood and tissues. Conversely, a non-living vaccine while able to enhance initial blood clearance and killing of virulent secondary challenge bacteria, was unable to alter the subsequent bacterial growth rate in the systemic organs, did not prevent the resurgence of extensive bacteraemia and failed to control the spread of the bacteria in the body.This work was supported by the Biotechnology and Biological Sciences Research Council [grant number BB/I002189/1].This is the published manuscript. It was originally published by PLOS One here: http://www.plospathogens.org/article/info%3Adoi%2F10.1371%2Fjournal.ppat.1004359

Key Role of the GITR/GITRLigand Pathway in the Development of Murine Autoimmune Diabetes: A Potential Therapeutic Target

BACKGROUND: The cross-talk between pathogenic T lymphocytes and regulatory T cells (Tregs) plays a major role in the progression of autoimmune diseases. Our objective is to identify molecules and/or pathways involved in this interaction and representing potential targets for innovative therapies. Glucocorticoid-induced tumor necrosis factor receptor (GITR) and its ligand are key players in the T effector/Treg interaction. GITR is expressed at low levels on resting T cells and is significantly up-regulated upon activation. Constitutive high expression of GITR is detected only on Tregs. GITR interacts with its ligand mainly expressed on antigen presenting cells and endothelial cells. It has been suggested that GITR triggering activates effector T lymphocytes while inhibiting Tregs thus contributing to the amplification of immune responses. In this study, we examined the role of GITR/GITRLigand interaction in the progression of autoimmune diabetes. METHODS AND FINDINGS: Treatment of 10-day-old non-obese diabetic (NOD) mice, which spontaneously develop diabetes, with an agonistic GITR-specific antibody induced a significant acceleration of disease onset (80% at 12 weeks of age). This activity was not due to a decline in the numbers or functional capacity of CD4(+)CD25(+)Foxp3(+) Tregs but rather to a major activation of 'diabetogenic' T cells. This conclusion was supported by results showing that anti-GITR antibody exacerbates diabetes also in CD28(-/-) NOD mice, which lack Tregs. In addition, treatment of NOD mice, infused with the diabetogenic CD4(+)BDC2.5 T cell clone, with GITR-specific antibody substantially increased their migration, proliferation and activation within the pancreatic islets and draining lymph nodes. As a mirror image, blockade of the GITR/GITRLigand pathway using a neutralizing GITRLigand-specific antibody significantly protected from diabetes even at late stages of disease progression. Experiments using the BDC2.5 T cell transfer model suggested that the GITRLigand antibody acted by limiting the homing and proliferation of pathogenic T cells in pancreatic lymph nodes. CONCLUSION: GITR triggering plays an important costimulatory role on diabetogenic T cells contributing to the development of autoimmune responses. Therefore, blockade of the GITR/GITRLigand pathway appears as a novel promising clinically oriented strategy as GITRLigand-specific antibody applied at an advanced stage of disease progression can prevent overt diabetes

The Dynamic Processing of CD46 Intracellular Domains Provides a Molecular Rheostat for T Cell Activation

Adequate termination of an immune response is as important as the induction of an appropriate response. CD46, a regulator of complement activity, promotes T cell activation and differentiation towards a regulatory Tr1 phenotype. This Tr1 differentiation pathway is defective in patients with MS, asthma and rheumatoid arthritis, underlying its importance in controlling T cell function and the need to understand its regulatory mechanisms. CD46 has two cytoplasmic tails, Cyt1 and Cyt2, derived from alternative splicing, which are co-expressed in all nucleated human cells. The regulation of their expression and precise functions in regulating human T cell activation has not been fully elucidated.Here, we first report the novel role of CD46 in terminating T cell activation. Second, we demonstrate that its functions as an activator and inhibitor of T cell responses are mediated through the temporal processing of its cytoplasmic tails. Cyt1 processing is required to turn T cell activation on, while processing of Cyt2 switches T cell activation off, as demonstrated by proliferation, CD25 expression and cytokine secretion. Both tails require processing by Presenilin/γSecretase (P/γS) to exert these functions. This was confirmed by expressing wild-type Cyt1 and Cyt2 tails and uncleavable mutant tails in primary T cells. The role of CD46 tails was also demonstrated with T cells expressing CD19 ectodomain-CD46 C-Terminal Fragment (CTF) fusions, which allowed specific triggering of each tail individually.We conclude that CD46 acts as a molecular rheostat to control human T cell activation through the regulation of processing of its cytoplasmic tails

The role of neutrophils in the upper and lower respiratory tract during influenza virus infection of mice

BACKGROUND: Neutrophils have been shown to play a role in host defence against highly virulent and mouse-adapted strains of influenza virus, however it is not clear if an effective neutrophil response is an important factor moderating disease severity during infection with other virus strains. In this study, we have examined the role of neutrophils during infection of mice with influenza virus strain HKx31, a virus strain of the H3N2 subtype and of moderate virulence for mice, to determine the role of neutrophils in the early phase of infection and in clearance of influenza virus from the respiratory tract during the later phase of infection. METHODS: The anti-Gr-1 monoclonal antibody (mAb) RB6-8C5 was used to (i) identify neutrophils in the upper (nasal tissues) and lower (lung) respiratory tract of uninfected and influenza virus-infected mice, and (ii) deplete neutrophils prior to and during influenza virus infection of mice. RESULTS: Neutrophils were rapidly recruited to the upper and lower airways following influenza virus infection. We demonstrated that use of mAb RB6-8C5 to deplete C57BL/6 (B6) mice of neutrophils is complicated by the ability of this mAb to bind directly to virus-specific CD8+ T cells. Thus, we investigated the role of neutrophils in both the early and later phases of infection using CD8+ T cell-deficient B6.TAP-/- mice. Infection of B6.TAP-/- mice with a low dose of influenza virus did not induce clinical disease in control animals, however RB6-8C5 treatment led to profound weight loss, severe clinical disease and enhanced virus replication throughout the respiratory tract. CONCLUSION: Neutrophils play a critical role in limiting influenza virus replication during the early and later phases of infection. Furthermore, a virus strain of moderate virulence can induce severe clinical disease in the absence of an effective neutrophil response

Cytomegalovirus microRNAs Facilitate Persistent Virus Infection in Salivary Glands

Micro (mi)RNAs are small non-coding RNAs that regulate the expression of their targets' messenger RNAs through both translational inhibition and regulation of target RNA stability. Recently, a number of viruses, particularly of the herpesvirus family, have been shown to express their own miRNAs to control both viral and cellular transcripts. Although some targets of viral miRNAs are known, their function in a physiologically relevant infection remains to be elucidated. As such, no in vivo phenotype of a viral miRNA knock-out mutant has been described so far. Here, we report on the first functional phenotype of a miRNA knock-out virus in vivo. During subacute infection of a mutant mouse cytomegalovirus lacking two viral miRNAs, virus production is selectively reduced in salivary glands, an organ essential for virus persistence and horizontal transmission. This phenotype depends on several parameters including viral load and mouse genetic background, and is abolished by combined but not single depletion of natural killer (NK) and CD4+ T cells. Together, our results point towards a miRNA-based immunoevasion mechanism important for long-term virus persistence

Modulation of IL-17 and Foxp3 Expression in the Prevention of Autoimmune Arthritis in Mice

©2010 Duarte et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.Background: Rheumatoid Arthritis (RA) is a chronic immune mediated disease associated with deregulation of many cell types. It has been reported that different T cell subsets have opposite effects in disease pathogenesis, in particular Th17 and Treg cells. Methodology and Findings: We investigated whether non-depleting anti-CD4 monoclonal antibodies, which have been reported as pro-tolerogenic, can lead to protection from chronic autoimmune arthritis in SKG mice – a recently described animal model of RA – by influencing the Th17/Treg balance. We found that non-depleting anti-CD4 prevented the onset of chronic autoimmune arthritis in SKG mice. Moreover, treated mice were protected from the induction of arthritis up to 60 days following anti-CD4 treatment, while remaining able to mount CD4-dependent immune responses to unrelated antigens. The antibody treatment also prevented disease progression in arthritic mice, although without leading to remission. Protection from arthritis was associated with an increased ratio of Foxp3, and decreased IL-17 producing T cells in the synovia. In vitro assays under Th17-polarizing conditions showed CD4-blockade prevents Th17 polarization, while favoring Foxp3 induction. Conclusions: Non-depleting anti-CD4 can therefore induce long-term protection from chronic autoimmune arthritis in SKG mice through reciprocal changes in the frequency of Treg and Th17 cells in peripheral tissues, thus shifting the balance towards immune tolerance.This work was funded by SUDOE, grant number IMMUNONET-SOE1/1P1/E014, and supported by a research grant from Fundação para a Ciência e Tecnologia (FCT), Portugal (FCT/POCI/SAU-MMO/55974/2004). JD, AA-D, and VGO are funded with scholarships from FCT (SFRH/BD/23631/2005, SFRH/BD/49093/2008, and SFRH/BPD/22575/2005)

Host-Based Th2 Cell Therapy for Prolongation of Cardiac Allograft Viability

Donor T cell transfusion, which is a long-standing approach to prevent allograft rejection, operates indirectly by alteration of host T cell immunity. We therefore hypothesized that adoptive transfer of immune regulatory host Th2 cells would represent a novel intervention to enhance cardiac allograft survival. Using a well-described rat cardiac transplant model, we first developed a method for ex vivo manufacture of rat host-type Th2 cells in rapamycin, with subsequent injection of such Th2.R cells prior to class I and class II disparate cardiac allografting. Second, we determined whether Th2.R cell transfer polarized host immunity towards a Th2 phenotype. And third, we evaluated whether Th2.R cell therapy prolonged allograft viability when used alone or in combination with a short-course of cyclosporine (CSA) therapy. We found that host-type Th2.R cell therapy prior to cardiac allografting: (1) reduced the frequency of activated T cells in secondary lymphoid organs; (2) shifted post-transplant cytokines towards a Th2 phenotype; and (3) prolonged allograft viability when used in combination with short-course CSA therapy. These results provide further support for the rationale to use “direct” host T cell therapy for prolongation of allograft viability as an alternative to “indirect” therapy mediated by donor T cell infusion

Common Gamma Chain Cytokines Promote Rapid In Vitro Expansion of Allo-Specific Human CD8+ Suppressor T Cells

Human CD8+ regulatory T cells, particularly the CD8+CD28− T suppressor cells, have emerged as an important modulator of alloimmunity. Understanding the conditions under which these cells are induced and/or expanded would greatly facilitate their application in future clinical trials. In the current study, we develop a novel strategy that combines common gamma chain (γc) cytokines IL-2, IL-7 and IL-15 and donor antigen presenting cells (APCs) to stimulate full HLA-mismatched allogeneic human CD8+ T cells which results in significant expansions of donor-specific CD8+CD28− T suppressor cells in vitro. The expanded CD8+CD28− T cells exhibit increased expressions of CTLA-4, FoxP3, and CD25, while down-regulate expressions of CD56, CD57, CD127, and perforin. Furthermore, these cells suppress proliferation of CD4+ T cells in a contact-dependent and cytokine-independent manner. Interestingly, the specificity of suppression is restricted by the donor HLA class I antigens but promiscuous to HLA class II antigens, providing a potential mechanism for linked suppression. Taken together, our results demonstrate a novel role for common γc cytokines in combination with donor APCs in the expansion of donor-specific CD8+CD28− T suppressor cells, and represent a robust strategy for in vitro generation of such cells for adoptive cellular immunotherapy in transplantation

Ubiquitous LEA29Y Expression Blocks T Cell Co-Stimulation but Permits Sexual Reproduction in Genetically Modified Pigs

We have successfully established and characterized a genetically modified pig line with ubiquitous expression of LEA29Y, a human CTLA4-Ig derivate. LEA29Y binds human B7.1/CD80 and B7.2/CD86 with high affinity and is thus a potent inhibitor of T cell co-stimulation via this pathway. We have characterized the expression pattern and the biological function of the transgene as well as its impact on the porcine immune system and have evaluated the potential of these transgenic pigs to propagate via assisted breeding methods. The analysis of LEA29Y expression in serum and multiple organs of CAG-LEA transgenic pigs revealed that these animals produce a biologically active transgenic product at a considerable level. They present with an immune system affected by transgene expression, but can be maintained until sexual maturity and propagated by assisted reproduction techniques. Based on previous experience with pancreatic islets expressing LEA29Y, tissues from CAG-LEA29Y transgenic pigs should be protected against rejection by human T cells. Furthermore, their immune-compromised phenotype makes CAG-LEA29Y transgenic pigs an interesting large animal model for testing human cell therapies and will provide an important tool for further clarifying the LEA29Y mode of action