Hepatitis C virus infection causes cell cycle arrest at the level of initiation of mitosis.

Chronic infection with the hepatitis C virus (HCV) is associated with increased risk for hepatocellular carcinoma (HCC). Chronic immune-mediated inflammation is likely to be an important factor in the development of HCV-associated HCC, but direct effects of HCV infection on the host cell cycle may also play a role. Although overexpression studies have revealed multiple interactions between HCV-encoded proteins and host cell cycle regulators and tumor suppressor proteins, the relevance of these observations to HCV-associated liver disease is not clear. We determined the net effect of these interactions on regulation of the cell cycle in the context of virus infection. Flow cytometry of HCV-infected carboxyfluorescein succinimidyl ester-labeled hepatoma cells indicated a slowdown in proliferation that correlated with abundance of viral antigen. A decrease in the proportions of infected cells in G(1) and S phases with an accumulation of cells in G(2)/M phase was observed, compared to mock-infected controls. Dramatic decreases in markers of mitosis, such as phospho-histone H3, in infected cells suggested a block to mitotic entry. In common with findings described in the published literature, we observed caspase 3 activation, suggesting that cell cycle arrest is associated with apoptosis. Differences were observed in patterns of cell cycle disturbance and levels of apoptosis with different strains of HCV. However, the data suggest that cell cycle arrest at the interface of G(2) and mitosis is a common feature of HCV infection.

Identification of T-cell epitopes in Francisella tularensis using an ordered protein array of serological targets.

Francisella tularensis is a Gram-negative intracellular bacterium that is the causative agent of tularaemia. Concerns regarding its use as a bioterrorism agent have led to a renewed interest in the biology of infection, host response and pathogenesis. A robust T-cell response is critical to confer protection against F. tularensis. However, characterization of the cellular immune response has been hindered by the paucity of tools to examine the anti-Francisella immune response at the molecular level. We set out to combine recent advances of genomics with solid-phase antigen delivery coupled with a T-cell functional assay to identify T-cell epitopes. A subset of clones, encoding serological targets, was selected from an F. tularensis SchuS4 ordered genomic library and subcloned into a bacterial expression vector to test the feasibility of this approach. Proteins were expressed and purified individually employing the BioRobot 3000 in a semi-automated purification method. The purified proteins were coupled to beads, delivered to antigen-presenting cells for processing, and screened with Francisella-specific T-cell hybridomas of unknown specificity. We identified cellular reactivity against the pathogenicity protein IglB, and the chaperone proteins GroEL and DnaK. Further analyses using genetic deletions and synthetic peptides were performed to identify the minimal peptide epitopes. Priming with the peptide epitopes before infection with F. tularensis LVS increased the frequency of antigen-specific CD4 T cells as assessed by intracellular interferon-γ staining. These results illustrate the feasibility of screening an arrayed protein library that should be applicable to a variety of pathogens.

Respiratory Francisella tularensis live vaccine strain infection induces Th17 cells and prostaglandin E2, which inhibits generation of gamma interferon-positive T cells.

Two key routes of Francisella tularensis infection are through the skin and airway. We wished to understand how the route of inoculation influenced the primary acute adaptive immune response. We show that an intranasal inoculation of the F. tularensis live vaccine strain (LVS) with a 1,000-fold-smaller dose than an intradermal dose results in similar growth kinetics and peak bacterial burdens. In spite of similar bacterial burdens, we demonstrate a difference in the quality, magnitude, and kinetics of the primary acute T-cell response depending on the route of inoculation. Further, we show that prostaglandin E(2) secretion in the lung is responsible for the difference in the gamma interferon (IFN-gamma) response. Intradermal inoculation led to a large number of IFN-gamma(+) T cells 7 days after infection in both the spleen and the lung. In contrast, intranasal inoculation induced a lower number of IFN-gamma(+) T cells in the spleen and lung but an increased number of Th17 cells in the lung. Intranasal infection also led to a significant increase of prostaglandin E(2) (PGE(2)) in the bronchoalveolar lavage fluid. Inhibition of PGE(2) production with indomethacin treatment resulted in increased numbers of IFN-gamma(+) T cells and decreased bacteremia in the lungs of intranasally inoculated mice. This research illuminates critical differences in acute adaptive immune responses between inhalational and dermal infection with F. tularensis LVS mediated by the innate immune system and PGE(2).

Dominance of the CD4(+) T helper cell response during acute resolving hepatitis A virus infection.

Hepatitis A virus (HAV) infection typically resolves within 4-7 wk but symptomatic relapse occurs in up to 20% of cases. Immune mechanisms that terminate acute HAV infection, and prevent a relapse of virus replication and liver disease, are unknown. Here, patterns of T cell immunity, virus replication, and hepatocellular injury were studied in two HAV-infected chimpanzees. HAV-specific CD8(+) T cells were either not detected in the blood or failed to display effector function until after viremia and hepatitis began to subside. The function of CD8(+) T cells improved slowly as the cells acquired a memory phenotype but was largely restricted to production of IFN-γ. In contrast, CD4(+) T cells produced multiple cytokines when viremia first declined. Moreover, only CD4(+) T cells responded during a transient resurgence of fecal HAV shedding. This helper response then contracted slowly over several months as HAV genomes were eliminated from liver. The findings indicate a dominant role for CD4(+) T cells in the termination of HAV infection and, possibly, surveillance of an intrahepatic reservoir of HAV genomes that decays slowly. Rapid contraction or failure to sustain such a CD4(+) T cell response after resolution of symptoms could increase the risk of relapsing hepatitis A.

A broadly applicable approach to T cell epitope identification: application to improving tumor associated epitopes and identifying epitopes in complex pathogens.

Epitopes are a hallmark of the antigen specific immune response. The identification and characterization of epitopes is essential for modern immunologic studies, from investigating cellular responses against tumors to understanding host/pathogen interactions especially in the case of bacteria with intracellular residence. Here, we have utilized a novel approach to identify T cell epitopes exploiting the exquisite ability of particulate antigens, in the form of beads, to deliver exogenous antigen to both MHC class I and class II pathways for presentation to T cell hybridomas. In the current study, we coupled this functional assay with two distinct protein expression libraries to develop a methodology for the characterization of T cell epitopes. One set of expression libraries containing single amino acid substitutions in a defined epitope sequence was interrogated to identify epitopes with enhanced T cell stimulation for a MHC class I epitope. The second expression library is comprised of the majority of open reading frames from the intracellular pathogen and potential biowarfare agent, Francisella tularensis. By automating aspects of this technology, we have been able to functionally screen and identify novel T cell epitopes within F. tularensis. We have also expanded upon these studies to generate a novel expression vector that enables immunization of recombinant protein into mice, which has been utilized to facilitate T cell epitope discovery for proteins that are critically linked to Francisella pathogenicity. This methodology should be applicable to a variety of systems and other pathogens.

Identification of a dominant CD4 T cell epitope in the membrane lipoprotein Tul4 from Francisella tularensis LVS.

Francisella tularensis is a gram-negative intracellular bacterium that is the causative agent of tularemia. Small mammals such as rodents and rabbits, as well as some biting arthropods, serve as the main vectors for environmental reservoirs of F. tularensis. The low infectious dose, ability to aerosolize the organism, and the possibility of generating antibiotic resistant strains make F. tularensis a prime organism for use in bioterrorism. As a result, some strains of F. tularensis have been placed on the CDC category A select agent list. T cell immune responses are thought to be a critical component in protective immunity to this organism. However, investigation into the immune responses to F. tularensis has been hampered by the lack of molecularly defined epitopes. Here we report the identification of a major CD4(+) T cell epitope in C57Bl/6 (B6) mice. The murine model of F. tularensis infection is relevant as mice are a natural host for F. tularensis LVS and exhibit many of the same features of tularemia seen in humans. Using T cell hybridomas derived from B6 mice that had either been inoculated with F. tularensis and allowed to clear the infection or which had been immunized by conventional means using purified recombinant protein in adjuvant, we have identified amino acids 86-99 of the lipoprotein Tul4 (RLQWQAPEGSKCHD) as an immunodominant CD4 T cell epitope in B6 mice. This epitope is a major component of both the acute and memory responses to F. tularensis infection and can constitute as much as 20% of the responding CD4 T cells in an acute infection. Reactive T cells can also effectively enter the long-term memory T cell pool. The identification of this epitope will greatly aid in monitoring the course of F. tularensis infection and will also aid in the development of effective vaccine strategies for F. tularensis.

Francisella tularensis-infected macrophages release prostaglandin E2 that blocks T cell proliferation and promotes a Th2-like response.

Francisella tularensis is a highly infectious bacterial pathogen, and is likely to have evolved strategies to evade and subvert the host immune response. In this study, we show that F. tularensis infection of macrophages alters T cell responses in vitro, by blocking T cell proliferation and promoting a Th2-like response. We demonstrate that a soluble mediator is responsible for this effect and identify it as PGE(2). Supernatants from F. tularensis-infected macrophages inhibited IL-2 secretion from both MHC class I and MHC class II-restricted T cell hybridomas, as well as enhanced a Th2-like response by inducing increased production of IL-5. Furthermore, the soluble mediator blocked proliferation of naive MHC class I-restricted T cells when stimulated with cognate tetramer. Indomethacin treatment partially restored T cell proliferation and lowered IL-5 production to wild-type levels. Macrophages produced PGE(2) when infected with F. tularensis, and treatment of infected macrophages with indomethacin, a cyclooxygenase-1/cyclooxygenase-2 inhibitor, blocked PGE(2) production. To further demonstrate that PGE(2) was responsible for skewing of T cell responses, we infected macrophages from membrane PGE synthase 1 knockout mice (mPGES1(-/-)) that cannot produce PGE(2). Supernatants from F. tularensis-infected membrane PGE synthase 1(-/-) macrophages did not inhibit T cell proliferation. Furthermore, treatment of T cells with PGE(2) recreated the effects seen with infected supernatant. From these data, we conclude that F. tularensis can alter host T cell responses by causing macrophages to produce PGE(2). This study defines a previously unknown mechanism used by F. tularensis to modulate adaptive immunity.

Interplay between TCR affinity and necessity of coreceptor ligation: high-affinity peptide-MHC/TCR interaction overcomes lack of CD8 engagement.

CD8 engagement is believed to be a critical event in the activation of naive T cells. In this communication, we address the effects of peptide-MHC (pMHC)/TCR affinity on the necessity of CD8 engagement in T cell activation of primary naive cells. Using two peptides with different measured avidities for the same pMHC-TCR complex, we compared biochemical affinity of pMHC/TCR and the cell surface binding avidity of pMHC/TCR with and without CD8 engagement. We compared early signaling events and later functional activity of naive T cells in the same manner. Although early signaling events are altered, we find that high-affinity pMHC/TCR interactions can overcome the need for CD8 engagement for proliferation and CTL function. An integrated signal over time allows T cell activation with a high-affinity ligand in the absence of CD8 engagement.