The Yin and Yang of inflammation.

Inflammation is an essential protective part of the body's response to infection, yet many diseases are the product of inflammation. For example, inflammation can lead to autoimmune disease and tissue damage, and is a key element in chronic health conditions such as heart disease, diabetes, rheumatoid arthritis, and also drives changes associated with aging. Animal models of infectious and chronic disease are important tools with which to dissect the pathways whereby inflammatory responses are initiated and controlled. Animal models therefore provide a prism through which the role of inflammation in health and disease can be viewed, and are important means by which to dissect mechanisms and identify potential therapies to be tested in the clinic. A meeting, "The Yin and Yang of Inflammation" was organized by Trudeau Institute and was held between April 4-6, 2014. The main goal was to bring together experts from biotechnology and academic organizations to examine and describe critical pathways in inflammation and place these pathways within the context of human disease. A group of ~80 scientists met for three days of intense formal and informal exchanges. A key focus was to stimulate interactions between basic research and industry.

Vaccination against murine gamma-herpesvirus infection.

The gamma-herpesviruses establish life-long latency in the host and are important human pathogens. T cells play a major role in controlling the initial acute infection and subsequently maintaining the virus in a quiescent state. However, the nature of the T-cell response to gamma-herpesvirus infection and the requirements for effective vaccination are poorly understood. The recent development of a murine gamma-herpesvirus (murine herpesvirus-68 [MHV-68]) has made it possible to analyze T-cell responses and test vaccination strategies in a small animal model. Intranasal infection with MHV-68 induces an acute infection in the lung and the subsequent establishment of long-term latency, which is associated with splenomegaly and an infectious mononucleosis-like syndrome. Here we review the T-cell response to different phases of the infection and the impact of vaccination against either lytic-cycle, or latency-associated T-cell epitopes.

Identification of quantitative trait loci controlling activation of TRBV4 CD8+ T cells during murine gamma-herpesvirus-induced infectious mononucleosis.

The murine gamma-herpesvirus, MHV-68, shares important biological and genetic features with the human gamma-herpesvirus, Epstein-Barr virus. Following intranasal infection, mice develop an infectious mononucleosis-like syndrome accompanied by increased numbers of activated CD8+ T cells in the blood. A consistent feature of the CD8+ T-cell activation is a marked increase in the frequency of cells expressing a TRBV4+ T-cell receptor. Previous studies suggested that the magnitude of TRBV4 expansion varied significantly among mouse strains, and was influenced by both MHC and non-MHC genes. Detailed analysis of strains with high (C57BL/6) or low (DBA/2) TRBV4 CD8+ T-cell expansion showed that differences in the degree of expansion were not a consequence of variation in genetic susceptibility to the viral infection. Rather, the magnitude of the TRBV4 CD8+ T-cell expansion correlated with differences in expression of the unidentified stimulatory ligand on activated, latently infected B cells. In the present study, analysis of TRBV4 expansion in C57BL/6, DBA/2, B6D2 F1 mice, BXD recombinant inbred strains, and the progeny of C57BL/6xDBA/2 F1 hybrids backcrossed to C57BL/6 demonstrated strong cumulative dominance of the low DBA/2 trait and moderately high heritability (h2 approximately 0.5). Two quantitative trait loci (QTLs) strongly associated with variance in TRBV4 expansion were identified using simple and composite mapping procedures. The first QTL is located on Chromosome (Chr) 17, near or proximal to H2. The second QTL is located on Chr 6 in a region spanning the Tcrb and Cd8a loci.

Latent antigen vaccination in a model gammaherpesvirus infection.

Vaccines that can reduce the load of latent gammaherpesvirus infections are eagerly sought. One attractive strategy is vaccination against latency-associated proteins, which may increase the efficiency with which T cells recognize and eliminate latently infected cells. However, due to the lack of tractable animal model systems, the effect of latent-antigen vaccination on gammaherpesvirus latency is not known. Here we use the murine gammaherpesvirus model to investigate the impact of vaccination with the latency-associated M2 antigen. As expected, vaccination had no effect on the acute lung infection. However, there was a significant reduction in the load of latently infected cells in the initial stages of the latent infection, when M2 is expressed. These data show for the first time that latent-antigen vaccination can reduce the level of latency in vivo and suggest that vaccination strategies involving other latent antigens may ultimately be successfully used to reduce the long-term latent infection.

Analysis of virus-specific CD4(+) t cells during long-term gammaherpesvirus infection.

Major histocompatibility complex class II-mediated antigen presentation after intranasal infection with murine gammaherpesvirus 68 differs in mediastinal lymph nodes and spleen. Evidence that virus-specific CD4(+) T cells were being stimulated was found as late as 6 to 8 months after infection, and cells specific for the viral gp150(67-83) and ORF11(168-180) peptides were maintained as a fairly stable proportion of the total response.

Persistent transmission of mouse hepatitis virus by transgenic mice.

Variation in susceptibility to viral infection is well documented across mouse strains. Specific combinations of viral strains and murine hosts may favor viral infection and disease, and could potentially allow the unexpected development of chronic, persistent, or latent infections. In some genetically modified strains of mice, immune function and perhaps other physiologic or metabolic systems may be substantially or marginally impaired. In the case study reported here, we document the apparent persistent transmission of mouse hepatitis virus (MHV) over a two-year period by MHV-seropositive transgenic mice. Transmission occurred via direct contact with seropositive mice and exposure to contaminated bedding. However, MHV was not detected at diagnostic laboratories by use of viral isolation or reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of tissues from MHV-seropositive animals. Our observation, together with the constantly expanding varieties of immune-impaired or poorly characterized murine hosts and the burgeoning dissemination of these animals throughout the biomedical research community, indicate that unexpected pathophysiologic presentations of common murine viral diseases may present new challenges to the biomedical research community in the future.

Factors controlling levels of CD8+ T-cell lymphocytosis associated with murine gamma-herpesvirus infection.

Intranasal infection of mice with murine gamma-herpesvirus 68 (MHV-68) elicits a striking CD8+ T-cell lymphocytosis following the establishment of latency, which includes a marked increased frequency of Vbeta4+ CD8+ T cells. The Vbeta4+ CD8+ T cells do not recognize a conventional viral peptide, but are stimulated by an uncharacterized ligand expressed on latently infected, activated B cells. The selective expansion of Vbeta4+ CD8+ T cells after MHV-68 infection is observed in all mouse strains examined, although the fold-increase varies widely, ranging from less than twofold to greater than 10-fold. The factors controlling the variation are currently undefined. In the current study, CD8+ T cell activation and Vbeta4+ CD8+ T-cell frequencies were analyzed in 18 inbred strains of mice. The data show that the magnitude of the Vbeta4+ CD8+ T-cell response correlates with the degree of CD8+ T cell-activation, and that both major histocompatibility complex (MHC) and non-MHC genes contribute to the magnitude of the activation. Furthermore, the magnitude of the response does not reflect major differences in susceptibility to viral infection and/or corresponding differences in the acute response. Rather the degree of Vbeta4+ CD8+ T cell activation may be determined by differences in levels of expression of the stimulatory ligand at the peak of latency.

Antigen expression during murine gamma-herpesvirus infection.

Gammaherpesviruses (gammaHV) establish a life-long latency in the host and are associated with a number of malignant human diseases. It is generally believed that T cells play a major role in controlling the initial acute infection and subsequently maintaining the virus in a quiescent state. However, the nature of the T cell response to gamma-herpesvirus infections is poorly understood. In the current report we took advantage of a mouse model of gammaHV infection (murine herpesvirus-68, MHV-68) to investigate the T cell response to different phases of the infection. Intranasal infection with MHV-68 induces an acute infection in lung epithelial cells and long-term latency in B cells. The kinetics of the CD8+ T cell response to different lytic cycle and latency-associated antigens was highly complex and distinct patterns of response could be identified. These responses were regulated by multiple factors including differences in temporal expression of the relevant antigens, differences in the presentation of antigen in different organs, and differential expression of antigen in different types of antigen presenting cells. For example, some antigens were expressed at distinct phases of the infection and in specific organs or subsets of antigen presenting cells. In addition, recent data suggest that in addition to B cells, both macrophages and dendritic cells harbor latent MHV-68 infection, adding further complexity to their role in controlling the T cell response to this infection.

Control of gammaherpesvirus latency by latent antigen-specific CD8(+) T cells.

The contribution of the latent antigen-specific CD8(+) T cell response to the control of gammaherpesvirus latency is currently obscure. Some latent antigens induce potent T cell responses, but little is known about their induction or the role they play during the establishment of latency. Here we used the murine gammaherpesvirus system to examine the expression of the latency-associated M2 gene during latency and the induction of the CD8(+) T cell response to this protein. M2, in contrast to the M3 latency-associated antigen, was expressed at day 14 after infection but was undetectable during long-term latency. The induction of the M2(91-99)/K(d) CD8(+) T cell response was B cell dependent, transient, and apparently induced by the rapid increase in latently infected cells around day 14 after intranasal infection. These kinetics were consistent with a role in controlling the initial "burst" of latently infected cells. In support of this hypothesis, adoptive transfer of an M2-specific CD8(+) T cell line reduced the initial load of latently infected cells, although not the long-term load. These data represent the first description of a latent antigen-specific immune response in this model, and suggest that vaccination with latent antigens such as M2 may be capable of modulating latent gammaherpesvirus infection.

Effect of Staphylococcus enterotoxin B on the concurrent CD8(+) T cell response to influenza virus infection.

Bacterial superantigens have potent in vivo effects. Respiratory viral infections are often associated with secondary bacterial infections, raising the likelihood of exposure to bacterial superantigens after the initiation of the anti-viral immune response. In this study, the general and V beta-specific effects of exposure to Staphylococcal enterotoxin B (SEB) during influenza virus infection on both the ongoing acute and the subsequent recall CD8(+) T cell responses were analyzed, using the well-characterized murine influenza model system and tetrameric MHC/peptide reagents to directly identify virus-specific T cells. The results show that although superantigen exposure during the primary viral infection caused delayed viral clearance, there was remarkably little effect of SEB on the magnitude or TCR repertoire of the ongoing cytolytic T cell response or on the recall response elicited by secondary viral infection. Thus, despite the well-characterized immunomodulatory effects of SEB, there was surprisingly little interference with concurrent anti-viral immunity.

Murine gamma-herpesvirus infection causes V(beta)4-specific CDR3-restricted clonal expansions within CD8(+) peripheral blood T lymphocytes.

Infection of mice with the gamma-herpesvirus MHV-68 results in lytic infection in the lung cleared by CD8(+) cells and establishment of lifelong latency. An Epstein-Barr virus (EBV)-like infectious mononucleosis (IM) syndrome emerges approximately 3 weeks after infection. In human IM, the majority of T cells in the peripheral blood are monoclonal or oligoclonal and are frequently specific for lytic or latent viral epitopes. However, a unique feature of MHV-68-induced IM is a prominent MHC haplotype-independent expansion of CD8(+) T cells, the majority of which utilize V(beta)4 chains in their alphabetaTCR. The ligand driving the V(beta)4 expansion is unknown, but the V(beta) bias and MHC haplotype independence raised the possibility that these cells were responding to a virally encoded or a virally induced endogenous superantigen (sAg). The aim of this study was to determine whether this rapidly proliferating subset is composed of polyclonally or clonally expanded T cells. Complementarity-determining region (CDR)-3 size analysis of V(beta)4(+)CD8(+) cells in infected mice demonstrated CDR3-restricted expansions in the V(beta)4 family as a whole. More refined analysis demonstrated major distortions in every J(beta) subfamily. V-D-J junctional region sequencing indicated that these CDR3 size-restricted expansions were composed of clonal or oligoclonal populations. The sequences were largely unique in individual mice, although evidence for 'public' or highly conserved T cell expansions was also seen between different mice. Taken together with previous studies showing an apparent MHC independence, the data suggest that a novel ligand, distinct from conventional sAg and peptide-MHC, drives proliferation of V(beta)4(+)CD8(+) T cells.

Latent murine gamma-herpesvirus infection is established in activated B cells, dendritic cells, and macrophages.

Intranasal infection of mice with the murine gamma-herpesvirus MHV-68 results in an acute lytic infection in the lung, followed by the establishment of lifelong latency. Development of an infectious mononucleosis-like syndrome correlates with the establishment of latency and is characterized by splenomegaly and the appearance of activated CD8+ T cells in the peripheral blood. Interestingly, a large population of activated CD8+ T cells in the peripheral blood expresses the V beta 4+ element in their TCR. In this report we show that MHV-68 latency in the spleen after intranasal infection is harbored in three APC types: B cells, macrophages, and dendritic cells. Surprisingly, since latency has not previously been described in dendritic cells, these cells harbored the highest frequency of latent virus. Among B cells, latency was preferentially associated with activated B cells expressing the phenotype of germinal center B cells, thus formally linking the previously reported association of latency gene expression and germinal centers to germinal center B cells. Germinal center formation, however, was not required for the establishment of latency. Significantly, although three cell types were latently infected, the ability to stimulate V beta 4+CD8+ T cell hybridomas was limited to latently infected, activated B cells.

T-cell vaccination alters the course of murine herpesvirus 68 infection and the establishment of viral latency in mice.

Diseases caused by gammaherpesviruses such as Epstein-Barr virus are a major health concern, and there is significant interest in developing vaccines against this class of viral infections. However, the requirements for effective control of gammaherpesvirus infection are only poorly understood. The recent development of the murine herpesvirus MHV-68 model provides an experimental tool to dissect the immune response to gammaherpesvirus infections. In this study, we investigated the impact of priming T cells specific for class I- and class II-restricted epitopes on the acute phase of the infection and the subsequent establishment of latency and infectious mononucleosis. The data show that vaccination with either major histocompatibility complex class I- or class II-restricted T-cell epitopes derived from lytic cycle proteins significantly reduced lung viral titers during the acute infection. Moreover, the peak level of latently infected spleen cells was significantly reduced following vaccination with immunodominant CD8(+) T-cell epitopes. However, this vaccination approach did not prevent the long-term establishment of latency or the development of the infectious mononucleosis-like syndrome in infected mice. Thus, the virus is able to establish latency efficiently despite strong immunological control of the lytic infection.

Requirement for CD4+ T cells in V beta 4+CD8+ T cell activation associated with latent murine gammaherpesvirus infection.

A CD8+ T cell lymphocytosis in the peripheral blood is associated with the establishment of latency following intranasal infection with murine gammaherpesvirus-68. Remarkably, a large percentage of the activated CD8+ T cells of mice expressing different MHC haplotypes express V beta 4+ TCR. Identification of the ligand driving the V beta 4+CD8+ T cell activation remains elusive, but there is a general correlation between V beta 4+CD8+ T cell stimulatory activity and establishment of latency in the spleen. In the current study, the role of CD4+ T cells in the V beta 4+CD8+ T cell expansion has been addressed. The results show that CD4+ T cells are essential for expansion of the V beta 4+CD8+ subset, but not other V beta subsets, in the peripheral blood. CD4+ T cells are required relatively late in the antiviral response, between 7 and 11 days after infection, and mediate their effect independently of IFN-gamma. Assessment of V beta 4+CD8+ T cell stimulatory activity using murine gammaherpesvirus-68-specific T cell hybridomas generated from latently infected mice supports the idea that CD4+ T cells control levels of the stimulatory ligand that drives the V beta 4+CD8+ T cells. As V beta 4+CD8+ T cell expansion also correlates with levels of activated B cells, these data raise the possibility that CD4+ T cell-mediated B cell activation is required for optimal expression of the stimulatory ligand. In addition, in cases of low ligand expression, there may also be a direct role for CD4+ T cell-mediated help for V beta 4+CD8+ T cells.

Lytic cycle T cell epitopes are expressed in two distinct phases during MHV-68 infection.

Murine herpesvirus-68 (MHV-68) is a type 2 gamma herpesvirus that productively infects alveolar epithelial cells during the acute infection and establishes long-term latency in B cells and lung epithelial cells. In C57BL/6 mice, T cells specific for lytic cycle MHV-68 epitope p56/Db dominate the acute phase of the infection, whereas T cells specific for another lytic cycle epitope, p79/Kb, dominate later phases of infection. To further understand this response, we analyzed the kinetics of Ag presentation in vivo using a panel of lacZ-inducible T cell hybridomas specific for several lytic cycle epitopes, including p56/Db and p79/Kb. Two distinct peaks of Ag presentation were observed. The first peak was at day 6 in the mediastinal lymph nodes and correlated with the initial pulmonary lytic infection. The second peak was at day 18 in both the mediastinal lymph nodes and spleen and correlated with the peak of latent infection. Interestingly, the p56 epitope was detected only in the mediastinal lymph nodes at day 6 after infection whereas the p79 epitope was predominantly presented in the spleen at day 18, suggesting that differential epitope presentation drives the switch in T cell responses to this virus. Phenotypic analysis of APCs at day 18 postinfection revealed that dendritic cells, macrophages, and B cells all presented lytic cycle epitopes. Taken together, the data suggest that there is a resurgence of lytic cycle Ags in the spleen, which explains the kinetics and specificity of the T cell response to MHV-68.

Apparent MHC-independent stimulation of CD8+ T cells in vivo during latent murine gammaherpesvirus infection.

Like EBV-infected humans with infectious mononucleosis, mice infected with the rodent gammaherpesvirus MHV-68 develop a profound increase in the number of CD8+ T cells in the circulation. In the mouse model, this lymphocytosis consists of highly activated CD8+ T cells strikingly biased toward V beta 4 TCR expression. Moreover, this expansion of V beta 4+CD8+ T cells does not depend on the MHC haplotype of the infected animal. Using a panel of lacZ-inducible T cell hybridomas, we have detected V beta 4-specific T cell stimulatory activity in the spleens of MHV-68-infected mice. We show that the appearance and quantity of this activity correlate with the establishment and magnitude of latent viral infection. Furthermore, on the basis of Ab blocking studies as well as experiments with MHC class II, beta2-microglobulin (beta2m) and TAP1 knockout mice, the V beta 4-specific T cell stimulatory activity does not appear to depend on conventional presentation by classical MHC class I or class II molecules. Taken together, the data indicate that during latent infection, MHV-68 may express a T cell ligand that differs fundamentally from both conventional peptide Ags and classical viral superantigens.

Murine gammaherpesvirus M2 gene is latency-associated and its protein a target for CD8(+) T lymphocytes.

Murine gammaherpesvirus 68 (MHV-68) infection of mice is a potential model with which to address fundamental aspects of the pathobiology and host control of gammaherpesvirus latency. Control of MHV-68 infection, like that of Epstein-Barr virus, is strongly dependent on the cellular immune system. However, the molecular biology of MHV-68 latency is largely undefined. A screen of the MHV-68 genome for potential latency-associated mRNAs revealed that the region encompassing and flanking the genomic terminal repeats is transcriptionally active in the latently infected murine B-cell tumor line S11. Transcription of one MHV-68 gene, that encoding the hypothetical M2 protein, was detected in virtually all latently infected S11 cells and in splenocytes of latently infected mice, but not in lytically infected fibroblasts. Furthermore, an epitope was identified in the predicted M2 protein that is recognized by CD8(+) T cells from infected mice and a cytotoxic T lymphocyte line that recognizes this epitope killed S11 cells, indicating that the M2 protein is expressed during latent infection and is a target for the host cytotoxic T lymphocyte response. This work therefore provides essential information for modeling MHV-68 latency and strategies of immunotherapy against gammaherpesvirus-related diseases in a highly tractable animal model.

Bacterial superantigens reactivate antigen-specific CD8+ memory T cells.

Superantigens stimulate naive CD4+ and CD8+ T cells in a TCR V beta-specific manner. However, it has been reported that memory T cells are unresponsive to superantigen stimulation. In this study, we show that staphylococcal enterotoxins (SE) can activate influenza virus-specific CD8+ memory cytotoxic T cells. In vivo SEB challenge of mice that had recovered from influenza virus infection (memory mice) resulted in the generation of vigorous influenza-specific cytotoxic T lymphocyte (CTL) activity and in vitro SEA or SEB stimulation of splenic T cells from memory mice, but not naive mice, also induced influenza-specific CTL. Analysis of the mechanism of activation suggested that although there may be a component of cytokine-mediated bystander activation, the CTL activity is largely generated in response to direct TCR engagement by superantigen. Moreover, influenza-specific CTL could be generated from purified CD8+ CD62L loCD44hi (memory phenotype) T cells cultured in the presence of T cell-depleted splenic antigen-presenting cells and SE. Purified CD8+ memory T cells also secreted lymphokines and synthesized DNA in response to superantigen. These results definitively demonstrate that CD8+ memory T cells respond to SE stimulation by proliferating and developing appropriate effector function. Furthermore, the data raise the possibility that otherwise inconsequential exposure to bacterial superantigens may perturb the CD8+ T cell memory pool.

Tuning into immunological dissonance: an experimental model for infectious mononucleosis.

Virus infections cause a much more profound perturbation of the lymphoid tissue than can be accounted for by the exigencies of the antigen-specific response. The extent of this 'immunological dissonance' is seen most dramatically in mice infected with a persistent gamma-herpesvirus, MHV-68. A profile of massive, continuing proliferation of both T and B cells in the lymph nodes and spleen leads to a dramatic increase in the prevalence of a CD62Llow CD8+ T cell subset in the blood, a pattern first detected two to three weeks after intranasal exposure to the inducing virus. This syndrome, which seems identical to human infectious mononucleosis (IM), persists for a further month or more. Part of the IM-like phase of MHV-68 infection reflects the selective expansion of Vbeta4+ CD8+ T cells, with the Vbeta4 effect being apparent for several different MHC class I H-2 types but not in mice that are deficient in MHC class II glycoprotein expression. Depleting CD4(+) T helper cells in MHV-68-infected mice leads to the decreased proliferation of the CD8+ T cells in the spleen and fewer CD62Llow CD8+ T lymphocytes than would be expected in peripheral blood, but fails to diminish the prominence of the V4beta+ CD8+ population. The results so far of this unique experimental mouse model of IM suggest that both cytokine-mediated effects and a viral superantigen are operating to promote the dramatic expansion and persistence of activated CD8+ T cells in the vascular compartment.