Infection of IFNγ receptor knockout (IFNγ R-/-) mice with murine gammaherpesvirus-68 (MHV-68) results in fibrosis in the lung, spleen, liver and lymph nodes. In the spleen, pathology involves an increase in the number of latently infected B cells that corresponds with a Th2 biased immune response, in which germinal centres become walled off and fibrosis dominates the splenic architecture. Remarkably, the spleen recovers from this pathology, and the starting point for this process is a loss of latently infected B cells. The aim of this project is to gain further understanding of the control of MHV-68 latent infection in the absence of IFNγ response. This project investigates: (1) the mechanisms that result in the loss of splenocytes, in particular the reduction of latently infected B cells; (2) the dynamics of macrophages in the induction, expression and recovery of fibrosis. Several approaches were employed to examine the hypothesis that the massive cell loss in IFNγR-/- spleen is caused by apoptosis. However, there was no evidence for excessive apoptosis throughout the development of fibrosis. Moreover, RT-PCR analysis showed that there was no significant increase in expression of viral genes associated with lytic infection. Hence it is unlikely that viral reactivation and subsequent lytic infection occurs. These data suggest apoptosis and viral reactivation are not the main mechanisms that cause splenic cell loss. Furthermore, B cell subpopulations and cells that express viral ORF73 in IFNγR-/- mice were examined using a recombinant virus. The ORF73-expressing cells are mainly germinal centre B cells and memory B cells. These two subpopulations undergo a drastic decrease in numbers during fibrosis, whereas naïve B cells, which are less susceptible to infection, maintain a relatively stable population. Therefore, the significant reduction of latently infected B cells appears to be related to the removal of germinal center B cells and memory B cells. Macrophages induced by Th2 cytokines are considered to be pro-fibrotic, and they are reported to have the potential to differentiate into myofibroblasts. In order to determine the role played by macrophages in MHV-68 induced fibrosis, transgenic mice with eGFP constitutively expressed in macrophages and dendritic cells were used. A different pattern of macrophage distribution in IFNγR-/- mice was observed compared to that in wild type mice. Moreover, the number of splenic macrophages changed dramatically in the spleen at different stages of fibrosis. The possibility that alternatively activated macrophages differentiate into myofibroblasts was investigated by co-staining with α-SMA antibody. However, no evidence was found that macrophages are one of the origins of myofibroblasts. This suggests macrophages may play other roles in regulating fibrosis rather than contributing directly to the formation of fibrosis.

Murine gammaherpesvirus 68 (MHV-68) is used as a model for the study of gammaherpesvirus infection and pathogenesis. In the left region of the genome MHV-68 encodes four unique genes, eight viral tRNA-like molecules (vtRNAs) and nine miRNAs. The vtRNAs have a predicted cloverleaf-like secondary structure like cellular tRNAs and are processed into mature tRNAs with the addition of 3’ CCA termini, but are not aminoacylated. Their function is unknown; however they have been found to be expressed at high levels during both lytic and latent infection and are packaged in the virion. The miRNAs are expressed from the vtRNA primary transcripts during latent infection. All herpesviruses examined to date have been found to express miRNAs. These are thought to aid the viruses in avoiding the host immune response and to establish and maintain latency. The aim of this project was to investigate the functions of the vtRNAs and miRNAs of MHV-68. MHV-76 is a natural deletant mutant lacking the unique genes, vtRNAs and miRNAs. This virus was previously used in our lab to construct two insertion viruses encoding vtRNAs1-5 and miRNAs1-6. The only difference between MHV-76 and the insertion viruses is therefore the vtRNAs and miRNAs. The B-cell line NS0 was latently infected with the various viruses and the infected cells characterised. In situ hybridisation and antibody staining showed that all viruses infect the same proportion of cells. The insertion viruses were confirmed to express the vtRNAs during latency by RT-PCR. In addition, using Northern blot analysis the insertion viruses were shown to express miRNA1 during lytic infection of fibroblast cells; however, not during latent infection of NS0 cells. The lack of miRNA1 expression during latency was confirmed using qRT-PCR and miRNAs3-6 were found to be expressed at a lower level than in MHV-68 infected cells. Replication and reactivation kinetics of latently infected NS0 cells showed that introduction of vtRNAs and miRNAs into MHV-76 causes a reduction in reactivation and production of lytic virus. To determine if the reduction in reactivation was caused by the miRNAs, they were introduced into infected cells by transfection. Transfection of miRNAs1-6 into MHV-76 infected cells or miRNA1 into insertion virus infected cells did not lead to an increase or decrease in reactivation. It was confirmed by qRT-PCR that the transfection did result in miRNA levels higher than in insertion virus infected cells. Further, down-regulation of miRNAs using a siRNA against DICER did not lead to a reduction in reactivation. This supports the hypothesis that the vtRNAs rather than the miRNAs are responsible for the reduction of reactivation seen in insertion virus latently infected cells. To determine the effect of the non-coding RNAs on protein expression, NS0 cells latently infected with MHV-76 and insertion virus were analysed using cleavable ICAT and 1-D PAGE cleavable ICAT. In an ICAT analysis the proteins are labelled and the levels of individual proteins in two samples can be compared using mass spectrometry. These techniques were optimised and several proteins with differences in expression between the viruses were identified. It was, however, difficult to determine any specific functions of the non-coding RNAs from the data.

Herpesviruses are ubiquitous in both humans and animals and can cause life-threatening disease. The discovery of murine gammaherpesvirus 68 (MHV-68), which has many similarities in genome and pathogenesis as the human pathogens Epstein-Barr virus and Kaposi’s sarcoma-associated herpesvirus, provides a model for further investigation of the pathogenesis of gammaherpesviruses. The M4 gene was found to be at the left end region of MHV-68 genome. The presence of the M4 protein is required during the early establishment of MHV-68 latency. However, the function of M4 protein remains unclear. The aim of this project was to investigate the function of the M4 protein in vitro and during infection. By using an ELISA, the recombinant M4 protein was shown to bind several Cxc-chemokines and stop the interaction between Cxcl4 and GAGs. The role of M4 protein during MHV-68 lytic infection and in the early establishment of latency was studied by comparing the pathogenesis of virus which does not express M4 (M4stop) and wild type virus (WT). Compared to WT infection, this study found that M4stop was decreased in the lungs at day 8 post infection (p.i.). At the same time point, the viral loads were higher in M4stop infected spleens, which was accompanied by increased expression of the CD4+ T cell activation marker PD-1 and the macrophage activation marker CD69. However, at day 14 p.i., the M4stop infected spleens had lower viral loads, and the expression of CD69 was decreased on CD4+, CD8+ T cells, B cells and macrophages. Furthermore, gene expression PCR arrays were used to investigate how cellular activation and inflammation were transcriptionally regulated. It has been found that the transcription of several genes, which are involved in germinal centre development, was lower in the spleens of WT infected mice at day 12 and 14 p.i. compared to day 10 p.i. of WT infection, as well as day 12 and 14 p.i. of M4stop infection. In addition, the percentage of germinal centre B cells was found to be higher in spleens infected with M4stop at day 10 p.i.. However, there was no difference in percentages of TFH and plasma cells in the spleens. Finally, in order to understand the role of IFN-γ in control of infection in M4stop infected mice, IFN-γR-/- mice were infected with M4stop and WT. Although there were differences in pathogenesis between WT and M4Stop virus infected IFN-γR-/- mice, there was no clear evidence that M4 function is involved in inhibiting IFN-γ pathways. In this study, we found M4 can disturb the interaction of chemokine and GAGs and might delay virus trafficking to the spleen, which could lead to a reduction of cellular activation. M4 may also impair the development of germinal centres at the beginning of latent infection in the spleens.

All mammalian cells encode microRNAs (miRNAs), which are small non-coding RNAs (~ 22 nucleotides) that control numerous physiological processes via regulation of gene expression. A number of viruses, in particular herpesviruses, also encode miRNAs. Gammaherpesviruses such as Epstein-Barr virus (EBV) and Kaposi’s sarcoma associated herpesvirus (KSHV) are associated with lymphoproliferative disorders and some types of cancer in humans. Gammaherpesvirus-encoded miRNAs are predicted to contribute to pathogenesis and virus life cycle by suppressing host and viral target genes. However, the exact functions of these miRNAs during virus infection in the natural host are largely unknown. Strict species specificity has limited research on the human gammaherpesviruses mainly to in vitro studies. Murine gammaherpesvirus 68 (MHV-68) encodes at least 15 miRNAs and provides a unique tractable small animal model to investigate in vivo gammaherpesvirus pathogenic features that are difficult to assess in humans. Following intranasal infection of lab mice, the virus undergoes primary lytic infection in the lung epithelial cells and then spreads to the spleen establishing latent infection in splenic B lymphocytes, macrophages, and dendritic cells. The peak of the latent viral load occurs in the spleen at 14 dpi and then it decreases over time, but the virus is not completely eliminated and the latent viral genomes remain in the host cells for lifetime and can reactivate to produce infectious virus under certain conditions. The aims of my project were to: (1) establish and develop quantitative reverse transcription polymerase chain reaction (qRT-PCR) assays for quantification of the MHV-68 miRNAs, (2) determine the miRNAs expression profiles during the two stages of virus infection (lytic and latent infection), (3) investigate the kinetics of the miRNAs expression during latency in vivo, (4) construct an MHV-68 miRNA mutant virus lacking 9 miRNAs (designated MHV-68.ΔmiRNAs), and (5) carry out thorough phenotypic characterisation of this mutant virus in order to determine the possible functions MHV-68 miRNAs in the context of natural host infection. It was found that the MHV-68 miRNAs expression pattern varied during different stages of infection, suggesting a differential regulation of the expression of these miRNAs depending on the phase of infection. In order to investigate the kinetics of miRNAs expression during latency in vivo, BALB/c mice were infected intranasally with MHV- 68 virus and spleens were harvested at days 10, 14, 21, and 32 post infection. The levels of miRNAs expression were determined by qRT-PCR in the splenocytes from infected mice. Interestingly, in contrast to the lytic MHV-68 protein coding genes, the expression of the miRNAs increased over time after 21 dpi, suggesting that the MHV-68-encoded miRNAs may play more fundamental roles during later stages of latent infection. In order to determine the potential roles of the MHV-68 miRNAs in virus pathogenesis, a miRNA mutant virus lacking the expression of 9 miRNAs, named MHV- 68.ΔmiRNAs, was constructed. The miRNA mutant virus replicated with the same kinetics as wild type virus in vitro and in vivo demonstrating that the deleted MHV-68 miRNAs are dispensable for virus lytic replication. To examine the roles of the miRNAs during virus latency, the MHV-68.ΔmiRNAs virus was characterised throughout a 49- day course of infection. Although the level of ex vivo reactivation of the MHV-68.ΔmiRNAs virus was comparable to that of the WT virus during the establishment of latency and as late as 28 dpi, the reactivation of the MHV-68.ΔmiRNAs virus was approximately 18-times higher than that of the WT virus at 49 dpi despite the similar levels of the genomic viral DNA loads at the same time-point. This suggests that the MHV-68 miRNAs suppress virus reactivation and promote maintenance of long-term latency. Moreover, the lytic viral gene expression levels were higher in splenocytes from the MHV-68.ΔmiRNAs-infected mice than the basal expression levels in the splenocytes from WT MHV-68-infected mice, suggesting that the MHV-68 miRNAs may suppress viral lytic gene expression during long-term latency in vivo and thus help the virus lay low.

ZTLs vermitteln die Eliminierung von infizierten und entarteten Zellen durch Apoptose. Neuste Erkenntnisse unserer Gruppe haben gezeigt, dass eine Subpopulation der CD8+ T-Zellen, anstelle der zytotoxischen Marker das Oberflächenmolekül CD40L exprimiert. Die Expression von CD40L ist bislang als Schlüsselmolekül für die CD4+ T-Zell vermittelte Hilfe bekannt, welche durch Bindung an den CD40 Rezeptor auf anderen Immunzellen induziert wird. Das von den CD4+ T–Zellen ausgehende CD40L Signal ist besonders für die T-Zell abhängige B-Zell Aktivierung und die Bildung von Keimzentren essentiell, in denen B-Zellen heranreifen und hochaffine Antikörper produzieren um den Organismus vor eindringenden Erregern zu schützen. Aufgrund der CD40L-assoziierten Helferfunktion sollte in dieser Arbeit untersucht werden, welche Auswirkungen die Interaktion von CD8+CD40L+ T-Zellen mit B Zellen hat. In in vitro Studien konnte gezeigt werden, dass 50% der antigen-spezifischen CD8+ T-Zellen nach Aktivierung durch B-Zellen CD40L hochregulieren. Sowohl auf RNA- als auch auf Proteinebene induzierten CD8+CD40L+ T-Zellen einen B-Zell Phänotyp, der stark dem von CD4+ T-Zellen stimulierten B-Zellen ähnelte. In Infektionsversuchen mit dem B-Zell-trophen Virus MHV-68 konnte gezeigt werden, dass transgene Mäuse mit CD40L defizienten CD8+ T-Zellen im Vergleich zu Kontrolltieren eine signifikante Reduktion der Keimzentrums-B-Zellen in den Lymphknoten der oberen Halsregion aufweisen. Eine genauere Betrachtung des B-Zell Repertoires von IgG Gedächtniszellen ergab jedoch, dass die Sequenzen der IGHJ3 Genfamilie bevorzugt für die Modifikation der CDR3 Region in Mäusen mit CD40L defizienten CD8+ T-Zellen verwendet wird, die eine entscheidende Rolle bei der Antigenerkennung spielt. Zusammengefasst kann mit dieser Arbeit zum ersten Mal gezeigt werden, dass CD8+CD40L+ T-Zellen Helferfunktionen durch Unterstützung der B-Zell Aktivierung und Bildung von Keimzentren übernehmen können. / CTLs are important for the elimination of infected and degenerated cells by inducing apoptosis of the target cells. Recently our group identified a sub-population of CD8+ T cells expressing CD40L instead of common CTL markers. To that date, transient CD40L expression on T cells has been only described as a function of activated CD4+ T cells, which displays this key molecule for CD4+ T cell mediated help by binding to the CD40 receptor on other immune cells. Particularly, CD40L signaling provided by CD4+ T cells is indispensable for T cell dependent B cell activation and GC responses, which generate B cells secreting high affinity antibodies that protect the host from invading pathogens. Due to its associated helper functions, this thesis aimed to dissect whether CD40L positive CD8+ T cells are restricted to cytotoxic killing or if this sub-population possesses similar properties as CD4+ T cells when interacting with B cells. In vitro co-culture experiments showed that 50% of murine antigen specific CD8+ T cells up-regulated CD40L upon activation by antigen presenting B cells. When compared to CD40L deficient CD8+ T cells, the interaction of CD8+ CD40L+ T cells induced remarkable changes in B cells on the RNA and protein level and triggered a B cell phenotype resembling that of B cells primed by CD4+ T cells. By the infection of mice with the B cell trophic virus MHV-68, it was found that E8IcrexCD40Lflox transgenic mice lacking CD40L only on matured CD8+ T cells, exhibited a significant decrease of GC B cells in superficial cervical lymph nodes at the acute state of infection compared to WT mice. A closer look into the memory B cell repertoire revealed a preferred usage of the murine IGHJ3 gene family that modifies the CDR3 and thus the recognition groove of the B cell antibody in E8IcrexCD40Lflox mice. In summary, this work provides sufficient evidence that CD8+ CD40L+ T cells adopt helper-like functions by supporting B cell activation and subsequent GC formation.

Viruses replicating in the respiratory tract (RT) triggers a wide- range of cytokines and chemokines that have antiviral and pro-inflammatory features, instigating an efficient virus- specific B and T cell response that aids in virus- clearance. The majority of antibody secreting cells (ASCs) localizing in the upper RT secrete IgA that can effectively neutralize viruses. In addition, elements of B cell memory are generated that can provide protection from re-infection. Studies examining these aspects, following murine gammaherpesvirus 68 (MHV-68) infection comprise chapter 2 of the dissertation work. Our studies demonstrate that following MHV-68 infection, unlike influenza infection, resulted in a generalized deficiency of virus-specific IgA induction and deficient B cell memory establishment in the respiratory tract. The studies indicate that these aspects of B cell response are regulated by features of virus- replication in the RT. These studies lead to the speculation that these features of B cell response may represent an evolutionary adaptation of viruses that establish long-term latency and are transmitted periodically after reactivation and shedding in secretions. Following cognate interactions with CD4+ T cells, the B cells undergo proliferation, isotype-switching and differentiate towards extrafollicular (low affinity, rapid) or germinal center pathway (high affinity). It is not clear what factors regulate these pathways of B cell differentiation, especially in the context of virus infection in the RT. Studies examining these aspects following influenza infection comprise chapter 3 of the dissertation work. Our studies establish a model for the investigation of host and viral factors that modulate the quality and effectiveness of the B cell response to influenza infection. The findings indicate that the strength of the extrafollicular B cell response depends on the nature of the infecting virus. We present evidence that this pathway of rapid antiviral antibody production relates to the production of non-specifically acting factors in the lung and also dependent of the cytokine profile of virus-specific CD4+T cells. In summary, the current dissertation findings point out to an influence of virus and host associated factors in regulating features of B cell response in the RT.