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Visualization of type I immunity using bicistronic IFN-gamma reporter mice in vitro and in vivo
(2006)
IFN-γ is the signature cytokine of Th1 and CD8+ effector cells generated in type I immune responses against pathogens, such as Influenza virus, Sendai virus and the intracellular protozoan parasite Toxoplasma gondii. Understanding the regulation of IFN-γ is critical for the manipulation of immune responses, prevention of immunopathology and for vaccine design. In the present thesis, IFN-γ expression by CD4+ and CD8+ T cells was characterized in detail and the requirement of IFN-γ receptor mediated functions for IFN-γ expression was assessed. Bicistronic IFN-γ-eYFP reporter mice, which allow direct identification and isolation of live IFN-γ expressing cells, were used to visualize IFN-γ expression in vitro and in vivo after infection with the afore mentioned pathogens. Expression of the IFN-γ-eYFP reporter by CD4+ and CD8+ T cells was broadly heterogeneous in vitro and in vivo after infection. Increased expression of the reporter correlated positively with the abundance of IFN-γ transcripts and IFN-γ protein production upon stimulation. eYFP reporter brightness reflected the potential for IFN-γ production, but actual secretion was largely dependent on antigenic stimulation. Increased expression of the reporter also correlated with enhanced secretion of additional proinflammatory cytokines and chemokines and cell surface expression of markers that indicate recent activation. Highly eYFP fluorescent cells were generally more differentiated and their anatomical distribution was restricted to certain tissues. The anatomical restriction depended on the pathogen. IFN-γ expressing CD4+ and CD8+ T cells were generated in IFN-γ receptor deficient reporter mice after infection with Sendai virus or Toxoplasma gondii. However, in the absence of IFN-γ receptor mediated functions, the frequency and brightness of the eYFP reporter expression was altered. Dual BM chimeric mice, reconstituted with wild-type and IFN-γ receptor deficient reporter BM, revealed a T cell-intrinsic requirement for the IFN-γ receptor for optimal IFN-γ expression. Reporter fluorescence intensities were regulated independently of IFN-γ receptor mediated functions. Finally, we propose a model for IFN-γ expression by CD4+ and CD8+ T cells. 2. SUMMARY 10 In summary, the expression of IFN-γ is differentially regulated in CD4+ and CD8+ T cells and after viral or protozoan infections. Additionally, the role of IFN-γ receptor mediated functions for the expression of IFN-γ was determined.
Glucocorticoids (GCs) are small lipophilic compounds that mediate a plethora of biological effects by binding to the intracellular glucocorticoid receptor (GR) which, in turn, translocates to the nucleus and directly or indirectly regulates gene transcription. GCs remain the cornerstone in the treatment for a number of hematological malignancies, including leukemia, lymphoma and myeloma. Extensive literature suggests that the efficacy of GCs stems from their ability to mediate apoptosis. Despite the enormous strides made in our understanding of regulated cell death, the exact mechanism by which GCs cause apoptosis is still unknown. The data obtained so far provide strong evidence that gene transactivation by the GR underlies the initiation phase of GC-induced thymocyte apoptosis. Furthermore, the multicatalytic proteasome, several members of the Bcl-2 family, changes in calcium flux as well as caspases have been identified as important players in the execution phase of GC-mediated cell death. However, the exact sequence of events in this process still remains elusive. A major problem of the current discussion arises from the fact that different cell types, such as thymocytes, peripheral T cells and lymphoma cells are compared without acknowledging their different characteristics and gene expression profiles. Although it is generally assumed that GCs induce apoptosis via a conserved mechanism, this is not supported by any data. In other words, it is possible that thymocytes, peripheral T cells and lymphoma cells may undergo cell death along different pathways. We therefore wondered whether a unique signal transduction pathway is engaged by GCs to initiate and execute cell death in all types of T lymphocytes or whether distinct pathways exist. Therefore, we compared the role of the proteasome, various caspases, the lysosomal compartment and other factors in GC-induced apoptosis of murine thymocytes and peripheral T cells as well as T-ALL lymphoma cells. Our findings show that the initiation phase of GC-induced apoptosis is similar irrespective of the differentiation state of the cell. Apoptosis in both thymocytes and peripheral T cells is mediated by the GR and depends on gene transcription. In contrast, the execution phase significantly differs between thymocyte and peripheral T cells in its requirement for a number of signal transduction components. Whilst in thymocytes, the proteasome, caspases 3, 8 and 9 as well as cathepsin B play an important role in GC-induced apoptosis, these factors are dispensable for the induction of cell death in peripheral T cells. In contrast, changes in the expression and intracellular location of Bcl-2 family members do not appear to contribute to GC-induced apoptosis in either cell type. Importantly, our observation that GC treatment of thymocytes leads to an activation of the lysosomal protease cathepsin B and that this is an essential step in the induction of cell death by GCs, is the first indication that a lysosomal amplification loop is involved in this process. Analysis of GC-induced apoptosis in several T-ALL cell lines further indicates that the signaling pathway induced by GCs in thymocytes but not in peripheral T cells is shared by all lymphoma cell-types analyzed. Given the therapeutic importance of high-dose GC-therapy for the treatment of hematological malignancies, this finding could potentially form a basis for new anti-cancer strategies in the future, which specifically target tumor cells whilst leaving peripheral T cells of patients untouched.
The work of the previous chapters describes the role of Nipah virus (NiV) V and W proteins regarding their role in interferon antagonism and regulation of viral replication. Previous publications have shown that NiV encodes IFN antagonist activity in its V, W and C protein (Park et al., 2003b; Rodriguez et al., 2002). In order to study the effect of both NiV proteins in the context of a virus infection, recombinant Newcastle disease viruses (rNDVs) expressing NiV V or NiV W were constructed. As a control virus served rNDV expressing NDV V proteins, which behaved like wildtype NDV. Growth kinetic experiments demonstrated that rNDVs expressing NiV V or W grew to higher titers than rNDV expressing NDV V in human A549 cells. This result suggested that both NiV V and W were able to render the avian virus, which normally does not replicate well in human cells, into a better growing virus. This hypothesis was supported by the fact that all rNDVs grew similarly in avian DF1 or Vero cells. When rNDV-infected A549 cells were specifically stained for NiV V or W protein it was observed that V is localized in the cytoplasm whereas W could be predominantly found in the nucleus. This observation was in agreement with previous studies reporting a nucleus export signal (NES) for NiV V and a nuclear localization signal (NLS) for NiV W (Rodriguez et al., 2004; Shaw et al., 2005). The specific localization of each NiV protein has also been shown to contribute to different functions in terms of IFN antagonism (Shaw et al., 2005). Here, NiV V and W proteins caused a severe attenuation of the immune response in rNDV-infected human A549 and dendritic cells. The transcription of type I interferons and ISGs was significantly downregulated in the presence of NiV V and W proteins. As a consequence of the transcriptional block, there was also an inhibition at the level of translation (as seen for A549 cells) and the secretion of IFNs and cytokines/chemokines (as seen for DCs). In contrast, NDV V protein induced a host immune response. Both NiV V and W also displayed a strong inhibitory effect on the function DCs. DCs represent a very important cell class because they link the innate immune response to the adaptive immune response (Banchereau & Steinman, 1998). By downregulating the production and secretion of important cytokines/chemokines that are important for the activation of B and T lymphocytes, NiV V and W were able to disrupt that link. Interestingly, NiV W seemed to be a stronger inhibitor than NiV V in both A549 cells and DCs. Overall, it was demonstrated that NiV V and W were able to prevent the induction of the innate and adaptive host immune response cascade by inhibiting the transcription of immune genes in DCs and A549 cells. The second part of this work addressed the question whether NiV V and W proteins have a regulatory role in viral replication. This has been previously reported for Nipah virus itself (Sleeman et al., 2008) and other viruses (Atreya et al., 1998; Horikami et al., 1996; Witko et al., 2006). In order to study the ability of the V and W proteins of NiV to regulate viral transcription and/or replication, an existing NiV minireplicon assay was used (Halpin et al., 2004). Here, it was shown that NiV V and W (but not C) proteins significantly downregulated NiV minireplicon activity. The common N terminal region was shown to harbor the inhibitory activity. Co-immunoprecipitation experiments showed that both NiV V and W (but not C) were able to interact with NiV N, one component of the NiV polymerase. This result was supported by immunofluorescence experiments that revealed co-localization of NiV N with V and W. The binding of NiV V or W to NiV N occurred via their N terminus and more specifically amino acids 1-50. This suggested that V and W might inhibit viral replication by interacting with the viral polymerase resulting in a loss of function. Exact mechanisms still have to be elucidated.
Measles is an extremely contagious vaccine-preventable disease responsible
for more than 90000 deaths worldwide annually. The number of deaths has
declined from 8 million in the pre-vaccination era to few thousands every year due
to the highly efficacious vaccine. However, this effective vaccine is still unreachable
in many developing countries due to lack of infrastructure, while in developed
countries too many people refuse vaccination. Specific antiviral compounds are not
yet available. In the current situation, only an extensive vaccination approach
along with effective antivirals could help to have a measles-free future. To develop
an effective antiviral, detailed knowledge of viral-host interaction is required.
This study was undertaken to understand the interaction between MV and
the innate host restriction factor APOBEC3G (A3G), which is well-known for its
activity against human immunodeficiency virus (HIV). Restriction of MV
replication was not attributed to the cytidine deaminase function of A3G, instead,
we identified a novel role of A3G in regulating cellular gene functions. Among two
of the A3G regulated host factors, we found that REDD1 reduced MV replication,
whereas, KDELR2 hampered MV haemagglutinin (H) surface transport thereby
affecting viral release. REDD1, a negative regulator of mTORC1 signalling
impaired MV replication by inhibiting mTORC1. A3G regulated REDD1
expression was demonstrated to inversely correlate with MV replication. siRNA
mediated silencing of A3G in primary human blood lymphocytes (PBL) reduced
REDD1 levels and simultaneously increased MV titres. Also, direct depletion of
REDD1 improved MV replication in PBL, indicating its role in A3G mediated
restriction of MV. Based on these finding, a new role of rapamycin, a
pharmacological inhibitor of mTORC1, was uncovered in successfully diminishing
MV replication in Vero as well as in human PBL. The ER and Golgi resident
receptor KDELR2 indirectly affected MV by competing with MV-H for cellular
chaperones. Due to the sequestering of chaperones by KDELR2, they can no longer
assist in MV-H folding and subsequent surface expression. Taken together, the two
A3G-regulated host factors REDD1 and KDELR2 are mainly responsible for
mediating its antiviral activity against MV.
A small percentage (1-5%) of the blood lymphocytes expresses alternative T-cell antigen receptor that uses g and d TCR rearranging genes. A subset of them expresses the Vg9Vd2 TCR. Those cells respond to self-nonpeptide and foreign antigens presented by unknown antigen-presenting molecules. Vg9Vd2 T cells also express Toll-like receptors and natural killer receptors that allow them to respond to other nonpeptide microbial components or to alterations in the expression of stress cell surface ligands such as NKG2D ligands. Vg9Vd2 T cells frequently are regulated by the expression of activating and/or inhibitory NKRs (iNKRs) that can fine-tune their activation threshold and the activating NKG2D receptor is one of the most studied until now. NKG2D, a C-type lectin receptor directed against MICA/MICB and UL16-binding protein (ULBP) molecules, have been reported a powerful co-stimulus for Ag-mediated activation of CD8 and Vg9Vd2 T cells. Indeed, NKG2D is recruited within the Vg9Vd2 TCR immunological synapse and enhances recognition by Vg9Vd2 T cells of Mycobacteria-infected DCs and various MICA/MICB or ULBP hemopoietic and non-hemopoietic tumors. The level of NKG2D is upregulated by inflammatory cytokines (e.g. IL-15), and NKG2D ligands are induced after a physical or genotoxic stress and/or along infection by intracellular pathogens. Therefore, NKG2D is a key stress sensor that strongly enhances recognition of altered or infected self by human gd T cells. Recent progress in the field supports the idea that gd T cells fulfill a role in the innate and adaptative immune response in different way of the conventional ab T cells. We demonstrated direct activation of Vg9Vd2 T cells by NKG2D ligation through the association with DAP10 adapter molecules and independently of TCR-Ag recognition, similar to the NKG2D-mediated activation of NK cells. Culture of peripherical blood mononuclear cells with immobilized NKG2D mAb or NKG2D ligand MICA induces up-regulation of CD69 and CD25 in NK and Vg9Vd2 T cells but not in CD8 T cells. Additionally, the ligation of NKG2D induces in Vg9Vd2 T cells the up-regulation of molecules typical for antigenpresenting cells, such as co-stimulator molecules (CD86) antigen presenting molecules (CD1a, HLA-DR), adhesion molecules (CD54), and activation molecules (CD69). Furthermore, NKG2D ligation in Vg9Vd2 T cells induces the production of cytokines such as TNF-a and chemokines such as, MIP-1a, but cannot induce the production of cytokines such as IL-6 or IFN-g and chemokines such as RANTES, MCP-1 and GM-CSF. In addition, NKG2D triggers the activation of the cytolytic machinery as efficient as CD3 stimulation as shown by measurement of the release of granules with esterase activity (BLT assay), perforin and the up-regulation of CD107a on the surface of Vg9Vd2 T cells. This NKG2D dependent cytolysis has been confirmed using purified Vg9Vd2 T cells, which kill MICA-transduced RMA cells but not the control cells. The TCR independence and NKG2D dependence of this killing is supported by mAb inhibition experiment. Finally, DAP 10, which mediates NKG2D signaling of human NK cells, is found in resting and activated Vg9Vd2 T cells. Moreover, data of intracellular signaling studies suggest an important role of Scr kinases in the NKG2D mediated killing and involvement of DAP-10-PI3K and PLCg 1 pathways as mayor proteins implicated in target cell lysis, and shows remarkable difference with the TCR signaling. The identification of these similarities in NKG2D function between NK and Vg9Vd2 T cells may be of interest for development of new strategies for Vg9Vd2 T cell-based immunotherapy in certain types of cancer and help to understand Vg9Vd2 T cell function in general.
Measles is an ancient disease with historical records as early as the 9th century.
Extensive study as well as advances in scientific knowledge of virology have led to
identification of the viral pathogen and subsequent development of an effective vaccine
leading to global efforts towards measles elimination. In 2018, around 140,000 deaths were
reported due to measles with incomplete vaccine coverage being one of the leading causes
of resurgence. Measles is highly contagious and often regarded as a childhood illness.
However, measles is associated with a number of complications and persistent infections
like subacute sclerosing panencephalitis (SSPE), which have brought into focus the need
for specific anti-viral therapies.
The aim of this study was to target host and viral factors to optimize anti-measles virus
therapy. Our approach was to test a panel of compounds known to inhibit host cell
functions or viral factors for their antiviral effect on measles replication. Primary human
lymphocytes, persistently infected NT2 cells and post-mitotic neurons were used as in vitro
model systems of acute, persistent and neuronal infection respectively to test the inhibitors.
Using the inhibitors Ceranib-2 and SKI-II to target the sphingolipid metabolism enzymes
acid ceramidase and sphingosine kinase in infected human primary lymphocytes, we
observed a decreased protein translational capacity mediated by mTORC1, EIF4E and
ribosomal protein S6 phosphorylation that probably contributes to the antiviral effect. In
the persistently infected neural NT2 cells and post-mitotic neurons derived from LUHMES
cells, we observed effective infection inhibition and viral clearance upon treatment with a
small non-nucleoside inhibitor (ERDRP-0519) specifically targeting the Morbillivirus
large polymerase. Other inhibitors such as Ribavirin and Favipiravir were less effective. To
conclude, 1) we identified a mTOR associated protein translation axis associated with the
sphingolipid metabolism, which affects measles virus replication and 2) In vitro
persistently infected neuronal and post-mitotic neuron models were successfully used as a
rapid method to test antivirals against measles virus.
Semaphorin receptors in the immunological synapse: regulation and measles virus-driven modulation
(2010)
Measles virus (MV) infection causes approximately 164,000 deaths per year worldwide (WHO, 2008). The main cause of death is MV-induced immunosuppression but the underlying mechanisms are not fully understood. It has been suggested that MV renders T cells dysfunctional by disrupting the integrity of actin dynamics while MV infection of dendritic cells results in their inability to sustain T cell activation. During neuronal development, semaphorins (SEMAs), especially SEMA3A, induce a collapse of growing dendrites via the binding to plexin-A1 (plexA1) and its coreceptor neuropilin-1 (NP-1). The collapse results from a disruption of actin dynamics. In this study, the roles of these three molecules were investigated in human immune cells and their possible role in MV induced immunosuppression. The present data have shown that plexA1 is an important component of human immunological synapse (IS). It translocated transiently to the surface of T cells after CD3/28 ligation and accumulated at the stimulatory interface between T cells and DCs (or CD3/28 coated beads). When plexA1 expression was inhibited (RNAi) or its function was disrupted (exogenous blocking or dominant negative expression), T cell expansion was reduced. Upon MV exposure, translocation of plexA1 and NP-1, another important component of IS, towards the stimulatory interface in T cells was abrogated. Moreover, MV infection interfered with plexA1/NP-1 turnover in maturing DCs and promoted early and substantial release of SEMA3A from these cells, particularly in the presence of allogenic T cells. As revealed by scanning electron microscopy, the release of SEMA3A caused a transient loss of actin-based protrusions on T cells. SEMA3A affected chemotactic migration of T cells and DCs, and reduced formation of allogenic DC/T cell conjugates. In conclusion, MV targeted SEMA receptor function both by disrupting their recruitment to the IS and by promoting a premature release of their repulsive ligand, SEMA3A. Both of which could contribute to MV-induced immunosuppression.
Gene expression in eukaryotic cells is regulated by the combinatorial action of numerous gene-regulatory factors, among which microRNAs (miRNAs) play a fundamental role at the post-transcriptional level. miRNAs are single-stranded, small non-coding RNA molecules that emerge in a cascade-like fashion via the generation of primary and precursor miRNAs. Mature miRNAs become functional when incorporated into the RNA induced silencing complex (RISC). miRNAs guide RISCs to target mRNAs in a sequence-specific fashion. To this end, base-pairs are usually formed between the miRNA seed region, spanning nucleotide positions 2 to 8 (from the 5' end) and the 3'UTR of the target mRNA. Once miRNA-mRNA interaction is established, RISC represses translation and occasionally induces direct or indirect target mRNA degradation. Interestingly, miRNAs are expressed not only in every multicellular organism but are also encoded by several viruses, predominately by herpesviruses. By controlling both, cellular as well as viral mRNA transcripts, virus-encoded miRNAs confer many beneficial effects on viral growth and persistence. Murine cytomegalovirus (MCMV) is a ß-herpesvirus and so far, 29 mature MCMV-encoded miRNAs have been identified during lytic infection. Computational analysis of previously conducted photoactivated ribonucleotide-enhanced individual nucleotide resolution crosslinking immunoprecipitation (PAR-iCLIP) experiments identified a read cluster within the 3' untranslated region (3'UTR) of the immediate early 3 (IE3) transcript in MCMV. Based on miRNA target predictions, two highly abundant MCMV miRNAs, namely miR-m01-2-3p and miR-M23-2-3p were found to potentially bind to two closely positioned target sites within the IE3 PAR-iCLIP peak. To confirm this hypothesis, we performed luciferase assays and showed that activity values of a luciferase fused with the 3'UTR of IE3 were downregulated in the presence of miR-m01- 2 and miR-M23-2. In a second step, we investigated the effect of pre-expression of miR-m01-2 and miR-M23-2 on the induction of virus replication. After optimizing the transfection procedure by comparing different reagents and conditions, plaque formation was monitored. We could demonstrate that the replication cycle of the wild-type but not of our MCMV mutant that harbored point mutations in both miRNA binding sites within the IE3-3'UTR, was significantly delayed in the presence of miR-m01-2 and miR-M23-2. This confirmed that miR-m01-2 and miR-M23-2 functionally target the major transcription factor IE3 which acts as an indispensable regulator of viral gene expression during MCMV lytic infection. Repression of the major immediate early genes by viral miRNAs is a conserved feature of cytomegaloviruses. The functional role of this type of regulation can now be studied in the MCMV mouse model.
Regulation of B lymphocyte terminal differentiation and death by the transcription factor Blimp-1
(2005)
B lymphocyte induced maturation protein-1 (Blimp-1) and X-box-binding protein-1 (XBP-1) are indispensible transcription factors required for B lymphocyte terminal differentiation into Ig secreting plasma cells. Occurrence of an unfolded protein response (UPR) and XBP-1 splicing, due to elevated Ig levels, are critical events during plasma cell generation. However, the upstream molecule sufficient to trigger these events remain elusive. Because ectopic expression of Blimp-1 in B cells is sufficient to generate plasma cells, it is plausible that Blimp-1 might be the upstream molecule, sufficient for the induction of UPR and XBP-1 splicing. The results from the current study indicate that ectopic expression of Blimp-1 or its N-terminal domain, in B cells, is sufficient to induce XBP-1 splicing, UPR and Ig (immunoglobulin) secretion. Further more Blimp-1 is able to directly repress the antiapoptotic gene A1, by binding to specific DNA elements in A1 promoter. This repression of A1 by Blimp-1 seems to be an important prerequisite for Plasma cell differentiation because ectopic expression of A1 in primary B cells resulted in reduced immunoglobulin secretion.
iNKT cells are a population of T cells with unique characteristics. In contrast to most αβ T cells which recognize peptides presented by highly polymorphic MHC molecules, iNKT cells are reactive to glycolipids presented by CD1d, a non-polymorphic MHC-I like molecule. Moreover, whereas MHC-restricted αβ T cells bear highly variable receptors (TCRs) formed after somatic recombination of the V(D)J gene segments, the TCR of iNKT cells is formed by an invariant α chain, which always contains the same gene segments: AV14 and AJ18; and a β chain of limited BV gene usage: BV8S2, BV7 or BV2, in the mouse. This invariant α chain is the reason for which these cells are named “i” and the NK part of their name refers to the expression of receptors typical of natural killer (NK) cells. iNKT cells recognize glycolipids of endogenous and microbial origin. After activation they secrete large amounts of very different cytokines such as IFN-γ and IL-4 and thus influence immune responses and pathological conditions. One of the most potent iNKT cell agonists, recognized by the semi-invariant TCR, is the synthetic glycolipid α-Galactosylceramide (α-Gal). iNKT cells can be visualized using CD1d-multimeric complexes loaded with α-Gal and flow cytometry, since this reagent has enough avidity to stain these cells. Interestingly, mouse iNKT cells can be stained with human α-Gal-loaded CD1d oligomers and human iNKT cells can also be visualized with mouse α-Gal-loaded CD1d oligomers, indicating a high degree of conservation of the recognition of α-Gal presented by CD1d through evolution. Previous studies showed that rats have the genes necessary to build semi-invariant TCRs: They have a CD1d homologue; one or two BV8S2 homologues and interestingly, up to ten AV14 gene segments, which are highly conserved when compared to the mouse genes. Importantly, it has been shown at least for two of these AV14 gene segments that they can produce invariant TCRα chains which, when coexpressed with BV8-containing β chains, react to α-Gal presented by rat CD1d. Furthermore, ex vivo stimulation of primary splenocytes with α-Gal results in the secretion of IL-4 and IFN-γ. Surprisingly, rat semi-invariant TCRs do not recognize α-Gal presented by mouse CD1d and accordingly, mouse α-Gal-loaded CD1d tetramers failed to stain a discrete population of rat iNKT cells. Taking all together, despite that strong evidence suggested that iNKT cells are present in the rat, the direct identification of such population and the analysis of CD1d-restricted immune responses were still pending for this species. Hence the work presented in this doctoral thesis was aimed to identify iNKT cells, to analyze their phenotype and also to study the distribution and function of CD1d in the rat. For these purposes, we produced essential reagents which were still lacking such as rat specific anti-CD1d monoclonal antibodies and rat CD1d oligomers. Importantly, two of three anti-rat CD1d monoclonal antibodies (all of them generated in our laboratory before this thesis was initiated) also recognized mouse CD1d and therefore allowed a direct comparison of CD1d expression between rat and mouse. Whereas CD1d distribution in the hematopoietic system was found to be extremely similar between these two species; in non-lymphatic tissues important differences were observed. Interestingly, CD1d protein was detected at not yet described sites such as the rat exocrine pancreas and rat and mouse Paneth cells. These monoclonal antibodies did not only allowed the analysis of CD1d expression, but also the first demonstration of the function of rat CD1d as an antigen presenting molecule, since cytokine release in response to α-Gal was blocked when they were added to ex vivo cultures of rat primary cells. Staining of primary rat iNKT cells (possible now with the newly generated rat CD1d oligomers) revealed interesting similarities with human iNKT cells. First, we observed that rat iNKT cells are only a minority among all NKR-P1A/B positive T cells. Human iNKT cells constitute also a very small proportion of NKR-P1A (CD161) expressing T cells, whereas in mice inbred strains which express NKR-P1C (NK1.1), most of NKRP1C expressing T cells are iNKT cells. Second, the majority of rat iNKT cells are either CD4 or DN and only a small proportion expresses CD8β. These findings are similar to humans and different to mice which lack CD8+ iNKT cells. Third, analysis of various inbred rat strains demonstrated different iNKT cell frequencies which correlated with cytokine secretion after α-Gal stimulation of primary cells. In comparison to mice, iNKT cell numbers are markedly reduced in rats. In F344 rats, inbred rat strain which released the highest cytokine amounts after α-Gal stimulation, approximately 0.25% and 0.1% of total liver and spleen lymphocytes, respectively, are iNKT cells. In contrast, in LEW rats iNKT cells were practically absent and neither IL-4 nor IFN-γ were detected after stimulation of primary cells with α-Gal. Once more, these frequencies are very close to those observed in humans. Last, as reported for human peripheral blood cells, rat iNKT cells could be easily expanded in vitro by adding α-Gal to cultures of intrahepatic lymphocytes, whereas the expansion of mouse iNKT cells was not possible using the same protocol. The presence of a multimember AV14 gene segment family in the rat is an intriguing characteristic. These AV14 gene segments are extremely homologous except in the CDR2α region. Based on the amino acid sequence of this region they have been divided into two different types: Type I and II. A specific tissue distribution of the different types was proposed in the first study where the presence of several AV14 gene segments was described. We also analyzed the AV14 gene segment usage in F344 and LEW inbred rat strains. In F344 rats we found no preferential usage of either AV14 gene segment type in the spleen and the liver but type II AV14 gene segments appeared more frequently in the thymus. In contrast, LEW rats show a preferential usage of type I AV14 gene segments in all three compartments analyzed: Thymus, spleen and liver. Taken all together, the usage of newly generated reagents allowed to gain novel insights into CD1d expression in the rat and in the mouse and to directly identify rat iNKT cells for the first time. The phenotypic and functional analysis of rat iNKT cells revealed numerous similarities with human iNKT cells. These are of special interest, since rats serve to investigate several pathological conditions including models for autoimmune diseases. The possibility now to analyze iNKT cells and CD1d-restricted T cell responses in the rat might help to understand the pathogenesis of such diseases. In addition, the uncomplicated in vitro expansion and culture of rat iNKT cells should facilitate the analysis of the immunomoldulatory capacities of these cells.
Protein kinase B (PKB), a serine threonine kinase, is highly involved in the regulation of cellular proliferation and survival. To characterize PKB’s function in lymphocyte development and activation, transgenic (tg) mice that express a membrane targeted constitutively active form of PKBa (myr PKB) in T and B cells were analysed. Thymocytes from myr PKB tg mice showed enhanced proliferation after T cell receptor (TCR) engagement compared to wild type (wt) mice. Astonishingly, myr PKB tg thymocytes were capable to proliferate in response to PMA only and were also less sensitive to inhibition by the calcineurin inhibitors CsA or FK506, which indicates the proliferative response of myr PKB tg T cells is relatively independent of calcium mobilisation and calcineurin activity. In addition, when TCR signalling was inhibited by the MEKinase inhibitor PD98059 or the Srckinase inhibitor PP1 myr PKB tg thymocytes again were more resistant to inhibition. Western blot analysis revealed myr PKB enhances activation of the kinases Lck, Raf and Erk after TCR/CD3 stimulation. Thus, myr PKB renders proliferative responses of thymocytes more sensitive to TCR signals by positive regulation of the Lck-Raf-MEK-Erk signalling pathway. Studies on the cellular location of the tg protein showed myr PKB is located in membrane socalled “lipid rafts”. Furthermore, we found that after TCR/CD3 ligation endogenous cytoplasmic PKB moves into “lipid rafts”, which highlights PKB as a crucial mediator of TCR proximal signalling events. Analysing three different TCR tg model systems for positive and negative selection of immature precursors in the thymus, we found myr PKB promotes positive selection of CD4+ but not CD8+ T cells. This most likely results from PKB’s positive cross-talk on Lck-Raf-Erk signalling, which is known to influence thymocyte selection and CD4/CD8-lineage choice. Furthermore, myr PKB enhances phosphorylation of glycogen synthase kinase 3 (GSK3), a negative regulator of the transcription factor NFAT (nuclear factor of activated T cells) and T cell activation, and of the adapter protein c-Cbl. Concerning negative selection, myr PKB enhanced (OT1 mice), reduced (HY mice) or had no influence (OT2 mice) on negative selection. Thus, myr PKB’s effect on negative selection strongly depends on the model system analysed and this most likely results from differences in TCR affinity/avidity and TCR specificity for MHC. 106 Peripheral CD4+ T cells from myr PKB tg mice showed enhanced production of both Th1 and Th2 cytokines. Furthermore, after TCR/CD3 stimulation in the presence of TGF-b1, wt CD4+ T cells showed a drastic inhibition of proliferation, whereas myr PKB tg CD4+ T cells proliferated even better, i.e. they were resistant to the inhibitory TGF-b1 signals. Expression of myr PKB in B cells leads to reduced Ca2+ flux and proliferation after BCR stimulation, but activation of Lyn, SLP-65, c-Cbl and GSK-3 were enhanced. When we analysed B cell subsets in myr PKB tg mice, a decrease in immature and mature B cells became obvious, whereas cell numbers for marginal zone (MZ) B cells were normal. In aged myr PKB tg mice we detected a very strong reduction of pro/pre and immature B cell populations in the bone marrow, indicating PKB is very important for maintenance of B cell development. Furthermore, myr PKB also lead to a strong reduction of peritoneal B-1 cells. However, expression of NFATc1, which is required for B-1 cell development, was comparable between wt and myr PKB tg B-1 cells. To analyse the effect of myr PKB on immunoglobulin production, mice were immunized with thymus dependent (TD) and independent (TI) antigens. In both cases, B cell responses were strongly elevated in myr PKB tg mice. Finally, RT-PCR analyses of in vitro expanded B cells revealed increased Blimp-1 and Notch3 expression in myr PKB tg B cells, which might be primary candidates involved in their enhanced effector function. In summary, this study clearly shows an important cross-talk between PKB and various critical signalling molecules downstream of the TCR and BCR. Thereby active PKB modulates and regulates the thresholds for thymocyte selection and T cell activation as well as for B cell development and function.
Lack of acid sphingomyelinase (ASM) activity, either through genetic deficiency or through pharmacological inhibition, is linked with increased activity and frequency of Foxp3+ regulatory T cells (Treg) among cluster of differentiation (CD) 4+ T cells in mice in vivo and in vitro1. Thus, pharmacological blockade of ASM activity, which catalyzes the cleavage of sphingomyelin to ceramide and phosphocholine, might be used as a new therapeutic mechanism to correct numeric and/ or functional Treg de-ficiencies in diseases like multiple sclerosis or major depression.
In the present study, the effect of pharmacological inhibition of ASM in humans, in vitro and in vivo, was analyzed. In the in vitro experiments, peripheral blood mono-nuclear cells (PBMC) of healthy human blood donors were treated with two widely prescribed antidepressants with high (sertraline, Ser) or low (citalopram, Cit) capaci-ty to inhibit ASM activity. Similar to the findings in mice an increase in the frequency of Treg among human CD4+ T cells upon inhibition of ASM activity was observed. For the analysis in vivo, a prospective study of the composition of the CD4+ T cell com-partment of patients treated for major depression was done. The data show that pharmacological inhibition of ASM activity was superior to antidepressants with little or no ASM-inhibitory activity in increasing CD45RA- CD25high effector Treg (efTreg) frequencies among CD4+ T cells to normal levels. Independently of ASM inhibition, correlating the data with the clinical response, i.e. improvement of the Hamilton rat-ing scale for depression (HAMD) by at least 50 per cent (%) after four weeks of treatment, it was found that an increase in efTreg frequencies among CD4+ cells dur-ing the first week of treatment identified patients with a clinical response.
Regarding the underlying mechanism, it could be found that the positive effect of ASM inhibition on Treg required CD28 co-stimulation suggesting that enhanced CD28 co-stimulation was the driver of the observed increase in the frequency of Treg among human CD4+ T cells. Inhibition of ASM activity was further associated with changes in the expression and shuttling of CTLA-4, a key inhibitory molecule ex-pressed by Treg, between cellular compartments but the suppressive activity of CTLA-4 through its transendocytosis activity was unaffected by the inhibition of ASM activity.
In summary, the frequency of (effector) Treg among CD4+ T cells in mice and in hu-mans is increased after inhibition of ASM activity suggesting that ASM blockade might beneficially modulate autoimmune diseases and depression-promoting in-flammation.
Molecular modelling and simulation are powerful methods in providing important in-formation on different biological systems to elucidate their structural and functional proper-ties, which cannot be determined in experiment. These methods are applied to analyse versa-tile biological systems: lipid membrane bilayers stabilized by an intercalated single wall carbon nanotube and retroviral proteins such as HIV protease and integrase. HIV-1 integrase has nuclear localization signals (NLS) which play a crucial role in nuclear import of viral preintegration complex (PIC). However, the detailed mechanisms of PIC formation and its nuclear transport are not known. Previously it was shown that NLSs bind to the cell transport machinery e.g. proteins of nuclear pore complex such as transportins. I investigated the interaction of this viral protein HIV-1 integrase with proteins of the nuclear pore complex such as transportin-SR2 (Shityakov et al., 2010). I showed that the transportin-SR2 in nuclear import is required due to its interaction with the HIV-1 integrase. I analyzed key domain interaction, and hydrogen bond formation in transportin-SR2. These results were discussed in comparison to other retroviral species such as foamy viruses to better understand this specific and efficient retroviral trafficking route. The retroviral nuclear import was next analyzed in experiments regarding the retroviral ability to infect nondividing cells. To accomplish the gene transfer task successfully, ret-roviruses must efficiently transduce different cell cultures at different phases of cell cycle. However, promising and safe foamy viral vectors used for gene transfer are unable to effi-ciently infect quiescent cells. This drawback was due to their inability to create a preintegra-tion complex (PIC) for nuclear import of retroviral DNA. On the contrary, the lentiviral vec-tors are not dependant on cell cycle. In the course of reverse transcription the polypurine tract (PPT) is believed to be crucial for PIC formation. In this thesis, I compared the transduction frequencies of PPT modified FV vectors with lentiviral vectors in nondividing and dividing alveolar basal epithelial cells from human adenocarcinoma (A549) by using molecular cloning, transfection and transduction techniques and several other methods. In contrast to lentiviral vectors, FV vectors were not able to effi-ciently transduce nondividing cell (Shityakov and Rethwilm, unpublished data). Despite the findings, which support the use of FV vectors as a safe and efficient alternative to lentiviral vectors, major limitation in terms of foamy-based retroviral vector gene transfer in quiescent cells still remains. Many attempts have been made recently to search for the potential molecules as pos-sible drug candidates to treat HIV infection for over decades now. These molecules can be retrieved from chemical libraries or can be designed on a computer screen and then synthe-sized in a laboratory. Most notably, one could use the computerized structure as a reference to determine the types of molecules that might block the enzyme. Such structure-based drug design strategies have the potential to save off years and millions of dollars compared to a more traditional trial-and-error drug development process. After the crystal structure of the HIV-encoded protease enzyme had been elucidated, computer-aided drug design played a pivotal role in the development of new compounds that inhibit this enzyme which is responsible for HIV maturation and infectivity. Promising repre-sentatives of these compounds have recently found their way to patients. Protease inhibitors show a powerful sustained suppression of HIV-1 replication, especially when used in combi-nation therapy regimens. However, these drugs are becoming less effective to more resistant HIV strains due to multiple mutations in the retroviral proteases. In computational drug design I used molecular modelling methods such as lead ex-pansion algorithm (Tripos®) to create a virtual library of compounds with different binding affinities to protease binding site. In addition, I heavily applied computer assisted combinato-rial chemistry approaches to design and optimize virtual libraries of protease inhibitors and performed in silico screening and pharmacophore-similarity scoring of these drug candidates. Further computational analyses revealed one unique compound with different protease bind-ing ability from the initial hit and its role for possible new class of protease inhibitors is dis-cussed (Shityakov and Dandekar, 2009). A number of atomistic models were developed to elucidate the nanotube behaviour in lipid bilayers. However, none of them provided useful information for CNT effect upon the lipid membrane bilayer for implementing all-atom models that will allow us to calculate the deviations of lipid molecules from CNT with atomistic precision. Unfortunately, the direct experimental investigation of nanotube behaviour in lipid bilayer remains quite a tricky prob-lem opening the door before the molecular simulation techniques. In this regard, more de-tailed multi-scale simulations are needed to clearly understand the stabilization characteristics of CNTs in hydrophobic environment. The phenomenon of an intercalated single-wall carbon nanotube in the center of lipid membrane was extensively studied and analyzed. The root mean square deviation and root mean square fluctuation functions were calculated in order to measure stability of lipid mem-branes. The results indicated that an intercalated carbon nanotube restrains the conformational freedom of adjacent lipids and hence has an impact on the membrane stabilization dynamics (Shityakov and Dandekar, 2011). On the other hand, different lipid membranes may have dissimilarities due to the differing abilities to create a bridge formation between the adherent lipid molecules. The results derived from this thesis will help to develop stable nanobiocom-posites for construction of novel biomaterials and delivery of various biomolecules for medi-cine and biology.
MDSCs are suppressive immune cells with a high relevance in various pathologies including cancer, autoimmunity, and chronic infections. Surface marker expression of MDSCs resembles monocytes and neutrophils which have immunostimulatory functions instead of suppressing T cells. Therefore, finding specific surface markers for MDSCs is important for MDSC research and therapeutic MDSC manipulation. In this study, we analyzed if the integrin VLA-1 has the potential as a novel MDSC marker. VLA-1 was expressed by M-MDSCs but not by G-MDSCs as well as by Teff cells. VLA-1 deficiency did not impact iNOS expression, the distribution of M-MDSC and G-MDSC subsets, and the suppressive capacity of MDSCs towards naïve and Teff cells in vitro. In mice, VLA-1 had no effect on the homing capability of MDSCs to the spleen, which is a major reservoir for MDSCs. Since the splenic red pulp contains collagen IV and VLA-1 binds collagen IV with a high affinity, we found MDSCs and Teff cells in this area as expected. We showed that T cell suppression in the spleen, indicated by reduced T cell recovery and proliferation as well as increased apoptosis and cell death, partially depended on VLA-1 expression by the MDSCs. In a mouse model of multiple sclerosis, MDSC injection prior to disease onset led to a decrease of the disease score, and this effect was significantly reduced when MDSCs were VLA-1 deficient. The expression of Sema7A by Teff cells, a ligand for VLA-1 which is implicated in negative T cell regulation, resulted in a slightly stronger Teff cell suppression by MDSCs compared to Sema7A deficient T cells. Live cell imaging and intravital 2-photon microscopy showed that the interaction time of MDSCs and Teff cells was shorter when MDSCs lacked VLA 1 expression, however VLA-1 expression had no impact on MDSC mobility. Therefore, the VLA-1-dependent interaction of MDSC and Teff cells on collagen IV in the splenic red pulp is implicated MDSC-mediated Teff cell suppression.
To analyze the role of protein kinase B(PKB)on developmental and functional aspects of T cells, we have generated transgenic mouse lines expressing a constitutively active form of PKB (myrPKB) in early stages of T cell development.Peripheral CD4+ T cells from PKB tg mice are hyperreactive, more efficient in producing th1 and th2 cytokines and show faster and CD28 co-stimulation independent cell cycle progression.Interestingly PKB tg T cells are resistant to CsA treatment in proliferation and cytokine production.Further analysis show PKB tg CD4+ T cells have a drastically reduced nuclear translocation of NFAT proteins and this is due to a direct interaction between PKB and NFAT. To study whether the negative regulatiopn of NFATs by PKB affects T cell development, we analyzed double tg mice expressing both, a constitutively active version of calcineurin (dCam) and myrPKB. dCam tg mice have a severe block in thymocyte development at the DN3 stage.But in the dCam/PKB double tg mice this developmental block is significantly rescued.This rescue of thymocyte development by PKB is due to the expression of RAG1 and subsequent TCRb chain expression. CsA treatment of neonatal thymic lobes from dCam mice restores normal thymocyte development, indicating involvement of NFATs in the severe block in dCam thymocyte development.Confocal studies clearly established that compared to dCam DN cells there is a significant reduction in the nuclear levels of NFATc1 and NFATc3 in dCam/PKB cells.Downregulation of nuclear NFAT levels by myrPKB thus seems to be an essential parameter in dCam cells to proceed with normal differentiation. In summary, the data from PKB tg peripheral CD4+ T cells and dCam/PKB double tg thymocytes clearly establish PKB as an important modulator of T cell development and function and PKB as a novel negative regulator of NFAT activation.
This study focuses on phosphoantigen specific Vg9Vd2 T cells which only exist in human and non-human primates. This population accounts for 1%-5% of peripheral blood T-lymphocytes but their frequency can rise to 50% of total blood T cells upon infection. Vg9Vd2 T cells can be activated by nonpeptide compounds with critical phosphate moieties which are termed as phosphoantigens. These include isopentenyl pyrophosphate (IPP), a key compound of isoprenoid synthesis in all organisms, and (E)-4-Hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP), a direct precursor of IPP in DOXP pathway which only exist in eubacteria, plants, apicomplexaen parasites. Its activity as phosphoantigen is at least 1000 fold higher than that of IPP. However, direct structural evidence of phosphoantigen binding to the TCR is missing so far. Moreover, Vg9Vd2 T cells have potent anti-tumor activity e.g. against the B-cell lymphoma Daudi, whose Vg9Vd2 T cell activating properties have been suggested to result from sensing of abnormal intracellular IPP levels by the Vg9Vd2 TCR or Vg9Vd2 TCR binding to other postulated ligands such as an ectopically expressed F1-ATPase or UL-16 binding protein 4 (ULBP4). Aminobisphosphonates and alkymines were hypothesized to activate Vg9Vd2 T cells indirectly by inhibiting the IPP consuming enzyme farnysyl pyrophosphates synthesis (FPPS) although off target effects of these drugs or a direct interaction with the Vg9Vd2 TCR could not be excluded. This thesis presents new approaches for the mechanistic analysis of Vg9Vd2 T cell activation. By employing retroviral transduction of FPPS specific shRNA, it shows that specific shRNA reduces expression of FPPS and is sufficient to convert hematopoietic and non-hematopoietic tumor cell lines into Vg9Vd2 T cell activators. FPPS knockdown cells activated Vg9Vd2 T cells as measured by increased levels of CD69 and CD107a, kill of FPPS knockdown cells and induction of IFN-γ secretion. The IPP-synthesis-inhibiting drug mevastatin reduced Vg9Vd2 T cell activation by FPPS knockdown cells or aminobisphosphonate treated cells but not activation by the phosphoantigen bromohydrin pyrophosphate (BrHPP). A reduced growth of the FPPS knockdown cells has not been observed which is different to what has been reported for aminobisphosphonate treated cells. Finally, the human B-cell lymphoma RAJI has been transduced with Tetracyclin-inducible FPPS specific shRNA and proven to gain and loose the capacity to activate Vg9Vd2 TCR transductants upon doxycylin provision or removal. Another approach for the analysis of Vg9Vd2 T cell activation is Vg9Vd2 TCR transduced mouse cell lines with specificity for phosphoantigens. In contrast to the previously used Vg9Vd2 TCR transduced Jurkat cells, these cells do not present phosphoantigens, and are therefore specially suited for analysis of phosphoantigen presentation. The response of the new TCR transductants to presumed Vg9Vd2 TCR ligands/activators such as phosphoantigens, aminobisphosphonates or FPPS knockdown cells, depended strongly on the expression of a rat/mouse CD28 molecule by the transductants and its ligation by the (CD80) counter receptor on the ligand-presenting cell. The response is likely to reflect recognition of cognate Vg9Vd2 TCR antigens since mutations in the TCR-δ chain CDR2 and 3 abolished this response but activation by TCR or CD3 specific antibodies. A major difference between TCR transductants and primary gd T cells, was the lacking response of TCR transductants to Daudi or IPP. In addition their sensitivity to other soluble phosphoantigens was about 100 fold weaker than that of primary cells, stimulation of both cell type to CD80 expressing FPPS knock down or aminobisphosphonates was similar. Finally, the transductants have also been used to analyze effects of over-expression or knockdown of enzymes of isoprenoid synthesis such as 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMG-CoA reductase or HMGR), mevalonate-5-pyrophosphate decarboxylase (MVD), isopentenyl pyrophosphate isomerase (IDI), geranyl-geranyl pyrophosphate synthase (GGPPS) but no clear effects have been found. In conclusion, this thesis supports the concept of Vg9Vd2 T cells being sensors of a dysregulated isoprenoid metabolism and established new tools to study ligand recognition and TCR mediated activation of this T cell population. These tools will be most useful to address following questions: 1) How does the dysregulation of isoprenoid metabolism affect tumor growth? 2) What is the correlation between the modulation of IPP levels and the Vg9Vd2 TCR binding or expression of other postulated ligands? 3) Are there any mevalonate pathway enzymes other than FPPS and HMGR, which play an important role in Vg9Vd2 T cells activation? 4) What is/are the putative phosphoantigen-presenting molecule(s)?
The respiratory system is amongst the most important compartments in the human body. Due to its connection to the external environment, it is one of the most common portals of pathogen entry. Airborne pathogens like measles virus (MV) carried in liquid droplets exhaled from the infected individuals via a cough or sneeze enter the body from the upper respiratory tract and travel down to the lower respiratory tract and reach the alveoli. There, pathogens are captured by the resident dendritic cells (DCs) or macrophages and brought to the lymph node where immune responses or, as in case of MV, dissemination via the hematopoietic cell compartment are initiated. Basic mechanisms governing MV exit from the respiratory tract, especially virus transmission from infected immune cells to the epithelial cells have not been fully addressed before. Considering the importance of these factors in the viral spread, a complex close-to-in-vivo 3D human respiratory tract model was generated. This model was established using de-cellularized porcine intestine tissue as a biological scaffold and H358 cells as targets for infection. The scaffold was embedded with fibroblast cells, and later on, an endothelial cell layer seeded at the basolateral side. This provided an environment resembling the respiratory tract where MV infected DCs had to transmigrate through the collagen scaffold and transmit the virus to epithelial cells in a Nectin-4 dependent manner. For viral transmission, the access of infected DCs to the recipient epithelial cells is an essential prerequisite and therefore, this important factor which is reflected by cell migration was analyzed in this 3D system.
The enhanced motility of specifically MV-infected DCs in the 3D models was observed, which occurred independently of factors released from the other cell types in the models. Enhanced motility of infected DCs in 3D collagen matrices suggested infection-induced cytoskeletal remodeling, as also verified by detection of cytoskeletal polarization, uropod formation. This enforced migration was sensitive to ROCK inhibition revealing that MV infection induces an amoeboid migration mode in DCs. In support of this, the formation of podosome structures and filopodia, as well as their activity, were reduced in infected DCs and retained in their uninfected siblings. Differential migration modes of uninfected and infected DCs did not cause differential maturation, which was found to be identical for both populations. As an underlying mechanism driving this enforced migration, the role of sphingosine kinase (SphK) and sphingosine-1-phosphate (S1P) was studied in MV-exposed cultures. It was shown in this thesis that MV-infection increased S1P production, and this was identified as a contributing factor as inhibition sphingosine kinase activity abolished enforced migration of MV-infected DCs. These findings revealed that MV infection induces a fast push-and-squeeze amoeboid mode of migration, which is supported by SphK/S1P axis. However, this push-and-squeeze amoeboid migration mode did not prevent the transendothelial migration of MV-infected DCs.
Altogether, this 3D system has been proven to be a suitable model to study specific parameters of mechanisms involved in infections in an in vivo-like conditions.
Investigations of Measles virus regulation on activation and function of antigen presenting cells
(2008)
Interaction with dendritic cells (DCs) is considered as central to immunosuppression induced by viruses, including measles virus (MV). Commonly, viral infection of DCs abrogates their ability to promote T cell expansion, yet underlying mechanisms at a cellular level are undefined. It appears that MV-WTF infection modulate DCs morphology and dynamic adhesion on extra cellular matrix proteins such as FN or ICAM-1. By morphological criteria, WTF-DCs resembled LPS-DCs, associated with their mature phenotype also adhered less efficiently to the FN or ICAM-1 support. Reduced adhesion could not be explained by a lack of 1-integrin expression or activation. Similarly, MV-DCs strongly resembled LPS-DCs in that levels of focal adhesion kinase phosphorylated at Y397 were high and not further enhanced upon FN ligation. Fascin, a downstream effector of integrin signaling was highly upregulated in LPS-DCs and moderately in WTF-DCs, and differences in its subcellular distribution were not observed between both cell cultures. Apparently, however, fascin associated less efficiently with PKC in WTF-DCs then in LPS-DCs. In line with findings for murine DCs, high motility of mature human DCs was found to require expression of Rac-GTPases. Human LPS-DCs and more so, DC transfected to express constitutively active Rac1 were the most motile DC-species analysed, confirming that migration of human DC also involved Rac activity. The velocity of WTF-DCs on FN is below that of LPS-DCs, indicating that maturation induced by WTF may be insufficient to completely promote integrin signaling which leads to Rac activation. The organisation of MV-DC/T cell interfaces was consistent with that of functional immune synapses with regard to CD3 clustering, MHC class II surface recruitment and MTOC location. These analyses are based in the selection of stable conjugates. Subsequently, however, neither contacts nor calcium flux can be stabilised and sustained in the majority of MV-DC/T cell conjugates and only promoted abortive T cell activation. Formation of spatially organised IS in T cells requites, prolonged contact durations. Therefore, aberrant distribution patterns of CD3 in these structures, if occurring, are not likely to contribute to the type of contacts predominating for WTF-DC/T cell interactions. It is also likely that transient interactions of less than 2 minutes may if at all, not efficiently support viral transmission to T cells. Transient interactions are typically observed with immature DCs in the absence of antigen, but this is not likely to be relevant in our allogenic system, which includes SA-loaded WTF-DCs. Thus, MV-infected DCs retain activities required for initiating, but not sustaining T cell conjugation and activation. This is partially rescued if surface expression of the MV glycoproteins on DCs is abolished by infection with a recombinant MV encoding VSV G protein instead, indicating that these contribute directly to synapse destabilisation and thereby act as effectors of T cell inhibition.
Primary contact with human polyomaviruses is followed by lifelong asymptomatic persistence of viral DNA. Under severe immunosuppression JCV activation may lead to unrestricted virus growth in the CNS followed by development of progressive multifocal leukoencephalopathy (PML). Besides the kidney and the brain, target cells of persistent infection were also found in the hematopoietic system. This included the presence of JCV genomes in peripheral blood cells (PBCs). In the attempt to understand the role of PBCs for the JCV infection in humans, we asked for the type of cells affected as well as for virus interaction with PBCs. Analysis of separated subpopulations by highly sensitive and specific polymerase chain reaction and Southern blot hybridization revealed the presence of JCV DNA mostly in circulating granulocytes. These cells have important functions in innate immunity and are professional phagocytes. This suggested that PCR amplified DNA might be the result of an extranuclear association of the virus due to membrane attachment or phagocytosis rather than JCV infection with presence of viral DNA in the nucleus. In the attempt to answer this question JCV DNA was subcellularly localized in the blood of 22 healthy donors by JCV specific fluorescence in situ hybridization (FISH). Granulocytes and peripheral blood mononuclear cells (PBMCs) were separated by Percoll gradient centrifugation. Intracellular JCV DNA was hybridized with Digoxigenin-labeled JCV specific DNA probes covering half of the viral genome. As the sensitivity of the anti-digoxigenin antibody system was lower than the PCR detection level, a chemical amplification step was included consisting of peroxidase labeled secondary antibody precipitating biotinylated tyramide followed by detection with streptavidin-Texas-Red and fluorescence microscopy. Comparison of the number of cells affected in healthy individuals with 15 HIV-1 infected patients with and without PML revealed that the rate of affected PBMCs was comparable in both groups (2.5±0.4 and 14.5±0.9 per 1000). In contrast, the rate of JCV positive granulocytes in the immunosuppressed group was 92.6±1.7% compared to 4±1.4% in healthy donors thus confirming that granulocytes are the major group of circulating cells affected by JCV and that HIV-1 associated immune impairment has an important effect on the virus-cell association. Localization revealed that JCV DNA was predominantly located within the cytoplasm, although hybridizing signals occasionally covered the nuclear compartment. The fluorescent glow of chemical amplification combined with classical fluorescence microscopy did not allow an unequivocal localization of viral DNA. However, confocal microscopy of 24 sections through single cells combined with FISH without chemical amplification confirmed cytoplasmic localization of JCV DNA in a large number of cells. Additionally, it clearly demonstrated that JCV DNA was also located in the nucleus and nuclear localization directly correlated with the number of cells affected. Calculation of the virus load in subcellular compartments revealed that up to 50% of the JCV genomes were located in the nucleus thus pointing to viral infection at least in the granulocytes of HIV-1 infected patients. This may contribute to the distribution of the virus from sites of peripheral infection to the CNS and may promote the development of active PML in the severely immune impaired patients.
Pancreatic cancer (PC) remains one of the most challenging solid tumors to treat with a high unmet medical need as patients poorly respond to standard-of-care-therapies. Prominent desmoplastic reaction involving cancer-associated fibroblasts (CAFs) and the immune cells in the tumor microenvironment (TME) and their cross-talk play a significant role in tumor immune escape and progression. To identify the key cellular mechanisms induce an immunosuppressive tumor microenvironment, we established 3D co-culture model with pancreatic cancer cells, CAFs, monocyte as well as T cells.
Using this model, we analysed the influence of tumor cells and fibroblasts on monocytes and their immune suppressive phenotype. Phenotypic characterization of the monocytes after 3D co-culture with tumor/fibroblast spheroids was performed by analysing the expression of defined cell surface markers and soluble factors. Functionality of these monocytes and their ability to influence T cell phenotype and proliferation was investigated.
3D co-culture of monocytes with pancreatic cancer cells and fibroblasts induced the production of immunosuppressive cytokines which are known to promote polarization of M2 like macrophages and myeloid derived suppressive cells (MDSCs). These co-culture spheroid polarized monocyte derived macrophages (MDMs) were poorly differentiated and had an M2 phenotype. The immunosuppressive function of these co-culture spheroids polarized MDMs was demonstrated by their ability to inhibit autologous CD4+ and CD8+ T cell activation and proliferation in vitro, which we could partially reverse by 3D co-culture spheroid treatment with therapeutic molecules that are able to re-activate spheroid polarized MDMs or block immune suppressive factors such as Arginase-I.
In conclusion, we generated a physiologically relevant 3D co-culture model, which can be used as a promising tool to study complex cell-cell interactions between different cell types within the tumor microenvironment and to support drug screening and development. In future, research focused on better understanding of resistance mechanisms to existing cancer immunotherapies will help to develop new therapeutic strategies in order to combat cancer.