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Herein we describe a novel APC population in mice, designated LAPCs. LAPCs are BM-derived myeloid leukocytes, distinctive from other immune cells. As APCs, LAPCs respond to various virus infections including VACV, CBV3 and influenza A virus. Notably, influenza virus-activated LAPCs capture Ag in the lungs, and migrate into the DLN and spleen with delayed kinetics compared to DCs. In the DLN, influenza virus-activated LAPCs co-localize with T cells and selectively induce Th2 effector cell polarization by cell-cell contact-mediated modulation of GATA-3 expression. In support of a role for LAPCs in anti-influenza T2 immunity, adoptive transfer experiments revealed that influenza virus-activated LAPCs selectively augmented Th2 effector T cell responses in the DLN, increased production of anti-influenza immunoglobulin (Ig) including IgE in peripheral blood and increased levels of IL-5 and eotaxin in BAL fluid in recipient influenza infected mice. LAPC recipient mice exhibited exacerbated pulmonary pathology, with delayed viral clearance and enhanced pulmonary eosinophilia. Collectively, these results highlight the importance of LAPCs as novel immuno-modulators of T2 immunity during influenza A virus infection, which is implicated in both immunoprotection and immunopathology. Subsequently, we examined the immuno-modulatory effect of type-I IFN, specifically IFN-on the immune response against pulmonary influenza virus infection. We have provided evidence that a single dose of IFN- (1×105U) augmented DC migration but inhibited LAPC migration into the DLN. mIFN- treatment skewed the immune balance toward T1 immunity, identified as enhanced T1 effector T cell responses (Th1 and CTL) but diminished T2 effector T cell responses (Th2) in influenza virus infected mice. Finally, IFN- treated mice showed accelerated viral clearance and diminished pulmonary eosinophilia in lung tissue compared to control mice. Taken together, these results suggest that anti-influenza T1 and T2 immunity may be modulated differently by DCs and LAPCs, respectively. Furthermore, these results support the therapeutic potential of type I IFNs, especially IFN-, as an alternative antiviral to control both viral replication and immunopathology induced by influenza A virus infection in humans.

CD8+ T-cell antigen sensitivity is critical for optimal control of persistent viral infections, including HIV-1. The devastating HIV-1 pandemic may be countered by development of a cytotoxic T lymphocyte based vaccine if qualities associated with protection can be defined at the molecular level. However, the heterogeneity of the total viral-specific CTL response confounds identification of protective correlates and T-cell sensitivity is no exception and remains controversial. To address this issue we reduced the heterogeneity of the HIV-1 CTL response to single units, generating 19 CTL clones that recognise the same HIV-1 derived epitope restricted by HLA B*08. Correlation of functional assays directly with the ability of each clone to control HIV-1 replication in vitro, the “viral suppression assay,” identified antigen sensitivity as a key quality for anti-viral efficacy. Remarkably, four clones from this panel, isolated from one individual, a long-term non-progressor, all used an identical TCR yet had distinct antigen sensitivity and suppressive activity. Two of these clones were characterised in detail, and had distinct cytokine profiles, regulated by epigenetic mechanisms, and differential expression of a group of cell surface receptors with the potential to modulate the signalling threshold to antigen. Expression of the TNFα locus of the high sensitivity clone with “Good” suppression was repressed by DNA methylation. Understanding how CTL qualities required for optimal control of HIV-1 replication differentiate and are then enriched in the total CTL response, and if repression of TNFα contributes to this process, will contribute to rational vaccine design. This is the first evidence that avidity maturation in CD8+ T cells with the same TCR affinity occurs in viral infections in humans as reported in the mouse. This suggests the induction of high sensitivity CTL will be critical for an effective HIV-1 vaccine, but offers hope that this can be achieved even in individuals without protective HLA alleles, by further exploration of peripheral avidity maturation and epigenetic regulation of the HIV-1 specific CD8+ T-cell response.

Mesenchymale Stammzellen (MSC) sind starke Suppressoren des Immunsystems. Für die immunsupressive Wirkung werden lösliche Faktoren propagiert. In der vorliegenden Arbeit wurde untersucht, ob neben den bekannten Sekretionsfaktoren stromaler Stammzellen wie RANK/RANKL/OPG, TNFα, IFNγ und verschiedene Interleukine, andere Sekretionsfaktoren eine immunmodulatorische Wirkung auf monozytäre Zellen haben. Als Sekretionsprodukt wurde hCyr61 untersucht, das im Überstand von mit TNFα behandelten humanen fetalen Osteoblasten (hFOB) nachgewiesen worden war. Das zur CCN-Familie gehörende Protein hCyr61 ist an der Angiogenese beteiligt, wird im Kallus während der Frakturheilung vermehrt exprimiert und hat, wie zwischenzeitlich nachgewiesen wurde, einen deutlichen Effekt auf die Differenzierung endothelialer Vorläuferzellen aus monozytären Zellen. In den hier beschriebenen Experimenten wurden sowohl monozytäre Zellen aus peripherem Blut (PBMC), als auch THP1-Zellen auf die Veränderung der CD14-, 80-, 83- und 86- Expression unter Stimulation mit und ohne Il-4, GM-CSF und hCyr61 untersucht. Die Expressionsänderung wurde mittels konventioneller PCR, real-time PCR und FACS-Analyse untersucht. In allen drei Nachweismethoden zeigte sich ein Verlust typisch monozytärer Marker wie CD14 unter Stimulation mit hCyr61. Der Verlust an CD14 scheint bei PBMC deutlicher als bei THP1-Zellen, was möglicherweise durch die oben beschriebene endotheliale Differenzierung erklärt wird. THP1-Zellen befinden sich bereits in einer höheren Differenzierungsstufe, sodass sie ihr monozytäres Commitment nicht vollständig verlieren. / Mesenchymal stem cells have a strong suppressive effect on the immune system. Soluble factors are discussed to be responsible for this effect. The aim of this dissertation was to investigate, whether there are other soluble factors with immunomodulatory effects on monocytic cells beside the already known factors like RANK/RANKL/OPG, TNFα, IFNγ and various interleukins. As a soluble factor found in the supernatant of humane fetal osteoblasts stimulated by TNFα, hCyr61 was investigated. This protein belongs to the CCN-family and it takes part in angiogenesis, was found in high concentrations in the callus during the healing process of a fracture and it effects strongly the differentiation of endothelial precursor cells from monocytic cells. The change of the expression of CD14, 80, 83 and 86 was investigated with and without stimulation by Il-4, GM-CSF and hCyr61 in monocytic cells from peripheral blood (PBMC) and THP1-cells. The results were verified by conventional PCR, real-time PCR and FACS analysis. All three methods showed a decrease of CD14 as typical monocyte marker in the presence of hCyr61. The decrease of CD14 in PBMC seems more striking then in THP1, which might be explained by endothelial differentiation mentioned above. THP1 cells have reached a higher level of differentiation, therefore they don´t lose their monocyte commitment anymore.