The role of novel costimulatory molecules of the B7-family in immune responses

Die vorliegende Arbeit beschäftigt sich mit einem Teilbereich der Immunologie, der T-Zell Aktivierung, und der Fragestellung, welche Rolle die Moleküle B7-H1 und B7-H3 der B7-Familie von Kostimulationsmolekülen hierbei übernehmen. Das Ziel der Arbeit war zum einen die Rolle von B7-H1 in humanen Glioblastomen und zum anderen die Bedeutung von B7-H3 im Muskel zu charakterisieren. Es konnte gezeigt werden, dass in vitro alle 12 untersuchten Gliomzelllinien sowohl B7-H1 mRNA Transkripte als auch B7-H1 Protein auf ihrer Zelloberfläche konstitutionell aufweisen. In Gegenwart von IFN-g zeigte sich eine vermehrte Expression von B7-H1 mRNA und B7-H1 Protein auf der Zelloberfläche. Auch in vivo konnte gezeigt werden, dass Glioblastome in histologischen Präparaten positiv für B7-H1 anfärbbar waren, wohingegen das umgebende normale Hirngewebe negativ hierfür war. Zur Untersuchung der funktionellen Bedeutung von B7-H1 für die Glioblastom - T-Zell - Interaktion wurden Kokulturversuche mit Gliomzellen und alloreaktiven CD4+ oder CD8+ T-Zellen durchgeführt. Es konnte gezeigt werden, dass B7-H1 auf Gliomzellen zu einer Inhibiton von CD4+ oder CD8+ T-Zellen führt, da in Gegenwart eines neutralisierenden Antikörpers gegen B7-H1 die Zytokinmengen von IFN-g, IL-2 und IL-10 im Überstand und die Expression des T-Zell-Aktivierungsmarkers CD69 auf den T-Zellen erhöht war. Somit stellt B7-H1 einen neu charakterisierten Mechanismus dar, mit dem sich Glioblastome einer Kontrolle durch das Immunsystem entziehen können. Im zweiten Teil der Arbeit konnte gezeigt werden, dass B7-H3 in Zelllysaten von humanen Myoblasten vorkommt und dass es sich auf der Zelloberfläche von diesen Zellen befindet. Zur Charakterisierung der funktionellen Bedeutung dieses Moleküls im Muskel wurden Transfektanten von einer Rhabdomyosarkomzelllinie, die phänotypische Charakteristika von Muskelzellen zeigt, mittels siRNA-Technologie hergestellt. Spezifische CD8+ T-Zellen, die zuvor in Anwesenheit der kompletten Blutlymphozyten mit Wildtyp-Zellen stimuliert worden waren, wurden auf ihre zytotoxische Aktivität bei den Transfektanten getestet. In den Ergebnissen zeigte sich, dass Zellen, die wenig B7-H3 auf ihrer Zelloberfläche haben, besser von CD8+ T-Zellen lysiert werden. Dieses Ergebnis lässt den Rückschluss zu, dass B7-H3 eine inhibitorische Wirkung auf die zytotoxische Aktivität von CD8+ Effektor T-Zellen hat und somit ein neues hemmendes kostimulatorisches Molekül in der Muskel-Immunzellinteraktion darstellt.The present paper deals with the aspect of T-cell activation in the immune response and the question which role is played by the molecules B7-H1 and B7-H3 of the B7-family of costimulatory molecules. The intention was to characterize on the one hand the role of B7-H1 in human glioblastoma and on the other hand the relevance of B7-H3 in muscle. The expression and the functional activity of B7-H1 in human glioma cells were investigated in vitro and in vivo. All twelve glioma cell lines constitutively expressed B7-H1 mRNA and protein in vitro. Exposure to IFN-g strongly enhanced B7-H1 expression. Immunohistochemical analysis of malignant glioma specimens revealed in vivo strong B7-H1 expression in all samples examined, whereas no B7-H1 expression could be detected on normal brain tissues. To elucidate the functional significance of glioma cell-related B7-H1 expression, coculture experiments of glioma cells with alloreactive CD4+ and CD8+ T-cells were performed. Glioma-related B7-H1 was identified as a strong inhibitor of CD4+ as well as CD8+ T-cell activation as assessed by increased cytokine production (IFN-g, IL-2, IL-10) and expression levels of the T-cell activation marker CD69 in the presence of a neutralizing antibody against B7-H1. Thus, B7-H1 expression represents a novel mechanism by which glioma cells evade immune recognition and destruction. In the second part of the paper it was shown that B7-H3 exists in cell lysates of human myoblasts and that it is expressed on the cell surface. To further characterize the functional role of this molecule in muscle, transfectants of a rhabdomyosarcoma cell line with phenotypical characteristics of muscle cells were established using siRNA technology. Specific CD8+ T-cells primed in the presence of PBMCs and wildtype rhabdomyosarcoma cells were tested for their capability of specific lysis of the transfectants. The results showed that transfectants with low B7-H3 cell surface expression were better lysed by CD8+ T-cells. In conclusion it was demonstrated that B7-H3 has an inhibitory effect on the cytotoxic activity of CD8+ T-cells and therefore represents a new inhibitory costimulatory molecule in muscle–immune interactions

Inhibition of Thrombopoietin/Mpl Signaling in Adult Hematopoiesis Identifies New Candidates for Hematopoietic Stem Cell Maintenance

<div><p>Thrombopoietin (Thpo) signals via its receptor Mpl and regulates megakaryopoiesis, hematopoietic stem cell (HSC) maintenance and post-transplant expansion. Mpl expression is tightly controlled and deregulation of Thpo/Mpl-signaling is linked to hematological disorders. Here, we constructed an intracellular-truncated, signaling-deficient Mpl protein which is presented on the cell surface (dnMpl). The transplantation of bone marrow cells retrovirally transduced to express dnMpl into wildtype mice induced thrombocytopenia, and a progressive loss of HSC. The aplastic BM allowed the engraftment of a second BM transplant without further conditioning. Functional analysis of the truncated Mpl <i>in vitro</i> and <i>in vivo</i> demonstrated no internalization after Thpo binding and the inhibition of Thpo/Mpl-signaling in wildtype cells due to dominant-negative (dn) effects by receptor competition with wildtype Mpl for Thpo binding. Intracellular inhibition of Mpl could be excluded as the major mechanism by the use of a constitutive-dimerized dnMpl. To further elucidate the molecular changes induced by Thpo/Mpl-inhibition on the HSC-enriched cell population in the BM, we performed gene expression analysis of Lin-Sca1+cKit+ (LSK) cells isolated from mice transplanted with dnMpl transduced BM cells. The gene expression profile supported the exhaustion of HSC due to increased cell cycle progression and identified new and known downstream effectors of Thpo/Mpl-signaling in HSC (namely TIE2, ESAM1 and EPCR detected on the HSC-enriched LSK cell population). We further compared gene expression profiles in LSK cells of dnMpl mice with human CD34+ cells of aplastic anemia patients and identified similar deregulations of important stemness genes in both cell populations. In summary, we established a novel way of Thpo/Mpl inhibition in the adult mouse and performed in depth analysis of the phenotype including gene expression profiling.</p></div

Comparison of dnMpl LSK cells and of CD34+ cells of aplastic anemia patient.

<p>(A) Expression scores of each gene in murine dnMpl mice and human RC and SAA patients CD34+ cells were compared. Negative scores in both cases (lower left quadrant) reflect the downregulation of genes typically associated with a healthy phenotype. Differentially expressed genes (lower right and upper left quadrant) may reflect species/disease differences. Score: (expression sample–expression control)/(SD sample + SD control). Gene lists referring to each quadrant in the supplements.</p

Gene expression analysis of LSK cells from dnMpl mice.

<p>(A) Schematic picture of the performed experiment. Wildtype Lin- BM cells were transduced with either GFP or dnMpl.IRES.GFP expressing vectors and transplanted into lethally irradiated wt recipients. Eight weeks after transplantation LSK cells were sorted using flow cytometry. (B) Representative FACS blots of the BM cells of GFP control and dnMpl.IRES.GFP transplanted mice. In control mice GFP positive and negative LSK cells were pooled, whereas for dnMpl.IRES.GFP mice GFP positive (47%) and negative (53%) LSK cells were separately subjected to transcriptome analysis. (C) Principal component analysis (PCA) on genes differentially expressed between dnMpl-GFP+ and control LSK cells. (D) Heatmap of selected genes found to be deregulated in the gene expression analysis. dnMpl positive and negative LSK cells from the same mice (dnMpl pos (4–6) and neg (7–9)) were compared to the expression in LSK of GFP control transplanted mice (control 1–3). (blue: donwregulated genes, red: upregulated genes). (E) Gene set enrichment analysis (GSEA) of dnMpl versus GFP control transplanted mice. The expression matrix of dnMpl positive and dnMpl negative cells from the same mice were either used in combination or separately for the GSEA. The normalized enrichment scores (NES) of gene sets are displayed that are significantly enriched in the dnMpl phenotype. Most of the gene sets were part of the gene set collection of the GSEA tool and depicted based on the following publications [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0131866#pone.0131866.ref013" target="_blank">13</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0131866#pone.0131866.ref015" target="_blank">15</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0131866#pone.0131866.ref034" target="_blank">34</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0131866#pone.0131866.ref035" target="_blank">35</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0131866#pone.0131866.ref046" target="_blank">46</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0131866#pone.0131866.ref047" target="_blank">47</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0131866#pone.0131866.ref054" target="_blank">54</a>] or based on the KEGG database.</p

dnMpl expression <i>in vivo</i> causes thrombocytopenia.

<p>(A) Blood cell counts of transplanted mice six and 12–14 weeks after transplantation. Platelet counts remained significantly reduced in the dnMpl group, whereas, the hematocrit was unchanged in both groups. Slightly reduced WBC counts were observed at late time points after transplantation in the dnMpl group. (*** p<0.005, Students t-test) HCT: hematocrit, WBC: white blood cells. (B) Correlation of dnMpl expression on leukocytes with platelet counts. Blood of mice transplanted with dnMpl or GFP control transduced BM cells was taken at different time points after transplantation, platelet counts and transgene expression were measured by automated blood cell counts or flow cytometry, respectively. dnMpl expression results in decreased platelet counts. With declining dnMpl expression the platelet counts adjust to physiological levels. (C) Thpo plasma levels were determined in the blood of transplanted mice by ELISA, 12 weeks after transplantation or untransplanted wildtype and <i>Mpl-/-</i> mice. (*p<0.05, **p<0.01, Students t-test). (D) Receptor uptake in 32D cells. 32D cells were transduced with either wtMpl or dnMpl expressing vectors and stimulated with 50ng/mL mThpo or not. Cells were then fixed at different time points after Thpo has been added and surface expression of either receptor was measured by flow cytometry using an anti-HA Biotin conjugated primary and Streptavidin-PE secondary antibody.</p

Cell cycle analysis and cell surface molecule expression on LSK cells in dnMpl mice.

<p>(A) Venn diagram showing the overlap of the leading edge genes from the Gene Set Enrichment Analysis comparing our dataset with the data of [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0131866#pone.0131866.ref015" target="_blank">15</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0131866#pone.0131866.ref034" target="_blank">34</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0131866#pone.0131866.ref054" target="_blank">54</a>]. (B) Expression of essential players in the cell cycle control. Displayed are the log2(fold-change) values determined based on the microarray analysis in comparison to control. (C) Cell cycle analysis of LSK cells of dnMpl and GFP control transplanted mice. Total BM cells were stained and gated for the LSK or LSK, CD34- cell population. Cell cycle status was determined by staining with Ki-67 and Hoechst 33342. A representative example of the flow cytometric analysis is shown (left picture) and the results summarized (right graph). Significantly less LSK and LSK, CD34- cells are within the G0 phase of the cell cycle (*p<0.05, n = 3). (D) Wild-type mice were transplanted with dnMpl or truncated (tr)CD34 (as control) expressing BM cells. At 16 weeks post transplantation total BM cells were stained for LSK cells, each BM sample was then splitted into three subsamples to stain for the different surface molecules: TIE2, EPCR (CD201), ESAM1. As controls, cell surface expression was also detected in untransplanted wt, <i>Mpl-/-</i> and <i>Thpo-/-</i> mice. The difference between the percentages of surface marker positive cells was significant, as indicated: *p<0.05, **p<0.01, ***p<0.005. (wt, <i>Mpl-/-</i>, <i>Thpo-/-</i>: n = 6; dnMpl, trCD34: n = 9).</p

Impact of dnMpl expression on the HSC compartment.

<p>(A) Survival curve of primary transplanted mice of the dnMpl or the control groups (experiment 1–3, dnMpl n = 25, control n = 20). Of experiment three, the BM was transplanted into two secondary recipients each (3 dnMpl donors, 2 cd-dnMpl donors, 3 GFP control donors). There was no difference in survival in primary recipients but a significantly reduced survival of secondary recipients (10 dnMpl recipient mice and 6 control recipient mice, p<0.05). (B) Percentage of LSK cells in primary recipients (dnMpl and trCD34 control black filled circles, cd-dnMpl and GFP control in grey filled circles) or in steady state hematopoiesis (wt, <i>Mpl-/-</i>, <i>Thpo-/-</i>). (*p<0.05, Students t-test). (C) Percentage of LSK-CD34 negative cells in primary recipients of GFP control (n = 3) or dnMpl mice (n = 5) (***p<0.005, Students t-test). (D) Histological analysis of the BM of dnMpl and control transplanted mice. Primary (1°) dnMpl chimeric mice had reduced numbers and smaller megakaryocytes similar to <i>Mpl-/-</i> mice. Secondary dnMpl recipient mice (2°) had a hypocellular BM in agreement with the symptoms of BM failure. BM sections were Hematoxylin/Eosin stained and microscopic images were taken at 200x magnification. (E) CD45.2 wildtype C57Bl/6 mice were transplanted with dnMpl, cd-dnMpl or GFP control transduced CD45.2 wildtype lin- BM cells (experiment 3). 19 weeks after the first transplantation, two dnMpl, two cd-dnMpl and two GFP mice were infused with a second graft of 2x10⁷ CD45.1 whole BM cells without further conditioning. The chimerism of CD45.1 in the blood leukocytes was analyzed over a period of 16 weeks. dnMpl conditioned mice allowed the engraftment of a second wt graft without further conditioning. (F) In experiment 5, CD45.2 wildtype C57Bl/6 mice were transplanted with dnMpl or GFP control transduced CD45.2 wildtype lin- BM cells, however, with intended lower chimerism. 19 weeks after the first transplantation mice were given a second graft of 2 x10⁷ CD45.1 whole BM cells without further conditioning. The chimerism of CD45.1 in the blood leukocytes was analyzed over a period of 20 weeks. While the two mice exhibiting the highest percentage of dnMpl expression (4 and 14%) allowed stable engraftment of the second graft, the mice with < 1% dnMpl expression in the periphery failed to do so and presented with the low engraftment levels similar to the four GFP control mice.</p

dnMpl does not transmit Thpo induced signals.

<p>(A) The gammaretroviral LTR vector encoded the full length or the intracellular truncated, dominant-negative (dn)Mpl cDNA. For detection of the Mpl proteins a hemagglutinin (HA)Tag was added at the N-terminus between the signal peptide and the ECD. The vector also co-expressed GFP using an internal ribosomal entry site (IRES). As control the retroviral vector only containing IRES.GFP or a truncated form of human CD34 was used. (LTR: long terminal repeat, ψ: packaging signal, SD: splice donor, SA: splice acceptor, wPRE: Woodchuck hepatitis virus posttranscriptional regulatory element, SP: signal peptide, ECD: extracellular domain, TMD: transmembrane domain, ICD: intracellular domain). (B) Western blot analysis of Mpl downstream signaling proteins in 32D cells that were transduced with wtMpl, dnMpl or GFP as a control. Transduced cells were stimulated with mThpo (20ng/mL), IL-3 (5ng/ml) or fixed without stimulation. Activation of STAT3 and STAT5 was analyzed by EMSA. No phosphorylation of ERK1/2, AKT and STATS was detected in dnMpl expressing 32D cells after Thpo stimulation similar to the GFP control transduced cells.</p