Targeting the Monocyte–Macrophage Lineage in Solid Organ Transplantation

textabstractThere is an unmet clinical need for immunotherapeutic strategies that specifically target the active immune cells participating in the process of rejection after solid organ transplantation. The monocyte-macrophage cell lineage is increasingly recognized as a major player in acute and chronic allograft immunopathology. The dominant presence of cells of this lineage in rejecting allograft tissue is associated with worse graft function and survival. Monocytes and macrophages contribute to alloimmunity via diverse pathways: antigen processing and presentation, costimulation, pro-inflammatory cytokine production, and tissue repair. Cross talk with other recipient immune competent cells and donor endothelial cells leads to amplification of inflammation and a cytolytic response in the graft. Surprisingly, little is known about therapeutic manipulation of the function of cells of the monocyte-macrophage lineage in transplantation by immunosuppressive agents. Although not primarily designed to target monocyte-macrophage lineage cells, multiple categories of currently prescribed immunosuppressive drugs, such as mycophenolate mofetil, mammalian target of rapamycin inhibitors, and calcineurin inhibitors, do have limited inhibitory effects. These effects include diminishing the degree of cytokine production, thereby blocking costimulation and inhibiting the migration of monocytes to the site of rejection. Outside the field of transplantation, some clinical studies have shown that the monoclonal antibodies canakinumab, tocilizumab, and infliximab are effective in inhibiting monocyte functions. Indirect effects have also been shown for simvastatin, a lipid lowering drug, and bromodomain and extra-terminal motif inhibitors that reduce the cytokine production by monocytes-macrophages in patients with diabetes mellitus and rheumatoid arthritis. To date, detailed knowledge concerning the origin, the developmental requirements, and functions of diverse specialized monocyte-macrophage subsets justifies research for therapeutic manipulation. Here, we will discuss the effects of currently prescribed immunosuppressive drugs on monocyte/macrophage features and the future challenges

T cell Allorecognition Pathways in Solid Organ Transplantation.

Transplantation is unusual in that T cells can recognize alloantigen by at least two distinct pathways: as intact MHC alloantigen on the surface of donor cells via the direct pathway; and as self-restricted processed alloantigen via the indirect pathway. Direct pathway responses are viewed as strong but short-lived and hence responsible for acute rejection, whereas indirect pathway responses are typically thought to be much longer lasting and mediate the progression of chronic rejection. However, this is based on surprisingly scant experimental evidence, and the recent demonstration that MHC alloantigen can be re-presented intact on recipient dendritic cells-the semi-direct pathway-suggests that the conventional view may be an oversimplification. We review recent advances in our understanding of how the different T cell allorecognition pathways are triggered, consider how this generates effector alloantibody and cytotoxic CD8 T cell alloresponses and assess how these responses contribute to early and late allograft rejection. We further discuss how this knowledge may inform development of cellular and pharmacological therapies that aim to improve transplant outcomes, with focus on the use of induced regulatory T cells with indirect allospecificity and on the development of immunometabolic strategies. KEY POINTS Acute allograft rejection is likely mediated by indirect and direct pathway CD4 T cell alloresponses.Chronic allograft rejection is largely mediated by indirect pathway CD4 T cell responses. Direct pathway recognition of cross-dressed endothelial derived MHC class II alloantigen may also contribute to chronic rejection, but the extent of this contribution is unknown.Late indirect pathway CD4 T cell responses will be composed of heterogeneous populations of allopeptide specific T helper cell subsets that recognize different alloantigens and are at various stages of effector and memory differentiation.Knowledge of the precise indirect pathway CD4 T cell responses active at late time points in a particular individual will likely inform the development of alloantigen-specific cellular therapies and will guide immunometabolic modulation

What Is Direct Allorecognition?

Direct allorecognition is the process by which donor-derived major histocompatibility complex (MHC)-peptide complexes, typically presented by donor-derived ‘passenger’ dendritic cells, are recognised directly by recipient T cells. In this review, we discuss the two principle theories which have been proposed to explain why individuals possess a high-precursor frequency of T cells with direct allospecificity and how self-restricted T cells recognise allogeneic MHCpeptide complexes. These theories, both of which are supported by functional and structural data, suggest that T cells recognising allogeneic MHC-peptide complexes focus either on the allopeptides bound to the allo-MHC molecules or the allo-MHC molecules themselves. We discuss how direct alloimmune responses may be sustained long term, the consequences of this for graft outcome and highlight novel strategies which are currently being investigated as a potential means of reducing rejection mediated through this pathway

Structure and Characterization of the cd30 gene

0. Titelseite und Inhaltsverzeichnis 2. Material und Methoden 7 1. Einleitung 5 2. Material und Methoden 7 2.1. Material 7 2.1.1. Enzyme, Vektoren, Nukleotide 7 2.1.2. Chemikalien 7 2.1.3. Stammlösungen und Nährlösungen 7 2.2. Methoden 8 2.2.1. Herstellung von Agarosegelen, Elektrophorese 8 2.2.2. Gel-Extraktion von DNA 8 2.2.3. Southern Blot 9 2.2.4. Markierung synthetischer Oligonukleotide mit [32P]-dCTP 9 2.2.5. Random priming von cDNA-Sonden 10 2.2.6. Markierung synthetischer Oligonukleotide mit Digoxigenin-markiertem dUTP 10 2.2.7. Detektion hybridisierter Digoxigenin-markierter Oligonukleotide 10 2.2.8. Hybridisierung von Membranen mit markierten Oligonukleotiden 11 2.2.9. Polymerasekettenreaktion (PCR) 12 2.2.10. Isolation genomischer -Phagen-Klone 12 2.2.11. Isolation genomischer CD30-Klone aus Cosmid-Banken 13 2.2.12. Isolation von cd30-Gen-Fragmenten mit Langstrecken- Polymerasekettenreaktion (LR-PCR) aus genomischen Genbanken 13 2.2.13. Präparation der DNA eines Phagenklons 16 2.2.14. Klonierung von DNA-Fragmenten in pBlueScript 19 2.2.15. Klonierung von PCR-Fragmenten in den pCR II-TOPO Vektor 19 2.2.16. Transformation von kompetenten E. coli Zellen (TOP10) 19 2.2.17. Plasmidpräparation 20 2.2.18. DNA-Sequenzierung 21 2.2.19. Auswertung der Sequenzdaten 22 3. Ergebnisse 24 3.1. Isolation und Klonierung von cd30-Gen-Fragmenten 24 3.2. Sequenzierung der cd30-Gen-DNA-Fragmente 27 3.3. Exon / Intron Struktur des cd30-Gens 29 3.4. Promoterregion des cd30-Gens 30 3.5. Mikrosatelliten-Sequenz 32 3.6. Karte des cd30-Gens 34 3.7. Vergleich des cd30-Gens mit der TNFR-Superfamilie 35 4. Diskussion 36 4.1. Analyse von Genbanken 36 4.2. Genstruktur des CD30 37 4.3. Promoterregion des cd30-Gens 38 4.3.1. Genetischer Polymorphismus 39 4.3.2. Transkriptionsfaktorbindungsstellen und Transkriptionsstartpunkt 40 5. Zusammenfassung 43 6. Anhang 44 6.1. Liste der Abkürzungen 44 7. Literaturverzeichnis 46 8. Lebenslauf 55 9. Danksagung 57Das CD30-Antigen ist ein Mitglied der Tumornekrosefaktor-Rezeptor-Familie (TNFR), das vorwiegend auf Tumorzellen des Morbus Hodgkin, des ALCL sowie des embyonalen Karzinoms des Hodens überexprimiert wird. Die Besonderheit des CD30-Antigens ist seine sehr restriktive Expression auf neoplastischen Zellen verschiedener Tumorerkrankungen auf der einen und die physiologischerweise auf nur wenige lymphatische beziehungsweise deziduale Zellen beschränkte Expression auf der anderen Seite. Nachdem gezeigt werden konnte, daß die restriktive Expression von CD30 auf transkriptioneller Ebene reguliert wird, wurde in der vorliegenden Arbeit die Genstruktur und die Promoterregion des cd30-Gens sequenziert und näher untersucht. Das cd30-Gen besteht aus 8 Exons und 7 Introns. Die Exons lassen sich strukturellen Domänen auf Proteinebene zuordnen. Das 1. Exon kodiert für das hydrophobe Leitpeptid, das 2. und 3. Exon beinhalten jeweils drei cysteinreiche Domänen des extrazellulären Rezeptorteils und sind einander zu 70% homolog. Die Transmembrandomäne befindet sich überwiegend im 4. Exon und der intrazelluläre Teil des CD30 wird von den Exonen 5 bis 8 kodiert. Eine Besonderheit von CD30 innerhalb der TNFR-Superfamilie ist die Aufteilung der 6 cysteinreichen Domänen auf zwei zu einander homologe Exons. Möglicherweise könnte diese Struktur durch partielle Duplikation eines Exon nach Verlust zweier Intronen während der Evolution von CD30 entstanden sein. Das Vorhandensein von drei cysteinreichen Domänen im murinen CD30 im Vergleich zu sechs cysteinreichen Domänen im humanen CD30 wäre hierdurch erklärbar. Der Promoter von CD30 besitzt keine TATA-Box und ist GC-reich. Auffällig ist im Promoterbereich von CD30 eine ATCC-Mikrosatellitensequenz. In Untersuchungen von CD30+ Zellinien und in normalem Gewebe konnte gezeigt werden, daß ein deutlicher Längenpolymorphismus besteht. Dieser ist bei CD30+ Zellinien stärker ausgeprägt als in Geweben mit physiologisch geringer CD30-Expression. Die beschriebene Mikrosatelliteninstabilität könnte bei der Onkogenese der CD30+-Tumoren durch Erhöhung der Promoteraktivität eine entscheidende Rolle spielen.The CD30 antigen is a member of the tumor necrosis factor receptor (TNFR) family which is overexpressed on the surface of the tumor cells of Hodgkin's lymphoma, anaplastic large cell lymphoma (ALCL), and embryonal carcinoma of the testis. In this study the entire cd30 gene which is more than 24 000 bp long and organized in eight exons was characterized by analyzing cosmid and phage clones from human placental libraries with long-range polymerase chain reaction (PCR) and sequencing. Differences to other genes of the TNFR family were detected in the region encoding the extracellular domain of the cd30 gene. In nearly all other TNFR genes, the coding region of each cysteine-rich repeat is interrupted by one intron, i.e., the 3-4 cysteine-rich repeats of these receptors are encoded by at least 4-5 exons, whereas the six cysteine-rich repeats of the cd30 gene are encoded by two exons, i.e., each of these exons encode three cysteine-rich repeats. The 5'-fanking regulatory region of the cd30 gene was analyzed for possible transcription factor binding sites. Sequence analysis showed that the cd30 promoter ist TATA-less and GC-rich. In addition a genetic polymorphism of tetranucleotide ATCC-repeats was found in the 5' part of the CD30 promoter. This region was amplified by PCR from seven CD30 overexpressing human lymphoid cell lines and five human tissues with an absent or very low CD30 expression. The amplification products showed length differences of more than 550 bp which was due to different numbers of ATCC-repeats. The number of the ATCC-repeats was higher in CD30+ cell lines than in normal tissues