Funktionale Charakterisierung der Effektor-Mechanismen humaner CD8+ T-Zell Memory-Subtypen

Abstract

Die Zytotoxizität von CD8+ T-Zellen ist für die langfristige Beseitigung von entarteten oder virusinfizierten Zellen von entscheidender Bedeutung. Während der Immunantwort werden verschiedene Effektor und Memory T Zell-Subtypen gebildet, um einen Erreger zu beseitigen sowie ihn bei einer erneuten Infektion zu erkennen und effizient abwehren zu können. Zytotoxische T Zellen können Tumorzellen mittels zweier unterschiedlicher Mechanismen eliminieren. Entweder durch die Freisetzung lytischer Granula, die Perforin und Granzyme enthalten, oder durch die Bindung der Liganden FAS L beziehungsweise TRAIL an Todesrezeptoren auf der Zielzelle. Durch diese Mechanismen werden sowohl apoptotische als auch nekrotische Zielzelllysen induziert. Mit Hilfe des FRET-basierten Apoptose-Sensors pCaspeR-GR ist es möglich, Apoptosen und Nekrosen in mikroskopischen Einzelzellanalysen zu detektieren und mechanistisch voneinander zu unterscheiden. Diese Analyseverfahren wurden von unserer Arbeitsgruppe entwickelt, um die Induktion und Kinetiken der Zielzellapoptose oder –nekrose durch zytotoxische Immunzellen zu unterscheiden und die zugrunde liegenden Mechanismen aufzuklären. Dazu wurde mit der Zelllinie NALM-6 pCaspeR ein Zielzellsystem für stimulierte CD8+ T-Zellen etabliert, das sowohl gegenüber Perforin- als auch Todesrezeptor-vermittelten Lysen sensibel ist. Bisher wurde nicht funktional untersucht, welches Repertoire an zytotoxischen Mechanismen verschiedene CD8 T-Zell-Subtypen verwenden, um Zielzellen zu lysieren. Moderne Tumortherapieansätze, wie der adoptive Transfer von modifizierten T Zellen, basieren auf den Effektorfunktionen der transferierten Zellen und könnten von neuen Kenntnissen zur Subtypen-abhängigen Verwendung der zytotoxischen Mechanismen profitieren. Ein wesentliches Ziel dieser Arbeit war es somit, die Subtypen-abhängige Zytotoxizität von CD8+ Effektor-Memory (TEM) und Central Memory (TCM) T-Zellen zu untersuchen.

The cytotoxicity of CD8+ T cells is crucial for long-term elimination of tumor- or virus-infected cells. CD8+ T cells differentiate into various subsets of effector and memory cells to eliminate pathogens and to ensure rapid recognition and defense in case of a reinfection. CD8+ T cells kill tumor cells by two different main cytotoxic mechanisms. One of these involves directed release of lytic granula, which contain perforin and granzymes, the other signals through ligation of the death receptor ligands FAS-L and TRAIL. These cytotoxic mechanisms can induce apoptotic or necrotic target cell lysis. A novel microscopic single-cell analysis facilitates the detection and differentiation of apoptosis and necrosis using the apoptosis sensor pCaspeR-GR. This analysis was developed by our group to differentiate the induction of apoptosis and necrosis of target cells and investigate the kinetics of underlying mechanisms. To this end, we established the cell line NALM-6 pCaspeR as a target cell system for CD8+ T cells to identify perforin- and death receptor mediated target cell lysis. So far, it has not been functionally investigated which repertoire of cytotoxic mechanisms is used by distinct subsets to lyse target cells. Modern approaches of tumor therapy, such as adoptive transfer of modified T cells, rely on a highly efficient effector function of transferred cells and would greatly benefit from new knowledge on subtype-dependent use of cytotoxic mechanisms. A major aim of this work therefore was the investigation of the subtype-dependent cytotoxicity of CD8+ effector-memory (TEM) and central-memory (TCM) T cells. The first part of this work describes a newly developed protocol to obtain CD8+ T cells in sufficient quantity and quality from peripheral blood and isolate expanded CD8+ subsets. Furthermore, the entire CD8+ T cell population was characterized by flow cytometry and the cytotoxic potential was investigated using in vitro cytotoxicity assays on single-cell and population level. In the second part of this work TEM und TCM subtypes were isolated by flow cytometric sorting to characterize their cytotoxic potential by a combination of descriptive analysis and the named killing assays. In summary, the descriptive and functional analysis of the CD8+ population display an expected elevated expression of the effector molecules granzyme b, FAS-L, Trail and IFNγ in TEM. Interestingly, TCM also reveal a broad expression of effector molecules that should enable lysis of target cells. To investigate the pace and interplay of distinct cytotoxic mechanisms, perforin-mediated lysis was inhibited using concanamycin A and death receptor-mediated lysis was inhibited by specific inhibitory antibodies. Both subtypes mainly rely on the perforin (± granzyme) -mediated mechanism to induce target cell death. By induction of primary necrosis (within the first hour of the assay) and apoptosis by perforin+granzyme (0-4 hours) this mechanism is clearly dominant. Apoptosis induced by FAS-L (from 2 hours on) and TRAIL (from 3 hours on) was slower and was also observed less frequently. In contrast to TCM, TEM display a higher expression of perforin as well as a higher efficiency of target cell lysis. For example TEM induce apoptosis and necrosis in 64,8% of target cells within two hours, in contrast TCM induce apoptosis only in 31,4% of target cells, while primary necrosis is not detectable at all. In parallel to a Summary - 9 - time-saving semi-automated quantification of necrotic and apoptotic lysis, an alternative manual analysis was established to facilitate the detection of primary necrosis and verify the semi-automated analysis. Combining single-cell analysis of target cells and the manual analysis of CTL activity, distinct phases of target cell lysis as well as the timeframe of apoptosis-induction were determined. Thus we could show that TEM induce apoptosis within 25,7 ± 43,05 minutes from target cell contact on, while TCM need 81,1 ± 78,9 minutes. Within the scope of this work the cytotoxic potential of TEM and TCM subtypes was characterized and the pace, the mode of cell death and the differential use of cytotoxic mechanisms were summarized for both subsets in a profile of lysis.