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Strauss, G., Knape, I., Melzner, I. & Debatin, K. M. Constitutive caspase activation and impaired death-inducing signaling complex formation in CD95-resistant, long-term activated, antigen-specific T cells. J. Immunol. 171, 1172-1182

University Children's Hospital and Institute of Pathology, University of Ulm, Ulm, Germany.
The Journal of Immunology (Impact Factor: 5.36). 09/2003; 171(3):1172-82. DOI: 10.4049/jimmunol.171.3.1172
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ABSTRACT Elimination of T cells during an immune response is mediated by activation-induced cell death (AICD) and CD95-mediated apoptosis. Chronic graft-vs-host disease and T cell-mediated autoimmune diseases are caused by the persistence of activated T cells that escaped tolerance induction by deletion or silencing. To mimic the in vivo situation of long-term activated T cells, we generated an in vitro system using HLA-A1-specific T cells, weekly restimulated by Ag. While short-term activated T cells (two to five rounds of stimulation) were CD95 sensitive and susceptible to AICD, T cells stimulated more than eight times acquired constitutive CD95 resistance and exhibited reduced AICD. Phenotypically, these long-term activated T cells could be identified as effector/memory T cells. The expression of the proforms of the CD95 receptor initiator caspases, caspase-8 and -10, and the effector caspase-3 was strongly decreased in these cells, and only active caspase fragments were detected. In contrast to short-term activated T cells, constitutive CD95 receptor clustering was observed on the cell surface, and caspase-8 was bound to the CD95 receptor in the absence of receptor triggering. After further cross-linking of CD95, additional formation of the death-inducing signaling complex (DISC) was strongly impaired. Reduced DISC formation in long-term activated T cells was associated with the loss of PTEN expression and the increased phosphorylation of protein kinase B. Inhibitors of phosphoinositol 3-kinase restored CD95 sensitivity and DISC formation in long-term activated T cells. These data suggest that defective CD95 signaling in effector/memory T cells may contribute to the apoptosis resistance toward physiological stimuli in T cells mediating tissue destruction in vivo.

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    • "FasL is cleaved from the cell surface by several members of the MMP family ( Kayagaki et al , 1995 ; Webb et al , 2002 ) . Treatment of activated T cells or tumor cell lines with MMP inhibitors in vitro leads to the accumulation of surface FasL ( Strauss et al , 2003 ) . Treat - ment of all three of CTCL cell lines used in the study with a potent broad - spectrum MMP inhibitor had little effect on the level of FasL on the cell surface ( not shown ) . "
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    ABSTRACT: By engaging Fas, Fas ligand (FasL) on activated T lymphocytes induces activation-induced cell death (AICD), and also triggers apoptosis of target cells during immune downregulation. We previously showed that within cutaneous T cell lymphoma (CTCL) lesions, malignant CD4(+) T cells expressing FasL accumulated, and were inversely distributed with CD8(+) T cells. We thus determined the responses of human CTCL cells to AICD and their cytotoxic to Fas(+) target T cells in vitro. CTCL cells expressing Fas were resistant to AICD following activation by CD3 monoclonal antibody (mAb) whereas still undergoing apoptosis if Fas was ligated to Fas mAb. CTCL cell lines, as well as Sezary Syndrome patients' peripheral blood lymphocytes, exhibited ratio-dependent cytotoxicity to Fas(+) Jurkat cells. The kinetic study showed that FasL surface expression was absent before activation, and its expression was low and/or delayed after activation. We therefore hypothesize that CTCL cells express functional FasL possibly contributing to bystander cytotoxicity within tumor infiltrates. In addition, decreased and/or delayed FasL surface expression following activation may in part contribute to their resistance to AICD. Both bystander cytotoxicity and resistance to AICD are likely to contribute to the loss of cytotoxic anti-tumor CD8(+) T cells as well as the accumulation of malignant T cells in CTCL.
    Journal of Investigative Dermatology 05/2005; 124(4):741-50. DOI:10.1111/j.0022-202X.2005.23657.x · 6.37 Impact Factor
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    ABSTRACT: In dieser Arbeit wurde die Depletion alloreaktiver T-Zellen mittels CD95L exprimierender Zellen mit dem Ziel einer möglichen Anwendung von Donor-Lymphozyten-Präparaten nach allogener Stammzelltransplantation (SCT) untersucht. Die Infusion von Donor-Lymphozyten soll vor allem nach haploidenter SCT, bei der hochaufgereinigte Stammzellen verabreicht werden, die benötigte Zeit der Immunrekonstitution verkürzen. Dem Ansatz, CD95L exprimierende Zellen zur Allodepletion zu verwenden, lag die Tatsache zugrunde, dass vor allem aktivierte T-Zellen sensitiv für CD95L-vermittelte Apoptose wurden, während nicht aktivierte T-Zellen eher eine Resistenz gegenüber CD95L aufwiesen. Unter Anwendung gemischter Lymphozytenkulturen zur allogenen Stimulation wurde untersucht, ob CD95L exprimierende Zellen in diesen Kultursystemen effizient Apoptose induzieren können. Dafür erfolgte die Apoptoseinduktion durch lentiviral CD95L transduzierte B-Zell-Linien oder Fibroblasten entweder simultan zur – oder sequentiell nach – allogener Aktivierung der T-Zellen. Es konnte gezeigt werden, dass sowohl allospezifische Proliferation als auch Zytotoxizität nach simultaner Aktivierung und Apoptoseinduktion durch die CD95L exprimierende B-Zell-Linie C1R-A1-CD95L vollständig inhibiert waren. Da diese Zell-Linie ausschließlich HLA-A1 spezifisch stimuliert – und Apoptose induziert, wurde hierdurch zwar die Effektivität der Apoptoseinduktion demonstriert, ohne jedoch klinisch praktikabel zu sein. Im nächsten Schritt wurde für eine potentielle klinische Anwendung ein sequentielles Kultursystem entwickelt, in dem eine EBV-transformierte B-Zell-Linie zur allogenen Stimulation verwendet und nachfolgend Apoptose durch CD95L-exprimierende Zellen induziert wurde. Auch in diesem System zeigten wir eine effektive Inhibition der Immunantwort: in vitro war keine residuale Alloreaktivität mehr nachweisbar. Der Vorteil des sequentiellen Kultursystems liegt in der universellen Einsetzbarkeit einer CD95L-exprimierenden Zell-Linie für die Depletion individueller, HLA-spezifisch stimulierter T-Zellen. Da CD4+CD25+FoxP3+ regulatorische T-Zellen (Treg) für die Unterdrückung alloreaktiver Immunreaktionen und GvHD (Graft-versus-host-disease) nach SCT von großer Bedeutung sind, wurde außerdem deren Präsenz und Funktion nach erfolgter Apoptoseinduktion analysiert. Sowohl im simultanen als auch im sequentiellen Kultursystem blieben funktionelle CD4+CD25+FoxP3+ Treg erhalten. Treg, die hier durch eine mäßiggradige CD25 Expression charakterisiert waren, erwiesen sich als resistent gegenüber CD95L vermittelter Apoptose und wurden in den Kulturen mit C1R-A1-CD95L sogar angereichert. Interessanterweise ging die Expression des Transkriptionsfaktors FoxP3, welcher für die Funktion von Treg essentiell ist, nicht konstant mit einer suppressorischen Wirkung einher. Erstmalig wurde Funktion und FoxP3-Expression nach Apoptoseinduktion untersucht und somit sichergestellt, dass es sich bei der residualen CD4+CD25+ Population tatsächlich um regulatorische T-Zellen handelte. Außerdem deuten unsere Ergebnisse auf eine antigenspezifische Funktion der erhaltenen Treg, was für eine klinische Anwendung von Bedeutung ist, da sonst das Risiko einer generellen Immunsuppression bestünde. Im Rahmen der Dissertation wurde eine Methode zur Depletion alloreaktiver T-Zellen mittels CD95L exprimierender B-Zell-Linien entwickelt, ohne dass regulatorische T-Zellen von der Apoptoseinduktion betroffen waren. Die Kombination aus selektiver Apoptoseinduktion allogen stimulierter T-Zellen und Suppression residualer Alloreaktivität durch Treg führte zu einer maximalen Effektivität des Systems und eröffnet vielversprechende Möglichkeiten für eine therapeutische Nutzung. Within the project as presented in this thesis, depletion of alloreactive human T-cells by CD95L-expressing cells was examined, aiming at transfer of donor-lymphocyte-infusions after allogenetic stem-cell-transplantation (SCT). Specifically after HLA-haploidentical SCT with highly purified stem cells, application of donor-lymphocytes could reduce the required time for immune-reconstitution of donor-derived T-cells. Using a mixed-lymphocyte-culture system for stimulation of HLA-alloreactive T-cells, the approach of CD95L expressing cells as stimulator cells for allodepletion was based on the fact that stimulated T-cells are highly sensitive towards CD95L-mediated apoptosis, while unstimulated T-cells are rather resistant. It was investigated, whether stimulation by CD95L-expressing cells could result in efficient apoptosis in simultaneous or sequential culture systems. To this extent, induction of apoptosis by lentivirally transducted CD95L-expressing B-cell-lines or fibroblasts was carried out either simultaneously to – or sequentially after – allogeneic activation of T-cells. It was demonstrated, that allospecific proliferation and cytotoxicity after simultaneous activation and induction of apoptosis were completely abolished by the CD95L-expressing B-cell-line C1R-A1-CD95L. Although we could demonstrate high effectivity of allodepletion, this system was not of clinical practicability because of solely HLA-A1 specific stimulation and apoptosis-induction by C1R-A1-CD95L. As a next step, a sequential culture system was developed for potential clinical use: an EBV-transformed B-cell-line was used for HLA-specific, allogeneic stimulation and apoptosis was induced in a second step by CD95L-expressing cells. In this system, we demonstrated effective inhibition of allogenetic immunoreactions after sequential activation and induction of apoptosis by CD95L: residual alloreactivitiy was abrogated. The advantage of this sequential culture system is the universal applicability of a CD95L-expressing cell-line for depletion of individually, HLA-specific allogeneic stimulated T-cells - thereby clinical applicability is given in principle. CD4+CD25+FoxP3+ regulatory T-cells (Treg) have been proven of great importance for prevention and amelioration of alloreactive immune reactions and Graft-versus-host-Disease after SCT. Therefore, presence and function of Treg was examined after stimulation and induction of apoptosis by CD95L-expressing cells. In both culture systems, simultaneous and sequential, functional CD4+CD25+FoxP3+ Treg were preserved. Treg with intermediate CD25 expression were resistant towards CD95L mediated apoptosis and even enriched after culture with C1R-A1-CD95L. Interestingly, expression of the transcription factor FoxP3 did not necessarily correlate with suppressive function. Function of Treg and expression of FoxP3 was examined after induction of apoptosis for the first time. It was proven that indeed residual CD4+CD25+ cells were regulatory T-cells, and, furthermore, our results indicate antigen-specific function of residual Treg. This is of great importance for potential clinical use to prevent the risk of an otherwise general immune-suppressive function by Treg. In summary, the work presented in this thesis provides a method for effective depletion of alloreactive T-cells without affecting regulatory T-cells by using a CD95L-expressing B-cell-line. The combination of selective induction of apoptosis in stimulated, HLA-alloreactive T-cells and suppression of residual alloreactivity by Treg resulted in a highly efficient system and opens promising options for therapeutic applications.
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    ABSTRACT: ENGLISH ABSTRACT: In just 25 years since the first reported cases in 1981, the number of Human Immunodeficiency virus (HIV) infected people has risen to 65 million, and over 25 million have died of acquired immunodeficiency syndrome (AIDS). Sub-Saharan Africa accounts for 67% of all people living with HIV and 72% of deaths in this region were AIDS related. Tuberculosis (TB) is one of the most common opportunistic infections in AIDS patients, particularly in developing countries, where 60 - 70% of TB cases occur in HIV-1-infected persons. HIV-1 is a high risk factor for the development of TB, the reactivation of a latent Mycobacterium tuberculosis infection and also progressive TB. CD8+ Cytotoxic T Lymphocytes (CTL) are pivotal in the host immune response to HIV infection. CTL are associated with resolution of acute infection and with reduction in viral load. Studies in macaques and humans indicate the importance of CTL in the control of HIV infection, where reduction in CD8+ T cell number has been correlated with progression to AIDS. The current study was a cross-sectional descriptive study of CD8+ T cells of HIV+ adult South Africans with and without TB co-infection (TB disease). The cohort consisted of anti-retroviral therapy (ART) naive patients and all CTL analyses were carried out on peripheral blood mononuclear cells (PBMCs). A total of 60 South African adults from the Western Cape were utilized in this study, including 15 healthy controls; 30 HIV+TB-individuals and 15 HIV+TB+ individuals. Expression of phenotypic, activation and functional markers were investigated by flow cytometry with the use of fluorochomeconjugated antibodies. The markers examined included the novel activation marker CD137, the CTL associated markers Perforin, Granzyme A, CD107a/b, Fas (CD95), and FasL (CD95L), intracellular cytokines IFN-y and TNF-a and the chronic HIV CTL dysfunction marker PD-1. HIV infection alone was associated with increased baseline expression of TNF-a, Perforin, Granzyme A, PD-1, Fas (CD95), and FasL (CD95L), but not CD137(4-1BB) or IFN-y as compared to uninfected controls. TB co-infection resulted in further increased baseline expression of TNF-a, perforin, PD-1, FasL (CD95L), as well as increased IFN-y. HIV-1 antigen (gag)-specific stimulation in vitro indicated that in HIV infection was associated with antigen-specific upregulation of activation and cytotoxicity markers CD137, IFN-y, TNF-a, Fas, FasL and CD107a/b. In TB co-infection a reduction in antigen-specific degranulation (CD107a/b up-regulation) and also Fas and FasL expression was observed. TB co-infection (in the form of active pulmonary TB) reduced antigen-specific CTL functional activity, but simultaneously there was an association with increased baseline PD-1 expression and also cytolytic marker expression (Fas, FasL, TNF-a). These cytolytic markers could be involved in non-antigen-specific bystander target cell death. The expression of the co-stimulatory molecule CD137 appeared to correlate with interferon-y production and levels of degranulation, confirming its usefulness as a putative surrogate marker of functional responsiveness. These data indicate that in addition to impacting on CD4 T cell function, TB co-infection leads to higher baseline expression of CTL-associated markers, but to dysfunctional antigen-specific CTL responses. AFRIKAANSE OPSOMMING: Slegs vyf en twintig jaar na die eerste berigte van die menslike immuniteitsgebrekvirus (MIV) in 1981, het die getal MIV-geinfekteerde individue gestyg tot 65 miljoen en het meer as 25 miljoen mense alreeds gesterf aan die verworwe immuniteitsgebrek sindroom (VIGS). Sub Sahara Afrika maak 67% uit van alle HIV gevalle en het `n MIVverwante doodsyfer van 72%. Een van die algemeenste opportunistiese infeksies in VIGS pasiente is Tuberkulose (TB). In ontwikkelende lande, veral, kom 60-70% van TB gevalle voor in MIV-1 geinfekteerde individue. MIV-1 is `n hoe risiko faktor vir die ontwikkeling van TB, die heraktivering van latente Mycobacterium tuberculosis infeksie en progressiewe TB. Die CD8+ sitotoksiese T Limfosiete (STL) se immuun reaksie teen `n MIV infeksie is noodsaaklik en word geassosieer met `n resolusie van die akute infeksie en `n afname in viruslading. Studies in die mens en macaque het getoon dat sitotoksiese T limfosiete belangrik is vir die beheer van MIV infeksies aangesien die afname in CD8+ sel getalle korreleer met die verloop tot VIGS. Hierdie deursnit-beskrywende studie het die CD8+ T selle van MIV+ volwasse Suid-Afrikaners, met of sonder`n TB mede-infeksie, ondersoek. STL analise is gedoen op die perifere bloed mono-nuklere selle (PBMS) van pasiente wat geen teen-retrovirale terapie (TRT) ontvang het nie. `n Totaal van sestig Suid-Afrikaanse volwassenes van die Wes-Kaap het deelgeneem aan die studie wat 15 gesonde kontroles; 30 MIV+TBen 15 MIV+TB+ individue ingesluit het. Die uitdrukking van fenotipiese, aktiverings en funksionele merkers is ondersoek deur middel van vloeisitometrie en fluorochroomgekonjugeerde teenliggaampies. Laasgenoemde het ingesluit die nuwe aktiversingsmerker CD 137, die STL geassosieerde merkers Perforien en Gransiem A, CD 107a/b, Fas (CD95) en FasL (CD95L), intrasellulere sitokiene IFN-y en TNF-a en PD-1, die merker vir chroniese MIV CTL disfunksie. Daar is gevind dat `n TB mede-infeksie (in die vorm van aktiewe pulmonere TB) die antigeen-spesifieke STL funksie verlaag en terselftertyd `n verhoging in die uitdrukking van PD-1 en sitolitiese merkers (Fas, FasL, TNF-a) bewerkstellig. Hierdie sitolitiese basislyn merkers is moontlik betrokke by die dood van nie-antigeen-spesifieke omstander teiken selle. Die uitdrukking van die mede-stimulatoriese molekule CD 137 blyk om te korreleer met die produksie van STL IFN-y en die vlakke van degranulasie. Dit bevestig die merker se bruikbaarheid as `n gewaande surrogaat merker vir funksionele reaksies. Die data toon verder dat `n TB mede-infeksie nie net `n effek het op die CD4 T sel funksie nie, dit lei ook tot `n verhoogde basislyn uitdrukking van STLgeassosieerde merkers, maar met disfunksionele antigeen-spesifieke STL reaksies. Hierdie studie het bepaal dat `n MIV infeksie verbind word met `n toename in die basislyn uitdrukking van TNF-a, Perforien, Gransiem A, PD-1, Fas (CD95) en FasL (CD95L). Dit is egter nie die geval wanneer die uitdrukking van CD 137 (4-1BB) of IFN-y vergelyk word met nie-geinfekteerde kontroles. `n TB mede-infeksie het `n verdere toename in die uitdrukking van TNF-a, Perforien, PD-1, FasL (CD95L) getoon, asook `n verhoging in IFN-y vanaf die basislyn. In vitro MIV-1 antigeen (gag)-spesifieke stimulasies het aangedui dat `n MIV infeksie met die antigeen-spesifieke op-regulasie van aktiverings en sitotoksiese merkers CD137, IFN-y, TNF-a, Fas, FasL en CD107a/b geassosieer word. In `n TB mede-infeksie, is `n verlaging van antigeen-spesifieke degranulasie (CD 107a/b op-regulasie) asook die uitdrukking van Fas en FasL waargeneem. Bibliography Thesis (MScMedSc (Pathology. Medical Virology))--University of Stellenbosch, 2010. The Poliomyelitis Research Foundation The National Health Laboratory Service
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