Selective depletion of regulatory T cells to enhance the CD8+ T cell response during a Friend retrovirus infection of mice

Zytotoxische CD8+ T-Zellen spielen bei der Kontrolle der akuten FV Infektion eine maßgebliche Rolle. Allerdings wird ihre Funktion im Verlauf der akuten Infektion von regulatorischen T-Zellen supprimiert, bevor sie in der Lage sind, die Virus-infizierten Zellen vollständig zu eliminieren. Nach Depletion von regulatorischen T-Zellen während der akuten Infektion wurde in Vorarbeiten in Form meiner Diplomarbeit eine Expansion von CD8+ Effektorzellen, die zytotoxische Moleküle exprimierten, nachgewiesen. Diese Analysen basierten ausschließlich auf der durchflusszytometrischen Bestimmung von absoluten Zellzahlen von Effektor T-Zellpopulationen. Um die funktionelle Aktivität der expandierten CD8+ T-Zellen bestimmen zu können, wurde im ersten Teil der vorliegenden Arbeit das zytotoxische Potential von CD8+ T-Zellen in vivo untersucht. Dazu wurde ein in vivo CTL-Test etabliert und eine Kinetik der CD8+ T-Zellzytotoxizität während der akuten FV-Infektion angefertigt. Mit diesen Untersuchungen wurde gezeigt, dass es eine Korrelation zwischen der Anzahl an CD8+ Effektor T-Zellen und der Stärke der detektierten zytotoxischen Aktivität in vivo gibt. Danach wurde die in vivo Zytotoxizität von CD8+ T-Zellen nach Depletion von regulatorischen T-Zellen bestimmt und eine verbesserte Zytotoxizität der CD8+ Effektor T-Zellen nachgewiesen. Außerdem zeigten die Untersuchungen der Zytokinproduktion von CD8+ Effektorzellen nach Depletion von regulatorischen T-Zellen während der akuten Infektion, dass signifikant mehr Zytokine in Abwesenheit von regulatorischen T-Zellen von CD8+ T-Zellen produziert wurden. Diese Daten beweisen erstmals, dass regulatorische T-Zellen in einer akuten Retrovirus-Infektion einen supprimierenden Einfluss auf die antiviralen Funktionen von CD8+ T-Zellen haben. Der Schwerpunkt dieser Arbeit lag auf der Untersuchung der chronischen Infektion mit Retroviren. In diesem Teil der Arbeit wurde der Funktionsverlust von CD8+ T-Zellen während der chronischen FV-Infektion, der durch die supprimierende Aktivität von regulatorischen T-Zellen verursacht wird, detailliert charakterisiert. Das Hauptaugenmerk lag dabei auf der Fragestellung, ob es in der chronischen Phase der FV-Infektion möglich ist, durch Depletion von regulatorischen T-Zellen die Dysfunktionalität der CD8+ Effektor T-Zellen aufzuheben. Die Resultate dieser Versuche zeigten, dass die Depletion von regulatorischen T-Zellen in der chronischen FV-Infektion zu einer erhöhten Anzahl an Virus-spezifischen CD8+ T-Zellen führte. Diese Zellen zeigten eine gesteigerte Proliferationsrate und eine erhöhte Produktion von Zytokinen und zytotoxischen Molekülen. Dies ließ auf eine verbesserte zytotoxische Aktivität der CD8+ Effektorzellen schließen, was mit einem in vivo CTL-Test bestätigt werden konnte. Die Aufhebung der durch regulatorische T-Zellen vermittelten Suppression von CD8+ Effektorzellen in der chronischen Infektionsphase ermöglichte also eine Reaktivierung der vorher dysfunktionalen Zellen. Eine Analyse der Viruslast zeigte, dass die Depletion von regulatorischen T-Zellen eine signifikante Reduktion der Viruslast in chronisch infizierten Tieren zur Folge hatte, die nicht nur kurzfristig war, sondern über einen längerfristigen Zeitraum Bestand hatte. Diese Ergebnisse verdeutlichen, dass die durch regulatorische T-Zellen vermittelte Immunsuppression ein signifikanter Faktor in der Etablierung und Aufrechterhaltung von chronischen viralen Infektionen ist. Immuntherapien, die sich auf eine Manipulation von regulatorischen T-Zellen konzentrieren, könnten also eine erfolgversprechende Strategie für die Behandlung von chronischen Infektionskrankheiten darstellen.Cytotoxic CD8+ T cells are critical for the control of acute FV infection. However, they are functionally suppressed by regulatory T cells, compromising the ability of CD8+ T cells to completely eliminate the virus-infected cells. On the basis of preliminary work performed during my diploma, it was observed that depletion of regulatory T cells during the acute infection resulted in an expansion of effector CD8+ T cell population with enhanced production of cytotoxic molecules. These analyses were exclusively based on determination of absolute cell numbers of effector T cell populations by Flow cytometry. To identify the functional activity of these expanded CD8+ T cells, the cytotoxic potential of CD8+ T cells was analyzed in the first part of the present work. For that purpose an in vivo CTL assay was established and firstly, the kinetics of the CD8+ T cell cytotoxicity in the course of the acute FV infection was obtained. These studies indicated a correlation of the numbers of CD8+ T cells with the magnitude of the detected cytotoxic activity in vivo. Secondly, the in vivo cytotoxicity of the CD8+ T cells after depletion of regulatory T cells was analyzed. After depletion of regulatory T cells, an enhanced cytotoxicity of CD8+ T cells was found. Correlating with that the analyses of cytokine production after depletion of regulatory T cells showed an enhanced production of cytokines by the CD8+ T cells. These results demonstrated that regulatory T cells suppress the antiviral functions of CD8+ T cells during the acute FV infection. The main focus of the present work was on the analysis of chronic FV infection. In this second part of the work, the dysfunction of the CD8+ T cells during chronic infection, caused by the immunosuppressive regulatory T cells, was characterized in detail. Therefore, the emphasis was on the question, if it could be possible to abrogate the dysfunctionality of the CD8+ T cells during the chronic phase of infection by depleting regulatory T cells. The results of these experiments showed enhanced numbers of virus-specific CD8+ T cells after transient depletion of regulatory T cells. These cells showed increased proliferation rates, an increased production of cytokines and cytotoxic molecules. These results indicated an improved cytotoxic activity of the effector CD8+ T cells, which was verified by analyzing cytotoxicity in an in vivo CTL assay. In this respect, reversal of regulatory T cell-mediated suppression probably allowed a regain of function of previously exhausted effector CD8+ T cells. A conclusive analysis of the viral loads after short term depletion of regulatory T cells showed a significant long term reduction of the viral setpoints in chronic infected mice. These results demonstrate that regulatory T cell-mediated immunosuppression can be a significant factor in the maintenance of chronic viral infections and that Treg-targeted immunotherapy could be a valuable component in therapeutic strategies to treat chronic infectious diseases

Woodchuck hepatitis virus core antigen-based DNA and protein vaccines induce qualitatively different immune responses that affect T cell recall responses and antiviral effects

AbstractT helper type 1 (Th1) immunity was considered to play a dominant role in viral clearance of hepadnaviral infection. However, pre-primed Th2 type responses were able to efficiently control hepadnaviral infection in animal models. We investigated how pre-primed Th1/2 responses control hepadnaviral replication using the newly established mouse models. DNA (pWHcIm, pCTLA-4-C) and protein vaccines based on the nucleocapsid protein (WHcAg) of woodchuck hepatitis virus (WHV) primed specific immune responses with distinct features. The pre-primed responses determined the characteristics of recall responses if challenged with a WHcAg-expressing adenoviral vector. Vaccination with pWHcIm and pCTLA4-C facilitated viral control in the hydrodynamic injection model and reduced WHV loads by about 3 and 2 logs in WHV-transgenic mice, respectively, despite of different kinetics of specific CD8+ T cell responses. Thus, pre-primed Th2-biased responses facilitate the development of CD8+ T cell responses in mice compared with naïve controls and thereby confer better viral control

Proteomic analysis of stratum corneum in Cutaneous T-Cell Lymphomas and psoriasis

Stratum corneum collected by tape stripping from 10 and 24 subjects with cutaneous T-cell lymphomas (CTCL) or psoriasis, respectively, were compared using quantitative label-free mass spectrometry analysis. A non-supervised statistical analysis (Posneg NMF) based on 352 differentially expressed proteins in both CTCL and psoriasis samples was able to separate the two disease groups and finally able to identify a set of 112 proteins that contributed most and significantly to the separation when compared to non-lesional samples. In addition, Luminex assay revealed that the increase in the amount of chemokines related to the inflammatory response, and immune cell infiltration and recruitment in lesional stratum corneum in CTCL, including CXCL8, CXCL9, CXCL10, CCL27, TNF and sICAM-1 was in agreement with published data on entire skin biopsies. Proteome analysis using quantitative methods including mass spectrometry and Luminex technology offered the possibility to investigate the relevant protein signature in CTCL and may be helpful to diagnose and investigate the efficacy of treatments in clinical investigations using non-invasive methods in future

Combining Regulatory T Cell Depletion and Inhibitory Receptor Blockade Improves Reactivation of Exhausted Virus-Specific CD8⁺ T Cells and Efficiently Reduces Chronic Retroviral Loads

<div><p>Chronic infections with human viruses, such as HIV and HCV, or mouse viruses, such as LCMV or Friend Virus (FV), result in functional exhaustion of CD8⁺ T cells. Two main mechanisms have been described that mediate this exhaustion: expression of inhibitory receptors on CD8⁺ T cells and expansion of regulatory T cells (Tregs) that suppress CD8⁺ T cell activity. Several studies show that blockage of one of these pathways results in reactivation of CD8⁺ T cells and partial reduction in chronic viral loads. Using blocking antibodies against PD-1 ligand and Tim-3 and transgenic mice in which Tregs can be selectively ablated, we compared these two treatment strategies and combined them for the first time in a model of chronic retrovirus infection. Blocking inhibitory receptors was more efficient than transient depletion of Tregs in reactivating exhausted CD8⁺ T cells and reducing viral set points. However, a combination therapy was superior to any single treatment and further augmented CD8⁺ T cell responses and resulted in a sustained reduction in chronic viral loads. These results demonstrate that Tregs and inhibitory receptors are non-overlapping factors in the maintenance of chronic viral infections and that immunotherapies targeting both pathways may be a promising strategy to treat chronic infectious diseases.</p></div

Transient depletion of regulatory T cells in transgenic mice reactivates virus-specific CD8+ T cells and reduces chronic retroviral set points

Although chronic infections with viruses such as HIV and hepatitis C virus have been associated with regulatory T cell (Treg)-mediated suppression of virus-specific CD8+ T-cell activity, no causal relationship between Tregs and chronic viral set points has been established. Using transgenic mice in which Tregs can be selectively ablated, we now show that transient depletion of Tregs during a chronic retroviral infection allows exhausted CD8+ T cells to regain antiviral functions, including secretion of cytokines, production of cytotoxic molecules, and virus-specific cytolytic activity. Furthermore, short-term Treg ablation resulted in long-term reductions in chronic virus loads. These results demonstrate that Treg-mediated immunosuppression can be a significant factor in the maintenance of chronic viral infections and that Treg-targeted immunotherapy could be a valuable component in therapeutic strategies to treat chronic infectious diseases

Viral loads in chronic infection after Treg depletion and/or blocking of inhibitory pathways.

<p>(<b>A</b>) Spleens of chronically FV-infected mice from the different treatment groups were analyzed for viral loads by infectious center assays 1 day after termination of treatment. Each dot represents an individual mouse. Data were pooled from 4 to 6 independent experiments with similar results. Statistically significant differences are indicated by asterisks (*<0.05; ***<0.0005; analysis of variance [ANOVA] nonparametric, Dunn's multiple comparison test). (<b>B</b>) To study long-term effects of the treatment spleens of mice from the different groups were analyzed for viral loads by infectious center assay at 21 days post treatment (dpt). Each dot represents an individual mouse.</p