An equilibrium between proinflammatory and anti-inflammatory immune responses is essential for maternal tolerance of the fetus throughout gestation. To study the participation of fetal tissue-derived factors in this delicate immune balance, we analyzed the effects of human chorionic gonadotropin (hCG) on murine Treg cells and Th17 cells in vitro, and on pregnancy outcomes, fetal and placental growth, blood flow velocities and remodeling of the uterine vascular bed in vivo. Compared with untreated CD4+CD25+ T cells, hCG increased the frequency of Treg cells upon activation of the LH/CG receptor. hCG, with the involvement of IL-2, also interfered with induced differentiation of CD4+ T cells into proinflammatory Th17 cells. In already differentiated Th17 cells, hCG induced an anti-inflammatory profile. Transfer of proinflammatory Th17 cells into healthy pregnant mice promoted fetal rejection, impaired fetal growth and resulted in insufficient remodeling of uterine spiral arteries, and abnormal flow velocities. Our works show that proinflammatory Th17 cells have a negative influence on pregnancy that can be partly avoided by in vitro re-programming of proinflammatory Th17 cells with hCG.

Το καρκίνωμα εκ πλακώδους επιθηλίου ή πλακώδες καρκίνωμα (ΠΚ) του δέρματος είναι ο δεύτερος πιο συχνός καρκίνος του δέρματος και εμφανίζεται συνήθως σε έδαφος ακτινικής υπερκεράτωσης (ΑΚ). Τα νεοπλασματικά κύτταρα εκφράζουν μια ποικιλία αντιγόνων προσελκύοντας με αυτό τον τρόπο λεμφοκύτταρα, τα διηθούντα τον όγκο λεμφοκύτταρα (Tumor Ιnfiltrating Lymphocytes-TILs) στο μικροπεριβάλλον του όγκου. Με βάση το ανοσοφαινοτυπικό τους προφίλ η πλειοψηφία των TILs εκφράζει το μόριο CD3 και αφορά σε Τ-λεμφοκύτταρα. Αυτά με την σειρά τους διαχωρίζονται σε CD8+ Τ-κυτταροτοξικά λεμφοκύτταρα και CD4+ Τ-λεμφοκύτταρα. Η έκφραση του δείκτη CD25+ επιτρέπει τον διαχωρισμό των CD4+ λεμφοκυττάρων σε δύο επιπλέον υποομάδες, τα T-επικουρικά (CD4+/CD25-) και τα Τ-ρυθμιστικά (CD4+/CD25+) κύτταρα. Ο πιο αξιόπιστος δείκτης για την ανίχνευση των Τ-ρυθμιστικών κυττάρων (Tregs) θεωρείται ο Forkhead box P3 (Foxp3). Ο αυξημένος αριθμός των TILs έχει συσχετισθεί με την πρόγνωση και τη θεραπευτική αντιμετώπιση σε μια ποικιλία νεοπλασμάτων, περιλαμβανομένων και καρκίνων του δέρματος όπως το μελάνωμα. Η μεθοδολογία αξιολόγησης της λεμφοκυτταρικής διήθησης, ωστόσο, δεν έχει ακόμα αποσαφηνισθεί και ποικίλλει στα διάφορα είδη νεοπλασμάτων. Στα πλαίσια της παρούσας διπλωματικής εργασίας η ανίχνευση των Τ-λεμφοκυττάρων έγινε με ανοσοϊστοχημική μέθοδο με τη χρήση των κατάλληλων αντισωμάτων (CD3, CD4, CD8, Foxp3). Η παρουσία των κυττάρων αξιολογήθηκε με τη χρήση φωτονικού μικροσκοπίου με δυο διαφορετικά ημιποσοτικά συστήματα βαθμολόγησης για όλους τους μελετηθέντες Τ-κυτταρικούς υποπληθυσμούς καθώς και με ένα τρίτο σύστημα ποσοτικής καταμέτρησης για τα Τregs. Σκοπός ήταν να γίνει σύγκριση των μεθόδων ώστε να βρεθεί η καταλληλότερη μεθοδολογία αξιολόγησης της λεμφοκυτταρικής διήθησης στο πλακώδες καρκίνωμα του δέρματος, να υπολογιστεί ο αριθμός των Tregs στις μελετούμενες οντότητες χρησιμοποιώντας το βέλτιστο σύστημα βαθμολόγησης και να αναζητηθούν διαφορές στην πυκνότητα των λεμφοκυτταρικών υποπληθυσμών μεταξύ των οντοτήτων, οι οποίες μπορεί να έχουν παθογενετική ή θεραπευτική κλινική σημασία. / Squamous cell carcinoma (SCC) of the skin is the second most common skin cancer. It usually develops in a background of actinic keratosis (AK). Neoplastic cells express a variety of antigens, attracting lymphocytes in the tumor microenvironment (Tumor Ιnfiltrating Lymphocytes-TILs). Based on the immunophenotypic profile, the majority of TILs expresses the CD3 molecule, so they are T-lymphocytes. These in turn are divided into CD8+ cytotoxic T-lymphocytes and CD4+ T-lymphocytes. The expression of CD25 enables the separation of CD4+ lymphocytes in two further subgroups, the T-helper (CD4+/CD25-) and T-regulatory (CD4+/CD25+) cells. The most reliable marker for the detection of T-regulatory cells (Tregs) is considered to be the Forkhead box P3 (Foxp3). The increased number of TILs is associated with prognosis and treatment in a variety of tumors, including skin cancers, such as melanoma. The evaluation methology of lymphocytic infiltration, however, is not yet clarified and varies in different types of tumors. In the context of this thesis, the detection of T-lymphocytes was performed by immunohistochemistry using the appropriate antibodies (CD3, CD4, CD8, Foxp3). The presence of the cells was evaluated using light microscopy with two different semiquantitative scoring systems, for all the T-lymphocyte subpopulations, as well as with a third system of quantification of Tregs. The purpose was to compare the methods in order to find the most suitable methodology for the evaluation of the lymphocytic infiltration in squamous cell carcinoma of the skin, to evaluate the number of Tregs in the studied entities using the best method and examine for differences in the density of lymphocyte subpopulations.

Differenzierungsentscheidungen von Zellen werden durch die Integration mehrerer Stimuli bestimmt. Die Differenzierung von Helfer-T-Zellen (Th-Zellen) ist hierfür ein gut untersuchtes Beispiel: reife Th-Zellen entwickeln sich beim Kontakt mit einem für sie spezifischen Antigen zu einem spezialisierten Subtyp, der von den in ihrer Umgebung vorhandenen Zytokinen abhängt und exprimieren dann einen spezifischen Mastertranskriptionsfaktor. Die häufigsten Th-Zell-Subtypen sind T-bet-exprimierende Th1-Zellen und GATA-3-exprimierende Th2-Zellen. Neuere Entdeckungen bezüglich der Plastizität von Th-Zell-Subtypen sowie die Existenz von T-bet+GATA-3+ Hybrid-Phänotypen haben die detaillierte Untersuchung vom Differenzierungsprozessen von Th-Zellen mit komplexer Zytokinsignale motiviert. Dazu haben wir systematisch die Zytokine IFN-g, IL-12 und IL-4 während der primären Differenzierung Th-Zellen titriert und Signaltransduktion und Zielgenexpression quantifiziert. Der Umfang und die Komplexität der Daten machten eine systematische Analyse notwendig, um involvierte Mechanismen genau zu identifizieren. Lineare Regressionsanalyse wurde verwendet, um die Netzwerktopologie zu extrahieren, wobei schon bekannte und zahlreiche neue Interaktionen vorausgesagt wurden. Die prognostizierte Netzwerktopologie wurde dann verwendet, um ein mechanistisches, mathematisches Modell der Zytokinsignalintegration zu entwickeln. Diese Methode hat ein hochgradig vernetztes regulatorisches Netzwerk inferiert. Bisher nicht beschriebene Funktionen von STAT-Proteine, die die Neuverkabelung des Netzwerkes während der Differenzierung vermitteln, wurden vorhergesagt. Ausgewählte neue Interaktionen wurden in gezielten genetischen Experimenten bestätigt. Während gegenseitige Inhibitionsmotive oft als kanonische digitale Schalter interpretiert werden, funktioniert das Th-Zell-Netwerk als ein Rheostat, der Variationen der Zytokinsignale in graduelle Expressionsänderungen der Mastertranskriptionsfaktoren übersetzt. Unsere Arbeit erklärt mechanistisch das beobachtete Kontinuum von Th-Zelldifferenzierungszuständen entlang der Th1-Th2-Achse und beschreibt eine quantitative Methode für die datenbasierte Inferenz zellulärer Netzwerke der Signalintegration. / Cell-fate decisions are governed by the integration of multiple stimuli. Th cell differentiation is a well-studied example thereof: mature Th cells differentiate into a specialised subtype upon encounter with their cognate antigen depending on the polarising cytokines present in their environment and start expressing specific master transcription factors. The most common Th cell subtypes are T-bet-expressing Th1 cells and GATA-3-expressing Th2 cells. Recent discoveries concerning the plasticity of Th cell subtypes as well as the existence of stable T-bet+GATA-3+ hybrid Th1/2 phenotypes have stimulated the detailed study of the differentiation process under different assumptions than the hitherto valid paradigm of single master transcription factor expression by using complex cytokine signals as inputs. Here, we developed a data-based approach for inferring the molecular network underlying the differentiation of T-bet- and/or GATA-3 expressing lymphocytes. We performed systematic titrations of the polarising cytokines IFN-g, IL-12 and IL-4 during primary differentiation of Th cells and quantified signal transduction as well as target-gene expression. The size and complexity of the dataset made a systematic analysis necessary to identify the mechanisms involved. To extract the network topology, we used linear regression analysis, retrieving known regulatory mechanisms and predicting numerous novel ones. This network topology was used to develop a mechanistic mathematical model of cytokine signal integration. This approach inferred a highly connected regulatory network. Previously undescribed functions of STAT proteins mediating network rewiring during differentiation were predicted. Selected new interactions were confirmed by experiments using gene-deficient cells. Importantly, while mutual-inhibition motifs are often considered canonical digital switches, the inferred Th-cell network acts as a rheostat, generating a continuum of differentiated states along the Th1-Th2 axis. This work explains the observed Th1-Th2 cell fate continuum mechanistically and provides a quantitative framework for the data-based inference of cellular signal integration networks.

Indiana University-Purdue University Indianapolis (IUPUI) / The transcriptional repressor BCL6 has been shown to be essential for the differentiation of germinal center (GC) B cells and follicular T helper (TFH) cells. The interaction of TFH and GC B cells is necessary for the development of high affinity antibodies specific for an invading pathogen. Germline BCL6-deficient mouse models limit our ability to study BCL6 function in T cells due to the strong inflammatory responses seen in these mice. To overcome this, our lab has developed a new BCL6 conditional knockout (cKO) mouse using the cre/lox system, wherein the zinc finger region of the BCL6 gene is flanked by loxP sites. Mating to a CD4-Cre mouse allowed us to study the effects of BCL6 loss specifically in T cells, without the confounding effects seen in germline knockout models. Using this cKO model, we have reaffirmed the necessity of BCL6 for TFH differentiation, including its role in sustained CXCR5 surface expression, a signature marker for TFH cells. This model also allowed us to recognize the role of BCL6 in promoting the expression of PD-1, another key surface marker for TFH cells. Without BCL6, CD4+ T cells cannot express PD-1 at the high levels seen on TFH cells. Our discovery of DNMT3b as a target for BCL6 suggests BCL6-deficient T cells have increased DNA methyltransferase activity at the PD-1 promoter. This data establishes a novel pathway for explaining how BCL6, a transcriptional repressor, can activate genes. Experiments with the BCL6 cKO model have also established a role for BCL6 in naïve CD4+ T cell activation. Furthermore, we did not observe increased differentiation of other helper T cell subsets, in contrast to what has been reported elsewhere with germline BCL6-deficient models. Unexpectedly, we found decreased T helper type 2 (Th2) cells, whereas mouse models with a germline mutation of BCL6 have increased Th2 cells. These results indicate that BCL6 activity in non-T cells is critical for controlling T cell differentiation. Finally, using an HIV-1 gp120 immunization model, we have, for the first time, shown BCL6-dependent GCs to be limiting for antibody development and affinity maturation in a prime-boost vaccine scheme.