Amino acid availability plays an important role in modulating the activity of T-cells. One of the pathways employed by T-cells to sense nutrient levels is the “mammalian target of rapamycin” (mTOR) pathway that is inhibited in response to nutrient depletion. Indoleamine 2,3-dioxygenase (IDO) is the first and rate-limiting enzyme along the tryptophan catabolising kynurenine pathway. T-cells are very sensitive to lack of this essential amino acid in their microenvironment and this confers strong immunomodulatory properties to cells expressing active IDO. It therefore has a significant physiological role as a homeostatic mechanism used in mammalian organisms to dampen excessive activation of the immune system but is also used as an immune evasion mechanism by many cancers. In this study, we investigated the IDO inhibitory properties and mechanism of action of the tryptophan metabolite 3-hydroxyanthranilic acid (3-HAA) that potentially forms a negative feedback loop in the kynurenine pathway. We studied the molecule in enzymatic assays, in live cells and discovered that it inhibits IDO in an indirect way via the formation of hydrogen peroxide. Secondly, we looked at the effects of tryptophan and its metabolites on T-cell proliferation and mTOR activity, and discovered a metabolite that inhibits T-cell proliferation. Lastly we examined mechanisms of T-cell suppression employed by myeloid derived suppressor cells (MDSCs), focusing on their ability to deplete amino acids from their microenvironment. We were able to exclude tryptophan consumption as a suppressive mechanism and established that by manipulating extracellular concentrations of several amino acids other than arginine and cysteine – that are known to be utilised by MDSCs - we were able to reduce their inhibitory properties. In summary, we have described in detail how 3-HAA inhibits IDO in in vitro assays, outlined how some tryptophan metabolites can inhibit T-cell proliferation, and clarified aspects of suppressive mechanism employed by MDSCs.

In der vorliegenden Arbeit wurde die Rolle des Zytokins IFN-gamma in einer Th1-vermittelten Entzündungsreaktion untersucht. Ausgangspunkt war die Beobachtung, dass die Blockade des als proinflammatorisch beschriebenen Zytokins IFN-gamma zu einer chronischen Entzündung führte. Ziel war also das Erkennen und Verstehen der Regulationsmechanismen, sowie die Identifizierung beteiligter Zelltypen und beteiligter Moleküle. Mit Hilfe von Knochenmarkschimären konnte gezeigt werden, dass die Zelle, die von Th1-Zellen sekrektiertes IFN-gamma erkannte, haematopoietischen Ursprungs war. Zum weiteren Verständnis der Regulationsmechanismen wurden Tiere unter IFN-gamma-Blockade mit Tieren, die einen Kontrollantikörper erhielten, verglichen. Mittels Immunhistochemie konnte in Kontrolltieren eine starke Expression von iNOS am Ort der Entzündung nachgewiesen werden, während in Tieren, in denen IFN-gamma blockiert wurde, keine iNOS Expression nachzuweisen war. Mit Hilfe von iNOS-defizienten Mäusen konnte gezeigt werden, dass NO tatsächlich funktionell essentiell in der IFN-gamma abhängigen Selbstlimitation der Th1-vermittelten Entzündungsreaktion war. Weitere Charakterisierung der iNOS-exprimierenden Zellen mittels FACS-Analyse ergab, dass iNOS-produzierende Zellen den Oberflächenmarker CD11b exprimierten. Diese iNOS-produzierenden Zellen waren fast ausschließlich am Ort der Entzündung zu finden. Die funktionelle in vitro Charakterisierung dieser Zellen nach ex vivo Isolierung ergab, dass diese Zellen die Proliferation von CD4+ T-Zellen supprimierten und deshalb als Myeloide-Suppressor-Zellen bezeichnet werden können. Der hier untersuchte IFN-gamma vermittelte Regulationsmechanismus scheint auf andere T-Zell-Systeme übertragbar zu sein, da IFN-gamma Blockade in einer durch CD8+ Effektor-T-Zellen vermittelten Entzündung auch zu einer Verlängerung der Entzündung führte. Zusammenfassend konnte gezeigt werden, dass das Zytokin IFN-gamma während der Effektorphase einer Immunreaktion wichtig für die Selbstlimitation ist. / The aim of the work was to understand the role of the cytokine IFN-gamma in a Th1 dependent inflammation. Starting point was the observation that the blockade of IFN-gamma, which is generally regarded as a proinflammatory cytokine, led to a more severe inflammation. We were therefore aiming at a better understanding of the mechanism of regulation, the identification of important cell types and downstream effector molecules. With the help of bone marrow chimeras we could show that the host cell which recognises IFN-gamma is of haematopoietic origin. For further understanding we compared animals under IFN-gamma neutralisation with control animals. Immunohistochemical staining revealed a strong expression of the enzyme iNOS in control animals whereas iNOS was merely detectable under IFN-gamma neutralisation. With the help of iNOS deficient animals we could show, that NO is indeed essential as downstream effector molecule. Further characterisation via FACS analysis showed that iNOS production was only observed among CD11b+ cells, roughly half of the iNOS expressing cells were also positive for GR-1. iNOS expression could only be detected at the site of inflammation. Functional in vitro characterisation of these cells after ex vivo isolation revealed that they suppressed the proliferation of CD4+ T cells. They can therefore be regarded as myeloid suppressor cells. To study whether the observed mechanisms of regulation are of any general importance, we looked at a DTH response mediated by CD8+ effector T cells and indeed we could observe a more severe inflammation under IFN-gamma neutralisation, although the effect was not quite as strong as in the Th1 mediated inflammation. In summary we could show, that the cytokine IFN-gamma is important in the limitation of the effector phase of an ongoing immune response.

Regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSC) have been extensively studied in the past 30-40 years. Their potent suppressive capacity shown in several pathological and clinical settings, such as cancer and transplantation, has made it evident that better understanding their development and function is critical. Specifically, Tregs play a pivotal role in preventing autoimmunity, graft-versus-host disease (GvHD), and organ graft rejection. We previously demonstrated that germline or induced SH2 domain-containing inositol 5-phosphatase (SHIP) deficiency in the host abrogates GvHD. Here we show that SHIP-deficiency promotes an increase of FoxP3+ cells in both the CD4+CD25+ and the CD4+CD25- T cell compartments with increased expression of Treg-associated markers. Importantly, SHIP-deficiency does not compromise Treg function. Interestingly, like conventional Tregs, SHIP-/- CD4+CD25- T cells are unresponsive to allogeneic stimulators and suppress allogeneic responses by T cells in vitro, and can mediate reduced lethal GvHD in vivo. Thus, SHIP limits the immunoregulatory capacity of CD4+ T cell, particularly in allogeneic settings. SHIP-deficiency expands the number of immunoregulatory cells in both the T lymphoid and myeloid lineages. Here, we examined if these increases are interrelated. Specifically, we found that myeloid specific SHIP-deficiency leads to expansion of both MDSC and Treg numbers. Conversely, T lineage specific ablation of SHIP leads to expansion of Treg numbers, but not expansion of MDSC, indicating an intrinsic role for SHIP in limiting Treg numbers. Interestingly, MDSC lack SHIP expression suggesting that another SHIP-deficient myeloid cell promotes MDSC and Treg expansion. Also, increased levels of G-CSF, a myelopoietic growth factor, in SHIP-/- mice may extrinsically promote MDSC expansion since we found that G-CSF is required for the expansion of splenic MDSC in mice with induced SHIP-deficiency. MDSC consist of two distinct subsets, granulocytic-MDSC (G-MDSC), and monocytic-MDSC (M-MDSC) that differ in morphology, phenotype, suppressive capacity and differentiation potential. Importantly, M-MDSC can further differentiate into dendritic cells, macrophages and preferentially into G-MDSC, in the presence of tumor-derived factors (TDF). The retinoblastoma gene (Rb1), a tumor suppressor gene and central regulator of the cell cycle and differentiation, has been shown to influence monocytic and neutrophilic lineage commitment and to limit myeloproliferative disease. Here, we examined the role of Rb1 in the biology of MDSC subsets in tumor-bearing mice. Firstly, M-MDSC expressed high levels of Rb1 which remained relatively stable in culture with GM-CSF. Conversely, freshly isolated G-MDSC initially expressed undetectable levels of Rb1 that increased over time in culture, which correlated with increased histone acetylation at the Rb1 promoter. This increased Rb1 expression and histone acetylation was accelerated by histone deacetylase inhibitors (HDACi) treatment, suggesting Rb1 expression may be controlled by histone modification. Furthermore, when treated with HDACi, M-MDSC did not differentiate into G-MDSC in culture, even with TDF present. Finally, induced Rb1 deficiency in vivo promoted an expansion of splenic CD11b+Ly6G+Ly6Clo cells, similar to G-MDSC in tumor-bearing mice. Although further studies are required, these results strongly suggest that Rb1, like SHIP, plays a role in MDSC accumulation, particularly G-MDSC in cancer.

[Truncated abstract] Breast cancer is the cause of significant suffering and death in our community. It is now estimated that the risk of developing breast cancer for an Australian woman before the age of 85 is 1 in 8, with this risk rising for unknown reasons. While mortality rates from breast cancer are falling due to increased awareness and early detection, few new treatments have been developed from an advanced understanding of the molecular basis of the disease. From decades of scientific research it is clear that estrogen (E2) has a large role to play in breast cancer. However, the basic mechanism behind E2 action in breast cancer remains unclear. E2 plays a fundamental role in breast cancer cell proliferation and is highly expressed in breast cancers, thus, it is important to understand both E2 and its receptor, the estrogen receptor (ER). The ER is a member of the nuclear receptor (NR) superfamily. The NR superfamily consists of a large group of proteins which regulate a large number of homeostatic proteins together with regulator proteins termed coregulators and corepressors. SRA (steroid receptor RNA activator) is the only known RNA coactivator and augments transactivation by NRs. SRA has been demonstrated to play an important role in mediating E2 action (Lanz et al., 1999; Lanz et al., 2003) and its expression is aberrant in many human breast tumors, suggesting a potential role in breast tumorigenesis (Murphy et al., 2000). Despite evidence that an alternative splice variant of SRA exists as a protein (Chooniedass-Kothari et al., 2004), it has been conclusively shown that SRA can function as an RNA transcript to coactivate NR transcription (Lanz et al., 1999; Lanz et al., 2002; Lanz et al., 2003). The precise mechanism by which SRA augments ER activity remains unknown. However, it is currently hypothesized that SRA acts as an RNA scaffold for other coregulators at the transcription initiation site. Several SRA stem loops have been identified as important for SRA function, including structure (STR) 1, 5 and 7 (Lanz et al., 2002; Zhao et al., 2007). Previously, I sought to identify SRA-binding proteins using a specific stem-loop structure of SRA (STR7) that was identified as both important for its coactivator function (Lanz et al., 2002) and also as a target for proteins from breast cancer cell extracts (Hatchell, 2002). From a yeast E. Hatchell Abstract iii III hybrid screen using STR7 as bait, I identified a novel protein which was named SLIRP (Patent Number: WO/2007/009194): SRA stem-Loop Interacting RNA-binding Protein (Hatchell, 2002; Hatchell et al., 2006). '...' This thesis demonstrates that SLIRP modulates NR transactivation, provides mechanistic insight into interactions between SRA, SRC-1, HSP-60 and NCoR and suggests that SLIRP may regulate mitochondrial function. These studies contribute significantly to the growing field of NR biology, and contribute more specifically to the elucidation of estrogen action in breast cancer. Furthermore, it lays a strong and exciting foundation for further studies to evaluate SLIRP as a biomarker and potential therapeutic target in hormone dependent cancers.

T cells chronically stimulated by their antigen often become dysfunctional and lose effector functions and proliferative capacity. This state of unresponsiveness is referred as T cell exhaustion. In order to investigate this, we developed a laboratory model, which allowed us to stimulate chronically in vivo a monoclonal population of CD4+ T cells. This model is based on the adoptive transfer of TCR transgenic CD4+ T cells specific for the male mHAg into male recipient mice. We found that systemic exposure to the male antigen modified deeply anti-male TcR-transgenic CD4+ T cells, plunging them into a state of functional unresponsiveness. Microarray analysis revealed that, in comparison with naive T cells, transferred T cells displayed a gene expression profile very similar to that of virus-specific exhausted CD8+ T cells. Moreover, like exhausted CD8+ T cells, exhausted CD4+ T cells lost their capacity to secrete IFN-ã as well as to proliferate in response to antigen stimulation, and T cell unresponsiveness was controlled by the engagement of programmed death receptor 1 (PD-1) present at the surface of T cells. <p>MicroRNAs are key molecules in shaping T cell function. In order to explore the possibility that chronic antigenic stimulation could shape the pool of microRNAs in exhausted anti-male CD4+ T cells that would account for specific changes in protein synthesis, we compared by microarray analysis the specific expression of microRNAs in naive CD4+ T cells and exhausted CD4+ T cells. Ninety five of them were found differentially expressed, among which, microRNA-155 (miR-155) displayed one of the highest changes. To identify the importance of miR-155 in T cell exhaustion, we analyzed miR-155-deficient CD4+ T cells after chronic exposure to systemic antigen. We found that, chronically-stimulated miR-155-/- CD4+ T cells were retained in a deeper state of unresponsiveness than miR-155+/+ CD4+ T cells. Furthermore, inhibition of PD-1/PD-L1 interaction did not promote antigen-dependent expansion of miR-155-deficient CD4+ T cells, nor did it stimulate T cell inflammation of several organs, contrary to what was observed in mice that received miR-155-sufficient CD4+ T cells. Thus, our observations demonstrated that miR-155 deficiency played a dominant role over PD-1-mediated inhibition of T cells and that miR-155 was required for restoring function in exhausted CD4+ T cells.<p>Next, we explored the mechanism by which exhausted miR-155-/- CD4+ T cells were kept in a deeper unresponsiveness state than miR-155+/+ counterparts. By comparative microarray analysis of gene expression between exhausted miR-155+/+ CD4+ T cells and miR-155-/- CD4+ T cells, heme oxygenase 1 (HO-1) was identified as a specific target of miR-155. Finally, inhibition of HO-1 activity restored the capacity of exhausted miR-155-/- CD4+ T cells to promote autoimmune inflammation in adoptively-transferred recipients. <p>Taken together, our study identified miR-155-mediated regulation of protein expression as a critical factor for restoring function in exhausted CD4+ T cells. Our results also present regulation of HO-1 expression in T cells as one of the mechanisms by which miR-155 promote T cell-driven inflammation. / Doctorat en Sciences biomédicales et pharmaceutiques / info:eu-repo/semantics/nonPublished

Mon travail de thèse a consisté à caractériser une sous-population de lymphocytes T régulateurs naturels de phénotype CD8+CD25+.<p>Nous avons purifié les CD8+CD25+ nTregs et vérifié par cytométrie de flux leur expression en FOXP3 et CTLA-4. Puis nous avons pu montrer que ces cellules possèdent des propriétés suppressives dans un test d’inhibition de la prolifération de lymphocytes T activés allogéniquement. Les lymphocytes CD8+CD25+ nTregs expriment les gènes FOXP3, CTLA-4, GARP et CCL-4 et les cytokines IL-10 et TGF-β. Par contre, les gènes CD28, ICOS, FOXO1 et Helios sont sous-exprimés dans les nTregs CD8+CD25+ par rapport aux lymphocytes T CD8+CD25-. <p>Nous avons établi une signature micro-ARN qui comprend 10 micro-ARNs différentiellement exprimés :7 micro-ARNs sous-exprimés "miR-9, -24, -31, -155, -210, -335 et -449 " et 3 micro-ARNs surexprimés " miR-214, -205 et -509". De plus, nous avons pu explorer la relevance biologique de cette signature micro-ARN en montrant dans un premier temps que les miRs "-31, -24, -210, -335" ciblent spécifiquement la région 3'UTR de FOXP3, de même les miR-9 et miR-155 ciblent la région 3'UTR de CTLA-4, et les miR-24, et -335 ciblent la région 3'UTR de GARP. Ceci a été fait par des expériences de co-transfections suivies d'une mesure de l'activité rapportrice luciférase. De plus, nous avons pu démontrer par des expériences de transduction lentivirale ex vivo, de cellules T primaires, que des micro-ARNs de la signature régulent l’expression de FOXP3, CTLA-4 et GARP dans les Tregs naturels CD8+CD25+ humains. <p>Cette étude montre l'importance des micro-ARNs dans la régulation post-transcriptionnelle des gènes impliqués dans la fonction régulatrice des lymphocytes T régulateurs.<p> / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

Chronic myeloid leukaemia (CML) is characterised by the constitutively active tyrosine kinase BCR-ABL. Standard treatment with tyrosine kinase inhibitors (TKI) in the chronic phase (CP) of CML conveys excellent long-term prognosis but is associated with side effects and costs. Treatment free remission (TFR) is possible in a proportion of patients discontinuing treatment after obtaining deep treatment responses but it is not fully known how to select the right patients for stopping attempts. Treatment of accelerated phase (AP) and blast crisis (BC) is more complicated and the prognosis more dismal. In this thesis, we have studied factors of importance for outcome in CML patients with focus on immunological factors and clinical management. In a cohort of 32 newly diagnosed CP-CML patients, evidence of active immune escape mechanisms were found. These declined with the course of TKI treatment and at the same time, effector lymphocyte responses were elicited. These anti-leukaemia immune responses might help in the long-term control of CML. Multiple plasma protein markers were also measured with three multiplex platforms in a smaller cohort of patients (n=14). Inflammatory cytokines and other plasma proteins were affected by TKI treatment and multiplexing seems useful for finding potential biomarkers with biologic or prognostic significance in CML. Patients progressing to AP/BC were studied in a population-based material from the Swedish CML register. Approximately 4% of TKI-treated CP-CML patients transformed to AP/BC within 2 years of diagnosis. Monitoring of treatment responses was suboptimal in 1/3 of these patients and the median survival was 1.4 years after diagnosis of AP/BC. Thus, minimising the risk of disease progression through strict adherence to guidelines for monitoring and treatment is essential. In a cohort of patients (n=50) discontinuing TKI treatment within a large European trial, musculoskeletal pain was reported by 30% of patients, starting within 1- 6 weeks of TKI discontinuation and spontaneously resolving over time in most cases. Patients (n=56) were also evaluated with a multiplex platform with a total of 162 inflammation- and cancer-related plasma proteins. No predictive protein biomarkers for successful TKI discontinuation could be found. However, profound effects of TKI-treatment were seen and plasma proteomics could be useful for understanding effects of long-term TKI-treatment.

T-cells are present in the immune system to fight against invaders. Once their job is done, suppressing their activity is an important step in maintaining a proper immune response. Myeloid derived suppressor cells (MDSCs) are immune cells that suppress T-cell activity. Currently, MDSCs are defined as a heterogeneous population of immature cells that are derived in the bone marrow and travel to the site of inflammation or cancer. Two major subtypes of MDSCs have been identified in mice and humans, monocyte-like MDSCs (M-MDSC) and granulocyte MDSCs (G-MDSC). G-MDSCs typically make up the majority of the total population of MDSCs but are less T-cell suppressive than M-MDSCs. One of the major problems in the study of MDSCs is that the current marker system for subtypes does not differentiate between precursor MDSCs (lacking suppressive ability) and functional MDSCs (those with suppressive ability). Therefore, using cancer models in mice, we investigated the development and potential to classify precursor MDSCs from functional MDSCs. While MDSCs have been highlighted as a target cell to inhibit in cancer, in other conditions, such as pregnancy, MDSCs have been shown to be beneficial in maintaining a normal pregnancy. Therefore, targeting the increase of MDSCs in abnormal pregnancy conditions like pre-eclampsia may act as a prevention or therapeutic strategy. Finally, it is known that many dietary components can act as modulators of immune cells. Specifically, the polyphenol like phytochemical, curcumin has been shown to act as an anti-inflammatory agent with the potential to modulate multiple immune cells. Therefore, we propose two different studies to investigate the potential of curcumin as either an inhibitor and/or promotor of MDSCs in a disease-specific context. Together the role of phytochemicals as immunomodulators of MDSCs is still very young, in part due to the complexity of phytochemicals themselves, but the studies cited here provide evidence that the field is ripe for additional questions to be asked.

No description available.

T cells play a pivotal role in controlling viral infection; however, the precise mechanisms responsible for regulating T‐cell differentiation and function during infections are incompletely understood. In this study, we demonstrated an expansion of myeloid‐derived suppressor cells (MDSC s), in particular the monocytic MDSC s (M‐MDSC s; CD 14+ CD 33+ CD 11b+ HLA ‐DR −/low), in patients with chronic hepatitis C virus (HCV ) infection. Notably, HCV ‐induced M‐MDSC s express high levels of phosphorylated signal transducer and activator of transcription 3 (pSTAT 3) and interleukin‐10 (IL ‐10) compared with healthy subjects. Blocking STAT 3 signalling reduced HCV ‐mediated M‐MDSC expansion and decreased IL ‐10 expression. Importantly, we observed a significant increase in the numbers of CD 4+ CD 25+ Foxp3+ regulatory T (Treg) cells following incubation of healthy peripheral blood mononuclear cells (PBMC s) with MDSC s derived from HCV ‐infected patients or treated with HCV core protein. In addition, depletion of MDSC s from PBMC s led to a significant reduction of Foxp3+ Treg cells developed during chronic HCV infection. Moreover, depletion of MDSC s from PBMC s significantly increased interferon‐γ production by CD 4+ T effector (Teff) cells derived from HCV patients. These results suggest that HCV ‐induced MDSC s promote Treg cell development and inhibit Teff cell function, suggesting a novel mechanism for T‐cell regulation and a new strategy for immunotherapy against human viral diseases.