Staphylococcus aureus (S. aureus) is a gram-positive bacterium that causes mastitis in bovines and leads to financial losses to the dairy industry. Although antibody response plays a role in immune defense against S. aureus, cellular responses are of interest for vaccine development. A vaccine that stimulates both antibody and cellular responses could promote memory cell formation and provide effective protection against S. aureus. The superantigens and virulence factors secreted by live S. aureus (LSA) can interfere with immune responses and memory cell formation. Because irradiation reduces the metabolic activity and secretion of proteins, including S. aureus superantigens and hemolysins, we hypothesized the irradiated S. aureus (ISA) could drive immune cell responses. Dendritic cells (DC) were co-cultured with lymphocytes to study the cellular responses to ISA and LSA. Dendritic cells present antigens and polarize lymphocytes into different helper T (Th) cell types that drive cellular immune responses. The DC loaded with either ISA or LSA induced increased mRNA transcription of Th17-related cytokines and cytotoxic effector memory cell formation during antigen recall experiments. Lymphocytes co-cultured with LSA-loaded DC exhibited a higher fold-change in interferon (IFN) γ mRNA compared to ISA-loaded DC, suggesting the secreted antigens and the metabolic activity of S. aureus play a role in Th1 polarization. Th1 polarization can drive excessive inflammation and suppress beneficial Th17 responses. Bovine DC were stimulated with a mutant α-toxin deletion S. aureus strain to evaluate if α-toxin-mediated NOD2 receptor signaling activates Th1 polarization in response to S. aureus, which revealed that NOD2 mRNA transcription in DC was independent of α-toxin and that the deletion of α-toxin had no effect on the transcription of the cytokine IL-12 or the production of IFNγ by lymphocytes, events that drive Th1 polarization, in co-cultures. The deletion of accessory gene regulator (agr), which controls α-toxin production, reduced IFNγ production in lymphocytes co-cultured with the S. aureus-loaded DC, indicating that agr controlled the ability of S. aureus antigens to drive the Th1 polarization of lymphocytes. Overall, this thesis demonstrates that ISA is a promising source of antigens that stimulate memory cells formation and Th17 polarization in bovine immune cells. The reduced Th1 cytokine response to S. aureus was not dependent on α-toxin, but other virulence factors controlled by agr should be screened to determine the source of Th1 stimulation. / Ph. D.

T follicular helper cells (T-FH) are a specialized subset of CD4 T cells that reside in B cell follicles and promote B cell maturation into plasma cells and long-lived memory B cells. During chronic infection prior to the development of AIDS, HIV-1 (HIV) replication is largely concentrated in T-FH. Paradoxically, T-FH numbers are increased in early and midstages of disease, thereby promoting HIV replication and disease progression. Despite increased T-FH numbers, numerous defects in humoral immunity are detected in HIV-infected individuals, including dysregulation of B cell maturation, impaired somatic hypermutation, and low quality of antibody production despite hypergammaglobulinemia. Clinically, these defects are manifested by increased vulnerability to bacterial infections and impaired vaccine responses, neither of which is fully reversed by antiretroviral therapy (ART). Deficits in T-FH function, including reduced HIV-specific IL-21 production and low levels of co-stimulatory receptor expression, have been linked to these immune impairments. Impairments in T-FH likely contribute as well to the ability of HIV to persist and evade humoral immunity, particularly the inability to develop broadly neutralizing antibodies. In addition to direct infection of T-FH, other mechanisms that have been linked to T-FH deficits in HIV infection include upregulation of PD-L1 on germinal center B cells and augmented follicular regulatory T cell responses. Challenges to development of strategies to enhance T-FH function in HIV infection include lack of an established phenotype for memory T-FH as well as limited understanding of the relationship between peripheral T-FH and lymphoid tissue T-FH. Interventions to augment T-FH function in HIV-infected individuals could enhance immune reconstitution during ART and potentially augment cure strategies.

ABSTRACT Although there is ample evidence linking chronic inflammation with cancer, the cellular mechanisms involved in early events leading to tumor development remain unclear. Myeloid cells are an intricate part of inflammation. They consist of mature cells represented by macrophages, dendritic cells and granulocytes and a population of Immature Myeloid Cells (IMC), which in healthy individuals are cells in transition to mature cells. There is a substantial expansion of IMC in cancer and many other pathological conditions which is associated with pathologic activation of these cells. As a result, these cells acquire the ability to suppress immune responses and are termed Myeloid-derived Suppressor Cells (MDSCs). Although the role of MDSC in immune suppression in cancer and tumor progression is well established, their contribution to tumor development is still uncertain. The fact that cells with MDSC phenotype and function are observed in chronic inflammation raised the possibility that these cells can contribute to initial stages of tumor development. To address this question, we used an experimental system where the number of IMC was regulated by the expression of S100A9 protein. In this project, we used two different models of chronic inflammation in S100A9 transgenic (S100A9tg) and S100A9 knock-out (S100A9KO) mice. In the first model, we created the conditions for topical accumulation of these cells in the skin in the absence of infection or tissue damage using S100A9tg mice. Accumulation of IMC in the skin resulted in a dramatic increase in the formation of skin tumors during epidermal carcinogenesis. Conversely, lack of myeloid cell accumulation in S100A9KO mice substantially reduced the formation of skin papillomas. The effect of IMC was not associated with immune suppression but with the recruitment of CD4+ T cells mediated by CCL4 chemokine released by activated IMC. Elimination of CD4+ T cells or blockade of CCL4 abrogated the increase in tumor formation caused by myeloid cells. Thus, this study implicates the accumulation of IMC as an initial step in facilitating of tumor formation, which can mediate the recruitment of CD4+ T cells via the release of CCL4 chemokine. In the second model, we used inflammation-associated lung cancer caused by the chemical lung carcinogen urethane in combination with exposure to cigarette smoke referred to throughout as CS. Exposure of mice to CS alone resulted in a significant accumulation of cells with typical MDSC phenotype in different organs; however, these cells lacked immune suppressive activity and could not be defined as bona fide MDSC. When CS was combined with the single dose of urethane, it led to the accumulation of immune suppressive cells. The expansion of MDSC followed the onset of lung tumors development. This suggests that MDSC in this model is not the preceding factor but rather a consequence of tumor formation. Further studies are necessary to determine the relevance of targeting these cells for cancer treatment and prevention.

Obesity and type 2 diabetes (T2D) are associated with insulin resistance and increased levels of inflammation markers, suggesting activation of the immune system. However, the link between this so called ??low-grade inflammation?? and insulin resistance is poorly understood. In this thesis we aimed to investigate the direct effects of insulin on immune cells, and if these effects are changed in the setting of insulin resistance. We showed that insulin has anti-inflammatory effects by shifting T cell differentiation into a T helper type 2 phenotype. This effect was lost in insulin resistant subjects, which resulted in a more pro-inflammatory T helper type 1 cell hyperpolarisation. We also demonstrated that the Th1/2 balance is related to the degree of insulin resistance, and varies accordingly in clinical models of increasing or decreasing insulin resistance. Furthermore, we demonstrated that in a very early stage of pre-diabetes, where normal glucose tolerance and insulin sensitivity are still preserved, we cannot detect any immune activation, but we see a blunted food response of the appetite suppressant hormone PYY. Whilst this could put subjects at risk for further weight gain and development of obesity and T2D, we also demonstrated for the first time that PYY itself has strong anti-inflammatory properties, and that a deficiency in PYY could result in promoting a pro-inflammatory environment. In summary, we could demonstrate strong evidence that both, insulin and PYY are potent anti-inflammatory hormones which modulate immune function, and the observed deficiency in these hormones could contribute to further increase in inflammation and disease progression. Further work is indicated in this area to better understand the sequence and mechanism of immune activation, which may open up new therapeutic avenues for prevention and treatment of T2D.

Indiana University-Purdue University Indianapolis (IUPUI) / The differentiation of naïve CD4+ T cells into subsets of T helper cells (Th) is an essential process that impacts host defense and the pathogenesis of immunemediated diseases. Signal transducers and activators of transcription (STAT) proteins, activated downstream of instructive cytokines, dictate and perpetuate the lineage decision of Th cells through both positive and negative effects. This is accomplished by regulating transcription factors, surface receptors and promoting epigenetic changes in gene expression through chromatin remodeling. Transforming growth factor-β1 (TGF-β1) can induce Foxp3 in developing Th cells and these Foxp3-expressing adaptive T regulatory cells (aTregs) are able to suppress inflammation in vitro and in vivo. To define the mechanism by which STAT proteins regulate Th cell pro- and anti-inflammatory phenotypes, we examined T cells deficient in Stat3, Stat4, and Stat6 as well as T cells expressing two STAT4 isoforms after being cultured in the presence or absence of TGF-β1 and cytokines known to be instructive in Th cell development. The negative effects of STAT proteins are demonstrated by our results indicating STAT3, STAT4 and STAT6 proteins activated downstream of the instructive cytokines IL- 6, IL-12 and IL-4, respectively, negatively regulate the development of TGF-β induced Foxp3 and aTreg development. STAT3, STAT4, and STAT6 utilize a vi Mark H. Kaplan, Ph.D., Chair common mechanism to inhibit aTreg generation by inhibiting STAT5, a positive regulator of Foxp3 expression, from binding to the Foxp3 gene. STAT proteins positively effecting inflammatory immunity are demonstrated by our analysis of STAT4 isoforms and their ability to regulate the production of proinflammatory cytokines downstream of IL-12. STAT4β, a STAT4 splice isoform that lacks a Cterminal domain, and STAT4α, a full-length isoform are both capable of mediating inflammatory cell development. However, STAT4β promotes greater inflammation in vivo than STAT4α independent of its ability to repress Foxp3. Instead, the inflammation correlates with STAT4 isoform-dependent expression of inflammatory cytokines. Thus, cytokine-stimulated STAT proteins orchestrate T helper cell pro- and anti-inflammatory cell phenotypes.

The events that trigger an autoimmune disease remain largely unknown. To study these events animal models are necessary because symptoms of autoimmune diseases are preceded by a long asymptomatic period in humans. Experimental autoimmune encephalomyelitis (EAE) is the best characterized model for cell mediated autoimmunity and an animal model for the human disease multiple sclerosis. EAE is induced in rodents by immunization with myelin antigens (Ags) together with adjuvants. After immunization, T cells are primed in the periphery by Ag presenting cells and subsequently invade the central nervous system where they mediate parenchymal inflammation, resulting in demyelination and clinical symptoms of an ascending paralysis. It is now generally recognised that the main cell type mediating EAE is the T helper type 17 (Th17) cell. Tolerance to EAE can be attained by DNA vaccination, but how the immune response against the myelin Ags is abrogated after DNA vaccination is not known. By employing short interfering RNA technology, induction of the innate immune signalling molecule interferon (IFN) -β was found to be necessary for the protective effect of DNA vaccination in EAE. In addition, DNA vaccination inhibited subsequent autoimmune Th17 cell responses. The Toll-like receptors (TLRs) of the innate immune system have evolved to recognise conserved molecular structures on microbes and signalling through them almost exclusively converge on the molecule MyD88. Signalling via MyD88 was found to be required for induction of EAE since mice deficient in this molecule did not develop disease. Upstream signalling via TLR4 and TLR9 had tolerogenic properties. In studies of Ag presentation in EAE, two major subtypes of dendritic cells (DCs) were examined. Plasmacytoid DCs were found to have a promoting role in the induction of EAE, partly via type 1 IFNs. Myeloid DCs had a redundant role in the induction phase of EAE, neither disease severity nor encephalitogenic Th17 responses were affected by their absence during priming. These studies further demonstrate that the cells and molecules of the innate immune system exhibit a crucial role in controlling the adaptive immune system which mediates tissue damage in autoimmune diseases.

The Tec kinase ITK is activated by TCR stimulation and also required for TCR downstream signaling. Previous studies have reported differential roles of ITK and another Tec family kinase RLK in CD4+ TH differentiation and effector function. However, these findings are confounded by the complex T cell developmental defects in Itk-/- mice. Furthermore, the function of ITK in tissue-resident T cells in the intestine and anti-viral immune response to a persistent infection has not been studied previously. In addition to T cells, recent studies have indicated an expression of ITK in ILC2, but not in other ILC subsets. Yet, the role of ITK in ILC2 has not been characterized. Here, I have examined the role of ITK and RLK in CD4+ TH subsets using a small molecule inhibitor PRN694. I found that PRN694 impaired TH1 differentiation in vitro, and PRN694 administration prevented TH1-mediated colitis progression in vivo. In an MHV68 infection model, Itk-/- mice failed to control viral replication in the intestine, while gut-homing of CD8+ T cells was greatly impaired. Finally, I found that ILC2 number was markedly reduced in the intestine of Itk-/- mice. Gut-specific defect of Itk-/- ILC2 is associated with a low availability of IL-2 in the intestine of Itk-/- mice. Collectively, these data suggest that ITK is important in T cell migration to the intestine and ILC2 homeostasis in the intestine, thereby contributing to the protective response to a latent virus and intestinal tissue homeostasis.

Le système immunitaire humain est majoritairement commandé par les cellules dendritiques et les lymphocytes T auxiliaires. Lorsque les cellules dendritiques détectent un pathogène, elles vont instruire les lymphocytes T auxiliaires afin qu’ils adoptent le phénotype approprié à la menace rencontrée. Les lymphocytes T auxiliaires peuvent être divisés en plusieurs sous-populations, caractérisées par la production de cytokines spécifiques. Chaque sous-population de lymphocyte T auxiliaire possède des fonctions propres et est impliquée dans l’élimination de pathogènes distincts. Si les réponses des lymphocytes T auxiliaires ne sont pas finement régulées, ils peuvent devenir pathogéniques, et dans ce cas, considérés comme cibles potentielles pour des thérapies. Dans ce contexte, j’ai concentré mon travail de doctorat sur l’étude de la diversité des sous- populations de lymphocytes T auxiliaires et de leur régulation. Premièrement, j’ai démontré que les cellules dendritiques activées par la TSLP sont capables d’induire la polarisation de lymphocytes T folliculaires. Ensuite, j’ai participé à la construction d’un modèle mathématique capable de prédire la réponse lymphocytaire T auxiliaire en fonction de signaux dérivés des cellules dendritiques. Ce modèle nous a permis d’identifier un rôle spécifique pour l’IL-12p70, dépendant du contexte IL-1, dans l’induction d’IL-17F sans IL-17A. Enfin, j’ai monitoré huit populations de lymphocytes T auxiliaires et folliculaires dans le sang périphérique de patients atteints de dermatite atopique traités par Dupilumab, une immunothérapie ciblant la sous-unité alpha du récepteur de l’IL-4 et j’ai pu montré que la diminution du pourcentage de lymphocytes Th17 correlait avec l’amélioration du score clinique EASI. Globalement, mon travail sur la diversité de phénotypes Th apporte une ressource mécanistique importante, avec une potentielle application en immunothérapie. / Human immunity is essentially driven by dendritic cells and T helper cells. When dendritic cells detect a pathogen, they will instruct T helper cells to adopt the adapted phenotype for the specific threat encountered. T helper cells are subdivided in multiple subsets, characterized by particular sets of cytokines. Each T helper subset has specific functions and is involved in the clearance of distinct pathogens. If T helper responses are not precisely regulated, they can become pathogenic, in this case T helper pathways can be considered as potential targets for therapy. In this context, I focused my PhD work on studying T helper cell subset diversity and regulation. First, I demonstrated the ability of TSLP-activated dendritic cell to induce T follicular helper cell polarization. Then I participated in building a mathematical model capable of predicting T helper cell response to dendritic-cell derived signals. This model allowed us to identify the specific role of IL-12p70, in an IL-1 context, to induce IL-17F without IL-17A. Finally, I monitered eight T helper and T follicular helper cell populations in peripheral blood from atopic dermatitis patients treated with Dupilumab, an immunotherapy targeting the IL-4 receptor alpha subunit, and was able to show a correlation between decrease of Th17 cell percentage and improvement of EASI clinical score. Overall, my work on Th phenotype diversity provides key mechanistic insight with potential application in immunotherapy.

According to the centers for disease control and prevention (CDC) and the world healthorganization (WHO), heart disease and immune related diseases such as diabetes and cancer areamong the leading causes of death around the world. Thus, the regulation of the function ofimmune cell plays a key role in health and disease. Calcium (Ca2+) ions play a critical role inimmune cell activation, function and in a robust immune response. Defects in Ca2+ signalinginfluences the development of cardiac disease, Alzheimer disease, immune cell metabolism,muscle dysfunction, and cancer. Each immune cell is unique in its activation and function,making it relevant to understand how activation of each type of immune cell is regulated. Herewe describe the role of the nBMP2 protein in macrophage activation and function and the role ofthe CD5 co-receptor in helper T cell activation and function.The nuclear bone morphogenetic protein 2 (nBMP2) is the nuclear variant of the bonemorphogenetic protein 2 (BMP2), a growth factor important in heart development, neurogenesis,bone, cartilage and muscle development. To better understand the function of nBMP2, transgenicnBMP2 mutant mice were generated. These mice have a slow muscle relaxation and cognitivedeficit caused in part by abnormal Ca2+ mobilization. Mutant nBMP2 mice also have an impairedsecondary immune response to systemic bacterial challenge. Here we have further characterizedmacrophage activation and function from mutant nBMP2 mice before and after bacterialinfection. We describe how nBMP2 influences the Ca2+ mobilization response and phagocytosisin macrophages, revealing a novel role of the nBMP2 protein in immune cell regulation.CD5 is a surface marker on T cells, thymocytes, and the B1 subset of B cells. CD5 isknown to play an important role during thymic development of T cells. CD5 functions as anegative regulator of T cell receptor (TCR) signaling and fine tunes the TCR signaling response.Here we describe our characterization of CD5 regulation of Ca2+ signaling in naïve helper Tcells. We also outline our findings examining how CD5-induced changes in helper T cellactivation influence other biological processes such as immune cell metabolism, the diversity ofthe gut microbiome, and cognitive function and behavior. Thus, this work elucidates theinfluence of the CD5 co-receptor on the functional outcomes in multiple systems when CD5 isaltered.

Protektive MHC II Allele sind sowohl für den Menschen als auch für die Maus beschrieben worden und verhindern die Entstehung von Autoimmunerkrankungen. Hier untersuche ich die differentielle Expression von MHC II Allelen auf den unterschiedlichen Antigen-praesentierenden Zellen als Wirkmechanismus, der das Typ 1-Typ 2 Gleichgewicht der T-Helferzellen beeinflusst. Ich konnte zeigen, dass die als protektiv geltenden murinen I-Ab und I-Ek Molekuele auf fast allen Knochenmark-Makrophagen fuer 5 - 8 Tage stark exprimiert werden und dass die Expression dann langsam abnimmt. Im Gegensatz dazu fanden wir eine etwa 100-fach schwaechere Expression des mit der Kollagen-induzierten Arthritis (CIA) assoziierten I-Aq. Diese Expression war von nur kurzer Dauer und nahm rasch ab. Eine aehnlich differentielle Expression konnten wir weder auf B- noch auf dendritischen Zellen (DZ) nachweisen. Zusätzlich konnte in in vitro Restimulationsexperimenten gezeigt werden, dass Makrophagen durch diese differentielle Expression die T-Zell-Zytokinantwort massgeblich beeinflussen. Unsere Ergebnisse deuten an, dass Makrophagen eines protektiven Haplotyps MHC II Molekuele in hoher Zahl exprimieren und damit bevorzugt Typ 1 Antworten hervorrufen, wohingegen eine niedrige MHC II Expression Typ 2 Antworten beguenstigt. Wir schliessen daraus, dass das Ausmass der MHC II Expression das Signal, welches von den T-Zellen zu den Makrophagen zurückgesendet wird, steuert und damit die Aktivitaet der Makrophagen reguliert. Dieser durch polymorphe, jedoch nicht-kodierende MHC II-Gensegmente hervorgerufene Effekt koennte bei der Empfaenglichkeit fuer Autoimmunerkrankungen sowohl beim Menschen als auch bei der Maus eine Rolle spielen. Tatsaechlich konnten wir auch auf menschlichen Monozyten und B-Zellen eine differentielle Expression von HLA II Genen nachweisen und sie scheint sich hier auf die nur gering-polymorphen DRB4 Gene, die sowohl mit dem Rheumatoide Arthritis (RA) assoziierten DR4 als auch mit dem neutralen DR7 koexprimiert werden, zu beschraenken. In meinem letzten Teil ziele ich darauf ab, das den Verlauf der RA und der CIA begleitende Typ 1 Übergewicht in ein Gleichgewicht mit Typ 2 zu revertieren. Dazu wurde ein Mausmodell etabliert, bei dem bereits polarisierte Typ 2 Th-Effektorzellen als Manipulatoren der für die Entstehung der CIA verantwortlichen, Kollagen-spezifischen Typ 1 Zellen eingesetzt wurden. Tatsaechlich konnte die CIA dann am effektivsten verhindert werden, wenn beide T-Zellpopulationen, die Manipulierer und die Kollagen-spezifischen, im selben Zellcluster aktiviert wurden. Diese Aktivierung im selben Zellcluster konnte dadurch erreicht werden, dass DZ gleichzeitig Kollagen und das fuer die Manipulierer spezifische Epitop praesentieren. / Protective/suppressive MHC class II alleles have been identified in man and mouse where they exert a disease-protective and immunosuppressive effect. As a mode of action we here investigate differential expression of MHC class II genes in different types of antigen-presenting cells impacting on the Type 1-Type 2 balance. We found that the murine I-Ab and I-Ek molecules, both well characterized as protective/suppressive, are expressed at a high level on almost all bone marrow derived macrophages for five to eight days after which expression slowly declines. In contrast, the collagen-induced arthritis (CIA) associated I-Aq-expression is lower, peaks over a shorter period and declines more rapidly. No differential expression could be detected on B cells or dendritic cells (DC). In addition, the differential MHC class II expression found on macrophages skews the cytokine response of T cells as shown by an in vitro restimulation assay. The results indicate that macrophages of the protective/ suppressive haplotypes express MHC class II molecules at a high level and exert Type 1 bias whereas low level expression favors a Type 2 response. We suggest that the extent of expression of the class II gene gates the back-signal from T cells and in this way controls the activity of macrophages. This effect mediated by polymorphic non-exon segments of MHC class II genes may play a role in determining disease susceptibility in mouse and man. Indeed, we also found differential expression of HLA II genes on human antigen presenting cells. However, in humans, differential expression affects both, B cells and monocytes and seems to be restricted to the non-polymorphic DRB4 gene coexpressed with the rheumatoid arthritis (RA) associated DR4 and the neutral DR7. We finally aimed at reverting the Type 1 bias characteristic of RA and CIA. A murine system was developed where polarized Type 2 cells were used to manipulate collagen II-specific type 1 T cells responsible for the development of collagen induced arthritis. The polarized inducer cells indeed exerted their maximum effect when the two T-cell populations were activated within the same cluster, implemented by allowing a single DC to present both their epitopes.