Research into the cause of autism continues without any clear-cut answers. However, recent studies suggest that abnormalities of the immune system are associated with this disorder and autism results from failure of the immune system to regulate itself. Proper immune regulation requires that the host have the appropriate number and percentage of each population of lymphocytes and monocytes. These populations can be distinguished from one another with monoclonal antibodies that react with unique protein structures on the cell surface designated as the "cluster of differentiation" (CD) antigens. This investigation studied the possibility that the immune abnormalities seen in autism are due to a change in the lymphocytes or monocytes in the subjects with autism . The autistic subjects as compared to age- and sex-matched control subjects exhibited several changes in their cell populations. These included a depression of total lymphocytes, CD2+ (total T cells), CD4+ (helper T cells), and CD4+CD45RA+ (the antigen-inexperienced, suppressor inducer subset of helper T cells). Also analyzed were the siblings of the autistic subjects. A reduced percentage of CD4+ cells was seen in the male siblings as compared to unrelated males. Analyses also compared the cells of mothers and fathers of the autistic subjects with controls of mothers and fathers of normal children. No differences were seen in any of the markers used. Findings in the literature show an increase in memory cells and a decrease in naive cells as a function of age. The data gathered in these experiments uphold this concept and are consistent with the idea that CD45RA and CDw29 are maturational states of helper T cells. The quantitation of different immune markers on lymphoid cells seems to have been useful in the further characterization and investigation of the immune mechanism relevant to the syndrome of autism. Differences in some of the cell types were observed and may account for some of the immune abnormalities seen in autism. These differences may be the result or the cause of the syndrome. Further investigation seems necessary before a direct pathological link can be found between the body's immunity and autism.

Regulation of the immune system and of uterine tissue homeostasis, growth, and remodelling are deeply intertwined during pregnancy and are essential for successful reproduction. Recent findings showed that tissue-resident innate lymphoid cells (ILCs) are crucial regulators of both physiology and pathology of the tissues they populate. Uterine natural killer (uNK) cells are a subtype of ILCs known to regulate trophoblast invasion, uterine vascular adaptation to pregnancy, and foetal growth. We recently described additional types of ILCs in the uterus of women and mice. However, the role of these ILCs during reproduction is unknown. Among them, group 2 ILCs (ILC2s) have been previously characterised in other tissues, in which they modulate immune cells and tissue homeostasis by producing type-2 cytokines and growth factors (i.e. IL-4, IL-5, IL-13, and Amphiregulin). Based on these premises, I hypothesized that uterine ILC2s (uILC2s) regulate uterine immune homeostasis and thus contribute to successful reproduction. To test this, I first characterised the uILC subtypes present in humans and mice at various stages of the reproductive cycle. Secondly, I addressed the functional role of uILC2s during pregnancy by taking advantage of a uILC2 knockout mouse model. My results show that uterine ILC2s represent < 1% and < 0.1% of murine and human uterine leukocytes, respectively. However, as they can quickly produce large amounts of cytokines, uILCs are capable of potently affect both other immune cells and the surrounding tissue. Indeed, I found that compared to other tissue-resident ILC2s, uILC2s produce high levels of IL-5 and Areg even in the absence of any stimulation. On the contrary, non-uterine ILC2s mainly produce IL-13, which is lowly expressed by uILC2s. To further characterize the tissuespecific properties of uILC2s, I then performed RNAseq on uILC2s isolated from virgin, midgestation, and term murine uterus, and I compared their transcriptomes with those of ILC2s from lung, intestine, and bone marrow. Interestingly, uILC2s specifically express granzymes and genes typical of regulatory T cells. Therefore, uILC2s have tissue-specific properties and are modulated during pregnancy. Furthermore, the ability of uILC2s to produce IL-5 and Areg suggests that they may be crucial in the regulation of uterine type-2 immunity. I then studied the phenotype of $Rora^{flox/flox}Il7ra^{cre/wt}$(ILC2KO) mouse models, as well as that of mice lacking the ILC2 activating cytokine IL-33 ($IL33^{cit/cit}$; IL33KO). I examined the immune microenvironment in both the myometrium and decidua in ILC2KO mice and found alterations in type-2 cytokines and myeloid cell homeostasis. In particular, in absence of ILC2s, IL-4 and IL-5 are dramatically reduced, IL-13 is absent, and decidual inflammatory cytokines IL1β and IL-6 are increased. Furthermore, uterine dendritic cells (uDC), uterine macrophages (uMac), and uterine neutrophils (uN) increase, while uterine eosinophils (uEo) are virtually absent. These results show that uILC2s regulate uterine type-2 immunity, suggesting that uILC2s could be important during pregnancy. Accordingly, I found that lack of uILC2s leads to insufficient spiral artery remodelling and restricted foetal growth. Type-2 cytokines and in particular IL-4 regulates alternative activation of Macrophages (Mac) and Dendritic Cells (DCs), which promote the development of an anti-inflammatory environment and facilitate tissue remodelling. I hypothesised that similar mechanisms occur in the uterus and that uILC2s have a central role in the polarisation of the immune response. To explore this, I studied in more detail the characteristics of uEo, uMac, and uDCs dissected from wild type and ILC2KO mice. I found a reduction in genes associated with alternative activation in uMac and uDCs in the uterus of pregnant ILC2KO mice. Additionally, I showed that uEo are the main producers of the IL-4. This demonstrates that uILC2s promote alternative activation of myeloid cell population by modulating the uterine immune microenvironment. I then assessed the role of uILC2s-dependent type-2 immunity in inflammatory pathology following a type-1 response to bacterial infection. When challenged with LPS, pregnant ILC2KO mice showed more pronounced foetal demise. Therefore, uILC2s regulate uterine type-2 immune homeostasis and this prevents inflammatory pathology. Collectively, my work advances our knowledge of the innate immune mechanisms that control physiological and pathological events during pregnancy. These findings have implications to the field of immunology of pregnancy and may lead to clinical progress in diagnosis and prevention of infection-induced abortion in human pregnancies.

Introduction: Atopic dermatitis (AD) is characterized by chronic pruritic relapsing eczematous lesions of the skin. Eosinophilic inflammation in AD is driven by activation of type 2 inflammatory cells including CD4+ T cells and type 2 innate lymphoid cells (ILC2s). We have shown that type 2 cytokines, namely interleukin (IL)-5 and IL-13, stimulate migration and terminal differentiation of eosinophil progenitor cells (EoPs). We propose that these cytokines are important drivers of tissue eosinophilia in AD lesional skin. This study aimed to quantify, by flow cytometry, cells that produce type 2 cytokines in lesional skin compared to peripheral blood from moderate-severe AD patients. Methods: In a cross-sectional study of patients with moderate-to-severe AD (n=16), type 2 inflammatory cells were enumerated in blood and cells extracted from excised skin biopsies. By flow cytometry, live, singlet CD45+cells were identified as ILC2 (lin-CD127+CD294+), EoP (CD34+125+), and CD4+ T cells (Lin+CD3+CD4+). Intracellular expression of type 2 cytokines (IL-5 and IL-13) were evaluated in each cell population. In addition, we developed a protocol to enumerate ILC2s by fluorescence immune-histochemistry in lesional versus non-lesional skin samples and skin biopsies taken 24h post-intradermal challenge with allergen versus diluent. Data are expressed as median (interquartile range [IQR]) unless otherwise stated. Cross compartmental comparisons were made using the Wilcoxon rank-sum test and where applicable, correlational analyses were performed using a Spearman’s rank-correlational test. Results: There was a significantly higher number of total ILC2s in lesional skin compared to blood from AD subjects (556 [99 – 5501] vs 235 [67 – 569] cells/mL, p=0.03). Similarly, IL-5+, IL-13+ ILC2s, were significantly greater in skin compared to blood (6 [1 – 666] vs 1 [1 – 19] cells/mL, p=0.03; 28 [1 – 1357] vs 1 [1 – 7] cells/mL, p=0.01, respectively). We found higher numbers of total and type 2 cytokine positive EoP in lesional skin biopsies from AD patients compared to blood (Total EoP: 815 [285 – 2794] vs 112 [46 – 247] cells/mL, p<0.01; IL-5+EoP: 36 [1 – 129] vs 1 [1 – 23] cells/mL, p=0.07; IL-13+EoP: 92 [10 – 182] vs 1 [1 – 8] cells/mL, p<0.01 and IL-5+IL-13+ILC2: 70 [1 – 158] vs 1 [1 – 12] cells/mL, p=0.02, respectively). In contrast, significantly higher numbers of total and type 2 cytokine positive CD4+ cells were found in blood compared to lesional skin biopsies from AD patients (Total CD4+: 1092 [650 – 1742] vs 58.3 [35.3– 152.4] x 103 cells/mL, p<0.01 and IL-5+IL-13+CD4+ cells: 13.5 x 103 [2.1 x 103 – 42.9 x 103] vs 3.8 x 103 [1.6 x 103 – 4.9 x 103] cells/mL, p=0.02, respectively). For IF staining, there was a significant higher number of ILC2s in lesional compared to non-lesional skin biopsies and biopsies taken 24h post allergen- compared to diluent challenge (1 [0 – 2] vs 0 [0 - 0] cells/mm2, p=0.008, and 2 [1 – 2] vs 0 [0 – 0] cells/mm2, p=0.0002, respectively). Interestingly, in sex analyses we found significantly greater levels of blood ILC2 in females compared to males, but this not was found in the skin. Importantly, we found a significant correlation between lesional skin levels of ILC2 measured by flow cytometry and clinical measures of disease severity/symptoms as reported/calculated from the Patient-Oriented Eczema Measure questionnaire (POEM) score (total ILC2: r=0.55, p=0.04; IL-13+ ILC2s, r=0.61, p=0.02 and IL-5+ IL-13+ ILC2s: r=0.75, p=0.002). Conclusions: Preferential increases in skin-resident ILC2 that produce a type 2 rich environment were found in AD subjects. These levels correlated with patient-oriented measure of disease severity. We propose that this increase may encourage recruitment of mature eosinophils and EoP and possibly drive localized differentiation of EoP into mature eosinophils that may drive the pathology of AD lesions. Furthermore, immunofluorescence staining may be a suitable alternative to flow cytometry for identification of ILC2 in the event of a low cell count. These techniques can be used in future studies that target ILC2 biology to fully understand the role of these cells in driving AD. / Thesis / Master of Science (MSc)

Over the past four years, a previously unrecognised family of innate effector cells has been identified. Their comprehensive functional capabilities range from lymphoid organogenesis, tissue remodelling, wound healing, immune protection and homeostasis to contribution to inflammation and allergic responses. Here we investigate the presence and function of type 2 innate lymphoid cells (ILC2) in the skin. We show that human ILC2 are resident in human skin and express RORA and GATA3, and skin homing receptors. ILC2 further infiltrate the skin after allergen challenge, where they produce the type 2 cytokines IL-5 and IL-13. Skin-derived ILC2 express the IL-33 receptor ST2, which is up-regulated during activation. Signalling via IL-33 induces type 2 cytokine and amphiregulin expression, and increases ILC2 migration. Atopic dermatitis (AD) is a chronic inflammatory skin disorder. Current evidence suggests that both skin barrier dysfunction and immune system abnormalities, particularly those of a type 2 phenotype, contribute to disease pathogenesis. We demonstrated that ILC2 are enriched in lesional skin biopsies from atopic patients and show higher expression of cytokine receptors, reflecting an activated phenotype. Down-regulation of E-cadherin is characteristic of filaggrin insufficiency, a cardinal feature of AD. Interestingly, E-cadherin binding to KLRG1 on human ILC2 dramatically inhibits IL-5 and IL-13 production. ILC2 may contribute to increases in type 2 cytokine production in the absence of the inhibitory E-cadherin ligation through this novel mechanism of barrier sensing. CRTH2, a receptor for prostaglandin D₂ (PGD₂), is expressed by human ILC2. However, the function of CRTH2 in these cells is unclear. We sought to determine the role of PGD₂ and CRTH2 in human ILC2 and compare it with that of the established ILC2 activators IL-25 and IL-33. PGD₂ binding to CRTH2 induced ILC2 migration and production of type 2 cytokines IL-4, IL-5, IL-13 and release of other pro-inflammatory cytokines IL-3, IL-8, IL-9, IL-21, GM-CSF, and CSF-1 in a dose-dependent manner. ILC2 activation through CRTH2 also upregulated the expression of IL-33 and IL-25 receptor subunits (ST2 and IL-17RA) suggesting a synergistic role. The effects of PGD₂ on ILC2 could be mimicked by the supernatant from activated human mast cells and inhibited by a CRTH2 antagonist. Therefore, PGD₂ can be considered as an important and potent activator of ILC2 through CRTH2 mediating strong inflammatory responses. Cell surface interaction mechanisms that activate ILC2 function are unknown. We observed the expression of NKp30 on ILC2 ex vivo and after culture. Using quantitative PCR we confirmed that ILCs express NKp30c splice variant, an immune-modulatory isoform. Incubation of ILC2 with the NKp30 ligand B7H6 and tumour cell lines expressing this protein induced production of type 2 cytokines. This interaction can be inhibited by NKp30 blocking antibodies. We further established that activation of NKp30 induces the canonical pathway of NFƙB signalling. Overall the work in thesis shows for the first time that ILC2 are resident in human skin and infiltrate rapidly after allergen challenge and in AD lesional skin. We have defined cytokine and lipid mediators that contribute to migration and activation of ILC2 and shown that KLRG1 and NKp30 act as inhibitory and activatory receptors respectively. The work defines novel pathways for barrier sensing and cutaneous inflammation, and identifies potential new targets for therapeutic intervention.

Les cellules lymphoïdes innées (ILC) représentent une famille de cellules hématopoïétiques récemment identifiée, qui joue un rôle essentiel dans la réponse immunitaire précoce via la production rapide de cytokines. Trois groupes - ou types - d’ILC ont été définis selon l’expression de certaines molécules membranaires ou intracellulaires, ainsi que la production différentielle de cytokines. Les ILC du groupe 1 (ILC1) expriment le facteur de transcription(FT) T-BET et sécrètent des cytokines inflammatoires de la réponse immune de type 1, l’IFN-? et le TNF-?. Les ILC2 sécrètent des cytokines associées à la réponse immune de type 2,notamment l’IL-5 et l’IL-13, et ce de façon dépendante du FT GATA-3. Enfin, les ILC3 se caractérisent par la production de cytokines telles que l’IL-17 et l’IL-22, et expriment le FTROR?t. J’ai étudié en utilisant des techniques de biologie moléculaire et cellulaire, et à partir d’échantillons sanguins et tissulaire de donneurs sains ou de patients atteints de maladies inflammatoires chroniques, la fonction de ces trois groupes d’ILC chez l’homme. Ces travaux ont permis la construction d’un nouveau modèle de développement de ces cellules à partir de précurseurs. / Innate lymphoid cells (ILC) represent a novel family of hematopoietic effectors that serve essential roles in early immune response by rapid cytokines production. Three distinct groups of ILC subsets have been described. Group 1 ILC include cytotoxic natural killer (NK) cells and other type-1 cytokines (IFN-? and TNF-?) producing cells that regulated by T-BET. Group 2 ILC (ILC2) express GATA-3 and ROR?, secrete type-2 cytokines, IL-5 and IL-13. Group 3 ILC (ILC3) utilize ROR?t to drive production of the TH17-associated cytokines, IL-17 and/or IL-22. In this thesis, I have performed series of experiments to uncover the developmental pathway and function of human ILC that may allow us to harness ILC in diverse clinical settings. First, I analyzed the phenotypic and functional heterogeneity of human peripheral blood ILC2. I found human IL-13+ ILC2 can acquire the capacity to produce IFN-?, thereby generating ÔplasticÕ ILC2. ILC2 cultures demonstrated that IFN-?+ ILC2 clones could be derived and were stably associated with increased T-BET expression. The inductive mechanism for ILC2 plasticity was mapped to the IL-12/IL-12R signaling pathway and was confirmed through analysis of patients with Mendelian susceptibility to mycobacterial disease (MSMD) due to IL-12R?1 deficiencies that failed to generate plastic ILC2. This IL-13+IFN-?+ ILC2 are detected ex vivo in gut tissues from CrohnÕs patients. Second, I identified and isolated ILC precursors (ILCP) in peripheral blood of healthy donors. This circulating ILCP can give rise to four lineages of mature ILC including cytotoxic NK cells and helper ILC1, 2 and 3 in vitro and in vivo. Transcirptomic and epigenetic analysis showed ILCP have ILC-committed transcription factor profiles but have mature ILC signature locus at the epigenetics poised states. We further identified ILCP in various tissues including fetal liver, cord blood, postnatal lung and tonsil. Our result proposed a new model of ÒILC-poiesisÓ where circulating ILCP serve as cellular substrates to generate mature ILC subsets in tissues. Understanding the role of IL-12 on driving ILC2 to ILC1 plasticity may allow us to target plastic ILC2 in various diseases. The identification and isolation of ILCP from circulating blood allow further transfer into clinical setting for cellular therapy, especially for various diseases that ILC has been shown to be importance including infection, allergy, cancer and metabolic diseases.

La polyarthrite rhumatoïde (PR) est une maladie inflammatoire chronique d’étiologie inconnue. Les mécanismes physiopathologiques de cette maladie mettent en jeu un réseau cellulaire et cytokinique dont l’étude permet de définir des cibles thérapeutiques potentielles. L’IL-33 est une cytokine impliquée dans plusieurs maladies inflammatoires mais son rôle dans l’inflammation chronique comme la PR reste peu étudié. L’objectif de ce travail a été d’étudier le rôle de l’IL-33 et son mode d’action dans deux modèles expérimentaux d’inflammation chronique, l’arthrite au collagène (AEC) et le psoriasis induit par l’imiquimod (IMQ) chez la souris. Nous avons montré pour la première fois que l’administration d’IL-33 recombinante pendant les phases précoce et tardive de l’AEC, permet d’inhiber presque totalement les signes cliniques de la maladie. Cet effet protecteur passe par le déclenchement d’une réponse immunitaire de type 2 y compris l'expansion des ILC2, des éosinophiles et des cellules Th2. En outre, nous avons démontré que l’administration d’IL-33 dans notre modèle induit l’expansion de lymphocytes T régulateurs ST2L+ et une activité suppressive accrue. Dans un second temps, nous avons démontré que malgré l’expression de l’IL-33 dans les articulations ou la peau de souris développant une AEC ou un psoriasis induit par imiquimod, l’IL-33 endogène n’est pas nécessaire pour le développement de ces deux pathologies. En outre, l’absence de cette cytokine ne modifie pas la réponse des lymphocytes T ni dans l’AEC et ni dans le psoriasis. L'ensemble de ces résultats suggère que cette cytokine n’est pas cruciale pour le développement de l'inflammation chronique, et que la mise au point de traitement ciblant l’axe IL-33 / ST2 doit être envisagée avec précautions. / Rheumatoid arthritis (RA) is a chronic inflammatory disease that is associated with severalmediators. The physiopathological mechanisms of this disease involve cellular and cytokinenetworks whose study helps to define potential therapeutic targets. IL-33 is a cytokine involved inmany inflammatory diseases although its role in chronic inflammation such as RA remainsunclear. The aim of this work is to study the role of IL-33 and its mode of action in twoexperimental models of chronic inflammation, collagen induced arthritis (CIA) and imiquimod(IMQ) induced psoriasis. First, we showed for that the administration of recombinant IL-33 duringthe early and late phases of CIA inhibits the clinical signs of the disease in mice C57BL/6. Thisprotective effect is associated with the response type 2 including expansion of ILC2, eosinophilsand Th2 cells. Furthermore, we demonstrated that access of IL-33 in our model induced TregST2L and promotes the acquisition of regulatory phenotypes of Tregs. These Tregs achieve animportant suppressive activity leading inhibition of CIA. Secondly, despite the expression of IL-33 in the joint and skin of mice with CIA and IMQ induced psoriasis, endogenous IL-33 is notnecessary for the development of these two chronic inflammation models. In addition, the absenceof this cytokine does not alter the T cell response in the CIA or psoriasis. [...]

A breakdown of intestinal homeostasis due to dysregulated immune responses against intestinal bacteria underlies the pathogenesis of inflammatory bowel disease (IBD) in genetically susceptible individuals. Amongst mucosal immune cells, innate lymphoid cells (ILCs) are a heterogeneous group of cells whose functions in pathogenic inflammatory processes in the intestine are beginning to emerge from experimental murine models. However, less is known about the role of ILCs in chronic intestinal inflammation in humans. In this thesis, human ILCs were examined in the context of IBD and potential mechanisms by which these cells may contribute to IBD pathogenesis were investigated. We identified phenotypically and functionally distinct ILC1, ILC2 and ILC3 populations in the human intestinal lamina propria and peripheral blood and found that ILCs enriched for expression of IL-17A and IFNγ accumulated in the inflamed intestine, potentially through increased in situ proliferation and chemokine-mediated recruitment from blood. Based on their in situ localization, we investigated potential functional interactions between ILCs and CD4+ T cells and found that a proportion of human ILCs in peripheral blood and the intestinal lamina propria expressed HLA-DR and co-stimulatory molecules. ILCs were capable of taking up and processing protein antigen at levels equivalent to B cells, but in contrast to monocytes, antigen-pulsed ILCs failed to activate antigen-specific memory CD4+ T cells in vitro. Reciprocal activation between ILCs and monocytes enhanced the antigen-presenting potential and bactericidal capacity of myeloid cells and induced upregulation of co-stimulatory ligand expression by ILCs. This innate activation loop resulted in an augmentation of CD4+ T cell activation. These findings extend our knowledge of the complex interactions between human ILCs and other key immune cell populations, and suggest mechanisms by which rare ILCs may contribute to the pathogenesis of IBD by augmenting myeloid cell and CD4+ T cell responses.

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