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Organ specific neoantigen (OSN) is shed from cancer of patients with metastatic lung cancer and is filtered into the urine. The OSN was purified and characterized by physicochemical methods. OSN activity was detected by the blocking of the tube LAI assay. Two different methods are described for isolating the OSN. By both procedures material was isolated that consisted of three to four polypeptide chains in the molecular weight range of 43,000 to 31,000 dalton and that had high OSN activity. Urinary protein isolated from normal subjects by the same methods did not have OSN activity and also lacked the 31,000 mol. wt. protein. Immunoprecipitation studies with specific antisera did not show the presence of (beta)(,2) microglobulin or HLA molecules in the OSN material. Furthermore, lung tumor associated antigens such as carcinoembryonic antigen, lactoferrin and (alpha)(,1) antichymotrypsin were absent from the final isolate. / Material containing the lung tumor OSN is glycosilated and has a pI of 5.0 to 6.0. By two dimensional gel electrophoresis the OSN material was heterogeneous showing many spots from a pH of 6.2 to 7.2 but all had a molecular weight of 31,000 dalton.

The cellular basis for genetically-determined differences in resistance to infection with Listeria monocytogenes has been investigated in Listeria-resistant and Listeria-sensitive mouse strains. Adoptive transfer of specific immunity to naive recipients of sensitive and resistant genotypes has shown that the gene (Lr) controlling the level of resistance is expressed in the macrophage, rather than in the T-cell, response. In resistant mice, shortening of promonocyte generation time (T(,G)), together with prompt monocytosis and decreased half-time (T(, 1/2)) of circulating monocytes is seen following infection or injection of listerial cell wall extract; furthermore, the enhanced antilisterial response of resistant mice is highly radiosensitive indicating participation of an immigrant, inflammatory macrophage. Studies in chimeras constructed between sensitive and resistant strains have shown that high resistance is a property of the host environment in which the macrophage precursors mature. Thus, high antilisterial resistance is attributed to an Lr gene product which promotes early arrival of large numbers of young mononuclear phagocytes at infective foci, which destroy the pathogen.

The transmembrane potential changes ((DELTA)(PSI)) induced by the sensitizing cancer extract in leukocytes from patients with cancer were measured as part of their transmembrane signalling. Cancer extracts induce (DELTA)(PSI) changes in sensitized leukocytes, initiating the cascade of reactions that lead to leukocyte nonadherence to glass. Monocytes armed with cytophilic antitumor antibodies recognized the organ-specific cancer neoantigen (OSN) in extracts of allogeneic cancer and released leukotriene mediators that affected themselves and bystander leukocytes. Whereas the monocyte response is not MHC restricted, the T-cell response in the LAI and (DELTA)(PSI) assay is limited to extracts of autologous cancer and restricted by class I MHC antigens. (DELTA)(PSI) changes are seldom observed in leukocytes from patients with advanced cancer to either OSN or leukotrienes. The evidence suggests that these leukocytes are activated in vivo and are therefore refractory to a second in vitro stimulus. Increasing intracellular cyclic AMP restores transmembrane signalling and LAI.

The mechanisms by which lupus anticoagulant and antiphospholipid autoantibodies cause hemostatic abnormalities in patients with systemic lupus erythematosus (SLE) are poorly understood. We have approached this problem by investigating the binding and functional effects of human hybridoma lupus anticoagulant, anti-phospholipid and anti-DNA autoantibodies derived from SLE patients on platelets and endothelial cells. Most lupus anticoagulant antibodies did not bind to intact platelets and endothelial cells in vitro, while many antiphospholipid and anti-DNA antibodies were reactive. A comparison of SLE and normal-derived autoantibodies demonstrated that platelet-binding autoantibodies derived from SLE patients exhibited greater antigen specificity and platelet cytotoxicity than similar antibodies derived from normal individuals. By Western blotting analysis, many SLE-derived polyspecific antibodies reacted specifically with individual platelet proteins, whereas normal-derived polyspecific antibodies did not. One SLE-derived antibody, 9604 was found to react with ADP-activated platelets but not resting platelets. The reactive components in platelets were reducible polypeptides of approximately 200,000 and 32,000 molecular weight. These data suggest that some SLE autoantibodies may be able to interact with platelets and result in cell lysis or dysfunction in vivo.

Previous studies have established that sensitivity (IgE antibody response) to Ra5S, a 5 kd protein of short ragweed pollen, is significantly associated with host possession of HLA-Dw2/DR2 (Bias et al., 1979; March et al., 1982a; Coulter, 1983). The present work was undertaken to determine a possible association between HLA and sensitivity to Ra5G, an Ra5S homologue from giant ragweed pollen. The DR2 allele was also implicated in sensitivity to Ra5G, on the basis of co-sensitivity to both proteins (Goodfriend et al., 1985). Further analysis with an enlarged RW allergic population demonstrated that sensitivity to Ra5G and Ra5S is associated with separate alleles: DRw52 and DR2 respectively. Results consistent with the same sensitivity/DR associations were obtained in immunoabsorption studies with sera from co-sensitive individuals. As HLA-DR2 and DRw52 have identical alpha but different beta chains, it was considered that IgE antibody responses to Ra5S and Ra5G are associated with distinct DR-beta genes.

Microglia are myeloid cells that are long-term residents of the central nervous system (CNS). The active lesions in the CNS in multiple sclerosis (MS) show a heterogeneous array of myeloid cells, derived from recruitment of microglia and from infiltration of blood-derived cells. The central theme of this thesis relates to the distinct properties of myeloid cells and how they participate to the MS disease process. We investigated in vitro properties of microglia derived from the human CNS using blood-derived monocytes as well as monocyte-derived dendritic cells (DCs) and macrophages as comparators. First, we examined the ability of microglia to acquire myeloid DC properties. Monocytes differentiated with granulocyte/macrophage-colony stimulating factor (GM-CSF), interleukin (IL)-4 and subsequently activated with lipopolysaccharide (LPS), developed the phenotype and function of stimulatory DCs. Similarly treated adult microglia displayed few markers associated with DC phenotype, had reduced ability to sustain CD4 T cell alloreactive responses and produced IL-10. These data suggest that microglia may selectively contribute to an anti- versus pro-inflammatory response. We next examined the migration response of monocytes, macrophages and microglia to extracellular ATP and to the chemokine CCL2. Using a trans-well migration assay, we found that human microglia migration was enhanced in the presence of ATP but not CCL2. Monocyte migration was also enhanced with CCL2 and low concentrations of ATP, however, high concentrations of ATP decreased their basal migration. ATP had only an inhibitory effect on macrophage migration and CCL2 had no effect. These data demonstrate that there can be selective recruitment of distinct myeloid cell subsets to injury sites. Finally, we studied whether the cytokine response of microglia could be modulated by FTY720, a CNS-permeable immunomodulatory agent in clinical trials for MS. The production of IL-12 by adult microglia stimulated with / Les microglies sont des cellules myeloïdes qui résident au sein du système nerveux central (SNC). Les lésions actives du SNC caractéristiques de la sclérose en plaque (SP) contiennent un mélange hétérogène de cellules myeloïdes, provenant de l'activation des microglies ainsi que de l'infiltration de cellules sanguines. Le thème de cette thèse est l'étude des propriétés particulières de chacun des types de cellules myeloïdes. Nous avons caractérisé les propriétés in vitro des microglies provenant du SNC humain par rapport à celles des cellules myeloïdes sanguines – soit les monocytes, ainsi que les macrophages et cellules dendritiques (CDs) provenant de monocytes. Tout d'abord, nous avons examiné la capacité des microglies d'acquérir les propriétés de CDs. Lorsque que des monocytes sont différenciés en utilisant le facteur stimulant de colonies macrophages-granulocytes (GM-CSF) en combinaison avec de l'interleukine (IL)-4 et que ce procédé est suivi d'une activation avec de la lipopolysaccharide (LPS), les monocytes acquièrent les attributs et fonctions de CDs. Les microglies traitées de la même façon n'ont pas exprimé les marqueurs de CDs, étaient moins aptes à déclencher des réponses alloréactives et ont produit de l'IL-10. Ces données nous suggèrent que les microglies soutiennent des réponses anti-inflammatoires. Nous avons ensuite étudié la réponse migratoire de microglies, monocytes et macrophages envers l'ATP extracellulaire et la chimiokine CCL2. En utilisant un système «trans-well», nous avons constaté que la migration de microglies augmentait en présence d'ATP mais non de CCL2. Les monocytes ont migré davantage en présence de basses concentrations d'ATP, cependant, les concentrations plus élevées d'ATP ont eu un effet inhibiteur. Le CCL2 n'a eu aucun effet sur les macrophages et l'ATP a eu seulement un effet inhibiteur. Ces observations suggèrent que des signaux sélectifs régularisent le recrut

Dr. Fournier's laboratory has generated a mouse strain (L31 mice) that spontaneously develops a CD8+ T cell-mediated demyelinating disease in the central nervous system. In this model of dysregulated costimulation, CD4+ T cells have a regulatory role. A subset of CD4+ regulatory T cells that express the transcription factor Foxp3 have been shown to regulate autoimmune responses. In order to investigate this population's role in disease development, the goal of my M.Sc research project was to functionally characterize the CD4+Foxp3+ regulatory T cell population in L31 mice.We found that regulatory T cells from L31 mice were impaired in their ability to suppress the proliferation of effector T cells in vitro. In part, this was because B7.2 (CD86) expression impeded regulatory T cell suppressive activity. However, regulatory T cells delayed the onset of neurological symptoms in vivo. Although L31 Treg are not suppressive in vitro, our in vivo data suggest that they have a regulatory function in L31 disease development. This dichotomy could provide insights into the mechanisms by which these regulatory T cells control disease development in L31 mice. / Le laboratoire du Dr. Fournier a généré une lignée de souris (les souris L31) qui développe de façon spontanée une maladie du système nerveux central qui conduit à la perte de la gaine de myéline et qui est dépendante de la presence de lymphocytes T. Dans ce modèle les lymphocytes CD8+ sont les cellules effectrices de la maladie tandis que les lymphocytes T CD4+ jouent un rôle régulateur. Il a été démontré qu'une sous-population de lymphocytes T CD4+ qui expriment le facteur de transcription Foxp3 est impliquée dans la regulation des réponses auto-immunes. Afin d'étudier le rôle de cette population dans le développement de la maladie neurologique des souris L31, le but de mon projet de recherche était de caractériser de façon fonctionelle cette sous-population de lymphocytes T CD4+ régulateurs des souris L31. Nous avons trouvé que les lymphocytes T régulateurs des souris L31 sont altérés dans leur capacité à supprimer la proliferation de cellules T effectrices in vitro. Ceci est dû en partie à leur expression elevée de la protein B7.2 (CD86). Cependant, les lymphocytes T régulateurs des souris L31 sont capables de prévenir le développement des symptômes neurologiques in vivo. Donc, bien que les lymphocytes T régulateurs des souris L31 ne sont pas suppresseurs in vitro, notre données in vivo suggèrent qu'ils ont une fonction régulatrice dans le développement de la maladie neurologique des souris L31. Cette dichotomie pourrait nous permettre de déterminer les mécanismes utilisés par ces cellules régulatrices pour contrôler le développement de la maladie neurologique auto-immune dans les souris L31.

The T-cell protein tyrosine phosphatase (TC-PTP) is a widely expressed enzyme found at significantly higher levels in immune cells. To determine the signaling pathways and specific cell types affected by the loss of TC-PTP, TC-PTP gene-targeted animals were generated in our laboratory. TC-PTP -/- null mice have severe defects in hematopoiesis, elevated levels of inflammatory cytokines, and die by five weeks of age. These data indicate that TC-PTP plays an important role in hematopoiesis and immune cell activation. However, the identification of any substrates that could explain the role of TC-PTP these processes was lacking. Therefore, studies leading to the identification of physiological substrates of TC-PTP were undertaken. / Work in this thesis demonstrates that TC-PTP targets two members of the Janus family of tyrosine kinases (JAKs), JAK1 and JAK3. Both these JAK family members bound to TC-PTP in in vivo substrate trapping experiments, and hyperphosphorylation of JAK1 was demonstrated in TC-PTP-/- macrophages after treatment with interferon (IFN)-gamma. TC-PTP -/- null mice also have increased numbers of macrophages in the spleen. Studies demonstrated that TC-PTP targets the receptor for the colony-stimulating factor-1 (CSF-1), the primary growth factor that controls the proliferation, differentiation, and survival of macrophages. TC-PTP-/- macrophages have prolonged activation of Erk after CSF-1 stimulation. The number of CSF-1 dependent colony forming cells (CFU-Cs) is increased in TC-PTP-/- null mice, and TC-PTP-/- null hematopoietic progenitor cells produce more committed mononuclear phagocytic precursors in vitro . These data indicate that the loss of TC-PTP results in increased CSF-1 signaling and increased macrophage differentiation. Therefore, two novel classes of substrates for TC-PTP have been identified---members of the JAK family (JAK1 and JAK3) and the CSF-1R. Both substrates play important roles in hematopoiesis and immune cell activation, and their identification provides insight into the mechanism of TC-PTP function in vivo.

The B-cell specific enzyme Activation-induced deaminase (AID) is responsible for the diversification of rearranged antibody genes in activated B cells during humoral immune responses. AID deaminates dC to dU at the Immunoglobulin (Ig) loci and the uracil thereby introduced is processed in an error-prone fashion by specific DNA repair enzymes leading to somatic hypermutation (SHM), which underpins the affinity maturation of the antibody response, and to the DNA breaks necessary for class-switch recombination (CSR), which changes the isotype class of the antibody from IgM to IgG, IgA or IgE. Structure-function analysis of the AID protein was undertaken to better refine the 3-dimensional model of the protein previously published (Patenaude et al., 2009) which was based on a similar protein APOBEC2 whose structure has been previously elucidated (Prochnow et al., 2007). However, the C-terminal domain of AID (residues 182-198) is unique to AID and its structural details are lacking. This region is further interesting because the C-terminal domain, encoded by exon 5 of Aicda gene, has been shown to be essential for several of its biological functions: CSR, cytoplasmic retention, and nucleo-cytoplasmic shuttling. To better understand this region, and to define and identify potential functional domains, structure-function analysis was undertaken by creating truncations and point mutants and systematically assessing them for catalytic, SHM, and CSR activities. From such studies, it was identified that alpha-helix 6 (α6) is structurally important for AID catalytic activity while the C-terminal domain is not just dispensable for catalytic activity, but mutants lacking a portion of or all of the C-terminal domain are hyperactive for both catalytic activity and somatic hypermutation. We speculated that the hyperactivity of these mutants could account for the apparent dominant negative effect observed in a subset of patients with Hyper IgM type II (HIGM-II) syndrome, an immunodeficiency characterized by a normal or elevated SHM and lack or reduced switched isotypes in serum. While most loss of function mutations in AID are transmitted as autosomal recessive traits resulting in AID deficiency, in a subset of patients suffering from HIGM-II, Aicda mutations are inherited in an autosomal dominant (AD) manner. While the heterozygous carriers of the recessive alleles are asymptomatic, AD patients, who express one normal protein of AID along with a C-terminally truncated protein, show a similar HIGM-II immunodeficiency to AID-deficient patients and AID-null mice. This would suggest that the truncated AID protein has a dominant negative effect on the full-length protein. Two non-mutually exclusive possible explanations for this effect have been proposed: the truncated protein could form oligomers with full-length AID protein and render them non-functional, or the truncated protein could compete with the full-length protein for cellular factors involved in nucleo-cytoplasmic shuttling and/or other factors. We have investigated these possibilities and propose the alternative explanation that the hyperactivity of the truncated proteins leads to increased off-target mutations, DNA double-strand breaks, clonal expansion defects, and presumably p53-dependant apoptosis in primary B-lymphocytes. Our results correlate well with the clinical symptoms presented by AD patients: B cells with normal SHM and absence of the lymphadenopathies characteristic of AID-deficient patients. Furthermore, we identify a novel domain in alpha-helix 6 containing positively charged arginines that seem to be necessary for the enzyme's biological function, by mediating the interaction with a DNA targeting factor. / L'enzyme Activation-Induced Deaminase (AID), qui se trouve uniquement dans des lymphocytes B, est responsable pour la diversification des gènes d'anticorps réarrangés dans les cellules B actives au cours de la réponse immunitaire humorale. AID est responsible pour la déamination des dC à dU dans les gènes d'immunoglobulines (Ig). L'uracile ainsi introduit est traitée d'une manière erreurs par des enzymes de réparation conduisant l'hypermutation somatique (SH), promouvant la maturation de l'affinité de la réponse immunitaire, et des l'ADN brisées nécessaire pour la commutation isotypique (CS), qui change la classe isotype de l'anticorps d'IgM à IgG, IgA ou IgE. Analyse structure-fonction de la molécule AID a été réalisée afin d' améliorer la modèle en 3-D de la protéine, précédemment publiée (Patenaude et al., 2009). Cependant, le domaine C-terminale de l'AID (les résidus 182-198) est unique à l'AID, mais ses détails structurels font absents. Cette région est plus intéressant parce que le domaine C-terminale, codée par le cinquième l'exon du gène AICDA, a été révélée essentielle pour plusieurs de ses fonctions biologiques: CS, la rétention cytoplasmique, et pour le transport nucléo-cytoplasmique. Afin de mieux comprendre cette région et à définir et à identifier des nouveaux domaines fonctionnels, un rapport structure-fonction a été réalisée par la création de troncatures et des mutants ponctuels et les évaluer systématiquement pour les activités catalytiques, SH, et CS. De ces études, on a constaté que l'hélice alpha 6 (α6) est structurellement importante pour l'activité catalytique d'AID alors que le domaine C-terminale n'est pas seulement superflu pour l'activité catalytique, mais des mutants manquant une partie ou de la totalité du domaine C-terminale sont hyperactifs pour l'activité catalytique et hypermutation somatique. Nous avons supposé que l'hyperactivité de ces mutants pourrait expliquer l'effet apparent dominante négative observée dans un sous-ensemble de patients avec syndrome d'Hyper-IgM de type II (HIGM-II), une immunodéficience caractérisée par l'hypermutation somatique normale ou élevée et l'absence ou réduit d'anticorps de types IgG, IgA, et IgE. Alors que la plupart des mutations affectant le gène de la protéine AID sont transmises comme récessive autosomique résultant de carences de la protéine AID, dans un sous-ensemble de patients souffrant de HIGM-II, les mutations sont héritées dans une manière autosomique dominante (AD). Alors que des porteurs hétérozygotes des mutations récessifs sont asymptomatiques, les patients qui héritent des mutations dans une manière autosomique dominante expriment une protéine normale AID avec une protéine tronquée de côté C-terminale souffrent un déficit immunitaire. Cela suggère que la protéine AID tronquée a un effet dominante négative sur la protéine normale. Deux explications possibles ont été proposées: la protéine tronquée pourrait former des oligomères avec protéine AID normale et les rendre non-fonctionnelles, soit la protéine tronquée pouvait rivaliser avec la protéine normale pour des facteurs cellulaires impliqués dans le transport nucléo-cytoplasmique ou pour des autres facteurs. Nous avons étudié ces possibilités et on propose que l'hyperactivité des protéines tronquées conduit à une augmentation des mutations dans les gènes d'immunoglobulines, résultant en les défauts expansion clonale à cause d'apoptose dépendante sur p53 dans les lymphocytes B primaires. Nos résultats sont bien corrélés avec les symptômes cliniques présentés par les patients atteints de HIGM-II: les cellules B avec SHM normale et l'absence contrairement à les patients atteints du syndrome HIGM récessif autosomique avec d'adénopathies lymphocytiques. Nous aussi identifions une domaine nouvelle en hélice alpha 6 contenant arginines chargés positives qui semblent être nécessaires pour la fonction biologique de l'enzyme.