T cell development in the thymus is a highly evolved process. Signal transduction is critical to mediate maturation of T cells. Expression of pre-T cell receptor (pre-TCR) and TCR complex on cell surface allows signal transduction that is critical for T cell maturation. Four CD3 chains, which are gamma, delta, epsilon, and zeta, are associated with the two types of receptor complexes and play essential roles in T cell activation. While CD3gamma-, epsilon-, or zeta-deficient mice show impaired pre-TCR function, CD3delta deficiency impairs thymocyte development at the double positive (DP) stage indicating that it is not required for pre-TCR-mediated double negative (DN) to DP transition. / In the present thesis, we have assessed the role of two highly homologous CD3 chains, CD3gamma and CD3delta, in the DN to DP transition. We describe two mouse models to show that CD3delta is required for pre-TCR-mediated DN to DP transition. We also show that the cytoplasmic domain of CD3gamma plays a critical role in the DN to DP transition depending on the genetic background.

The interferon (IFN) regulatory factors (IRF) are transcriptional mediators of the cellular innate immune defense. Two of these factors, IRF-3 and IRF-7, are the principal inducers of type I IFN (IFN-alpha and -beta), which are key players in the antiviral response. Type I IFNs also exert strong antiproliferative and antiangiogenic effects and are widely used as adjuvants for therapy against cancer. When properly activated, macrophages can be tumoricidal making them attractive additions to standard cancer therapies. To this end, tolerance and activity of human autologous IFN-gamma-activated macrophages (MAK cells) have been assessed in pilot trials in cancer patients. In the present study, we tested the hypothesis that activation of IRF-3 and IRF-7 with subsequent type I IFN production may potentiate the antitumor functions of macrophages. Adenoviral vectors were generated to deliver constitutively active forms of IRF-3 (Ad-F3) or IRF-7 (Ad-F7) into human macrophages. Gene expression profiles of Ad-F3 or Ad-F7 transduced macrophages were assessed by microarray analysis. While IRF-3 strongly upregulated the expression of pro-apoptotic Noxa, IRF-7 poorly induced Noxa but upregulated Mcl-1- a Noxa-binding apoptosis inhibitor. Ad-F3 triggered rapid apoptosis and activation of the mitochondrial apoptotic pathway in macrophages. Active IRF-7 induced the expression of tumoricidal molecules (TRAIL, IFN-alpha and -beta), chemokines and genes involved in antigen processing/presentation. Ad-F7 macrophages exerted a cytostatic activity on different cancer cell lines and had an increased capacity to cross-present antigen. In conclusion, Ad-F3 or Ad-F7 appears to differentially regulate apoptosis and antitumor properties of macrophages, with active IRF-7 leading to the acquisition of antitumor effector functions. / Les facteurs régulateurs d'interferons (IRF) sont les médiateurs transcriptionnels de la réponse immunitaire innées. Deux de ces facteurs, IRF-3 et IRF-7, induisent l'expression des interferons de type I (IFN-alpha et -beta) importants pour la réponse antivirale et antitumorale. Les IFNs ont également des effets antiprolifératifs et antiangiogénics et sont couramment utilisés comme adjuvants en thérapie contre le cancer. Après activation, les macrophages presentment des activités antitumorales ce qui en font une alternative prometteuse au thérapie standard. À cet effet, la tolérance et l'activité des macrophages autologues humains activés à l'IFN-gamma (cellules MAK) ont été évaluées dans des essais pilotes chez des patients souffrants du cancer. Afin d'étudier le role d'IRF-3 et IRF-7 dans la regulation des fonctions antitumorales des macrophages, des vecteurs adénoviraux ont été produits pour livrer les formes constitutivement actives d'IRF-3 (Ad-F3) ou d'IRF-7 (Ad-F7) dans les macrophages humains. L' expression des gènes dans les macrophages transduits avec Ad-F3 ou Ad-F7 a été mesurée par microarray. IRF-3 induit fortement l'expression du gène pro-apoptotic Noxa. En revanche, IRF-7 induit faiblement Noxa mais induit Mcl-1 - un inhibiteur de l'apoptose qui interagit avec et inhibe Noxa. La transduction avec Ad-F3 induit l'activation rapide de la voie apoptotique mitochondriale dans les macrophages. La forme active d'IRF-7 induit l'expression des molecules tumoricidales (TRAIL et IFN-alpha et -beta) de chemokines et de genes impliqués dans la presentation d'antigènes. De plus, les macrophages transduits avec Ad-F7 exercent une activité cytostatique sur différentes lignées cancéreuses et une capacité de presentation-croisée accrue. En conclusion, Ad-F3 ou Ad-F7 semblent réguler l'apoptose et les propriétés anti-tumorales des macrophages de manière différente, l'expression d'IRF-7 menant à l'acquisition de fonctions e

T-dependent activation of B cell proliferation is crucial to humoral immune responses. Regulation of this activation is required to limit responses. The cyclin dependent kinase (CDK) inhibitor (CKI) p21cip-1/waf-1 is implicated in inhibition of cell cycle progression. This thesis describes experiments that investigated the role of p21cip-1/waf-1 in regulating B cell proliferation. C57Bl/6 splenic B cells were stimulated to enter cell cycle by crosslinking CD40. p21cip-1/waf-1 expression was upregulated by CD40 stimulation of B cells, and superinduced by co-crosslinking intercellular adhesion molecule 1 (ICAM-1)/CD54, major histocompatibility complex (MHC) I or MHC II with CD40. Treatments that superinduced p21 cip-1/waf-1 inhibited CD40-stimulated proliferation and induced apoptosis. Apoptosis was abrogated in p21cip-1/waf-1-/- B cells, which otherwise responded equivalently to controls. By contrast, B cells from p27cip-1/waf-1-/- mice showed the same induction of cell death as wild type mice. These results show that MHC and ICAM-1, specifically modulate CD40-mediated signals and this process involves p21cip-1/waf-1.

The maternal immune system exerts a key role in determining the outcome of mammalian pregnancy. It has been proposed that the high rate of early embryo loss (20--30%) in DBA/2-mated CBA/J female mice may be the consequence of a maternal non-speck immune rejection response to the allogeneic conceptus. Early embryo resorption in mice has been associated with the activation of natural killer like cells, macrophages, and production of TH1 inflammatory cytokines. To investigate and detect the earliest events in the activation of decidual NK cells and macrophages in initiating early embryo loss, gene expression analysis of two NK cell molecular markers, IFNgamma and perforin mRNA, and two macrophage specific cytokines, TNFalpha and NOS mRNA, was performed. Since IFNgamma is also produced by CD4+ T cells, cell labeling with an anti-NK antibody (DX5), cell sorting, and RT-PCR gene expression analysis were performed to confirm the source of IFNgamma mRNA in the decidua and spleen of DBA/2-mated CBA/J female. The role of the innate response during implantation, normal pregnancy, and early pregnancy loss will be discussed. (Abstract shortened by UMI.)

Although considered an autoimmune disease, the mechanisms underlying oligodendrocyte/myelin injury in multiple sclerosis (MS) remain to be established. The aim of this study was to evaluate the capacity and requirements for natural killer (NK) cells to induce cytotoxicity of autologous human adult central nervous system derived oligodendrocytes (OLs) as measured by cytotoxicity assays (51Cr release). Levels of cytotoxicity directed at either heterologous OLs or U251 glioma cell targets correlated with progressive enrichment for NK cells and were dependent on exposure of the NK cells to IL-2. We found that the IL-2 treated enriched NK cell preparations were capable of mediating significant cytotoxicity of autologous OLs. These results suggest IL-2 activated NK cells can induce autologous OL injury, bypassing putative protective effects of self-MHC class I molecules. The inflammatory milieu in MS lesions could provide conditions required for NK cell activation.

Experimental autoimmune encephalomyelitis (EAE) is a central nervous system (CNS) disease, with symptoms reminiscent of Multiple Sclerosis. During disease, the CNS is infiltrated by immune cells, with increased expression of MHC antigens and Th1 cytokines. / We asked whether antigen presentation in the CNS controls the bias towards Th1 cytokine in mice. We examined the capability of microglia to act as antigen presenting cells. We found that they stimulated T cell responses in vitro, but supported increased production of Th1 cytokines only. We also found that IL-12 mRNA was upregulated during peak disease, produced by resident microglia. We therefore propose that microglia, through their production of IL-12, influence the cytokine response in the CNS to produce the observed Th1 bias. / We investigated the effect of deletion of IFN-gamma on the ability to induce EAE in genetically-resistant (BALB/c) mice. IFN-gamma-/- mice were susceptible to induction of EAE, and cellular infiltrates in the CNS showed increases in message for CD3 and TNF-alpha. Therefore, IFN-gamma converts an otherwise EAE-susceptible mouse strain to become EAE-resistant. / We examined whether the negative effect of IFN-gamma on generation of encephalitogenic T cells in the periphery might differ from its role in the CNS. We interbred IFN-gamma -/- mice to mice transgenically overexpressing IFN-gamma in the CNS. Levels of IFN-gamma in CNS of the resulting mice were insufficient to induce spontaneous disease. These mice will be useful tools for studying the role of IFN-gamma in CNS disease. / During EAE, T cells of many specificities infiltrate the CNS. The function of those that do not recognize CNS antigens ("bystanders") is unclear. We transferred Ovalbumin-reactive (bystander) T cells to mice prior to EAE onset. Flow cytometry analysis showed that the vast majority of myelin basic protein-reactive T cells in the CNS were memory/effector cells, but only very few bystander T cells showed an activated/memory/effector phenotype. As a whole population, the bystander cells did not upregulate their expression of CD44 or IL-2Ralpha, or production of IFN-gamma, thus are probably not contributing to the ongoing immune response during disease.

In this study, biochemical techniques were used with immunological techniques to dissect the specific T cell reactivities to the PG molecule. Two T cell lines and two T cell hybridomas that belong to the T helper subset TH1 were isolated. The line JY.A recognized only the human fetal PG while the other line (JY.D) and the two T cell hybridomas (TH5 and TH14) recognized PG of all the different origins tested, except the rat chondrosarcoma and the mouse PG. The three latter T cells were found to recognize immunodominant T cell epitopes on the G1 domain of PG and cross-reacted with link protein. The presence of KS chain(s) close to T cell epitopes restricted the reactivity of the two T cell hybridomas. The location of one T cell hybridoma epitope (TH5) was contained within a peptide of twenty eight amino acids in the B region of G1. T cell line JY.A can induce early pathological change characteristic of rheumatoid arthritis when injected in the knee joint of naive BALB/c mice.

The properties shared by neoplastic and embryonic cells have long been recognized and it has been postulated that both cell types may express a common set of molecules not found in adult tissues of the same histologic type. This study has utilized a rabbit antiserum raised against human fetal spleen to identify 'oncofetal' antigens of the human lymphoreticular system. One molecule found to have oncofetal properties was hemoglobin F (HbF). A radioimmunoassay was developed to measure the very low concentrations of HbF present in the adult circulation and an extensive clinical trial was undertaken to determine the possible value of detecting elevated HbF levels in various pathological conditions. In addition, 2 other antigenic systems, present on the surface of lymphoid cells, were identified. Some preliminary information on the nature and distribution of these antigens is presented

No description available.

The current management of malignant gliomas is unsatisfactory compared to other solid tumors. Expected median survival is less than one year with even the best of care. At some point in their illness, most patients with neurological tumors are capable of mounting an immune response to their disease. This study focused on the humoral immune response of brain tumor patients by preparing human-human B cell hybridomas from autologous peripheral blood lymphocytes and a human myeloma-like cell line, designated as TM-H2-SP2. Eighteen fusions were successfully performed, and 15.8% of all microwells screened contained human immunoglobulin with anti-tumor activity. Five hybridomas, designated as BT27/1A2, BT27/2A3, BT32/A6, BT34/A5, and BT54/B8 were selected for detailed study. All five produced monoclonal IgM in a range of 2.4-44 $ mu$g/ml, had a similar (but not identical) pattern of reactivity against a panel of human tumor cell lines, and did not react with normal human astrocytes. All five human monoclonal antibodies (HmAbs) recognized a subpopulation of tumor cells based on multiparameter flow cytometric studies. Cell sorting experiments suggested that the identified subpopulation may share certain properties with hypothetical tumor stem cells. Preliminary antigen characterization indicated that the HmAbs are directed to cell surface glycolipids. These HmAbs possess certain properties of reactivity that suggest potential roles for them in the future diagnosis and clinical management of human malignant gliomas.