Most mouse strains express either NK1.1 or NK2.1 NK-specific antigens. We have produced and characterized a mAb, 4LO3311, detecting the NK2.1 antigen expressed in most mouse strains. Strain-dependent variations in the frequency of splenic NK2.1$ sp+$ cells and in the cell surface density of NK2.1 were observed by flow cytometry, suggesting that 4LO3311 mAb actually detects a subset rather than all NK cells. In confirmation of this hypothesis, treatment of mice with 4LO3311 mAb had little effect on splenic NK cell activity although NK2.1$ sp+$ cells were no longer detected. Moreover, remaining NK cells in mAb-treated mice were totally resistant to further in vitro mAb plus complement treatment. Evidence of the high heterogeneity of the NK cell population was obtained in B6C3F$ sb1$ hybrid mice in which a relatively large subset of NK1.1$ sp-$ NK2.1$ sp-$ cells was detected in addition to those expressing either NK1.1, NK2.1, or both antigens. On the basis of the expression of several NK-specific and non-specific markers, NK2.1$ sp+$ cells appear themselves heterogeneous. Interestingly, immobilized 4LO3311 mAb induces granule exocytosis from IL-2-stimulated NK cells, thus identifying the NK2.1 antigen as a putative triggering structure. The capacity of NK cells to lyse anti-NK2.1 producing hybridoma cells is inhibited in the presence of soluble 4LO3311 mAb, whereas lysis of NK-susceptible targets is increased. The activation of the lytic activity through 4LO3311 mAb binding is Fc-independent and does not even require bivalency of the mAb. These findings are consistent with the NK2.1 antigen being involved in NK cell signaling. On the basis of the lytic activity of NK and related MHC non-restricted cells in selected Recombinant Inbred mouse strains of the AXB/BXA series and of the expression of NK-specific markers on their spleen cells, we finally established that, in this system, the lytic activity level of NK cells depends on variable combinations of at least 3 ef

Experimental Allergic Encephalomyelitis (EAE) is an autoimmune demyelinating disease of the central nervous system (CNS) with pathological and clinical features, including patterns of remission and relapse, reminiscent of the human disease Multiple Sclerosis (MS). EAE can be induced by immunization with myelin proteins in genetically susceptible rodents and can be adoptively transferred with CD4$ sp+$ T cells. In this thesis the mechanisms governing EAE induction in SJL/J mice were examined by flow cytometric analysis of the migration into the CNS of CD4$ sp+$ T cells labelled with a lipophilic fluorescent dye (PKH2). Selective retention was demonstrated within the CNS of myelin basic protein (MBP)-reactive CD4$ sp+$ T cell blasts, which were of a CD44$ sp{ rm high},$ CD45RB$ sp{ rm low},$ memory/effector phenotype and which expressed mRNA for IL-2 and IFN-$ gamma$ but not for IL-4. Mechanisms of immunoregulation were examined during remission and in mice preimmunized with irradiated T cells (T-cell-vaccination). A loss of CD4$ sp+$ T cells from the CNS was observed in animals that had remitted, but neither reversion of memory/effector phenotype nor increased numbers of regulatory CNS CD8$ sp+$ cells were observed. In vaccination experiments, evidence was obtained for rescue of background activation levels and enhanced proliferative responses to antigens within lymph nodes of vaccinated animals. The effector and regulatory mechanisms described in this thesis may further facilitate the development of effective T-cell-vaccination protocols for the control of cell-mediated autoimmune disease.

The study presented in this thesis is an investigation of M$ phi$ effector mechanisms that mediate the pathogenesis of acute graft-versus-host disease (GVHD). Since the time that the GVH reaction was first described the mechanisms underlying the development of the disease have not been characterized. We have examined the state of M$ phi$ activation in nonirradiated B6AF1 mice injected with either 60 $ times$ 10$ sp6$ (acute GVHD) or 30 $ times$ 10$ sp6$ (nonlethal GVHD) parental B6 lymphoid cells. TNF-$ alpha$ and nitric oxide (NO) generation by M$ phi$ occurs following priming by IFN-$ gamma$ and triggering by LPS. During the early phase of acute GVHD, administration of normally sublethal amounts of LPS triggered release of significant amounts of TNF-$ alpha$ into the serum resulting in death of the animals within 36 hours. The amount of serum TNF-$ alpha$ produced following LPS injection increased during the course of GVHD and was significantly greater in acute GVH reactive mice. Normal animals treated with the same dose of LPS neither died nor produced detectable amounts of serum TNF-$ alpha$. In vitro studies demonstrated that M$ phi$ undergo priming for both TNF-$ alpha$ and NO production during the developing GVH reaction and that priming was greater during acute GVHD than nonlethal GVHD. As a result of M$ phi$ priming during acute GVHD, M$ phi$ mediated the selective release of iron from $ sp{59}$Fe,$ sp{51}$Cr dual-labelled target cells and expressed cytostatic activity when triggered by LPS. NO generation and cytostasis mediated by M$ phi$ from acute GVH-reactive animals were inhibited by NMMA. Endogenous bacterial-derived LPS was detected in the livers and spleens of acute GVH reactive animals and appeared subsequently the serum coincident with the onset of mortality. Immunohistochemical staining for TNF-$ alpha$ during acute GVHD demonstrated TNF-$ alpha$ in the splenic red pulp and liver, however, the hepatic portal infiltrates that characterize acute / Our results demonstrate the entry of bacterial-derived LPS during the acute GVH reaction and that as a result of M$ phi$ priming, LPS in the transplant recipient acts as a trigger for TNF-$ alpha$ and NO production by M$ phi$. The presence of enteric Gram-negative bacteria is a risk factor for development of acute GVHD due to the triggering effect of LPS on M$ phi$ that are primed following bone marrow transplantation. The development of acute GVHD therefore involves the excessive priming of M$ phi$ followed by contact with LPS leading to the activation of NO and TNF-$ alpha$-mediated mechanisms of tissue injury, weight loss, septic shock and death of the transplant recipient.

The control of B lymphocyte production in bone marrow is of central importance in maintaining a supply of new B cells for primary humoral immune responses throughout life, while its perturbation may underlie states of immunodeficiency, neoplasia and autoimmunity. Molecules expressed by developing B cells and stromal cells within bone marrow, as well as systemic factors, may all play key roles in regulating proliferation and survival of precursor B cells in bone marrow. Candidate regulatory molecules investigated in the present work include surrogate light chains (SL) of immunoglobulin (Ig) expressed intracellularly by precursor B cells before and during the synthesis of CI heavy (H) chains of IgM, and interleukin (IL)-1, a systemic pleiotropic macrophage-derived factor. In vivo administration of radiolabeled mAbs specific for SL and IL-1 receptors type I and type II revealed that both SL and IL-1 receptors are normally expressed in bone marrow, displayed on the surface of lymphoid precursors and stromal cells, respectively. Ex vivo analysis of the DNA content of SL/$ mu$H$ sp+$ pre-B cells revealed a low incidence of apoptosis. Systemic administration of rIL-1 followed by analysis of precursor B cell dynamics using double immunofluorescence labeling and stathmokinetic techniques revealed that systemic IL-1 can either stimulate or depress B lymphopoiesis in a dose-dependent manner. Stimulation of cytoplasmic (c) $ mu sp+$ pre-B cell proliferation following activation of systemic macrophages by injecting sheep red blood cells (SRBC) is partially abrogated by rIL-1ra. Bone marrow IL-1 receptors, assayed by binding of radiolabeled anti-IL-1R monoclonal antibodies to plasma membrane fractions, undergo changes in levels of expression following SRBC administration. The work supports the hypotheses that surface SL is involved in the positive selection of precursor B cells with productively-rearranged $ mu$ chain genes, and suggests that the elevated proliferative level o

No description available.

This investigation was carried out in order to determine the specificity of a panel of monoclonal antibodies previously generated against murine pregnancy-associated splenic natural suppressor cells and to characterize the cell surface target molecule(s) on cells that are recognized by these antibodies. The monoclonal antibody from clone 2C1.1 was purified on anti-rat Ig affinity column and used to partially characterize the antigenic determinant recognized by it. A 50 KDa target molecule was detected on pregnancy spleen cells enriched for Natural Suppressor activity but not on similar cells from the virgin animals. Experiments employing a cellular ELISA technique demonstrated that the antibody bound specifically to NS cells. It is proposed that the naturally occurring pregnancy-associated suppressor cells as defined by our specific anti-NS monoclonal antibodies may have physiological relevance in maintaining a state of immunological equilibration between the maternal and fetal environments by non-specifically downregulating potentially deleterious maternal anti-fetal immune reactions.

No description available.

No description available.

Bone marrow transplantations frequently result in a graft-versus-host reaction (GVHR) even when donors and recipients are HLA matched. / In the present work with mice, an experimental protocol was designed to verify whether or not a GVHR ensuing immunosuppression can occur even in the absence of a difference at the level of the major histocompatibility antigens. / Injection of lymphoid cells from donors histoincompatible with recipients at the level of one or multiple minor antigens induced a GVHR resulting in a marked decrease of humoral response of the recipients, and also a lack of ability of their lymphocytes to respond to mitogens. / This immunosuppression was associated with the presence of suppressor cells in the spleen of these animals. Furthermore histopathological studies demonstrated that the thymuses from animals experiencing a late GVHR induced across multiple minor histocompatibility antigens, showed signs of dysplasia.

To define the borders of the domain in polyomavirus middle T antigen required for transformation, mutants of recombinant plasmids encoding the viral oncogene were constructed and tested for their capacity to transform Rat-1 cells. Several different mutations which were predicted to affect the structure of the amino terminus of middle T antigen rendered the DNA incapable of transforming Rat-1 cells as measured by the focus assay. The middle T antigens encoded by these non-transforming mutants were efficiently synthesized but failed to associated with protein kinase activity. By contrast, mutants that were not predicted to affect the structure of the amino terminus of middle T antigen transformed Rat-1 cells efficiently, and their encoded middle T antigens were associated with a kinase activity comparable to that of wt middle T antigen. The data suggest that the amino-terminal 100 amino acid residues of middle T antigen constitute part of a domain that is required for activation of the pp60$ sp{ rm c-src}$ kinase activity and for cellular transformation. / To determine the consequences of simultaneous overexpression of both middle T antigen and pp60$ sp{ rm c-src}$ in the same cells, two recombinant adenoviruses which individually encode middle T antigen and pp60$ sp{ rm c-src}$ were used to co-infect 293 cells. Evidence suggests that the capacity of middle T antigen to activate the intrinsic kinase activity of pp60$ sp{ rm c-src}$ is reduced when pp60$ sp{ rm c-src}$ is overexpressed. / To study the role of small T antigen in cellular transformation by polyomavirus, Rat-1 derived cell lines were established by co-transfection of a cDNA encoding only small T antigen together with a gene encoding resistance to G418. A number of these cell lines exhibited growth in agarose in the presence of a high serum concentration, but failed to induce tumours in isogenic rats. Unlike middle T antigen, small T antigen failed to detectably associate with pp60$ sp{ rm c-src}$ when both proteins were overproduced in 293 cells, suggesting that the mechanisms of action of small T antigen and middle T antigen are different. (Abstract shortened by UMI.)